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https://openalex.org/W4200364985	https://zaguan.unizar.es/record/117367/files/texto_completo.pdf	English	null	Mindfulness-Based Program for Anxiety and Depression Treatment in Healthcare Professionals: A Pilot Randomized Controlled Trial	Journal of clinical medicine	2,021	cc-by	11,748	Córdoba and Guadalquivir, Institute Maimónides of Research Córdoba (IMIBIC), Reina Sofía University Hospital, University of Córdoba, 14001 Cordoba, Spain; langel.perula.sspa@juntadeandalucia.es 4 Carlos Castilla del Pino Health Center, Healthcare District of Córdoba and Guadalquivir, Institute Maimónides of Research Córdoba (IMIBIC), Reina Sofía University Hospital, University of Córdoba, 14001 Cordoba, Spain; aroldanvi@gmail.com Citation: Santamaría-Peláez, M.; González-Bernal, J.J.; Verdes-Montenegro-Atalaya, J.C.; Pérula-de Torres, L.Á.; Roldán-Villalobos, A.; Romero-Rodríguez, E.; Hachem Salas, N.; Magallón Botaya, R.; González-Navarro, T.d.J.; Arias-Vega, R.; et al. Mindfulness-Based Program for Anxiety and Depression Treatment in Healthcare Professionals: A Pilot Randomized Controlled Trial. J. Clin. Med. 2021, 10, 5941. https:// doi.org/10.3390/jcm10245941 Academic Editor: Laurent Boyer Received: 10 November 2021 Accepted: 13 December 2021 Published: 17 December 2021 p y p g p p j 4 Carlos Castilla del Pino Health Center, Healthcare District of Córdoba and Guadalquivir, Institute Maimónides of Research Córdoba (IMIBIC), Reina Sofía University Hospital, University of Córdoba, 14001 Cordoba, Spain; aroldanvi@gmail.com 5 Healthcare District of Córdoba and Guadalquivir, Institute Maimónides of Research Córdoba (IMIBIC), Reina Sofía University Hospital, University of Córdoba, 14001 Cordoba, Spain; emromerorodriguez@gmail.com 6 Mediterráneo-Torrecardenas Health Center, 04009 Almería, Spain; nurhachem@gmail.com 7 IIS-Aragon—Group B21-R17, Family and Community Medicine Teaching Department of Zaragoza Sector 1, Institute of Health Carlos III—REDIAPP 06/18, University of Zaragoza, 50018 Zaragoza, Spain; med000764@gmail.com 8 Emergency Service, University Hospital of Torrecardenas, 04009 Almería, Spain; teresagonzalezn@gmail.com 9 Castello Health Center (Madrid), Institute Maimónides of Research Córdoba (IMIBIC), Reina Sofía University Hospital, University of Córdoba, 14001 Cordoba, Spain; mrav98@gmail.com 10 Family and Community Medicine Teaching Department of Jaen, 23007 Jaen, Spain; f i j l d @j t d d l i 8 Emergency Service, University Hospital of Torrecardenas, 04009 Almería, Spain; teresagonzalezn@gmail.com 9 Castello Health Center (Madrid), Institute Maimónides of Research Córdoba (IMIBIC), Reina Sofía University Hospital, University of Córdoba, 14001 Cordoba, Spain; mrav98@gmail.com p y p g 10 Family and Community Medicine Teaching Department of Jaen, 23007 Jaen, Spain; franciscoj.valverde.sspa@juntadeandalucia.es j p j 11 Department of Educational Sciences, University of Burgos, 09001 Burgos, Spain; laminguez@ubu.es 11 Department of Educational Sciences, University of Burgos, 09001 Burgos, Spain; laminguez@ubu.es 12 Department of Psychology, Faculty of Teacher Training College, University of Extremadura, 11 Department of Educational Sciences, University of Burgos, 09001 Burgos, Spain; laminguez@ubu.es 12 Department of Psychology, Faculty of Teacher Training College, University of Extremadura, 10071 Caceres, Spain; bleon@unex.es * Correspondence: jejavier@ubu.es (J.J.G.-B.); mjgonzalez@ubu.es (J.G.-S.) Abstract: In primary health care, the work environment can cause high levels of anxiety and depres- sion, triggering relevant expert and individual change. Mindfulness-Based Stress Reduction (MBSR) programs reduce signs of anxiety and depression. The purpose of this sub-analysis of the total project, was to equate the effectiveness of the standard MBSR curriculum with the abbreviated version in minimizing anxiety and depression. This randomized controlled clinical trial enrolled 112 mentors and resident specialists from Family and Community Medicine and Nurses (FCMN), distributed across six teaching units (TU) of the Spanish National Health System (SNHS). Experimental group participants received a MBRS training (abbreviated/standard). Article Mindfulness-Based Program for Anxiety and Depression Treatment in Healthcare Professionals: A Pilot Randomized Controlled Trial Mirian Santamaría-Peláez 1 , Jerónimo Javier González-Bernal 1,* , Juan Carlos Verdes-Montenegro-Atalaya 2, Luis Ángel Pérula-de Torres 3 , Ana Roldán-Villalobos 4, Esperanza Romero-Rodríguez 5, Nur Hachem Salas 6, Rosa Magallón Botaya 7 , Teresa de Jesús González-Navarro 8, Raquel Arias-Vega 9, Francisco Javier Valverde 10, María Jiménez-Barrios 1, Luis Alberto Mínguez 11, Benito León-del-Barco 12 , Raúl Soto-Cámara 1 and Josefa González-Santos 1,* 1 Department of Health Sciences, University of Burgos, 09001 Burgos, Spain; mspelaez@ubu.es (M.S.-P.); mjb0007@alu.ubu.es (M.J.-B.); rscamara@ubu.es (R.S.-C.) 1 Department of Health Sciences, University of Burgos, 09001 Burgos, Spain; mspelaez@ubu.es (M.S.-P.); mjb0007@alu.ubu.es (M.J.-B.); rscamara@ubu.es (R.S.-C.) 2 Family and Community Medicine Teaching Department of Burgos, 09006 Burgos, Spain; juancarlosverdesm@yahoo.es 3 Multi-Professional Teaching Unit for Family and Community Care of Córdoba, Healthcare District of Córdoba and Guadalquivir, Institute Maimónides of Research Córdoba (IMIBIC), Reina Sofía University Hospital, University of Córdoba, 14001 Cordoba, Spain; langel.perula.sspa@juntadeandalucia.es 4 Carlos Castilla del Pino Health Center Healthcare District of Córdoba and Guadalquivir Institute 3 Multi-Professional Teaching Unit for Family and Community Care of Córdoba, Healthcare District of Córdoba and Guadalquivir, Institute Maimónides of Research Córdoba (IMIBIC), Reina Sofía University Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine 1. Introduction The level of health perceived by workers is closely related to the psychosocial compo- nents of the work environment. These components directly affect a person’s professional life, harming their physical and mental health, as well as the quality of life, contributing to the emergence and manifestation of various pathologies [1]. g p g The psychosocial environment of health professionals and especially primary health care personnel is characterized by a high degree of self-perceived stress and tremendous emotional and psychological demands. For this reason, these experts have a greater risk of developing anxiety and depressive disorders than the rest of the population [2,3]. In addition, the coronavirus pandemic has had a major impact on both these health experts’ quality of life and psychological health [4]. Some of the stressors are inherent in the health profession, such as long working hours, unpredictable work, dealing with pain, suffering, and death, or supporting families. However, there are other external components that can increase the degree of stress experi- enced by professionals, such as high load of work, staff shortages, and patients who are continuously asking for solutions for their problems and inconveniences, the greatest need for understanding, insufﬁcient time to continue learning and recycling their professional specialty, or a feeling of poor support from their managers [5–7]. In addition, as a side effect of the pandemic coronavirus disease, fear of infection, the likelihood of transmitting the disease to loved ones, conﬁnement, and even voluntary isolation increase the likelihood of stressful situations [8,9]. Regarding stress, the main effects on health professionals are burnout, anxiety dis- orders, and lateral violence [10]. Previous studies have shown that health care activities involve inherent stress that can damage high levels of cognitive functions, particularly attention and memory, and lead to anxiety and depression, with relevant individual and professional effects, including a decrease in patient satisfaction, decrease in job satisfaction, increase in medical errors, interpersonal interaction disruption, substance abuse, and other mental health problems [11–16]. Depression is a mood disorder that is accompanied by a loss of interest and a continu- ous sadness feeling. Several studies have reported a positive signiﬁcant correlation between the occurrence of signs of anxiety and mood and the level of job stress. In addition, there is also an important association between depressive disorders and other relevant health conditions, particularly chronic ones, such as a somatization disorder, chronic fatigue, and psychotropic drug consumption [17–20]. Depression and anxiety levels were measured with the Goldberg Anxiety and Depression Scale (GADS) at three different time periods during the analysis: before (pre-test) and after (post-test) participation, as well as 3 months after the completion of intervention. Taking into account the pre-test scores as the covariate, an adjusted analysis of covariance (ANCOVA) showed signiﬁcant depletion in anxiety and depression in general (F (2.91) = 4.488; p = 0.014; η2 = 0.090) and depression in particular (F (2, 91) = 6.653; p = 0.002; η2 = 0.128 at the post-test visit, maintaining their effects for 3 months (F (2.79) = 3.031; p = 0.050; η2 = 0.071—F (2.79) = 2.874; p = 0.049; η2 = 0.068, respectively), which is associated with the use of a standard training program. The abbreviated training program did not have a signiﬁcant effect on Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. 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/jcm J. Clin. Med. 2021, 10, 5941. https://doi.org/10.3390/jcm10245941 J. Clin. Med. 2021, 10, 5941 2 of 16 the level of anxiety and depression. The standard MBSR training program had a positive effect on anxiety and depression and promotes long-lasting effects in tutors and resident practitioners. New research is needed to demonstrate the effectiveness of abbreviated versions of training programs. Keywords: anxiety; depression; mindfulness; MBSR; primary care; mentors; resident intern specialists 1. Introduction Some researchers have shown the need of interventions to improve primary care specialists’ psychological health, especially during the early stages of pandemic coronavirus disease [21,22]. Once mindfulness training programs were compared with active control conditions, they proved to be a useful technique to reduce perceived stress and signs of anxiety and depression in health experts, improving different indices of psychological health and comfort, including quality of life, chronic pain, or emotional distress [23–28]. The results of a meta-analysis that included 209 clinical trials by Khoury et al. showed that mindfulness-based training was more effective in minimizing the severity of patients’ signs of anxiety and depression than the waiting list, psycho-education, therapeutic support, relaxation techniques, and imagery or suppression procedures. In addition, after a follow- up period of between 3 weeks and 3 years, and a median of 28 weeks after meditation, J. Clin. Med. 2021, 10, 5941 3 of 16 3 of 16 it was observed that the effects obtained after the mindfulness-based training program, remained over time [29]. Additionally, this class of interventions works through changes at speciﬁc points in psychopathology, such as cognitive function, emotional deregulation, and interpersonal effectiveness [25,30,31]. Kabat-Zinn deﬁned mindfulness originally in 1979 as the ability of paying attention to the end in the present moment without evaluating the development of one’s own experiences from moment to moment [32]. This type of meditation practice is based on attention’s self-regulation and consciousness in order to enhance mental processes’ control and increase personal well-being [33,34]. p g Self-compassion is understood as the function of responding to oneself in moments of failure or discomfort with instruction, decency, and understanding. This aspect is important for health professionals because they need to know how to respect and confess themselves in order to convey these emotions to the people to whom they provide their services. Self-compassion is an element of resilience associated with less psychopathology, stress, and greater comfort. It is related to mindfulness and is constantly included in training programs in order to improve the interaction between doctor and patient [35,36]. p g p p The MBSR training program, developed by Kabat-Zinn [37], is made up of eight group sessions per week lasting 2.5 h all together, and 45 min of practice at home, 6 days per week. The results of various meta-analyses on this program have shown useful results for mental and physical health in various clinical populations [38,39]. 2.1. Study Design The Mindfulness Teaching Units (MINDTU) study was a controlled multicenter empir- ical, cluster-randomized, open-label, pragmatic study of equal effectiveness with 3 parallel groups [44]. Its goal was to establish the impact of mindfulness and self-compassion program (MBRS) on mentors and resident FCMN professionals’ burnout and work stress. p g p The MINDTU clinical trial protocol specifying data collection procedures was previ- ously disclosed [44] and also registered with the ClinicalTrials.gov web portal maintained by the US National Library of Medicine under ID number NCT03629457. y y In this paper, a sub-analysis of participants’ levels of anxiety and depression is shown as part of secondary results from a clinical trial. 2.2. Recruitment and Participants This research was developed in Primary Care, and participants were selected in the next 6 Teaching Units (TU) of the SNHS: Almería, Burgos, Córdoba, Jaén, Ponferrada, and Zaragoza Sector I. The TUs were selected according to the population density of each territory, distributed across the Spanish geography. All mentors (n = 297) and internal specialist residents (n = 595) in FCMN at these units constituted the population under analysis. Eligibility criteria included being active and signing the informed consent form after receiving analysis data. Individuals who had followed a mindfulness module or seminar of minimum 4 weeks, who frequently practiced mindfulness techniques, were on long-term sick work leave due to pathology, or had a long-term illness, or with mental disorders that discourage the development of interventions were excluded. The analysis was disseminated through the communication options that exist in all 6 TUs. The likely participants were recruited through a face-to-face meeting of 1 h, where the objective, methodology, and voluntary nature of the analysis were described. They were invited to participate in the analysis, ﬁlling out, and signing the commitment term and the free and informed consent form. 2.3. Sample Size The feasible mean modiﬁcation in Mindfulness Five Facet Questionnaire (FFMQ) score, as the principal variable in this analysis, were used for the sample size estimation a priori. It was assumed a 0.05 alpha risk, a 0.20 beta risk, and a standard deviation (SD) of ± 20 points in bilateral contrast, with an expected drop-out rate of 25% throughout follow-up. There was a requirement of 140 participants (38 in each group) in order to detect the minimum difference of ≥15 points in the FFQM score of the experimental groups (EG) when they were compared to the control group (CG). The results of previous similar investigations were used to calculate this estimation [45,46]. In addition, the effects of the analysis type of its design were further examined once the sample measurement was calculated. Therefore, to obtain the same power between intra and intergroup variance, a multiplication factor of 1.7 was applied, assuming a cluster size of 15 and an intragroup correlation coefﬁcient < 0.05, similar to the most common in randomized clinical trials in Primary Care [47,48]. Assuming this value, 132 subjects, 44 in each group, and 22 for each TU, were considered sufﬁcient to detect clinically signiﬁcant differences in the primary variable of the study. 1. Introduction However, this program requires a signiﬁcant amount of time to complete the training, which is an obstacle for many people and limits its use in many situations [40]. Various investigations have tried to lessen the duration of these programs to 4 weeks for maintaining their effectiveness. In a systematic review, an abbreviated MBSR program was shown to be as effective as the standard training for maintaining the psychological management of health professionals. However, the samples of the different studies with which the abbreviated four-week MBSR program was implemented were reduced (16–20 participants), and none of them were mentors and resident specialists in Family and Community Medicine and Nurses (FCMN) [23]. The Spanish National Health System (SNHS) adopted the residency system for the postgraduate education of health specialists, such as Medicine and Nursing. This system is made up of Teaching Units (TU), which are the set of human resources and support materials, teaching, and research, necessary for regulated training in Health Sciences dis- ciplines, regardless of their ownership. After passing an annual national examination, licensed doctors and nurses have the possibility to complete a training period, ranging from 2 to 5 years, to become specialists in a special area of Medicine or Nursing; during this period, they are called resident internal specialists. Along the training process, they are expected to participate and progressively assume responsibilities in the diverse areas of competence of their own speciﬁc specialty program. The mentor is a key ﬁgure in this process. He is a professional with high experience and knowledge about patient care and he altruistically and voluntarily supervises resident internal specialist duties and activities [41–43]. Consequently, mentors and resident in-house specialists share responsi- bilities and expectations for education and learning similar to clinical practice. There are few studies conducted providing enough evidence on the effectiveness of the MBSR abbre- viated interventions in health specialists, especially in mentors and resident specialists in FCMN. Therefore, the objective of this sub-analysis was to analyze the effectiveness of two MBRS programs, an abbreviated and a standard one, on anxiety and depression in mentors and resident specialists in FCMN. J. Clin. Med. 2021, 10, 5941 4 of 16 2.4. Procedure and Randomisation The assessment and measurements of the participants were taken at three time points. In the baseline or initial evaluation visit (pre-test) carried out one week before the interven- tions began, the study variables of all participants from the different groups were collected. Consequently, in the ﬁnal evaluation (post-test), all participants were assessed again which took place 8 weeks after the baseline evaluation for those of the CG and EG2 participants and 4 weeks for the EG1. Furthermore, EG1 and EG2 participants were reassessed in the follow-up visit, 3 months after the intervention had ﬁnished, in order to verify the J. Clin. Med. 2021, 10, 5941 5 of 16 5 of 16 conservation or not of their outcomes over time. At the beginning of the study, there were 892 professionals available to participate; however, since many of them (n = 727) did not meet the inclusion criteria required to participate in the study or refused to participate in it, the ﬁnal sample was of a total of 165 participants (Figure 1). Figure 1. Flow participants’ chart through the research. Figure 1. Flow participants’ chart through the research. Every single one of the TUs analyzed was established as an independent cluster, and it was assigned randomly to the CG (2 TUs), the EG1 (2 TUs), or the EG2 (2 TUs). Partici- pants’ selection was performed in each TU and was also stratiﬁed by type of participant (66 mentors and 66 resident intern specialists). 2.5. Blinding Strategy Due to the intervention’s nature, it was not possible for the participants to be blinded. However, to prevent possible cross-contamination between groups, different strategies were used to obtain the highest possible level of blindness. The person who performed the training sessions in the EGs was different from the investigator who conducted the evaluation visits. In addition, both the researcher who conducted the evaluation visits and the one who carried out the statistical data analysis were blinded to the group to which the participants belonged. Clear guidelines were administered to all participants in order not to reveal, along the assessment sessions, the group to which each TU had been randomly assigned. J. Clin. Med. 2021, 10, 5941 6 of 16 2.6. Interventions EG1 and EG2 training programs were based on the Jon Kabat-Zinn’s MBSR training program developed at the University of Massachusetts Medical Center [49,50]. Mindful self-compassion (MSC) program practices were also incorporated to complement the inter- ventions [51–53]. The different sessions of each group were adapted to the characteristics of the health care professionals and differed in terms of the duration and time dedicated to the exercises [49,50]. EG1 participants belonged to an abbreviated training program, which consisted of 4 weekly sessions lasting 150 min and 15 min of daily practices at home. On the other hand, a standard MBSR program, consisting of 8 (one per week) sessions of 150 min with the daily practice for 30 min at home, was applied to the EG2 participants. All ses- sions were carried out in groups, looking for their practical application in the participants’ personal and/or professional ﬁeld. To this purpose, moments of collective exploration were alternated with other silent moments as the best systems for dealing with difﬁcult and complex situations. Power of emotions, perceived reality, knowledge of mindfulness, tolerance to emotional tension and stress, resilience, use of mindful communication, one- self-care, the management of time, and mindfulness integration into everyday life were some of the aspects that were promoted in the different sessions. The developed activities in each session were detailed in the study protocol previously [44]. Both training programs were incorporated in the TU and they were developed by the same accredited instructors. The therapist collected in a manual the standardized and uniform methodological criteria to follow, in order to make any variability associated with the instructors avoided. The par- ticipants in the CG received no intervention, but they attended to a 1 h information session, where the researchers explained their part in the research and they were invited to fulﬁll the assessment both in the pre- and post-test, coinciding with EG2. Participants agreed not to receive any intervention and not to participate in the activities of mindfulness training sessions or meditation techniques while the study was conducted. Once the ﬁeld work was ﬁnished, they were offered the chance to beneﬁt from a shortened training program. 2.7. Main Outcomes—Instruments 2.7. Main Outcomes—Instruments Participant’s anxiety and depression were the principal outcomes of the research, so, they were evaluated both in the pre-test, follow-up, and post-test evaluation sessions. y p p p Psychological symptoms of depression and anxiety were measured with the GADS [54]. Two subscales make up this test: one for the detection of anxiety (Goldberg Anxiety Scale, GAS) and the other for the detection of depression (Goldberg Depression Scale, GDS). Each subscale contains nine dichotomous questions answered by yes/no; where the ﬁrst four are mandatory, while the lasting ﬁve are only answered only in the case of any of the earlier ones are afﬁrmative. The evaluator asks the participant for the different symptoms included in the GDAS, alluding to the prior 15 days. For each afﬁrmative answer 1 point is added, not scoring in case of the negative one. The score of each subscale ranges from 0 to 9, while the GDAS from 0 to 18. When there is an afﬁrmative answer in four or more GAS’ items, the person is considered to have anxiety; regarding depression, if the person gives two or more afﬁrmative answers on the GDS it is considered that they have depression [54–56]. Montón Franco et al. validated GADS in Spanish population; it showed an adequate level on internal consistency with a Cronbach’s alpha of 0.81 for GADS, 0.74 for GAS, and 0.70 for GDS [57]. The participants’ attendance to person-to-person sessions was monitored so that the adherence to the training program could be measured. In addition, the participants had to write in a daily personal notebook or journal whether they did the exercises at home and show to the instructor in each session for supervision. All data of the participants that held an adequate adherence level were include in the statistical analysis. For this, it was considered that those participants had assisted to at least 3 of the 4 sessions in EG1 and 6 of the 8 sessions in EG2 had had an adequate adherence. q During the pre-test evaluation, information about the social and demographic vari- ables, like age, sex, job category (nurse or physician), type of participant (mentor or internal J. Clin. Med. 2021, 10, 5941 7 of 16 7 of 16 resident specialist), workplace (clinic or hospital), time that they had been working in the SNHS, was also collected to evaluate potential predictor or confounding effects. 2.9. Ethical Considerations This research’ protocol was accepted by the Clinical Research Ethics Committee of the Reina Sofía University Hospital in Córdoba (Spain) with reference 3845. Before entering the study, all participants knew the objective and its possible risks and beneﬁts. The written and signed declaration of consent was granted by each person in accordance with the Declaration of Helsinki recommendations. The data collected were not used for purposes different than those exposed in the written declaration of consent or was passed on to others outside the investigation. The participants’ data conﬁdentiality was guaranteed in accordance with the Organic Law 3/2018 of 5 December, on the Protection of Personal Data and Guarantee of Digital Rights (Law 14/2007 of 3 July), on Biomedical Research, and Regulation 2016/679 of the European Parliament and of the Council of 27 April 2016, on the General Data Protection of Natural Persons in the Processing of Personal and Free Circulation of these data. 2.8. Procedure for Data Collection, Management and Monitoring In the evaluation and the follow-up assessment visits, the measurement and data collection were conducted by same researcher, who had been speciﬁcally trained for these tasks previously. Another member of the research team carried out the randomization process, different from the one responsible for the later statistical data analysis. Each participant was assigned a unique alphanumeric code to make easier the identiﬁcation of the data collected in the different evaluations within the study. This code consisted of 6 numbers, which corresponded to the participant’s date of birth (day/month/year in dd/mm/yy format), as well as the ﬁrst two initial letters of their name and surname. A database was created, which only researchers from the investigation group involved in the statistical analysis had access. All the questionnaires used a double data entry procedure in order to keep the lowest error rate. The principal researcher was the responsible for adapting the procedures to the protocol and performing the weekly study monitoring and the database cleaning and debugging. 2.7. Main Outcomes—Instruments A more detailed description of these variables can be found in the clinical trial protocol [44]. 2.10. Statistical Analysis The results were analyzed on an intent-to-treat basis, in order to copy with with- drawals, losses, deviation from the protocol, or anything that happened after randomiza- tion. Continuous variables were expressed in mean and SD, while categorical variables were summarized as frequencies distribution and percentages. In continuous variables, the Kolmogorov–Smirnov test was used in order to verify the normality criteria; the Runs test was used to contrast the assumption of randomization; and the Levene test was used to evaluate the homoscedasticity’s equality. In all cases, p > 0.05 was observed, being justiﬁed the use of parametric tests. The comparability between the three groups in the pre-test evaluation, in terms of sociodemographic variables, was assessed with chi-squared test or Student’s t test for independent samples. The one-way variance analysis (ANOVA) test was used to analyze the MBRS training program effect on the different study groups participants’ anxiety and depression. To compare the mean anxiety and depression scores in each group over time, a repeated measures ANOVA test was performed. The absence or presence of sphericity was calculated using the Mauchly’s W test, performing the Greenhouse–Geisser correction when necessary. Multiple comparisons were performed with the Bonferroni correction. The squared eta coefﬁcient (η2) was calculated for the estimation of interventions’ effect side on the anxiety and depression levels; the results were interpreted in consonance with these criteria: if 0 ≤η2 < 0.05, no effect; if 0.05 ≤η2 < 0.26, minimal effect; if 0.26 ≤η2 < 0.64, moderate effect; and if η2 ≥0.64, strong effect [58]. Finally, in order to eliminate the effect that could be attributable to variables that were not J. Clin. Med. 2021, 10, 5941 8 of 16 8 of 16 included in this research’ design and to analyze the effects of the intervention, the changes between the CG and EG in post-test and follow-up punctuations were compared by a covariance analysis test (ANCOVA), with the pre-test scores of the dependent variables as covariate and the intervention groups as a ﬁxed factor. The contrasting hypothesis estab- lished as the limit of statistical signiﬁcance an alpha risk of 0.05. The data were analyzed using the SPSS Statistics software for Windows, version 25.0 (IBM SPSS Inc., Chicago, IL, USA) and MLwiN software, version 3.0 (Center for Multilevel Modelling, University of Bristol, Bristol, UK, 2019). 3.1. Baseline Aspects of the Participants in this Research Out of the 165 participants that were included in the study (63 in CG, 39 in EG1, and 63 in EG2) there were 38 losses due to the refusal to a continuous participation in the research and 15 because due to an inappropriate program adherence. This dropout rate was higher in EG2 (n = 26) than in EG1 (n = 15), being the main reason a refusal to continue to participate in the program. As a result, 112 participants completely ﬁnished the research and were included for the analysis of the data, 51 belonged to the CG, 24 to the EG1, and 37 to the EG2. (Figure 1). g Table 1 shows the participants’ baseline social and demographic characteristics related to the study group. Most of the participants were women (n = 86, 76.79%), and the mean age of the sample was 40.61 years (SD ± 12.61); 84.82% of participants (n = 95) were Primary Care workers, being the physician the most represented category of professionals (n = 95; 84.82%). The mean time that they had been working was 12.88 (SD ± 13.15) years. The sample showed a uniform distribution uniformly to mentors and resident intern specialist (62 versus 50). In the initial evaluation, statistically signiﬁcant differences between the three groups were found regarding work experience, job category and age. Table 1. Baseline participants’ aspects. Variable Total n = 112 CG n = 51 EG1 n = 24 EG2 n = 37 p-Value Age (in years) 41.61 ± 12.61 40.34 ± 13.22 47.66 ± 13.67 35.73 ± 12.04 <0.001 Sex Male 26 (23.21) 11 (21.57) 6 (25.00) 9 (24.32) 0.978 Female 86 (76.79) 40 (78.43) 18 (75.00) 28 (75.68) Occupation Physician/medicine 95 (84.82) 41 (80.39) 20 (83.33) 34 (91.89) 0.165 Nurse 17 (15.18) 10 (19.61) 4 (16.67) 3 (8.11) Professional type Tutor 50 (44.64) 24 (47.06) 15 (62.50) 11 (29.73) <0.001 Resident 62 (55.36) 27 (52.94) 9 (37.50) 26 (70.27) Workplace Health Center 95 (84.82) 40 (78.43) 22 (91.67) 33 (89.19) 0.217 Hospital 17 (15.18) 11 (21.57) 2 (8.33) 4 (10.81) Work experience (years) 12.88 ± 13.15 13.13 ± 12.95 19.49 ± 13.91 8.91 ± 11.06 <0.001 Values expressed in mean ± standard deviation or frequencies (percentages). Abbreviations: CG: Control Group; EG1: Experimental Group, 4 weeks; EG2; Experimental Group, 8 weeks. Table 1. Baseline participants’ aspects. Table 1. Baseline participants’ aspects. J. Clin. Med. 2021, 10, 5941 9 of 16 3.2. Anxiety and Depression 3.2. Anxiety and Depression In baseline evaluations, it was found no statistically signiﬁcant differences between the CG, EG1, and EG2 in the GADS (p = 0.500), GAS (p = 0.495), and GDS (p = 0.615), which indicated the equivalence between the groups before starting the MBRS training programs. However, when comparing the scores of the post-test evaluation, statistically signiﬁcant differences were observed in the GADS and GDS, with a weak effect size (η2 = 0.079 and η2 = 0.114, respectively). In both cases, these differences were observed between CG and EG2, with higher mean scores in the ﬁrst of them, according to the results of the pairwise comparisons by Bonferroni test. Likewise, in the follow-up evaluations, the differences in the mean scores of the GADS, GAS, and GDS were also statistically signiﬁcant, with a weak effect size (η2 < 0.26). The CG participants showed higher mean scores in these three variables with respect to those of EG2 (Table 2). Inter-group comparison of GADS, GAS, and GDS at different evaluation moments. One-way ANOVA. Table 2. Inter-group comparison of GADS, GAS, and GDS at different evaluation moments. One-way ANOVA. Group Assessment CG EG1 EG2 F p-Value η2 Mean SD Mean SD Mean SD GADS Pre-test 8.20 4.28 7.10 5.20 7.68 4.55 0.697 0.500 0.009 Post-test 7.82 * 4.64 5.82 5.51 4.82 * 3.88 5.227 0.007 0.079 Follow-up 8.35 * 4.19 6.41 5.63 5.18 * 3.72 5.725 0.004 0.095 GAS Pre-test 5.20 2.59 4.53 2.99 4.96 2.77 0.705 0.496 0.009 Post-test 4.64 2.68 3.57 2.94 3.46 2.70 2.645 0.075 0.042 Follow-up 5.15 * 2.41 4.12 3.08 3.54 * 2.57 4.238 0.017 0.072 GDS Pre-test 3.00 2.27 2.56 2.56 2.71 2.13 0.487 0.615 0.006 Post-test 3.17 * 2.35 2.25 2.81 1.36 * 1.51 7.823 0.001 0.114 Follow-up 3.19 * 2.28 2.29 2.88 1.64 * 1.60 5.252 0.007 0.088 * p-value < 0.05 in post-hoc Analysis (Bonferroni test) between CG and EG2. Abbreviations. CG: Control Group; EG1: Experimental Group, 4 weeks; EG2: Experimental Group, 8 weeks; SD: Standard deviation; GADS: Goldberg Anxiety and Depression Scale; GAS: Goldberg Anxiety Scale; GDS: Goldberg Depression Scale. Table 2. Inter-group comparison of GADS, GAS, and GDS at different evaluation momen * p-value < 0.05 in post-hoc Analysis (Bonferroni test) between CG and EG2. Abbreviations. 3.2. Anxiety and Depression ANOVA for $ p-value < 0.05 in post-hoc Analysis (Bonferroni test) between pre-test and post-test. Abbreviations. SD: Standard deviation; MS: Mean Square; CG: Control Group; EG1: Experimental Group, 4 weeks; EG2: Experimental Group, 8 weeks; GADS: Goldberg Anxiety and Depression Scale; GAS: Goldberg Anxiety Scale; GDS: Goldberg Depression Scale. mparison between groups: post-test–follow-up punctuations with pre-test scores control. ANCOVA. Table 4. Comparison between groups: post-test–follow-up punctuations with pre-test scores control. ANCOVA. Evaluation Variable Source Type III Sum of Square df MS F p-Value η2 Post-test GADS Pre-test GADS 656.61 1 656.61 49.880 <0.001 0.354 CG/EG1/EG2 118.15 2 59.08 4.488 0.014 0.090 Error 1197.91 91 13.16 GAS Pre-test GAS 195.23 1 195.23 37.249 <0.001 0.388 CG/EG1/EG2 24.53 2 12.26 2.340 0.102 0.128 Error 476.95 91 5.24 GDS Pre-test GDS 166.40 1 166.40 57.660 <0.001 0.388 CG/EG1/EG2 38.40 2 1.92 6.653 0.002 0.128 Error 262.62 91 2.87 Follow-up GADS Pre-test GADS 413.79 1 413.80 29.497 <0.001 0.272 CG/EG1/EG2 85.04 2 42.52 3.031 0.050 0.071 Error 1108.26 79 14.03 GAS Pre-test GAS 107.98 1 107.98 18.644 < 0.001 0.191 CG/EG1/EG2 25.78 2 12.89 2.226 0.115 0.053 Error 457.52 79 5.79 GDS Pre-test GDS 108.94 1 108.94 32.063 <0.001 0.289 CG/EG1/EG2 19.53 2 9.77 2.874 0.049 0.068 Error 268.42 79 3.40 Abbreviations. df: Degrees of Freedom; SD: Standard deviation; MS: Mean Square; CG: Control Group; EG1: Experimental Group, 4 weeks; EG2: Experimental Group, 8 weeks; GADS: Goldberg Anxiety and Depression Scale; GAS: Goldberg Anxiety Scale; GDS: Goldberg Depression Scale. Table 4. Comparison between groups: post-test–follow-up punctuations with pre-test scores control. Abbreviations. df: Degrees of Freedom; SD: Standard deviation; MS: Mean Square; CG: Control Group; EG1: Experimental Group, 4 weeks; EG2: Experimental Group, 8 weeks; GADS: Goldberg Anxiety and Depression Scale; GAS: Goldberg Anxiety Scale; GDS: Goldberg Depression Scale. 3.2. Anxiety and Depression CG: Control Group; EG1: Experimental Group, 4 weeks; EG2: Experimental Group, 8 weeks; SD: Standard deviation; GADS: Goldberg Anxiety and Depression Scale; GAS: Goldberg Anxiety Scale; GDS: Goldberg Depression Scale. In the intragroup comparisons, a signiﬁcant reduction in the mean scores of the post- test evaluation in relation to those of the pre-test in the GADS and GDS was observed in the subjects that attended to the standard program, with signiﬁcant and minimal effect sizes (η2 < 0.26). However, no statistically signiﬁcant differences were obtained in the mean anxiety and depression scores at the follow-up evaluation in relation to pre or post-test one (Table 3). ( ) Table 4 summarizes the comparison between CG, EG1, and EG2 in the post-test and follow-up punctuations, with pre-test scores control, using ANCOVA. This analysis showed statistically signiﬁcant differences in the GADS and GDS variables, between different groups, results that conﬁrm the previous intergroup comparisons. Thus, differences, primarily in EG2, may be ascribed to the MBRS training program (Table 4). J. Clin. Med. 2021, 10, 5941 10 of 16 Table 3. Intra-group comparison of GADS, GAS, and GDS at the same evaluation moment. ANOVA for repeated measures. Variable Group Pre-Test Post-Test Follow-Up MS F p-Value η2 Mean SD Mean SD Mean SD GADS CG 8.20 4.28 7.82 4.64 8.35 4.19 6.131 0.806 0.451 0.025 EG1 7.10 5.20 5.82 5.51 6.41 5.63 7.314 0.850 0.437 0.050 EG2 7.68 $ 4.55 4.82 $ 3.88 5.18 3.72 33.722 3.224 0.040 0.123 GAS CG 5.20 2.59 4.64 2.68 5.15 2.41 1.939 0.750 0.476 0.023 EG1 4.53 2.99 3.57 2.94 4.12 3.08 4.843 1.120 0.339 0.065 EG2 4.96 2.77 3.46 2.70 3.54 2.57 10.597 2.194 0.123 0.087 GDS CG 3.00 2.27 3.17 2.35 3.19 2.28 1.768 0.752 0.476 0.023 EG1 2.56 2.56 2.25 2.81 2.29 2.88 0.961 0.622 0.543 0.037 EG2 2.71 $ 2.13 1.36 $ 1.51 1.64 1.60 6.514 3.583 0.036 0.135 $ p-value < 0.05 in post-hoc Analysis (Bonferroni test) between pre-test and post-test. Abbreviations. SD: Standard deviation; MS: Mean Square; CG: Control Group; EG1: Experimental Group, 4 weeks; EG2: Experimental Group, 8 weeks; GADS: Goldberg Anxiety and Depression Scale; GAS: Goldberg Anxiety Scale; GDS: Goldberg Depression Scale. comparison of GADS, GAS, and GDS at the same evaluation moment. ANOVA for repeated measures. Table 3. Intra-group comparison of GADS, GAS, and GDS at the same evaluation moment. 4. Discussion In this research, a standard MBRS program effects among mentors and resident intern specialists of FCMN were examined; these effects were also compared to those of another abbreviated one. The potential beneﬁts of both training programs on anxiety and depression were been studied. The main results showed an improvement in the GADS and GDS scores in the participants who attended to the standard program, with the J. Clin. Med. 2021, 10, 5941 11 of 16 11 of 16 effects’ maintenance over time. However, the abbreviated training program on anxiety and depression levels showed no signiﬁcant impact. MBRS training programs have proven to be very useful in emotional regulation im- provement; and to the depletion of anxiety and depression levels, and post-traumatic stress disorders [59–61], characteristic symptoms during the COVID-19 pandemic, especially among health care workers [33]. Several studies have also shown an increase in the con- sciousness level, an improvement in coping strategies in times of stress, a greater emotion control, and a signiﬁcant anxiety and depression levels reduction when these interventions were performed [62–64]. Goyal et al. completed a systematic review of 47 clinical trials, where the aim was to analyze mindfulness and meditation training programs efﬁcacy on stress in the general population, and concluded that these techniques gave moderate evidence of improved anxiety at 8 weeks (η2 = 0.38; 95% conﬁdence interval (CI) 0.12–0.64) and at 3–6 months (η2 = 0.22; 95% CI 0.02–0.43), as well as depression at 8 weeks (η2 = 0.30; 95% CI 0.00–0.59) and at 3–6 months (η2 = 0.23; 95% CI 0.05–0.42) [27]. On the other hand, a 38 randomized clinical trial meta-analysis performed by Spinelli et al. focused on the effect of mindfulness activities on qualiﬁed and trained health care professionals [65]. In this review it was highlighted that mindfulness training program helped signiﬁcantly to reduce anxiety (Hedge’s g = 0.47; 95% CI 0.27–0.67) and depression (Hedge’s g = 0.41; 95% CI 0.26–0.57) at post-intervention. Most of the research to date has demonstrated the multiple beneﬁts of standard 8-week MBRS programs on healthcare professionals [23,65]. Some authors have analyzed these training programs’ effectiveness when implemented in a shorter period of time, such as 4 or 3 weeks [50,66–69]. In all cases, a signiﬁcant reduction in anxiety and depression levels was observed, maintaining their effects at 9 months follow-up [50]. 4. Discussion 2021, 10, 5941 12 of 16 12 of 16 program effects on psychiatric and depressive symptoms and negative effects in 84 primary healthcare professionals, and observed a statistically signiﬁcant depletion in all of the variables analyzed after the intervention [76]. In this kind of training program, the effects that are achieved in the short term must be considered with the same importance as their maintenance along time. Fortney et al., observed the continuity of signiﬁcant reductions in depression levels after a 9-month follow-up [50]. In the study by Lane et al., the mindfulness training program not only reduced negative symptoms such as depression in a sample of 200 subjects with different professions, but its beneﬁts were maintained over time 3 months after the intervention had already ended [40]. These results are in line with those obtained in this study, since the decrease in depression levels obtained in EG2 in the post-test were maintained during the three subsequent months. In order to objectively analyze the psycho-physiological effects of MBSR and assess the changes pre- and post-intervention, different studies have suggested the use of the event-related brain potentials or EEG [33,77]. This sub-analysis is part of a global project in which variables such as levels of mindfulness, self-compassion, and self-perceived empathy in these professionals were also analyzed. However, unlike the results obtained in the levels of anxiety and depression, it was observed that the abbreviated program of 4 weeks did not produce improvements on the mentioned aspects; although, the standard program of 8 weeks did allow improvements in mindfulness and self-pity to be obtained [78]. The present study is innovator in the comparison of the effects of an abbreviated MBSR and MSC training program with a standard one on anxiety and depression levels in mentors and resident intern specialists in Spain. However, these ﬁndings must be taken into account among the context of their strengths and limitations. Among the principal strengths, there were: the possibility of determining the causal relationship between variables through the longitudinal methodology; the guarantee of obtaining valid information and reducing the probability of information biases thanks to the use of instruments that were validated for the Spanish population; the baseline anxiety and depression levels were equivalent so that all participants were considered to start from a similar situation, as well as the evaluation of the continuity of the effect over time. 4. Discussion However, the number of studies comparing the effectiveness of abbreviated and standard interventions in health professional is very limited. De Marzo et al. compared the effectiveness of an 8-week MBRS training program and a 4-week-abbreviated version for well-being improvement in a sample of Health Sciences undergraduate students, and concluded that both types of programs worked in the same way [70]. Kriakus et al. performed a meta-analysis with the aim to update the latest data related to the efﬁcacy of mindfulness practice among health care professionals, additional evidence was provided that these programs were effective in improving psychological aspects, anxiety, and depression levels [23]. Furthermore, a standard 8-week training program showed the same effectiveness as an abbreviated 4-week one when decreasing these symptoms. The ﬁndings of this study revealed that only the standard training program improves the depression levels, with the maintenance of its effects at the 3-month follow-up. p In this research, no improvements in anxiety levels were observed in either of the two EGs. Consistent with these ﬁndings, two clinical controlled trials reported non-signiﬁcant effects in anxiety [71,72]. These results do not coincide with those obtained by other authors, who have demonstrated the positive effects of an intervention based on mindfulness in minimizing anxiety symptoms. Irving et al., completed a systematic re-view in which the important beneﬁts of MBSR training program on the improvement of well-being and stress resistance were examined amongst healthcare professionals and concluded that these types of interventions reduced anxiety [73]. In a quasi-experimental trial, Barbosa et al. analyzed the impact of the 8-week MBSR training program on students from ﬁve healthcare degrees and observed a signiﬁcant decline in anxiety levels at weeks 8 and 11 compared with initial evaluation [74]. In another study, a sample of primary care physicians participated in an abbreviated MBRS training program, improving their indicators of anxiety [50]. g p g p g y In the same line with the results of the present research, different researchers have concluded that the standard MBRS training program can reduce the depressive symptoms of the participants. A randomized controlled trial by Song et al., examined the effects of an 8-week MBRS program on depression levels in a sample of 50 Korean nursing students, and showed a signiﬁcantly greater decreases in depression measures [75]. In the same way, Pizutti et al. evaluated the Breathworks’ Mindfulness for Stress 8-week training J. Clin. Med. 4. Discussion Furthermore, this research also has certain limitations that may have inﬂuenced the obtained results, and decreased its representativeness. Despite the random assignment of the TUs to the different groups so that the contamination risk could be minimized, statistically signiﬁcant differences were found between the three groups related to age, job category, and time that they had been working in the SNHS. In addition, the ﬁnal sample size was inferior to that previously calculated at the beginning due to the COVID-19 pandemic epidemiological situation, which may have affected to the results obtained. This aspect must be into account in future research. On the other hand, the dropout rate due to refusal to continue participating in the program was high, being more pronounced in GE2 than in GE1. This could be explained by the differences in the sociodemographic characteristics of the participants between GE1 and GE2 in relation to the years of experience working and in the professional category. Because the EG participants had fewer years of experience, prevailing residents over tutors, the opposite occurring in the EG1. These characteristics could have inﬂuenced the motivation of the participants, which will be taken into account for future research. An analysis based on an intention-to-treat was carried out as the characteristics of the people that did not respond differ from those of the respondents, and therefore can control for this selection bias. Although a representative sample of Spanish mentors and general practitioners was available in FCMN, the overrepresentation of primary care workers, women, and physicians reduced the study results generalizability. Although the CG participants were not offered a theoretical practical session of either mindfulness or meditation, it could not be guaranteed that they would remain inactive during the period in which the study was carried out to evaluate the expected results’ differences when comparing this group with the EG J. Clin. Med. 2021, 10, 5941 13 of 16 13 of 16 5. Conclusions Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions An 8-week MBSR training program, aimed at tutors and resident specialists in FCMN, produced a signiﬁcant improvement in levels of anxiety and depression in general, and depression in particular, maintaining these effects for three months. However, a 4-week version was not associated with signiﬁcant changes in anxiety or depression levels. More representative and larger investigations must be conducted to support the effectiveness of abbreviated MBSR and MSC programs for primary care professionals, which could also increase adherence and accessibility to such training. Author Contributions: Conceptualization, J.C.V.-M.-A., and L.Á.P.-d.T.; methodology, J.C.V.-M.-A., L.Á.P.-d.T., A.R.-V., E.R.-R., N.H.S., R.M.B., T.d.J.G.-N., R.A.-V., and F.J.V.; software, M.S.-P., J.J.G.-B., M.J.-B., L.A.M., B.L.-d.-B., R.S.-C.; and J.G.-S.; validation, M.S.-P., J.J.G.-B., M.J.-B., L.A.M., B.L.-d.-B., R.S.-C.; and J.G.-S.; formal analysis, M.S.-P., J.J.G.-B., M.J.-B., L.A.M., B.L.-d.-B., R.S.-C.; and J.G.-S.; investigation, J.C.V.-M.-A., L.Á.P.-d.T., A.R.-V., E.R.-R., N.H.S., R.M.B., T.d.J.G.-N., R.A.-V., and F.J.V.; resources, J.C.V.-M.-A., and L.Á.P.-d.T.; data curation, M.S.-P., J.J.G.-B., M.J.-B., L.A.M., B.L.-d.-B., and J.G.-S.; writing—original draft preparation, M.S.-P., J.J.G.-B., M.J.-B., R.S.-C.; and J.G.-S.; writing— review and editing, M.S.-P., J.J.G.-B., M.J.-B., L.A.M., B.L.-d.-B., and J.G.-S.; visualization, M.S.-P., J.J.G.-B., J.C.V.-M.-A., L.Á.P.-d.T., A.R.-V., E.R.-R., N.H.S., R.M.B., T.d.J.G.-N., R.A.-V., F.J.V., J.J.G.-B., M.J.-B., L.A.M., B.L.-d.-B., R.S.-C.; and J.G.-S.; supervision, L.Á.P.-d.T.; project administration, J.C.V.- M.-A., and L.Á.P.-d.T.; funding acquisition, J.C.V.-M.-A., and L.Á.P.-d.T. All authors have read and agreed to the published version of the manuscript. Funding: The project has received funding from DGA group (B17-17R) and the Network for Preven- tion and Health Promotion in Primary Care (REDIAPP) grant from the “Instituto de Salud Carlos III” of the Spanish Ministry of Economy and Competitiveness, co-ﬁnanced with European Union ERDF funds (RD16/0007/0005). This project has received an “Isabel Fernández, 2017” scholarship from the Andalusian Society of Family and Community Medicine (SAMFyC, Ref. 153/17). In addition, it has received funding in the call for research and innovation projects in the ﬁeld of Primary Care of the Andalusian Health Service (Ref. AP-0155-2018). The funding source had no inﬂuence on the design of the study, data collection and analysis, or the writing of the manuscript. 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https://openalex.org/W2725551539	https://link.springer.com/content/pdf/10.1007%2Fs11661-017-4169-8.pdf	English	null	Microstructural and Phase Composition Differences Across the Interfaces in Al/Ti/Al Explosively Welded Clads	Metallurgical and materials transactions. A, Physical metallurgy and materials science	2,017	cc-by	7,814	Microstructural and Phase Composition Differences Across the Interfaces in Al/Ti/Al Explosively Welded Clads DAGMARA MALGORZATA FRONCZEK, ROBERT CHULIST, LIDIA LITYNSKA-DOBRZYNSKA, GABRIEL ALEJANDRO LOPEZ, ANNA WIERZBICKA-MIERNIK, NORBERT SCHELL, ZYGMUNT SZULC, and JOANNA WOJEWODA-BUDKA DAGMARA MALGORZATA FRONCZEK, ROBERT CHULIST, LIDIA LITYNSKA-DOBRZYNSKA, GABRIEL ALEJANDRO LOPEZ, ANNA WIERZBICKA-MIERNIK, NORBERT SCHELL, ZYGMUNT SZULC, and JOANNA WOJEWODA-BUDKA The microstructure and phase composition of Al/Ti/Al interfaces with respect to their localization were investigated. An aluminum-ﬂyer plate exhibited ﬁner grains located close to the upper interface than those present within the aluminum-base plate. The same tendency, but with a higher number of twins, was observed for titanium. Good quality bonding with a wavy shape and four intermetallic phases, namely, TiAl3, TiAl, TiAl2, and Ti3Al, was only obtained at the interface closer to the explosive material. The other interface was planar with three intermetallic compounds, excluding the metastable TiAl2 phase. As a result of a 100-hour annealing at 903 K (630 C), an Al/TiAl3/Ti/TiAl3/Al sandwich was manufactured, formed with single crystalline Al layers. A substantial diﬀerence between the intermetallic layer thicknesses was measured, with 235.3 and 167.4 lm obtained for the layers corresponding to the upper and lower interfaces, respectively. An examination by transmission electron microscopy of a thin foil taken from the interface area after a 1-hour annealing at 825 K (552 C) showed a mixture of randomly located TiAl3 grains within the aluminum. Finally, the hardness results were correlated with the microstructural changes across the samples. DOI: 10.1007/s11661-017-4169-8 / The Author(s) 2017. This article is an open access publication I. INTRODUCTION to 57 vol pct NiAl particulate,[3] new shapes of materi- als,[4,5] and integrally bonded composites.[6–9] These advantages make the methods suitable for a large number of technological applications.[1] AMONG the high-energy rate forming methods that use the energy from an explosion, three main techniques can be distinguished: powder compaction, explosive forming, and explosive welding (EXW).[1] These meth- ods are economically beneﬁcial and allow nanostruc- tured materials to be obtained, for example, consolidated samples from nanometric copper pow- ders,[2] Ni/NiAl metal-matrix composites containing up g pp Many of the studies presented in the literature are focused speciﬁcally on the EXW process. As conﬁrmed by various attempts, the joining of ﬂat surfaces[6–9] or tubes[10–12] can be easily performed by this method. Furthermore, the process can be carried out both under water[13–16] and under air atmospheres.[6–11,17,18] This method is steadily increasing its potential applications and improving the obtained results. As a ﬁrst example, the works completed by Gulenc et al.[19] in producing wire-reinforced multilayer composites are discussed. In this case, EXW was used to join two commercially pure aluminum plates with a steel mesh in-between, and the ﬁnal product showed better mechanical properties than conventional aluminum clads. According to the authors, these composites have applications in the space and automobile sectors, as well as for other structural applications.[19,20] For alloys, detailed studies on an explosively welded bilayered CuCrZr alloy/316LN-IG steel performed by Wang et al.[21] proved that this material, for applications in harsh environments, suc- cessfully qualiﬁed as an enhanced heat ﬂux ﬁrst wall of DAGMARA MALGORZATA FRONCZEK, ROBERT CHULIST, LIDIA LITYNSKA-DOBRZYNSKA, ANNA WIERZBICKA-MIERNIK, and JOANNA WOJEWODA-BUDKA are with the Institute of Metallurgy and Materials Science Polish Academy of Sciences, 25 Reymonta Street, 30-059 Krako´ w, Poland. Contact emails: d.fronczek@imim.pl; dfronczek@gmail.com GABRIEL ALEJANDRO LOPEZ is with the Departamento de Fı´sica Aplicada II, Facultad de Ciencia y Tecnologı´a, Universidad del Paı´s Vasco, Apdo. 644, 48080 Bilbao, Vizcaya, Spain. NORBERT SCHELL is with the Institute of Materials Research, Helmholtz- Zentrum Geesthacht, Max-Planck-Strasse 1, 21502 Geesthacht, Germany. ZYGMUNT SZULC is with the High Energy Technologies Works ’Explomet’, 100H Oswiecimska Street, 45-641 Opole, Poland. Manuscript submitted December 7, 2016. Article published online June 28, 2017 DAGMARA MALGORZATA FRONCZEK, ROBERT CHULIST, LIDIA LITYNSKA-DOBRZYNSKA, ANNA WIERZBICKA-MIERNIK, and JOANNA WOJEWODA-BUDKA are with the Institute of Metallurgy and Materials Science Polish Academy of Sciences, 25 Reymonta Street, 30-059 Krako´ w, Poland. JEOL is a trademark of Japan Electron Optics Ltd., Tokyo. JEOL is a trademark of Japan Electron Optics Ltd., Tokyo. 3 p The manufacturing of EXW clads requires a number of parameters to be controlled, such as the collision angle, impact velocity, detonation pressure, and geom- etry, with respect to the type of bonded materi- als.[28,34–39] However, the problem becomes even more complicated when more than only one interface is obtained. Therefore, in this manuscript, the simplest case with only two interfaces obtained in three-layered Al/Ti/Al clads is discussed. Signiﬁcant changes in the microstructure and phase composition of both interfaces are presented. The inﬂuence of interface localization with respect to the explosive material and the neighbor- ing regions is also studied. quality for the EBSD measurements, the following equipment and parameters were used for the electrolytic etching: a Struers electropolishing LectroPol-5 quality for the EBSD measurements, the following equipment and parameters were used for the electrolytic etching: a Struers electropolishing LectroPol-5 Struers is the world’s leading materialographic solution supplier. Struers ApS, Pederstrupvej, 84 Ballerup, Denmark. machine, a Struers electrolyte A3, an electrolyte tem- perature of 283 K (10 C), a voltage of 35 V, a specimen temperature of 77 K (196 C), a polishing time of 15 seconds, and a ﬂow rate of 15. The structural information for the intermetallic phases present at particular interfaces was investigated using high-energy synchrotron radiation (87.1 keV, k = 0.014235 nm) and the P07 beamline at DESY§ in I. INTRODUCTION Contact emails: d.fronczek@imim.pl; dfronczek@gmail.com GABRIEL ALEJANDRO LOPEZ is with the Departamento de Fı´sica Aplicada II, Facultad de Ciencia y Tecnologı´a, Universidad del Paı´s Vasco, Apdo. 644, 48080 Bilbao, Vizcaya, Spain. NORBERT SCHELL is with the Institute of Materials Research, Helmholtz- Zentrum Geesthacht, Max-Planck-Strasse 1, 21502 Geesthacht, Germany. ZYGMUNT SZULC is with the High Energy Technologies Works ’Explomet’, 100H Oswiecimska Street, 45-641 Opole, Poland. Manuscript submitted December 7, 2016. Article published online June 28, 2017 METALLURGICAL AND MATERIALS TRANSACTIONS A 4154—VOLUME 48A, SEPTEMBER 2017 Trident energy-dispersive X-ray spectrometer (EDS) produced by EDAX* and a TSL electron backscatter the International Thermonuclear Experimental Reactor component. Another impressive example is the produc- tion of Al/Ti clads, which were successfully obtained with not only 2[22] or 3[23] layers, but 21[24] or even 40 layers.[25] The EXW technique can also be used in the production of multilayered materials with signiﬁcantly diﬀerent properties, for example, metallic-intermetallic laminate composites. These composites belong to a new class of materials, whose properties combine the large ductility of the metal matrix and the high hardness of intermetallic phases.[26] Such versatile materials are used in the production of electrical wires and industrial machinery (Al/Cu),[7,27] platters in the aerospace (Al/ Ti)[24] and automobile (Al/Mg)[28] industries, and com- posites for medical applications (Ti/Al2O3/NiCr).[29] EDAX is the global leader in Energy Dispersive X-ray Spec- troscopy, Electron Backscatter Diﬀraction, Wavelength Dispersive X-ray Spectrometry and X-ray Fluorescence systems. EDAX Inc., 91 McKee Drive, Mahwah, NJ. diﬀraction (EBSD) system, a PHILIPS XL30 equipped with a LINK ISIS EDS system (Oxford Instruments), §DESY (Deutsches Elektronen-Synchrotron) is one of the world’s leading accelerator centres. Notkestr. 85 Hamburg, Germany. diﬀraction (EBSD) system, a PHILIPS XL30 equipped with a LINK ISIS EDS system (Oxford Instruments), *Oxford Instruments is a leading provider of high technology tools and systems for research and industry. We design and manufacture equipment that can fabricate, analyse and manipulate matter at the atomic and molecular level. Oxford Instruments, Tubney Woods Abingdon, Oxfordshire, Great Britain. Generally, the EXW process can be divided into three basic stages: detonation of an explosive material; defor- mation and acceleration of the ﬂyer plate; and, ﬁnally, collision of the clads.[1] The EXW process used to join aluminum and titanium clads leads to the occurrence of several Al-Ti intermetallic phases. Alternatively, further annealing causes the growth of a thermodynamically favored TiAl3 intermetallic phase.[23,25,30–33] and a JEOL JSM 7000F. To obtain a proper surface and a JEOL JSM 7000F. To obtain a proper surface JEOL is a trademark of Japan Electron Optics Ltd., Tokyo. II. MATERIALS AND METHODS During DSC measurements, DSC analyses were performed in a DSC 404 F1 Pegasus, NETZSCH§§. During DSC measurements, similar tendency in wave parameters (various wave- lengths) was reported by Mousavi et al. in explosively welded stainless steel with steel.[40] The authors explain this morphology by the change of impact angle. It should be noted that our upper interface was of good quality without any pores. Alternatively, the interface located further from the explosive materials (lower interface) was rather planar and no diﬀerence in contrast in the backscattered electrons (BSE) imaging mode was found, indicating that chemical homogeneity across the interface was observed (Figure 2(b)). Never- theless, a few cracks were present within the titanium layer (Figure 2(d)), which did not demonstrate any tendency for further propagation. A waveless interface is typical for welding modes close to the lower boundary of the welding window.[41] The morphology and shape of the interfaces strongly depend on the initial param- eters of the EXW.[12] According to Shiran et al.,[12] higher explosive forces promote wavy-mode interfaces. Taking that into account, it can be concluded that the middle Ti clad considerably damped the impact force, thereby reducing its power. However, although the force arriving to the lower interface was not strong enough to induce waviness, it was suﬃcient to form the lower joint with a ﬂat interface. The use of a thicker Ti clad would probably suppress the welding process of the three- layered material and the lower joint would not be formed, or proneness to some delamination could be expected. It should also be noticed that some reports for three-layered setups, such as Al/Cu/Al,[42] Zr/Ti/ steel,[43,44] and Al/steel/Al,[45] have shown a wavy mode for both interfaces. §§The NETZSCH Group consists of three Business Units under the umbrella of the Erich NETZSCH GmbH & Co. Holding KG. The Analyzing & Testing business unit of the NETZSCH Group develops and manufactures a complete high-precision instrument line for ther- mal analysis and thermophysical properties measurement, as well as oﬀering world class commercial testing services in their laboratories. Erich NETZSCH GmbH & Co. Holding KG, Gebru¨ der- Netzsch-Straße 19, Selb, Germany. the samples were placed in aluminum crucibles and protected against oxidation with ﬂowing argon. After heating at a rate of 20 K (20 C)/min, the samples were annealed at 825 K (552 C) for 1 hour. II. MATERIALS AND METHODS ( ) Finally, Vickers hardness measurements were carried out on polished samples using a microhardness tester from CSM– Instruments at room temperature. A sharp g p p y Taking that into account, it can be concluded that the middle Ti clad considerably damped the impact force, thereby reducing its power. However, although the force arriving to the lower interface was not strong enough to induce waviness, it was suﬃcient to form the lower joint with a ﬂat interface. The use of a thicker Ti clad would probably suppress the welding process of the three- layered material and the lower joint would not be formed, or proneness to some delamination could be expected. It should also be noticed that some reports for three-layered setups, such as Al/Cu/Al,[42] Zr/Ti/ steel,[43,44] and Al/steel/Al,[45] have shown a wavy mode for both interfaces. –Anton Paar TriTec SA develops, manufactures and sells instru- ments to characterize mechanical properties of surfaces. The company has been a global leader in this market for more than 30 years, ﬁrst under the name of LSRH, CSEM and then CSM Instruments SA. Anton Paar GmbH has acquired CSM Instruments SA in November 15, 2013. Anton Paar TriTec SA Rue de la gare, 4 Peseux, Switzerland. needle tip with a load of 0.09807 N was used for all indentations. The force was maintained for a dwell time of 15 seconds. The obtained results were averaged from at least ﬁve indentation measurements. Another important factor that inﬂuences the shape of the interface (wavy with or without vortexes or ﬂat) is the distance from the explosive material. This parameter was studied for multilayered Ti/Al clads in the works of Bataev et al.,[24] Pavliukova et al.,[33] and Lazurenko et al.,[25] or for sandwiches of low-carbon steel setups in works of Bataev et al.[46] In all these mentioned works, the examination was focused on the wavy mode of the interface, whereas the chemical composition was only analyzed in the vortex areas in the upper interfaces. –Anton Paar TriTec SA develops, manufactures and sells instru- ments to characterize mechanical properties of surfaces. The company has been a global leader in this market for more than 30 years, ﬁrst under the name of LSRH, CSEM and then CSM Instruments SA. Anton Paar GmbH has acquired CSM Instruments SA in November 15, 2013. Anton Paar TriTec SA Rue de la gare, 4 Peseux, Switzerland. II. MATERIALS AND METHODS Two cold-rolled plates of A1050 (further referred to as Al) (150 mm 9 240 mm 9 1 mm) and one of Ti Gr. 2 (further referred to as Ti) (150 mm 9 240 mm 9 0.8 mm) were explosively welded in a parallel system under an air atmosphere by the Explomet Company, as schematized in Figure 1(a). A thin layer of silicon was placed on top of the aluminum plate to prevent erosion. The detonation velocity was about 1900 to 1950 m/s. From the state obtained directly after welding, a three-layered material was cut into pieces from the central part in order to obtain samples of 6 mm 9 12 mm 9 2.8 mm in size (Figure 1(b)). One part of the samples was annealed at 903 K (630 C) for 100 hours in a vacuum. Subsequently, abrasive papers and diamond polishing pastes mixed with alumina were used to obtain mirror-ﬁnished cross sections. §DESY (Deutsches Elektronen-Synchrotron) is one of the world’s leading accelerator centres. Notkestr. 85 Hamburg, Germany. Hamburg. In order to ensure that the signal coming from only one of the interfaces was collected, the samples were ground either from the upper or lower side into smaller pieces (red dashed lines in Figure 1(b)). Electron transparent thin foils for transmission elec- tron microscopy (TEM) were cut from the properly selected regions of the bonds using a FEI Quanta 3D Dualbeam focused ion beam. Details of the microstruc- ture were revealed using a FEI TECNAI G2 FEG super TWIN (200 kV) microscope equipped with a Phoenix EDS, also manufactured by EDAX. The surface observations were carried out using scanning electron microscopy (SEM), with an FEI Quanta 3D ﬁeld emission gun (FEG) equipped with a Diﬀerential scanning calorimetry (DSC) was applied in conjunction with TEM to investigate the eﬀect of a high-temperature treatment on the thin foils. The METALLURGICAL AND MATERIALS TRANSACTIONS A VOLUME 48A, SEPTEMBER 2017—4155 Fig. 1—(a) Schematic setup of the explosively welded Al/Ti/Al system and (b) a photograph of the sample used in experiments. Fig. 1—(a) Schematic setup of the explosively welded Al/Ti/Al system and (b) a photograph of the sample used in experiments. Fig. 1—(a) Schematic setup of the explosively welded Al/Ti/Al system and (b) a photograph of the sample used the explosively welded Al/Ti/Al system and (b) a photograph of the sample used in experiments. DSC analyses were performed in a DSC 404 F1 Pegasus, NETZSCH§§. A. Microstructure A typical interface obtained in the state directly after EXW is presented in Figure 2. Depending on the welding conditions, i.e., collision angle and distance from the detonation point, diﬀerent interface morpholo- gies were obtained. In the case of the interface located closer to the explosive material (upper interface), a wavy mode was achieved (Figure 2(a)). The average values of the amplitude were determined to be between 5 and 20 lm, with a varied wavelength of 20 to 350 lm. A y pp As can be seen in Figure 3, the upper interface region is not uniform in composition and a zone with some particles in the Al clad can be distinguished. Such an eﬀect is shown in the works of Greenberg et al.[47,48] and 4156—VOLUME 48A, SEPTEMBER 2017 METALLURGICAL AND MATERIALS TRANSACTIONS A in intensely mixed node regions by means of SEM/EDS revealed the presence of TiAl2, TiAl, and TiAl3 phases (examples in the tables in Figure 3). However, the SEM/ EDS measurements have to be supported by other analytical techniques, since, as reported in the literature, an accelerating voltage of 12 kV induces an interaction volume of about 1.2 lm3, which implies that signals coming from diﬀerent phases might be included.[30] is known as granulating fragmentation (GF). GF was observed in both wavy and planar interfaces. It is also observed for other systems, i.e., copper-tantalum or iron-silver.[48] It can also be noticed in the same ﬁgure that the upper interface possesses intermetallic phase-rich areas. However, in contrast to our previous studies, they did not form peninsula- or island like structures.[30,31] According to the Ti-Al phase equilib- rium diagram,[49] three stable phases, namely, Ti3Al, TiAl, and TiAl3, or a metastable TiAl2 phase, can be formed. The chemical composition analysis carried out Comparison of the upper and lower interfaces con- ﬁrmed that dissimilar scenarios occurred on each interface, and as a result, diﬀerent morphologies, Fig. 2—SEM/BSE images presenting the inﬂuence of the collision angle on the interface shape: (a) the wavy mode with (c) swirled intermetallic phases and (b) the ﬂat mode with (d) noticeable cracks within the titanium layer. Fig. 2—SEM/BSE images presenting the inﬂuence of the collision angle on the interface shape: (a) the wavy mode with (c) swirled intermetallic phases and (b) the ﬂat mode with (d) noticeable cracks within the titanium layer. Fig. A. Microstructure 3—SEM/BSE micrograph of the upper interface of the clad together with the results of the EDS analysis. Fig. 3—SEM/BSE micrograph of the upper interface of the clad together with the results of the EDS analysis. VOLUME 48A, SEPTEMBER 2017—4157 METALLURGICAL AND MATERIALS TRANSACTIONS A microstructures, and chemical compositions were obtained. As mentioned previously, the upper interface, because of its closer proximity to the explosive material, was exposed to a greater impact force and, as a consequence, apart from the waviness, was character- ized by a signiﬁcantly more complex microstructure with swirled intermetallics, as well as intermediate phases scattered within the Al clad. Evidently, the energy available is used partly to plastically deform the clad, and there is a fraction that is transformed into heat, which promotes the nucleation and growth of other phases. This phenomenon has also been reported in the past,[12] with an increase in the explosive force being the reason for higher plastic deformation of ingoing mate- rials, correlated with an increase of the intermetallic layer thickness within the 321 austenitic stainless steel/ 1230 aluminum alloy assembly. microstructural evolution. It is expected that the inter- metallic phase growth will be enhanced on the upper interface, in comparison with the lower one, during the annealing process due to several reasons. According to the observation cited previously, a larger amount of energy should have been stored as deformation in the upper interface upon EXW, in comparison with the lower one. In addition, the energy barrier needed for nucleation should have already been overcome in the former. Furthermore, a stronger grain reﬁnement of the aluminum is observed near the upper interface, which provides more available paths for grain boundary diﬀusion and, in turn, enhances the growth process. Of course, this also depends strongly on the type of grain boundaries and is only true for high-angle grain boundaries (HAGBs). ( ) For a more detailed characterization, the crystallo- graphic information on the microstructure of the joined metals in the region close to the interfaces was acquired using the EBSD technique. As a result, two EBSD maps, collected for the upper and lower interface regions, are shown in Figure 4. In both Al clads, some wavy alignment of elongated grains was distinguished; how- ever, the wave character is much more pronounced within the upper Al (ﬂyer) plate. A remarkable grain reﬁnement can be observed in areas directly adjacent to the joint. A. Microstructure 4158—VOLUME 48A, SEPTEMBER 2017 METALLURGICAL AND MATERIALS TRANSACTIONS A interfaces, the reﬂections mainly coming from Al and Ti can be distinguished (Figures 7(a) and (b)). The samples for synchrotron measurements were separated along the titanium clad in order to investigate each interface independently (red dashed lines in Figure 1(b)). Since the two pieces cut from the samples did not have the same thickness of titanium clad, diﬀerences in the titanium reﬂection intensities between Figures 7(a) and (b) existed. Ege et al.[50] reported that ﬂat interfaces are usually more homogenous and contain no intermetallic phases compared to wavy ones, where intermetallics are only present within the molten areas. This occurs due to an adiabatic rise of temperature, which favors the formation of intermetallics. Moreover, Ghaderi et al.[51] concluded that melting or diﬀusion did not occur at ﬂat interfaces in EXW commercially pure A1100 with AZ31 Mg alloy. tendency was detected for the Ti clad (41.2 to 37.9 pct). This conﬁrms what was stated previously, namely, that a higher number of HAGBs in the upper region promote more intensive growth of the intermetallic layer during annealing because of the enhanced grain boundary diﬀusion. Additionally, a higher fraction of boundaries can be observed in the misorientation proﬁle at angles of 29 deg and close to 60 deg, indicating a high fraction of HAGBs and twin boundaries, respectively. The twin- type boundaries can be easily ascribed using the Brandon criterion, including peaks close to 55, 58, and 61 deg. g All of these microstructural features strongly suggest that EXW deforms the upper interface of Al and Ti clads more strongly, since more small grains, HAGBs, and twin boundaries were generated close to this interface. It must be stated that the EBSD map of the area located very close to the upper interface (Figure 4(a)) was similar to that obtained in our previous work (Figure 7 in Reference 29), where very ﬁne grains of mostly aluminum and intermetallics were present. The lower interface had a more uniform shape (Figure 4(b)), but again, very ﬁne grains of aluminum and intermetallic phases were observed. However, a further analysis with a diﬀerent intensity scale in the middle of the diﬀraction patterns showed some additional reﬂections, which were attributed to intermetallic phases. As can be seen, three phases at both interfaces were detected, i.e., Ti3Al, TiAl, and TiAl3 (Figures 7(c) and (d)). A. Microstructure This reﬁned region mainly consisted of Al crystallites and some intermetallics, which upon anneal- ing at high temperature would grow. The shape of the titanium grains is more equiaxed, as can be clearly seen in Figure 4. Therefore, each interface represents a diﬀerent initial condition for the heat treatments and directly aﬀects the Fig. 4—SEM/EBSD images presenting the microstructure of the (a) upper and (b) lower interfaces of the Al/Ti/Al sample. g The EBSD map revealed a strongly twinned microstructure with a much higher amount of twins and secondary twins close to the upper interface area than observed in the neighborhood of the lower inter- face. Additionally, from the grain size distribution for Al plotted in Figure 5, it can be clearly seen that in the region shown in the EBSD maps (up to 150 lm from the interface), more small grains were present in the Al upper clad than in the Al lower one (volume fraction of grains smaller than 10 lm equal to 72.3 pct vs 68.4 pct for the upper and lower parts, respectively). This tendency was even more pronounced in the Ti regions, where the upper part contained signiﬁcantly more twin boundaries (64.6 vs 42.7 pct, respectively). Moreover, the EBSD analysis shows that the average grain sizes for Al are 8.9 and 10.1 lm, while for Ti, they are 9.3 and 12.4 lm, for the upper and lower interface regions, respectively. In general, the reﬁnement of the grains, resulting from large plastic deformation, is more intense in the material located closer to the explosive material; however, this eﬀect is more intense for titanium than for aluminum. This remark is consistent with the observa- tions discussed previously. Fig. 4—SEM/EBSD images presenting the microstructure of the (a) upper and (b) lower interfaces of the Al/Ti/Al sample. p Figure 6 shows the grain boundary distribution of aluminum and titanium clads located close to the upper and lower interfaces. Grain boundaries with misorien- tation angles lower than 5 deg were not taken into consideration. A higher fraction of low-angle grain boundaries (LAGBs, less than 15 deg) was observed for the lower Al region than for the upper one (39.5 pct compared to 36.0 pct, Figures 6(a) and (c)). The same Fig. 4—SEM/EBSD images presenting the microstructure of the (a) upper and (b) lower interfaces of the Al/Ti/Al sample. A. Microstructure Additionally, in the upper interface, TiAl2 was observed (Figure 7(d)). There are other metastable phases that have been found in the literature, such as Ti3Al5 and h-TiAl2, as shown by Lazurenko et al.[25] and Palm et al.[52]; however, these were not observed in the current study. As presented in Detailed structural information for the phases present in both interface areas was collected using a monochro- matic synchrotron X-ray beam (87.1 keV). By analyzing the diﬀraction patterns taken from the upper and lower g. 5—Histogram of grain size distribution (diameters) calculated from EBSD maps presented in Fig. 4 for the upper ((a) Al and (b) Ti) and wer ((c) Al and (d) Ti) interfaces. Fig. 5—Histogram of grain size distribution (diameters) calculated from EBSD maps presented in Fig. 4 for the upper ((a) Al and (b) Ti) and lower ((c) Al and (d) Ti) interfaces. Fig. 5—Histogram of grain size distribution (diameters) calculated from EBSD maps presented in Fig. 4 for the upper ((a) Al and (b) Ti) and lower ((c) Al and (d) Ti) interfaces. METALLURGICAL AND MATERIALS TRANSACTIONS A VOLUME 48A, SEPTEMBER 2017—4159 METALLURGICAL AND MATERIALS TRANSACTIONS A VOLUME 48A, SEPTEMBER 2017—4159 METALLURGICAL AND MATERIALS TRANSACTIONS A our previous work,[30] a small intensity of the analyzed reﬂections indicates a minor volume fraction of inter- t lli i th l Additi ll i t t lli intermetallic occurrence and growth. Hence, enhanced initial conditions for further intermetallic development i d d b h t t t t d t th Fig. 6—Misorientation distribution of grain boundaries calculated from EBSD maps presented in Fig. 4 for the upper ((a) Al and (b) Ti) and lower ((c) Al and (d) Ti) interfaces. Fig. 6—Misorientation distribution of grain boundaries calculated from EBSD maps presented in Fig. 4 for the upper ((a) Al and (b) Ti) and lower ((c) Al and (d) Ti) interfaces. our previous work,[30] a small intensity of the analyzed reﬂections indicates a minor volume fraction of inter- metallics in the sample. Additionally, intermetallic crystallites were not randomly oriented and some degree of texture was present. This was the reason why the approach proposed by Saksl et al.[53] to estimate the volume fraction for each of the phases could not be applied. intermetallic occurrence and growth. Hence, enhanced initial conditions for further intermetallic development induced by heat treatment appeared at the upper interface. A. Microstructure Two main facts can be mentioned as having been more pronounced at the upper interface after EXW, namely, a thicker area with reﬁned grains and a larger amount of HAGBs, which are direct conse- quences of more extreme conditions during the process. As commented previously, a larger number of nuclei or places for heterogeneous nucleation (more reﬁned grains) at the upper interface neighborhood may lead to a decrease of the incubation time during the inter- metallic phase growth. Of course, these diﬀerences will depend on the parameters of the EXW. However, once this time is overcome, the growth rate should be similar for the same phase. B. Heat Treatment Thermally activated interdiﬀusion of elemental Ti and Al was observed after annealing for 100 hours at 903 K (630 C) under a vacuum atmosphere, and the growth of a continuous TiAl3 intermetallic layer between Al and Ti clads was noticed (Figure 8). Figures 8(b) and (c) show the distribution of Al and Ti, conﬁrming the chemical homogeneity of both layers. For the upper and lower interfaces, the width of the TiAl3 layer reached 235.3 ± 23.9 and 167.4 ± 19.9 lm, respectively. As expected, the localization of the interfaces in regard to the explosive materials plays a crucial role in We now compare the results obtained in the current work with those reported in the literature concerning the growth of phases upon annealing. According to the present work, the location within the multilayered composites plays an important role, as diﬀerent condi- tions of EXW occur on each interface. Hence, each METALLURGICAL AND MATERIALS TRANSACTIONS A 4160—VOLUME 48A, SEPTEMBER 2017 ig. 7—X-ray synchrotron diﬀraction of (a) lower and (b) upper interfaces of the Al/Ti/Al sample. (c) and (d) Enlarged central parts ﬀraction patterns with diﬀerent intensity scale demonstrate the low intensity reﬂections. Fig. 7—X-ray synchrotron diﬀraction of (a) lower and (b) upper interfaces of the Al/Ti/Al sample. (c) and (d) Enlarged central parts of the diﬀraction patterns with diﬀerent intensity scale demonstrate the low intensity reﬂections. work, both interfaces were of good quality without pores or hollows, in contrast to the interface in two-layered Ti/Al shown in Figure 5(e) in Reference 31. Both types of clads were manufactured with the use of a detonation velocity in the range of 1900 to 1950 m/ s. This conﬁrms that the other initial manufacturing conditions, such as collision angle or detonation pres- sure, also play a crucial role in determining strongly not only the microstructure of the obtained platter but also the growth velocity of the intermetallic phase at the interface during heat treatment. interface should be considered separately, especially when many layers, for example, 6[32] or 40,[25] are investigated. However, this fact was not clearly empha- sized in the works of Foadian et al.[32] and Lazurenko et al.[25] These authors did not indicate which interfaces were taken into consideration for the kinetics calcula- tions. Therefore, it can be presumed that the data were averaged. B. Heat Treatment Moreover, an external load may also have a strong inﬂuence on the intermetallic layer growth, meaning calculations for samples with and without compression should be conducted individually for each case, which was not applied in Reference 25. Thus, the examination of the growth kinetics for each interface separately is planned for future works. Upon annealing, twins are annihilated in Ti during grain growth by twin boundary motion and grain boundary migration. The annealing process also induces a growth of two single crystalline Al layers (Figure 8(a)). Both Al layers possess a random orientation, and no common high index plane or axis between them was p y p In our previous work, the intermetallics layer was 95.7 ± 9.9 lm in width for two-layered Ti/Al samples annealed under the same conditions.[30] In the current METALLURGICAL AND MATERIALS TRANSACTIONS A VOLUME 48A, SEPTEMBER 2017—4161 Fig. 8—(a) SEM/EBSD image presenting the microstructure of the upper and lower interfaces of the Al/Ti/Al sample after annealing at 903 K (630 C) for 100 h in a vacuum and maps showing the distribution of elemental (b) Al and (c) Ti across the sample (a). Fig. 8—(a) SEM/EBSD image presenting the microstructure of the upper and lower interfaces of the Al/Ti/Al sample after annealing at 903 K (630 C) for 100 h in a vacuum and maps showing the distribution of elemental (b) Al and (c) Ti across the sample (a). found. A single crystalline layer of Al has barely been described in the literature.[54–56] However, it does not exclude composites containing single crystals from future studies conﬁrming its potential application. average grain size changed from about 150 to 220 nm. The SADP analysis revealed only TiAl3 (Figure 9(f)), which is thermodynamically and kinetically most favored.[57,58] Thus, it can be concluded that the annealing mainly causes TiAl3 growth at the cost of other intermetallic phases, since only this phase was observed after annealing. C. Thin Foil Investigation An electron transparent thin foil was cut from the upper interface, in particular, from the region where intermetallic crystallites were randomly distributed within the reﬁned Al grains. Bright-ﬁeld (BF) and dark-ﬁeld (DF) images obtained from this region show many ﬁne grains (up to 600 nm in size, Figures 9(a) and (b)). The electron diﬀraction pattern conﬁrmed the presence of intermetallic grains randomly distributed within Al (Figure 9(c)). The phase analysis completed by a selected area diﬀraction pattern (SADP) showed two stable phases, namely, Ti3Al and TiAl, and one metastable phase, TiAl2, while the TiAl3 phase was not detected. The TiAl2 intermetallic phase was also detected in our previous work concerning two-layered Ti/Al samples,[30,31] although in other works on the Ti/ Al system, it was not observed.[22,24,32–34] D. Vickers Hardness Test The average hardness values of the parent Al and Ti clads are determined to be 34 and 180 HV, respectively. The Vickers hardness proﬁle measured for the cross section of the Al/Ti/Al sample is presented in Figure 10. The average hardness values for the aluminum base plate, titanium ﬂyer plate, and aluminum ﬂyer plate were 34, 187, and 36 HV, respectively. The component hardness only slightly increased due to the EXW process. Conversely, a signiﬁcant increase in hardness values for both Cu and Al explosively welded clads was observed in the work of Honarpisheh et al.[42] Cu and Al, as starting materials, had 26 and 54 HV, and after EXW, 50 to 52 HV and 102 to 103 HV, respectively. Mamalis et al.[59] explained the increase in hardness values by the stress waves and the intense thermal impact, which seems not to play a signiﬁcant role in the current study. Subsequently, the investigated thin foil was annealed for 1 hour at 825 K (552 C) in a calorimeter in order to observe the inﬂuence of postheating on structural heterogeneity and phase changes. Upon annealing, the lamella microstructure changed signiﬁcantly and grain growth became more signiﬁcant (Figure 9(d)). As can be seen in the DF images in Figures 9(b) and (e), the The microstructure in both aluminum clads changed signiﬁcantly from the typical rolled materials due to the high-speed crash of plates, i.e., plastic deformation. In METALLURGICAL AND MATERIALS TRANSACTIONS A 4162—VOLUME 48A, SEPTEMBER 2017 Fig. 9—TEM images of microstructure of the interface area in the state directly after EXW ((a) BF and (b) DF) and annealing ((d) BF and (e) DF) with corresponding SADPs ((c) and (f), respectively). Fig. 9—TEM images of microstructure of the interface area in the state directly after EXW ((a) BF and (b) DF) and annealing ((d) BF and (e) DF) with corresponding SADPs ((c) and (f), respectively). both plates, the elongated grains caused hardness varia- tion. A decrease of hardness in the area neighboring the interface in the Al ﬂyer plate was detected. The same phenomenon was detected in explosively welded carbon steel/Zr 700 samples in the work of Paul et al.[60] When the hardness measurements were performed within the melted regions, a visible decrease in hardness was detected. without separation or cracks around edges or diagonals. The intermetallic regions were too small for the Vickers hardness measurements. IV. SUMMARY AND CONCLUSIONS The three intermetallic phases, Ti3Al, TiAl, and TiAl3, were detected at both interfaces using TEM and synchrotron diffraction, while the metastable TiAl2 was only detected in the upper interface. The fraction of intermetallics in the entire system is minimal. If we compare the zones corre- sponding to the upper and lower interfaces, the amount of intermetallics is higher at the interface close to the explosive material, exhibiting no regu- larity in their distribution. pp 3. Considerable differences in morphology and the chemical composition between upper and lower interfaces appeared in the state directly after EXW. The three intermetallic phases, Ti3Al, TiAl, and TiAl3, were detected at both interfaces using TEM and synchrotron diffraction, while the metastable TiAl2 was only detected in the upper interface. The fraction of intermetallics in the entire system is minimal. If we compare the zones corre- sponding to the upper and lower interfaces, the amount of intermetallics is higher at the interface close to the explosive material, exhibiting no regu- larity in their distribution. ACKNOWLEDGMENTS The authors thank High Energy Technologies Works ‘Explomet’ (Opole, Poland) for the provision of the good quality Al/Ti/Al clads. Samples were exam- ined in the Accredited Testing Laboratories, Institute of Metallurgy and Materials Science, Polish Academy of Sciences (Krakow). Part of the SEM observations were performed in the frame of the Erasmus+ pro- gramme. y 4. An ulterior annealing treatment performed at 903 K (630 C) for 100 hours in a vacuum allowed receiving single crystal of Al in the obtained Al/ TiAl3/Ti/TiAl3/Al system. Furthermore, the inter- faces had similar chemical composition after heat y 4. An ulterior annealing treatment performed at 903 K (630 C) for 100 hours in a vacuum allowed receiving single crystal of Al in the obtained Al/ TiAl3/Ti/TiAl3/Al system. Furthermore, the inter- faces had similar chemical composition after heat IV. SUMMARY AND CONCLUSIONS The aim of the current work was to relate the microstructure and phase composition changes with the localization of the interface regarding the explosive material placement. The simpler case of two interfaces (Al/Ti/Al) joined by EXW was examined. The eﬀect of subsequent annealing was also studied. A comprehen- sive examination revealed signiﬁcant diﬀerences between the two interfaces (upper and lower), with the following conclusions. g It should be noted that the Ti clad, placed between the Al plates, was characterized by a large scatter of hardness values. The increase occurred due to the presence of deformation twins formed as a result of plastic deformation after the intense shock. The lower hardness values can be correlated to areas without twins. Consequently, the strength of the obtained Ti clad is larger than that for the rolled one.[61] As can be seen in Figure 10, all indentations were square shaped VOLUME 48A, SEPTEMBER 2017—4163 METALLURGICAL AND MATERIALS TRANSACTIONS A Fig. 10—Vickers hardness proﬁle through the Al/Ti/Al sample with the SEM images of the indentations. Fig. 10—Vickers hardness proﬁle through the Al/Ti/Al sample with the SEM images of the indentations. 1. Al possesses a slightly finer microstructure in the area closer to the upper interface than the lower interface. The same tendency, but more pro- nounced, was obtained for titanium. Additionally, a more intense plastic deformation caused a higher number of HAGBs and twin boundaries in the Ti clad, in the area adjacent to the upper interface. treatment composed of mainly the TiAl3 phase; however, an enormous difference for the continuous intermetallic layer thickness was measured, reaching values of 235.3 and 167.4 lm for upper and lower interfaces, respectively. p y 5. Vickers hardness measurements did not reveal significant differences related to the localization of the hardness measurements for the upper and lower plates of aluminum. A large scatter of hardness values obtained for Ti was attributed to the presence of deformation twins formed due to plastic deformation during the EXW process. 2. A higher fraction of LAGBs in the Al clad was found in the region close to the lower interface, and the same tendency was obtained in Ti. Additionally, more HAGBs with misorientation of about 29 deg, twinning, and secondary twinning were observed in the upper interface in Ti. pp 3. Considerable differences in morphology and the chemical composition between upper and lower interfaces appeared in the state directly after EXW. REFERENCES 35. K. Hokamoto, Y. Ujimoto, and M. Fujita: Mater. Trans., 2004, vol. 45, pp. 2897–2901. 1. T.Z. Blazynski: Explosive Welding, Forming and Compaction, Springer, Dordrecht, 1983. pp 36. J.R. Feng, P.W. Chen, K.D. Dai, E.F. An, and Y. Yuan: Mater. Sci. Forum, 2014, vol. 767, pp. 114–19. p g 2. A.R. Farinha, R. Mendes, J. Baranda, R. Calinas, and M.T. Vieira: J. Alloys Compd., 2009, vol. 483, pp. 235–38. 37. R. Mendes, J.B. Ribeiro, and A. Loureiro: Mater. Des., 2013, vol. 51, pp. 182–92. 3. J. Bystrzycki, J. Paszula, R. Trebinski, and R.A. Varin: J. Mater. 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OPEN ACCESS 29. B. Wang, F. Xie, B. Wang, and X. Luo: Mater. Sci. Eng. C, 2015, vol. 50, pp. 324–31. 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 re- production in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. pp 30. D.M. Fronczek, J. Wojewoda-Budka, R. Chulist, A. Sypien, A. Korneva, Z. Szulc, N. Schell, and P. Zieba: Mater. Des., 2016, vol. 91, pp. 80–89. pp 31. D.M. Fronczek, R. Chulist, L. Litynska-Dobrzynska, Z. Szulc, P. Zieba, and J. Wojewoda-Budka: J. Mater. Eng. Perform., 2016, vol. 25, pp. 3211–17. 32. F. Foadian, M. Soltanieh, M. Adeli, and M. Etminanbakhsh: Metall. Mater. Trans. A, 2014, vol. 45A, pp. 1823–32. 33. D.V. Pavliukova, V.I. Mali, A.A. Bataev, P.S. Yartsev, T.S. 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VOLUME 48A, SEPTEMBER 2017—4165 METALLURGICAL AND MATERIALS TRANSACTIONS A VOLUME 48A, SEPTEMBER 2017—4165
W2912948586.txt	null	fr		Description une espéce nouvelle du pliocène inférieur algérien /	null	1,907	public-domain	624	DESCRIPTION d'une ESPÈCE NOUVELLE DU PLIOCÈNE INFÉRIEUR ALGÉRIEN PAR lie Gal de LAMOTHE et Pis, OA1ITZEABERO (Extrait du Journal de Conchyliologie, Vol, LV) PARIS Direction et Rédaction : ■ . Administration: H. FISCHER F. R. dr RUDE VAL, Éditeur 51, Boulevard Saint-Michel (Ve) 4, Hue Antoine Dubois (VIe) 190? V V - . % rioôiijz - L^ si- ,i Extrait du Journal de Conchyliologie, Vol. LV, 1907, p. 358 ».f r • - DESCRIPTION • » D’UNE * ESPÈCE • • , NOUVELLE DU PLIOCÈNE HNFÉRIEtJlt ALGÉRIEN Par le Gal dk Lamothe et Ph. Dautzenbkrg * Gibbula Fighkuri n. sp. (Fig 1, 2, 3, grossies 3 fois 1/2) Testa solida, trochiformis, satlateac profunde umbilicata. Spira conoidea, versus apicem paululum attenuata. Anfr. embryonalis déficit. Anfr. normales 6 1/2 primi subplani, ultimi convexiusculi sutura conspicua ac subcanaliculata juncti, a liris transversis (in anfr. penultimo 7) ac costulis longitudinalibus obliquis tenuissimisque eleqanter granulatim decussati. In anfr. ultimo lirulae transversae aliquot intercé¬ dant. Anfr. ultimus ad peripheriam carinatus et minutissime crenulatus. Basis sat convexa, concentrice multilirata : lirae alternatim debiliores. (Jmbilicus primum sat late infundibuliformis, deinde vero angustus ac pervius. Apertura subquadrata, marginibus callo tenui junctis. Columella arcuata, superne callosa breviterque reflexa, inferne attenuata et cum marginem basalem angulum efformans. Labrum simplex, acutum et medio conspicue angulatum. Color albidus (?) flammulis longitudinalibus juscis (?), latis, haud numerosis depictus. Altit. 10, diam. 11 mm. Apertura 4.5 mm. alta, 6 mm. lata. Coquille solide, trochiforme, assez largement ombili¬ quée. Spire conoïde, un peu atténuée vers le sommet. Tour embryonnaire détruit; 61/2 tours normaux, les supé¬ rieurs plans, les deux derniers légèrement convexes, sé¬ parés par une suture bien visible et subcanaliculée. Sur¬ face ornée de cordons décurrents (7 sur l’avant-dernier — 359 — tour) et de costules longitudinales obliques formant, par leur entrecroisement, un réseau très délicat, à mailles quadrangulaires, et pourvu de petites granulations sur les » points d’intersection. On observe sur le dernier tour quel¬ ques cordons très fins qui s’intercalent entre les autres. Ce tour est pourvu, à la périphérie, d’une carène très déli¬ catement denticulée par les extrémités des costules lon¬ gitudinales. Base assez convexe, ornée de nombreux cor dons concentriques, alternativement plus forts et plus faibles. Ombilic d’abord assez large et infundibuliforme, se transformant ensuite en une perforation étroite et pro¬ fonde. Ouverture subquadrangulaire, bords reliés par une Fig. 1-2. — Gibbula Ficheuri de Lamothe et Dautzeoberg, grossi 3 fois 1/2. callosité mince et appliquée. Columellearquée, calleuse, et un peu réfléchie dans le haut, rétrécie dans le bas où elle forme un angle avec le bord basal. Labre simple, tran¬ chant, anguleux à l’endroit où aboutit la carène périphé¬ rique. Coloration blanchâtre (?) avec des traces de flammules longitudinales brunes (?), très larges et peu nombreuses. Gisement. — Douéra, fossile de la buanderie de 1 Hos¬ pice, 1898 ; marne glauconieuse de la base du Pliocène inferieur. Par sa sculpture, cette espèce rappelle le Calliostoma miliare Brocchi, tandis que par sa forme générale et son — 360 — ombilic, elle ressemble au Gibbula umbilicaris Linné; mais nous ne connaissons aucune forme dont elle se rapproche¬ rait suffisamment pour permettre une comparaison dé¬ taillée. Nous prions M. le professeur Ficheur, de la Fa¬ culté des Sciences d’Alger, de vouloir bien en accepter la dédicace et nous profitons de cette circonstance pour le remercier des nombreux et intéressants documents qu’il a bien voulu nous communiquer et qui nous ont été fort utiles pour l’établissement d’une liste des Mollusques fos¬ siles du Pliocène d’Algérie. Gal de L. et Ph. D. ■ : i. ' • . . a ' . •'\ 1 ■î •' ■: "• j ». « ; - . . . . A •. :j . . ■' ■ . ; ■- ". ; »
https://openalex.org/W4231048951	https://www.qeios.com/read/FENY8B/pdf	English	null	Clindamycin	Definitions	2,020	cc-by	65	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Clindamycin National Cancer Institute National Cancer Institute Qeios ID: FENY8B · https://doi.org/10.32388/FENY8B Source National Cancer Institute. Clindamycin. NCI Thesaurus. Code C377. A semisynthetic broad spectrum antibiotic produced by chemical modification of the parent compound lincomycin. Clindamycin dissociates peptidyl-tRNA from the bacterial ribosome, thereby disrupting bacterial protein synthesis. (NCI04) Qeios ID: FENY8B · https://doi.org/10.32388/FENY8B 1/1
https://openalex.org/W4244446960	https://www.qeios.com/read/VDEN0D/pdf	English	null	HNF1A wt Allele	Definitions	2,020	cc-by	103	Qeios · Definition, February 8, 2020 Open Peer Review on Qeios HNF1A wt Allele National Cancer Institute National Cancer Institute Qeios ID: VDEN0D · https://doi.org/10.32388/VDEN0D Source National Cancer Institute. HNF1A wt Allele. NCI Thesaurus. Code C97826. Human HNF1A wild-type allele is located in the vicinity of 12q22-qter; 12q24.2 and is approximately 25 kb in length. This allele, which encodes hepatocyte nuclear factor 1- alpha protein, is involved in both transcriptional regulation and DNA binding. Mutation of the gene is associated with familial hepatic adenoma, maturity-onset diabetes of the young type 3 and diabetes mellitus insulin-dependent type 20. Qeios ID: VDEN0D · https://doi.org/10.32388/VDEN0D 1/1
https://openalex.org/W2139084667	https://europepmc.org/articles/pmc3294510?pdf=render	English	null	<i>Salmonella enterica</i>Pulsed-Field Gel Electrophoresis Clusters, Minnesota, USA, 2001–2007	Emerging infectious diseases	2,010	cc-by	6,495	Salmonella enterica Pulsed-Field Gel Electrophoresis Clusters, Minnesota, USA, 2001–2007 Joshua M. Rounds, Craig W. Hedberg, Stephanie Meyer, David J. Boxrud, and Kirk We determined characteristics of Salmonella enterica pulsed-ﬁ eld gel electrophoresis clusters that predict their being solved (i.e., that result in identiﬁ cation of a conﬁ rmed outbreak). Clusters were investigated by the Minnesota De- partment of Health by using a dynamic iterative model. Dur- ing 2001–2007, a total of 43 (12.5%) of 344 clusters were solved. Clusters of >4 isolates were more likely to be solved than clusters of 2 isolates. Clusters in which the ﬁ rst 3 case isolates were received at the Minnesota Department of Health within 7 days were more likely to be solved than were clusters in which the ﬁ rst 3 case isolates were received over a period >14 days. If resources do not permit investigation of all S. enterica pulsed-ﬁ eld gel electrophoresis clusters, in- vestigation of clusters of >4 cases and clusters in which the ﬁ rst 3 case isolates were received at a public health labora- tory within 7 days may improve outbreak investigations. The development of molecular subtyping by pulsed- ﬁ eld gel electrophoresis (PFGE) has revolutionized Salmo- nella spp. surveillance. The National Molecular Subtyping Network for Foodborne Disease Surveillance (PulseNet) provides state and local public health department labora- tories with standardized methods to subtype Salmonella serovars and normalize PFGE patterns against a global reference standard provided by the Centers for Disease Control and Prevention (CDC) (6,7). Molecular subtyping enhances case deﬁ nition speciﬁ city, enabling outbreaks to be detected and controlled at an earlier stage, and enabling detection of geographically dispersed outbreaks (8–10). Although the beneﬁ ts of molecular subtyping, speciﬁ - cally by PFGE, in foodborne disease outbreak detection and investigation have been well established, there is no consensus about when a PFGE cluster warrants further in- vestigation and almost no quantitative analysis about char- acteristics of PFGE clusters that indicate a common source will be identiﬁ ed (11–15). Cluster size and the number of days from receipt of the ﬁ rst cluster case isolate to the third case isolate received by the public health laboratory were predictors of a source of infection being identiﬁ ed for List- eria monocytogenes clusters in France (16). Salmonella enterica Pulsed-Field Gel Electrophoresis Clusters, Minnesota, USA, 2001–2007 The objective of this study was to determine characteristics of Salmonella PFGE clusters that could serve as useful predictors for their being solved (i.e., result in identiﬁ cation of a conﬁ rmed outbreak). This information could help public health agen- cies with limited resources prioritize investigation of Sal- monella PFGE clusters. S S almonellosis is a major foodborne illness that results in ≈1.4 million infections, 15,000 hospitalizations, and 400 deaths each year in the United States (1,2). Salmonella infections are primarily of foodborne origin but can also occur through contact with infected animals, humans, or their feces (3). The epidemiology of salmonellosis is com- plex largely because there are >2,500 distinct serotypes (serovars) with different reservoirs and diverse geographic incidences (4). Changes in food consumption, produc- tion, and distribution have led to an increasing frequency of multistate outbreaks associated with fresh produce and processed foods (5). Author afﬁ liations: Minnesota Department of Health, St. Paul, Min- nesota, USA (J.M. Rounds, S. Meyer, D.J. Boxrud, K.E. Smith); and University of Minnesota School of Public Health, Minneapolis, Minnesota, USA (C.W. Hedberg) RESEARCH RESEARCH Author afﬁ liations: Minnesota Department of Health, St. Paul, Min- nesota, USA (J.M. Rounds, S. Meyer, D.J. Boxrud, K.E. Smith); and University of Minnesota School of Public Health, Minneapolis, Minnesota, USA (C.W. Hedberg) Inclusion and Exclusion Criteria Cluster serovar diversity was examined by categoriz- ing the 17 most frequent serovars into highly clonal or low clonality serovars on the basis of the Simpson diversity in- dex (23). Serovars with a Simpson index score <0.90 were considered highly clonal, and serovars with a Simpson index score >0.90 were considered to have low clonality. Cluster investigation thresholds were examined by comparing the percentage of outbreak clusters meeting a threshold, cluster investigation positive predictive value, and estimated inter- view burden in hours per year for various investigational thresholds. The time required to interview each patient with a Salmonella infection by using the MDH standard ques- tionnaire was recorded for a 6-month period in 2008, and the median interview time was calculated. Laboratory-conﬁ rmed cases of nontyphoidal Salmo- nella enterica infection among Minnesota residents with specimen collection dates from January 1, 2001, through December 31, 2007, for which isolates were received and subtyped by MDH PHL were included in the study. Isolates not received through routine surveillance (i.e., testing was requested or conducted by MDH as a part of an ongoing investigation) were excluded from the analysis. Solved clusters were included if they were detected and identiﬁ ed solely on the basis of investigation of cases identiﬁ ed through submission of isolates to MDH for rou- tine laboratory surveillance. Solved clusters for which a call to the MDH foodborne disease hotline (www.health. state.mn.us/divs/idepc/dtopics/foodborne/reporting.html) (e.g., from the public or a healthcare provider) directly con- tributed to the identiﬁ cation of an outbreak were excluded from analysis. Secondary clusters, deﬁ ned as clusters in which the cases were part of a conﬁ rmed outbreak that had been previously identiﬁ ed, were also excluded from Materials and Methods Salmonella infections are reportable to the Minnesota Department of Health (MDH) by state law (17). Clinical laboratories are required to forward all Salmonella isolates to the MDH Public Health Laboratory (PHL). PFGE sub- Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 1678 Salmonella enterica PFGE Clusters typing after digestion with XbaI is conducted on all iso- lates as soon as they are received according to PulseNet protocols (18). PFGE subtypes are uploaded into the na- tional PulseNet database (6). All Minnesota residents with a culture-conﬁ rmed Salmonella infection are routinely in- terviewed as soon as possible by MDH staff with a standard questionnaire about symptom history, food consumption, and other potential exposures occurring in the 7 days before onset of illness. The questionnaire contains detailed food exposure questions, including open-ended food histories and objective yes/no questions about numerous speciﬁ c food items, as well as brand names and purchase locations. Clusters are investigated by using an iterative model in which suspicious exposures identiﬁ ed during initial case- patient interviews are added to the standard interview for subsequent cases (19–21). Similarly, initial cluster case- patients may be reinterviewed to ensure uniform ascertain- ment of the suspicious exposures. This iterative approach is used to identify exposures for further evaluation with formal hypothesis testing, product sampling, or product tracing (19). analysis. Clusters that were part of a probable outbreak (an epidemiologic evaluation suggested, but did not conﬁ rm, a common source of infection) were also excluded. Study Variables Variables incorporated into the analysis were cluster year, cluster size, cluster case density, cluster serovar, clus- ter subtype, and cluster serovar diversity. Cluster size was deﬁ ned as the number of cases in each cluster and was cat- egorized into cluster sizes of 2, 3, 4, and >5. For clusters in which a common source was identiﬁ ed, only cases received before the cluster was solved were included. Cluster case density was deﬁ ned as the number of days from receipt date of the ﬁ rst cluster isolate at MDH PHL to the receipt date of the third cluster isolate and was categorized into cluster case densities of 0, 1–7, 8–14, and >14 days (16). Cluster serovar was coded as a categorical variable on the basis of serovar frequency. Serovars representing >20% of all isolates (Typhimurium and Enteritidis) were categorized as very common, those representing 3%–20% (Newport, Heidelberg, and Montevideo) as common, and those representing <3% (all other serovars) as uncom- mon. The relationship between common and uncommon PFGE subtypes and solving a cluster was examined for serovars Typhimurium and Enteritidis. For serovar Typh- imurium, clusters with CDC PFGE subtype designations JPXX01.0003, JPXX01.0410, and JPXX01.0111 (each representing >8% of all Typhimurium isolates) were categorized as common, and all other subtypes were cat- egorized as uncommon. For serovar Enteritidis, clusters with CDC PFGE subtype designations JEGX01.0004 and JEGX01.0030 (each representing >20% of all Enteritidis isolates) were categorized as common, and all other sub- types were categorized as uncommon. A cluster was deﬁ ned as >2 cases of salmonellosis in different households with isolates of the same serovar and PFGE subtype and with specimen collection dates within 2 weeks (22). Thus, a single cluster would be ongoing as long as a new isolate was collected within 2 weeks after the most recent isolate in the cluster. A cluster was considered solved if the epidemiologic evaluation of that cluster resulted in the identiﬁ cation of a common source of infection for those cases and consequently the documentation of a conﬁ rmed outbreak. Therefore, the terms solved cluster and conﬁ rmed outbreak are equivalent and used interchangeably. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 Results During 2001–2007, a total of 4,154 nontyphoidal Sal- monella isolates from Minnesota residents were received at MDH through routine surveillance; they represented 98% of reported Salmonella cases (n = 4,235, incidence 11.78 cases/100,000 person-years). PFGE subtyping was per- formed for 4,018 (97%) isolates, which were included in the study. Among these isolates, 194 Salmonella serovars were observed. The 6 most common S. enterica serovars were Typhimurium, 1,004 (25%); Enteritidis, 822 (20.5%); Newport, 314 (7.8%); Heidelberg, 223 (5.6%); Montevi- deo, 121 (3.0%); and Saintpaul, 81 (2.0%) (Figure 1). represented 502 (12.5%) isolates. Twenty-two (34%) out- breaks were excluded from analysis (6 were multistate out- breaks in which only 1 case was identiﬁ ed in Minnesota; in 7 outbreaks, a hotline call contributed to identiﬁ cation of the outbreak; 1 was an outbreak was not detected by PFGE; 4 were outbreaks that did not have cases that met the cluster deﬁ nition; and 4 outbreaks were considered probable). The remaining 43 outbreaks, representing 287 (7%) isolates, were included in the analysis and were composed of 35 foodborne, 6 person-to-person, and 2 animal contact out- breaks. Of these 43 outbreaks, 30 (70%) involved 1 facility (restaurant, daycare center, school) or event and therefore were classiﬁ ed as point source. Thirteen (30%) involved commercially distributed food items at multiple points of sale (grocery stores, restaurants) and therefore were classi- ﬁ ed as non–point source. The median cluster size of point source outbreaks was 3 cases, and the median cluster size of non-point source outbreaks was 5 cases (p<0.01, by Wilcoxon rank-sum test). The median cluster density was 6 days for point source and non-point source outbreaks (p = 0.74 by Wilcoxon rank-sum test). The frequency of PFGE subtypes was examined in detail for serovars Typhimurium and Enteritidis. The 3 most common subtypes of serovar Typhimurium were JPXX01.0003, 107 (11%); JPXX01.0410, 87 (9%); and JPXX01.0111, 85 (8%). The 3 most common subtypes of serovar Enteritidis were JEGX01.0004, 309 (38%); JEGX01.0030, 181(22%); and JEGX01.0005, 106 (13%). Serovar diversity was examined by comparing Simp- son diversity indices for the 17 most frequent serovars (Table 1). Javiana, Newport, Agona, Infantis, and Typh- imurium were low clonality serovars. Heidelberg, Hadar, Enteritidis, Thompson, and I 4,5,12:I:– were highly clonal serovars. The frequency of PFGE subtypes was examined in detail for serovars Typhimurium and Enteritidis. 1,000 600 800 1,000 lates 200 400 600 800 1,000 No. isolates 0 200 400 600 800 1,000 Typ Ent New Hei Mon Sai S. I 4 Inf Ago Mue Ora Par Tho Bra Had Jav Ana No. isolates 0 200 400 600 800 1,000 Typ Ent New Hei Mon Sai S. I 4 Inf Ago Mue Ora Par Tho Bra Had Jav Ana No. isolates S. enterica serovars 0 Typ Ent New Hei Mon Sai S. I 4 Inf Ago Mue Ora Par Tho Bra Had Jav Ana 0 Typ Ent New Hei Mon Sai S. I 4 Inf Ago Mue Ora Par Tho Bra Had Jav Ana S. enterica serovars Figure 1. Frequency of the 17 most common Salmonella enterica serovars among clinical case isolates submitted to the Minnesota Department of Health, 2001–2007. Typ, Typhimurium; Ent, Enteritidis; New, Newport; Hei, Heidelberg; Mon, Montevideo; Sai, Saintpaul; S.I4, S.I 4,5,12:I: –; Inf, Infantis, Ago, Agona; Mue, Muenchen; Ora, Oranienburo; Par, Paratyphi B var. L; Tho, Thompson; Bra, Braenderup; Had, Hadar; Jav, Javiana; Ana, Anatum. Results The 3 most common subtypes of serovar Typhimurium were JPXX01.0003, 107 (11%); JPXX01.0410, 87 (9%); and JPXX01.0111, 85 (8%). The 3 most common subtypes of serovar Enteritidis were JEGX01.0004, 309 (38%); JEGX01.0030, 181(22%); and JEGX01.0005, 106 (13%). Serovar diversity was examined by comparing Simp- son diversity indices for the 17 most frequent serovars (Table 1). Javiana, Newport, Agona, Infantis, and Typh- imurium were low clonality serovars. Heidelberg, Hadar, Enteritidis, Thompson, and I 4,5,12:I:– were highly clonal serovars. Statistical Analysis A descriptive analysis was conducted to characterize the frequency of Salmonella serovars and subtypes. Man- tel-Haenszel χ2 test for trend was used to characterize tem- Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 1679 RESEARCH 1,000 600 800 1,000 lates 200 400 600 800 1,000 No. isolates 0 200 400 600 800 1,000 Typ Ent New Hei Mon Sai S. I 4 Inf Ago Mue Ora Par Tho Bra Had Jav Ana No. isolates 0 200 400 600 800 1,000 Typ Ent New Hei Mon Sai S. I 4 Inf Ago Mue Ora Par Tho Bra Had Jav Ana No. isolates S. enterica serovars poral trends in the number of Salmonella clusters that were solved. Two-sided Wilcoxon rank-sum tests were used to compare the median cluster size and cluster density of point source and non–point source outbreaks. Univariate analy- sis was performed to calculate odds ratios (ORs) and 95% conﬁ dence intervals (CIs) characterizing the crude asso- ciations between Salmonella cluster serovar, cluster PFGE subtype, cluster serovar diversity, cluster size, and cluster case density and a cluster being solved. Mantel-Haenszel χ2 tests for trend and interaction terms were used to inves- tigate the linear nature of the relationship between cluster size, cluster case density, and the outcome. SAS software version 9.1 (SAS Institute, Cary, NC, USA) was used for descriptive and univariate analysis. An α value <0.05 was considered signiﬁ cant. Cluster Serovar and Cluster Serovar Diversity Cluster Serovar and Cluster Serovar Diversity solved than clusters of 2 cases, but the difference was not statistically signiﬁ cant. There was statistical evidence of a nonlinear relationship between cluster size and solving the cluster (Wald χ2 for interaction 5.0, p = 0.03). The dose re- sponse between cluster size and solving a cluster plateaued after a cluster size of 4. Clusters of the common Salmonella serovars Newport, Heidelberg, and Montevideo had 2.7× higher odds of be- ing solved than did clusters of the very common serovars Enteritidis and Typhimurium (Table 2). The proportion of uncommon serovar clusters that were solved did not dif- fer signiﬁ cantly from the proportion of very common or common serovar clusters that were solved (Table 2). Low clonality serovar clusters were not signiﬁ cantly more likely to be solved than highly clonal serovar clusters (OR 1.6, 95% CI 0.8–3.1). Cluster Case Density The proportion of clusters solved increased signiﬁ - cantly as the density of cluster cases increased (Mantel- Haenszel χ2 for trend, 12.7, p<0.001) (Table 2). The odds of solving a cluster if the ﬁ rst 3 case isolates were received on the same day were 25.8× higher than the odds of solv- ing a cluster in which the ﬁ rst 3 case isolates were received during a period >14 days (Table 2). The odds of solving a cluster if the ﬁ rst 3 case isolates were received within 1–7 days were 5.0× higher than the odds of solving a cluster in which the ﬁ rst 3 case isolates were received during a period >14 days. Clusters in which the ﬁ rst 3 case isolates were received within 8–14 days were 2.8× more likely to be solved than clusters in which the ﬁ rst 3 case isolates were received during a period >14 days, but the difference was not statistically signiﬁ cant (Table 2). There was statis- tical evidence of a nonlinear relationship between cluster case density and solving the cluster (Wald χ2 for interac- tion, 6.96, p<0.01). Cluster Subtype No signiﬁ cant associations between the subtype fre- quency of a cluster and a cluster being solved were ob- served. Uncommon serovar Enteritidis subtype clusters were not signiﬁ cantly more likely to be solved than were common clusters (OR 1.4, 95% CI 0.4–5.1). Uncommon serovar Typhimurium subtype clusters were not signiﬁ - cantly more likely to be solved than were common clusters (OR 0.9, 95% CI 0.3–3.2). Cluster Size The probability of a cluster being solved increased sig- niﬁ cantly as the number of cluster cases increased (Mantel- Haenszel χ2 for trend 13.7, p<0.001) (Table 2). The odds of solving a cluster of >5 cases were 3.8× higher than the odds of solving a cluster of 2 cases. Clusters of 4 cases were 3.9× more likely to be solved than were clusters of 2 cases. Twenty-four percent of clusters with >4 cases were solved (Table 2). Clusters of 3 cases were 2.1× more likely to be Temporal Trends During 2001–2007, a total of 376 Salmonella PFGE clusters were detected; they represented 1,399 (35%) iso- lates. Thirty-two (8.5%) clusters were excluded from anal- ysis (21 secondary clusters, 7 clusters in which a hotline call directly contributed to identiﬁ cation of an outbreak, and 4 probable outbreak clusters). Forty-three (12.5%) of the 344 clusters included in the analysis were solved. During the study period, the median number of Salmo- nella isolates subtyped per year was 567 (range 507–662 isolates). The median number of Salmonella clusters per year was 50 (range 44–57 clusters). The median number of conﬁ rmed Salmonella outbreaks per year was 6 (range 4–8 outbreaks). There were no statistically signiﬁ cant trends in the proportion of Salmonella clusters that resulted in identi- ﬁ cation of a conﬁ rmed outbreak (p = 0.20) (Figure 2). During 2001–2007, a total of 65 conﬁ rmed Salmonella outbreaks involving Minnesota cases were identiﬁ ed; these Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 1680 Salmonella enterica PFGE Clusters Table 1. Salmonella enterica serovar diversity identified by pulsed-field gel electrophoresis among case isolates submitted to the Minnesota Department of Health, 2001–2007* Serovar No. isolates No. PFGE subtypes observed Serovar isolates represented by most common subtype, % Serovar isolates represented by 2 most common subtypes, % Serovar isolates represented by 3 most common subtypes, % Simpson index† Heidelberg 223 46 57 62 66 0.67 Hadar 48 20 48 54 58 0.77 Enteritidis 822 80 38 60 73 0.79 Thompson 57 23 42 53 58 0.81 I 4,5,12:I:– 78 25 31 50 60 0.86 Braenderup 53 30 26 36 43 0.92 Oranienburg 63 26 21 32 41 0.93 Anatum 46 22 17 33 46 0.93 Paratyphi B var. L 60 35 22 37 43 0.93 Montevideo 121 59 22 30 36 0.94 Muenchen 73 50 21 25 27 0.96 Saintpaul 81 44 17 26 32 0.96 Typhimurium 1,004 285 11 20 28 0.96 Infantis 75 43 9 17 24 0.97 Agona 74 48 10 16 22 0.98 Newport 314 143 10 15 19 0.98 Javiana 48 41 6 11 15 0.99 PFGE, pulsed-field gel electrophoresis. †Calculated as 1 – D = (6n(n – 1))/(N(N – 1)), where n is number of isolates of each subtype and N is total number of isolates of a serovar. A value of 1 indicates infinite diversity, and a value of 0 indicates no diversity. Temporal Trends ca serovar diversity identified by pulsed-field gel electrophoresis among case isolates submitted to the Health, 2001–2007 oresis. /(N(N – 1)), where n is number of isolates of each subtype and N is total number of isolates of a serovar. A value of 1 alue of 0 indicates no diversity , p g p †Calculated as 1 – D = (6n(n – 1))/(N(N – 1)), where n is number of isolates of each subtype and N is total number of is indicates infinite diversity, and a value of 0 indicates no diversity. Cluster Serovar and Cluster Serovar Diversity Discussion During the study period, 344 Salmonella PFGE clus- ters were identiﬁ ed and 43 (13%) were solved. Cluster size and cluster case density were the most useful predictors of a cluster being solved. The proportion of clusters that were solved increased as the number of cases in the cluster in- creased (up to 4 cases). The association was not linear and the percentage solved did not increase further for clusters with >5 cases. The observed association is logical because as the number of cluster cases increases, the amount of epidemiologic data available for evaluation also increases. Our results suggest that public health ofﬁ cials should not wait to investigate Salmonella clusters if >4 cluster cases have been received. Figure 2. Temporal trends in number of Salmonella enterica isolates, number of clusters, and number of clusters solved (i.e., result in identiﬁ cation of a conﬁ rmed outbreak), Minnesota, USA, 2001–2007. The ability to solve a cluster of cases of Salmonella in- fection was also strongly associated with the density of the cluster cases. The proportion of clusters that were solved increased as the density of the cluster cases increased, but this relationship was not linear. This association is also logical. Dense clusters increase the likelihood that the clus- ter cases are epidemiologically linked rather than unrelated sporadic cases. In addition, dense clusters also likely signal larger outbreaks. Our results demonstrated a clear increase in the success of solving clusters in which the ﬁ rst 3 case isolates were received within 7 days. questionnaire. Interview times did not vary between inter- viewers. The median interview time was 27 minutes (range 13–56 minutes). Therefore, conducting standard interviews of all cases in the 344 clusters of >2 cases (n = 1,182 [31%] cases) required an estimated 76 interview hours/year. This threshold detected all 43 outbreaks identiﬁ ed through rou- tine laboratory surveillance during the study period and re- sulted in a cluster investigation positive predictive value (percentage of clusters investigated that were solved) of 13% (Table 3). Other cluster investigation thresholds had outbreak detection sensitivities of 53%–81% and positive predictive values of 23%–28% (Table 3). In theory, PFGE subtyping is less useful for recog- nizing clusters of unusual serovars worth investigating. In the current study, clusters of the common serovars Newport, Montevideo, and Heidelberg were statistically Table 2. Cluster Investigation Threshold During June–December 2008, 10 MDH staff inter- viewed 214 persons with Salmonella infections and re- corded the time required to complete the MDH standard Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 1681 RESEARCH 30 40 50 60 300 400 500 600 700 No. clu solates No. isolates No cl sters 0 10 20 30 40 50 60 0 100 200 300 400 500 600 700 2001 2002 2003 2004 2005 2006 2007 No. clusters No. isolates No. isolates No. clusters No. clusters solved 0 10 20 30 40 50 60 0 100 200 300 400 500 600 700 2001 2002 2003 2004 2005 2006 2007 No. clusters No. isolates No. isolates No. clusters No. clusters solved Figure 2. Temporal trends in number of Salmonella enterica isolates, number of clusters, and number of clusters solved (i.e., result in identiﬁ cation of a conﬁ rmed outbreak), Minnesota, USA, 2001–2007. 30 40 50 60 300 400 500 600 700 No. clu solates No. isolates No cl sters 0 10 20 30 40 50 60 0 100 200 300 400 500 600 700 2001 2002 2003 2004 2005 2006 2007 No. clusters No. isolates No. isolates No. clusters No. clusters solved 0 10 20 30 40 50 60 0 100 200 300 400 500 600 700 2001 2002 2003 2004 2005 2006 2007 No. clusters No. isolates No. isolates No. clusters No. clusters solved aenszel Ȥ2 test result for trend (p<0.001). compared with clusters of 3 cases odds ratio 1 87 95% confidence interval 0 52 6 66 aenszel Ȥ2 test result for trend (p<0.001). compared with clusters of 3 cases odds ratio 1.87, 95% confidence interval 0.52–6.66. ¶Significant Mantel-Haenszel Ȥ2 test result for trend (p<0.001). #Clusters of 4 cases compared with clusters of 3 cases odds ratio 1.87, 95% confidence interval 0.52–6.66. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 vars Typhimurium and Enteritidis. vars Newport, Heidelberg, and Montevideo. , s the number of days from receipt of first cluster case to third case received at the Minnesota Department of Health Public other serovars. gnificant Mantel-Haenszel Ȥ2 test result for trend (p<0.001). t f 4 d ith l t f 3 dd ti 1 87 95% fid i t l 0 52 6 66 1682 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 Ȥ (p ) usters of 4 cases compared with clusters of 3 cases odds ratio 1.87, 95% confidence interval 0.52–6.66. † yp ‡S. enterica serovars Newport, Heidelberg, and Montevideo. §All th Discussion It has been suggested that uncommon serovar clusters may be associated with uncommon food vehicles, which makes them more difﬁ cult to solve by using stan- dard methods (24). The relationship between serovar fre- quency and the likelihood of solving a cluster is unclear and warrants further study. all persons with Salmonella infection or investigate all small clusters. Rather, they must balance the time required for these efforts and the ability to detect outbreaks (25). more likely to be solved than clusters of the very com- mon serovars Enteritidis and Typhimurium. However, clusters of uncommon serovars were not more likely to be solved than were clusters of common or very common serovars. It has been suggested that uncommon serovar clusters may be associated with uncommon food vehicles, which makes them more difﬁ cult to solve by using stan- dard methods (24). The relationship between serovar fre- quency and the likelihood of solving a cluster is unclear and warrants further study. Incorporating a cluster investigation threshold on the basis of cluster size and cluster case density can decrease the number of unsuccessful cluster investigations and con- serve public health resources. However, this approach would also reduce the number of outbreaks that would be identiﬁ ed. One reason for this ﬁ nding is that outbreaks that are manifested as smaller, less dense clusters would not be investigated. Another potential disadvantage of a cluster threshold approach is that delay of interviews until a cluster is solved can decrease the quality of exposure information obtained and therefore the likelihood that the cluster will be solved (12). The limited number of solved clusters prevented mul- tivariate analysis from being used to characterize the inde- pendent effect of predictors and possible effect modiﬁ cation between predictors. However, comparing the magnitude of the estimated effect of cluster size and cluster case density suggests that cluster case density may be a more useful pre- dictor of a cluster being solved. Four conﬁ rmed outbreaks during the study did not meet the cluster deﬁ nition, and many conﬁ rmed outbreaks had cases that were outside the cluster deﬁ nition. This ﬁ nd- ing is an important reminder that lack of temporal clus- tering does not eliminate the possibility of an outbreak. Increasing the period covered by a cluster deﬁ nition will yield the beneﬁ t of solving more outbreaks. Discussion Univariate association between Salmonella enterica serovar frequency, cluster size, cluster density, and cluster being solved, Minnesota, USA, 2001–2007* Characteristic No. (%) solved clusters No. unsolved clusters Odds ratio (95% confidence interval) Serovar Very common† 22 (10) 203 Referent Common‡ 11 (23) 37 2.74 (1.23–6.13) Uncommon§ 10 (14) 61 1.51 (0.68–3.37) Total 43 (13) 301 Cluster size¶ 2 16 (8) 194 Referent 3 8 (15) 47 2.06 (0.83–5.11) 4 7 (24) 22 3.86# (1.43–10.40) >5 12 (24) 38 3.83 (1.68–8.74) Total 43 (13) 301 Cluster density, d** 0 5 (71) 2 25.8 (3.42–195.37) 1–7 16 (33) 33 5.01 (1.33–18.89) 8–14 11 (22) 40 2.84 (0.73–11.07) >15 3 (9) 31 Referent Total 35 (25) 106 A solved cluster is one that results in identification of a confirmed outbreak. †S. enterica serovars Typhimurium and Enteritidis. ‡S. enterica serovars Newport, Heidelberg, and Montevideo. §All other serovars. ¶Significant Mantel-Haenszel Ȥ2 test result for trend (p<0.001). Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 Salmonella enterica PFGE Clusters Table 3. Comparison of Salmonella enterica cluster investigation thresholds, Minnesota, USA, 2001–2007 Cluster investigation threshold No. isolates represented in clusters All Salmonella isolates, % (n = 3,803†) Estimated interview time, h/y‡ No. (%) outbreak clusters meeting threshold Cluster investigation PPV All clusters (n = 344) 1,182 31 76 43 (100) 13 Clusters >3 cases (n = 152) 778 20 50 35 (81) 23 Clusters >4 cases (n = 83) 601 16 39 23 (53) 28 Clusters with a density of 0–14 d§ (n = 119) 633 17 41 32 (74) 27 Clusters >4 cases or with a density of 0–7 d§ (n = 100) 652 17 42 28 (65) 28 PPV, positive predictive value. †A total of 215 isolates associated with excluded clusters were removed from study isolate total (n = 4,018). ‡Based on a 27-min median interview time per case-patient. §Density defined as the number of days from receipt of first cluster case isolate to third case isolate received at the Minnesota Department of Health Public Health Laboratory. f Salmonella enterica cluster investigation thresholds, Minnesota, USA, 2001–2007 more likely to be solved than clusters of the very com- mon serovars Enteritidis and Typhimurium. However, clusters of uncommon serovars were not more likely to be solved than were clusters of common or very common serovars. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 Discussion However, more resources will be expended conducting unsuccessful clus- ter investigations. The results of this study suggest that the use of a 2-week cluster window is sufﬁ ciently sensitive to detect most outbreaks. However, in practice, MDH epide- miologists do not use a strict 2-week cluster window when investigating clusters. Instead, all persons with Salmonella infection are interviewed and cases with matching PFGE patterns are often compared even if the second case is re- ceived >2 weeks after the ﬁ rst case. The 22 conﬁ rmed outbreaks that were excluded from the analysis demonstrate the value for national collabora- tion such as PulseNet and use of outbreak detection meth- ods in addition to PFGE clustering within a given state. Six outbreaks were solved in which Minnesota only had 1 case, which demonstrated the utility of molecular subtyp- ing in detecting geographically dispersed outbreaks. For 7 conﬁ rmed outbreaks, a call placed to the MDH foodborne illness hotline contributed to identiﬁ cation of the outbreak and demonstrated the utility of complaint systems in de- tecting outbreaks. Interviewing all persons with Salmonella infection required a median of 27 minutes per person with Salmo- nella infection when the MDH standard questionnaire was used. By extrapolation, MDH staff spent ≈244 hours/year conducting routine interviews of persons with Salmonella infections. This ﬁ gure does not include time spent attempt- ing to reach persons, gathering demographic information from clinicians, or reinterviewing persons for cluster in- vestigations. We recommend interviewing all persons with Salmonella infection and investigating all PFGE clusters to identify as many outbreaks as possible. However, many health departments do not have the resources to interview The potential utility of the cluster investigation thresh- olds reported is based on the characteristics of the population of Minnesota and MDH surveillance methods: conducting real-time PFGE subtyping of all Salmonella isolates, inter- viewing all case-patients in real time by using a detailed exposure questionnaire from a central location for the en- tire state, and investigating clusters by using an iterative model (19–21). These factors aid in the timeliness of out- break detection and investigation in Minnesota. These re- erging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 1683 RESEARCH 6. Swaminathan B, Barrett TJ, Hunter SB, Tauxe RV. CDC PulseNet Task Force. PulseNet: the molecular subtyping network for food- borne bacterial disease surveillance, United States. Emerg Infect Dis. 2001;7:382–9. Acknowledgments We thank Jeff Bender; the Public Health Laboratory staff; and the Foodborne, Vectorborne, and Zoonotic Diseases Unit staff at the Minnesota Department of Health for their contribu- tions to the study. 14. Hedberg CW, Besser JM. Commentary: cluster evaluation, PulseNet, and public health pracitce. Foodborne Pathog Dis. 2006;3:32–5. DOI: 10.1089/fpd.2006.3.32 15. Council to Improve Foodborne Outbreak Response (CIFOR). Guide- lines for foodborne disease outbreak response. Atlanta: Council of State and Territorial Epidemiologists; 2009. This study was supported in part by a cooperative agreement (U50/CCU51190) with the Centers for Disease Control and Pre- vention as part of the Emerging Infections Program, Foodborne Diseases Active Surveillance Network (FoodNet). 16. Hedberg CW, Jacquet C, Goulet V. Surveillance of listeriosis in France, 2000–2004: evaluation of cluster investigation criteria. Presented at the 16th International Symposium on Problems of Listeriosis. Savan- nah (GA) USA; 2007 Mar 20–23 [cited 20101 Jul 8]. http://www.aphl. org/profdev/conferences/proceedings/Documents/2007_ISOPOL/ Surveillance_of_Listeriosis_in_France.pdf Mr Rounds is an epidemiologist with the Minnesota Depart- ment of Health. His research interests include evaluating public health surveillance methods to improve outbreak investigations and disease control efforts. 17. Reportable disease rule. Minnesota Department of Health. 2009 Jun 23 [cited 2010 Jul 8]. http://www.health.state.mn.us/divs/idepc/ dtopics/reportable/rule/rule.html 18. Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swami- nathan B, et al. Standardization of pulsed-ﬁ eld gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonel- la, and Shigella for PulseNet. Foodborne Pathog Dis. 2006;3:59–67. DOI: 10.1089/fpd.2006.3.59 Discussion sults may not be applicable in jurisdictions in which PFGE is not conducted in real time or batching of PFGE isolates occurs. Additional studies at the national level and in other states are needed to understand surveillance characteristics in other states and determine useful predictors of multistate clusters being solved. 7. Swaminathan B, Barrett TJ, Fields P. Surveillance for human Salmo- nella infections in the United States. J AOAC Int. 2006;89:553–9. 8. Tauxe RV. Molecular subtyping and the transformation of pub- lic health. Foodborne Pathog Dis. 2006;3:4–8. DOI: 10.1089/ fpd.2006.3.4 Although successful cluster investigations will de- pend on the experience and ability of public health staff involved, this study demonstrates the increased probabil- ity of a cluster being solved as the number of cases in a cluster increases and as the cluster density increases. Spe- ciﬁ cally, investigation of PFGE clusters of >4 Salmonella case isolates and clusters in which the ﬁ rst 3 cases were received at the MDH PHL within 1 week yielded a major beneﬁ t in terms of outbreak identiﬁ cation. These results establish a benchmark for surveillance of Salmonella in- fections, and may provide a basis for investigating clus- ters of Salmonella cases for public health agencies with limited resources. 9. Allos BM, Moore MR, Grifﬁ n PM, Tauxe RV. Surveillance for spo- radic foodborne disease in the 21st century: the FoodNet perspective. Clin Infect Dis. 2004;38(Suppl 3):S115–20. DOI: 10.1086/381577 10. Barrett TJ, Gerner-Smidt P, Swaminathan B. Interpretation of pulsed-ﬁ eld gel electrophoresis patterns in foodborne disease inves- tigations and surveillance. Foodborne Pathog Dis. 2006;3:20–31. DOI: 10.1089/fpd.2006.3.20 11. Buehler JW, Hopkins RS, Overhage JM, Sosin DM, Tong V; CDC Working Group. Framework for evaluating public health surveillance systems for early detection of outbreaks: recommendations form the CDC Working Group. MMWR Recomm Rep. 2004;53(RR-5):1–11. 12. Hedberg CW, Greenblatt JR, Matyas BT, Lemmings J, Sharp DJ, Skibicki RT, et al. Timeliness of enteric disease surveillance in 6 US states. Emerg Infect Dis. 2008;14:311–3. DOI: 10.3201/ eid1402.070666 13. Lynch M, Painter J, Woodruff R, Braden C. Surveillance for food- borne-disease outbreaks—United States, 1998–2002. MMWR Sur- veill Summ. 2006;55(SS10):1–42. 24. Lynch MF, Tauxe RV, Hedberg CW. The growing burden of food- borne outbreaks due to contaminated fresh produce: risks and op- portunities. Epidemiol Infect. 2009;137:307–15. DOI: 10.1017/ S0950268808001969 25. Hoffman RE, Greenblatt J, Matyas BT, Sharp DJ, Esteban E, Hodge K, et al. Capacity of state and territorial health agencies to prevent foodborne illness. Emerg Infect Dis. 2005;11:11–6. References 1. Voetsch AC, Van Gilder TJ, Angulo FJ, Farley MM, Shallow S, Mar- cus R, et al. FoodNet estimate of the burden of illness caused by nontyphoidal Salmonella infections in the United States. Clin Infect Dis. 2004;38(Suppl 3):S127–34. DOI: 10.1086/381578 19. Smith KE, Medus C, Meyer SD, Boxrud DJ, Leano F, Hedberg CW, et al. Outbreaks of salmonellosis in Minnesota (1998 through 2006) associated with frozen, microwaveable, breaded, stuffed chicken products. J Food Prot. 2008;71:2153–60. 2. Mead PS, Sultsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, et al. Food-related illness and death in the United States. Emerg Infect Dis. 1999;5:607–25. DOI: 10.3201/eid0505.990502 20. Centers for Disease Control and Prevention. Multistate outbreak of Salmonella infections associated with frozen pot pies—United States, 2007. MMWR Morb Mortal Wkly Rep. 2008;57:1277–80. 3. Salmonellosis. In: Heymann DL, Thuriaux MC, editors. Control of communicable diseases manual. 18th ed. Washington: United Book Press; 2004. p. 469–73. 21. Hedican E, Hooker C, Jenkins T, Medus C, Jawahir S, Leano F, et al. Restaurant Salmonella Enteritidis outbreak associated with an asymptomatic infected food worker. J Food Prot. 2009;72:2332–6. 4. Olsen SJ, Bishop R, Brenner FW, Roels TH, Bean N, Tauxe RV, et al. The changing epidemiology of salmonella: trends in serotypes isolated from humans in the United States, 1987–1997. J Infect Dis. 2001;183:753–61. DOI: 10.1086/318832 22. Bender JB, Hedberg CW, Boxrud DJ, Besser JM, Wicklund JH, Smith KE, et al. Use of molecular subtyping in surveillance for Salmonella enterica serotype Typhimurium. N Engl J Med. 2001;344:189–95. DOI: 10.1056/NEJM200101183440305 5. Salmonella infections. In: Pickering LK, Baker CJ, Kimberlin DW, Long SS, editors. Red book: 2006 report of the Committee on Infec- tious Diseases. 27th ed. Elk Grove Village (IL): American Academy of Pediatrics, 2006. p. 584–89. 23. Boxrud D, Pederson-Gulrud K, Wotton J, Medus C, Lyszkowicz E, Besser J, et al. Comparison of multiple-locus variable-number tan- dem repeat analysis, pulsed-ﬁ eld gel electrophoresis, and phage typ- ing for subtype analysis of Salmonella enterica serotype Enteritidis. J Clin Microbiol. 2007;45:536–43. DOI: 10.1128/JCM.01595-06 1684 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 11, November 2010 Salmonella enterica PFGE Clusters Salmonella enterica PFGE Clusters Address for correspondence: Joshua M. Rounds, Acute Disease Investigation and Control Section, Minnesota Department of Health, PO Box 64975, St. Paul, MN 55164, USA; email: joshua.rounds@state. mn.us Address for correspondence: Joshua M. References Rounds, Acute Disease Investigation and Control Section, Minnesota Department of Health, PO Box 64975, St. Paul, MN 55164, USA; email: joshua.rounds@state. mn.us 25. Hoffman RE, Greenblatt J, Matyas BT, Sharp DJ, Esteban E, Hodge K, et al. Capacity of state and territorial health agencies to prevent foodborne illness. Emerg Infect Dis. 2005;11:11–6. 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https://openalex.org/W4319453024	https://zenodo.org/records/7620950/files/1_num%20sent%20to%20ijesrt.pdf	Latin	null	ANALYSIS OF POLLUTANT CONCENTRATION IN SURFACE WATERS USING ONE - DIMENSIONAL ADVECTION DIFFUSION EQUATION WITH TEMPORALLY VARYING COEFFICIENTS	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	7,631	IJESRT: 12(1), January, 2023 IJESRT: 12(1), January, 2023 ISSN: 2277-9655 International Journal of Engineering Sciences & Research Technology (A Peer Reviewed Online Journal) Impact Factor: 5.164 IJESRT Chief Editor Executive Editor Dr. J.B. Helonde Mr. Somil Mayur Shah ernational Journal of Engineering Sciences & Research Technology (A Peer Reviewed Online Journal) Impact Factor: 5.164 Technology (A Peer Reviewed Online Journal) Impact Factor: 5.164 IJESRT Chief Editor Executive Editor Dr. J.B. Helonde Mr. Somil Mayur Shah Chief Editor Dr. J.B. Helonde Mail: editor@ijesrt.com Website: www.ijesrt.com ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 ABSTRACT The aim of this study is to provide an analysis on pollutant concentration in surface waters using one – dimensional advection diffusion equation with temporally varying coefficients. Numerical and analytical solutions are obtained for one - dimensional Advection Diffusion Equations with variable coefficients in a finite medium. Finite Difference and Laplace Transforms Methods are applied to solve the Advection Diffusion Equation with temporally varying coefficients. Absolute error obtained from comparing analytical and numerical solutions at different points reveals that the numerical scheme is accurate. Simulations based on the validated numerical scheme are obtained. Simulations on the effect of dispersion and velocity coefficients (based on Peclet number) on pollutant concentration show that concentration increases around the source point and gradually decreases with increasing distance from the source point. It further shows that concentration is higher for Peclet number much greater than one as compared to Peclet numbers much less than or equal to one. Effect of temporally varying velocity and dispersion coefficients on pollutant concentration is also presented. The findings show that concentration is higher for exponentially decreasing dispersion in an exponentially accelerating flow and lower for exponentially increasing dispersion in an exponentially accelerating flow. KEYWORDS: Advection Diffusion Equation, Finite Difference Method ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 schemes. They observed that Crank Nicolson scheme is the most accurate scheme and that FTCSCS and CNS show good rate of convergence. An analytical and numerical solution of a one - dimensional ADE with uniformly and exponentially increasing forms of sources was determined by Manitcharoen & Pimpunchat (2020). Laplace transforms was used to solve the ADE with constant coefficients; and explicit finite difference techniques to solve the ADE with spatially varying coefficients. Numerical approximations were compared against relative error values. They concluded that the analytical and numerical solutions agreed to a higher degree. Alebrahem (2017) applied Forward Time Centered Space (FTCS) to solve a non - trivial transport problem using different time step (∆t) and space size (∆x). While imposing Dirichlet boundary conditions, it was observed that the results were converging to the exact solution if dispersion coefficient, ∆t and ∆x are small. Ahmed (2012) developed a new Finite Difference scheme based on mathematical combination between Siemieniuch and Gradwell approximations for time and Dehgans approximation for spatial differences. The new scheme was used to determine the numerical solution for a one - dimensional ADE with constant and variable coefficients. The numerical solutions were compared with some available analytical solutions and showed a good agreement. Tenth order Finite Difference Scheme in space and a fourth order Runge kutta scheme in time was applied by Gurarslan et al (2010) to solve a one - dimensional ADE. Numerical experiments demonstrated that the schemes were efficient and had higher order accuracy for Pe ≤5. Yip (2021) studied pollutant transport in a straight narrow channel using upwind finite difference method. They used explicit, backward and central differences to discretize the time scale, the advection term and the diffusion terms respectively. The numerical model was validated against an existing analytical solution and the results showed good agreement between the analytical and numerical solutions. The validated model was applied to different cases of pollutant release mechanisms involving continuous and instantaneous pollutant releases, further observing that this model could capture the physics of the problem and be able to provide valuable information on the time and spatial evolution of pollutant concentration in both cases. When comparing the performance of the forward time centered space and the Crank Nicolson schemes for advection diffusion equation with various velocity and dispersion parameters, Johari et al (2018) observed that Crank Nicolson schemes were better than the forward time centered space in terms of accuracy. A numerical solution of the one dimensional ADE using standard and non- standard finite difference schemes was also obtained by Appadu (2013). The researcher used explicit Lax Wendroff, Crank Nicolson and a non - standard scheme to determine the solution to the ADE subject to specified initial and boundary conditions. It was observed that Crank Nicolson method was the most efficient method followed by the non - standard finite difference scheme. In a study aimed at comparing the accuracy of four numerical methods in solving a one-dimensional ADE, Kaya & Gharehbaghi (2014) used Finite Difference Method (FDM), Fourth Order Finite Difference Method (FOFDM), Finite Volume Method (FVM) and Differential Quadrature Method (DQM) in implicit conditions. They deduced that DQM provided better accurate results followed by FOFDM. FDM produced worst results. Huang et al (1997) on the other hand developed a third order numerical scheme with upwind weighting for solving the solute transport equation. This scheme yielded very accurate solutions near sharp concentration fronts, thereby showing its ability to eliminate numerical dispersion. However, the scheme was found to suffer from numerical oscillations, and could be avoided by employing upwind weighting techniques in the numerical scheme. Solutions obtained after upwind weighting were free of numerical oscillations and exhibited negligible numerical dispersion. In most studies on numerical solution of one - dimensional ADE we encountered, researchers have used FDM to solve the ADE with constant coefficients subject to various initial and boundary conditions. Though Ahmed (2012) considered spatially varying dispersion and velocity coefficients, their study did not explore temporally varying coefficients. Our current study determines pollutant concentration using Finite Difference Method with discretization based on forward time central space, centered scheme (FTCSCS). 2.1 Model Formulation 1. INTRODUCTION The transport of pollutants in natural streams such as rivers may be as a result of two processes namely dispersion and advection. These processes are prescribed in a mathematical model (Advection Diffusion Equation) that describes how pollutants are transported from one region to another. The Advection Diffusion Equation (ADE) is a parabolic partial differential equation based on conservation of mass and Fick’s first law. It distinguishes two transport modes; the advective transport as a result of pollutant molecules being carried by the bulk motion of the fluid and transport due to hydrodynamic dispersion. Hydrodynamic dispersion is the combination of molecular diffusion and mechanical dispersion. The effects of advection and hydrodynamic dispersion in transportation of pollutant molecules are represented in the ADE as advection and dispersion coefficients. The velocity and dispersion coefficients of the ADE may be considered to be constant, spatially varying, temporally varying, or spatially and temporally varying. In this study, temporally varying velocity and dispersion coefficients are considered. Solutions of partial differential equations that describe transport processes have been obtained in various studies using numerical methods such as Finite Difference Method (FDM), Finite Element Method (FEM) and Finite Volume Method (FVM). http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology Andallah & Khatun (2020) obtained a numerical solution for the one - dimensional ADE using explicit centered difference and Crank Nicolson schemes (CNS) for a prescribed initial condition. They established that Crank Nicolson scheme is unconditionally stable using von Neumann stability criterion. They further performed error estimation of forward time central space, centered scheme (FTCSCS), forward time backward space, centered space (FTBSCS) and Crank Nicolson schemes to determine the accuracy and rate of convergence of these three [1] 2. MATERIALS AND METHODS Modeling and simulation of pollutant concentration from a point source into surface waters can be broken down into the following steps: Formulation of the model (describing the geometry of the domain, introducing sources, sinks and dispersion characteristics of the entire domain, introducing the appropriate boundary conditions), solving the model using finite differences and simulation of results. In our study, both numerical and analytical solutions of the model have been determined. Analytical solutions are obtained to validate the numerical results. Simulation of results based on the validated numerical results is presented. Substituting Equations (2) and (3) in Equation (1) yields 𝜕𝑐(𝑥,𝑡) 𝜕𝑡 −𝐷0𝑓1(𝑚𝑡) 𝜕2𝑐(𝑥,𝑡) 𝜕𝑥2 + 𝑈0𝑓2(𝑚𝑡) 𝜕𝑐(𝑥,𝑡) 𝜕𝑥 = 𝜆(𝑡)𝛿(𝑥− 𝑆) (4) But 𝛿(𝑥 − 𝑆) = 0 for 𝑥≠𝑆 (Duffy, 2001), Equation (4) becomes 𝜕𝑐(𝑥,𝑡) 𝜕𝑡 −𝐷0𝑓1(𝑚𝑡) 𝜕2𝑐(𝑥,𝑡) 𝜕𝑥2 + 𝑈0𝑓2(𝑚𝑡) 𝜕𝑐(𝑥,𝑡) 𝜕𝑥 = 0 (5) 𝜕𝑐(𝑥,𝑡) 𝜕𝑡 −𝐷0𝑓1(𝑚𝑡) 𝜕2𝑐(𝑥,𝑡) 𝜕𝑥2 + 𝑈0𝑓2(𝑚𝑡) 𝜕𝑐(𝑥,𝑡) 𝜕𝑥 = 𝜆(𝑡)𝛿(𝑥− 𝑆) (4) But 𝛿(𝑥 − 𝑆) = 0 for 𝑥≠𝑆 (Duffy, 2001), Equation (4) becomes 𝐷0𝑓1(𝑚𝑡) 𝜕2𝑐(𝑥,𝑡) 𝜕𝑥2 + 𝑈0𝑓2(𝑚𝑡) 𝜕𝑐(𝑥,𝑡) 𝜕𝑥 = 𝜆(𝑡)𝛿(𝑥− 𝑆) (4) (4) But 𝛿(𝑥 − 𝑆) = 0 for 𝑥≠𝑆 (Duffy, 2001), Equation (4) becomes 𝜕𝑐(𝑥,𝑡) 𝜕𝑡 −𝐷0𝑓1(𝑚𝑡) 𝜕2𝑐(𝑥,𝑡) 𝜕𝑥2 + 𝑈0𝑓2(𝑚𝑡) 𝜕𝑐(𝑥,𝑡) 𝜕𝑥 = 0 (5) = 0 for 𝑥≠𝑆 (Duffy, 2001), Equation (4) becomes 1(𝑚𝑡) 𝜕2𝑐(𝑥,𝑡) 𝜕𝑥2 + 𝑈0𝑓2(𝑚𝑡) 𝜕𝑐(𝑥,𝑡) 𝜕𝑥 = 0 (5) 𝜕𝑐(𝑥,𝑡) 𝜕𝑡 −𝐷0𝑓1(𝑚𝑡) 𝜕2𝑐(𝑥,𝑡) 𝜕𝑥2 + 𝑈0𝑓2(𝑚𝑡) 𝜕𝑐(𝑥,𝑡) 𝜕𝑥 = 0 (5) Further assuming that no pollution has occurred at some initial time; there is a continuous mass injection of pollutants at the point x = 0 and that there is zero concentration gradient at the downstream. The initial and boundary conditions used include Further assuming that no pollution has occurred at some initial time; there is a continuous mass injection of pollutants at the point x = 0 and that there is zero concentration gradient at the downstream. The initial and boundary conditions used include 𝑐(𝑥, 0) = 0; 0 ≤ 𝑥 ≤ 𝑙; 𝑡 > 0 (6) 𝑐(0, 𝑡) = 𝐶0; 𝑡 > 0 (7) 𝜕𝑐(𝐿,𝑡) 𝜕𝑥 = 0; 𝑡 > 0 (8) 𝑐(𝑥, 0) = 0; 0 ≤ 𝑥 ≤ 𝑙; 𝑡 > 0 𝑐(0, 𝑡) = 𝐶0; 𝑡 > 0 𝜕𝑐(𝐿,𝑡) 𝜕𝑥 = 0; 𝑡 > 0 (6) (7) (8) (8) 2.2 Solution of Mathematical Model 2.2 Solution of Mathematical Model We start by rewriting Equations (5) – (8) in non – dimensional form. We use the following dimensionless quantities: 𝑐∗= 𝑐 𝐶0 ; 𝑡∗= 𝑡𝐷0 𝐿2 ; 𝑥∗= 𝑥 𝐿; 𝑃𝑒= 𝐿𝑈0 𝐷0 (9) 𝑐∗= 𝑐 𝐶0 ; 𝑡∗= 𝑡𝐷0 𝐿2 ; 𝑥∗= 𝑥 𝐿; 𝑃𝑒= 𝐿𝑈0 𝐷0 (9) Where 𝐶0 is the input concentration, 𝐿 is the length scale of the spatial domain, 𝑡∗ is the time scale and Peclet number (𝑃𝑒) is the ratio of advective and diffusive fluxes. 2.1 Model Formulation http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology [2] IJESRT is licensed under a Creative Commons Attribution 4.0 International License. Model Formulation [2] ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 Transport of pollutants in rivers is commonly described using processes of advection and diffusion. Based on the assumptions that: The flow conditions are unsteady; transport dominantly occurs along the longitudinal direction; pollutants are soluble compounds which may be subjected to advection and diffusion processes, pollutants are conservative in nature and pollutants emanate from discrete locations, the process of transportation of pollutants in surface waters is mathematically described using Advection - Diffusion Equation (ADE) 𝜕𝑐(𝑥,𝑡) 𝜕𝑡 −𝐷(𝑥, 𝑡) 𝜕2𝑐(𝑥,𝑡) 𝜕𝑥2 + 𝑈(𝑥, 𝑡) 𝜕𝑐(𝑥,𝑡) 𝜕𝑥 = 𝜆(𝑡)𝛿(𝑥− 𝑆) (1) 𝜕𝑐(𝑥,𝑡) 𝜕𝑡 −𝐷(𝑥, 𝑡) 𝜕2𝑐(𝑥,𝑡) 𝜕𝑥2 + 𝑈(𝑥, 𝑡) 𝜕𝑐(𝑥,𝑡) 𝜕𝑥 = 𝜆(𝑡)𝛿(𝑥− 𝑆) (1) Where 𝑐(𝑥, 𝑡) is the pollutant concentration. 𝐷(𝑥, 𝑡) and 𝑈(𝑥, 𝑡) are the dispersion and velocity coefficients respectively. 𝜆(𝑡)𝛿(𝑥− 𝑆) represents the source term where 𝜆(𝑡) is the source intensity and 𝛿(𝑥− 𝑆) is a function that mathematically represents a point source. Here S is pollutant source location and 𝛿 is Dirac delta function. In our study, both dispersion and velocity coefficients are dependent on time only thus the pollutant dispersion and velocity parameters in Equation (1) can be written in terms of initial dispersion 𝐷0 and uniform velocity coefficients 𝑈0 as 𝐷(𝑡) = 𝐷0𝑓1(𝑚𝑡) (2) 𝑈(𝑡) = 𝑈0𝑓2(𝑚𝑡) (3) 𝐷(𝑡) = 𝐷0𝑓1(𝑚𝑡) 𝑈(𝑡) = 𝑈0𝑓2(𝑚𝑡) (2) (3) Where 𝑓𝑖(𝑚𝑡), 𝑖= 1,2 is a function that describes the temporal dependence of the velocity and dispersion coefficients, m is the unsteady parameter whose dimension is inverse of time variable t. 2.2.1 Numerical Solution We then approximate the difference equation into finite difference equations Using Taylor series expansion for the first and second order We compute 𝑐(𝑥𝑖, 𝑡𝑗) for 𝑖= 1, 2, … … . . , 𝑛+ 1; 𝑗= 1,2, … … . , 𝑚+ 1. We then approximate the difference equation into finite difference equations. Using Taylor series expansion for the first and second order derivatives, the combination gives rise to either Explicit or Implicit schemes. In this study, an explicit centered difference scheme (Forward time Central Space, Centered Scheme (FTCSCS)) is used to determine the solution. 2.2.1 Numerical Solution 2.2.1 Numerical Solution 2.2.1 Numerical Solution Finite difference method is applied to solve the dimensionless Equation (10) subject to the initial and boundary conditions in Equations (11) – (13). We discretize the domain by dividing the solution domain in the xt - plane into equal meshes with grid points, each mesh with step size ∆𝑥∗ by ∆𝑡∗. The interval [0, 𝑙] is divided into 𝑛 equal parts by the points 𝑥1 ∗, 𝑥2 ∗, … … … … … , 𝑥𝑛 + 1 ∗ with step length ∆𝑥∗= 𝑥𝑛+1 ∗ − 𝑥1∗ 𝑛 . Similarly, the time interval is divided into m equal parts by the points 𝑡1 ∗, 𝑡2 ∗, … … … … … , 𝑡𝑚 + 1 ∗ with time step ∆𝑡∗= 𝑡𝑚+1 ∗ − 𝑡1∗ 𝑚 . We compute 𝑐∗(𝑥𝑖, 𝑡𝑗) for 𝑖= 1, 2, … … . . , 𝑛+ 1; 𝑗= 1,2, … … . , 𝑚+ 1. We then approximate the difference equation into finite difference equations. Using Taylor series expansion for the first and second order derivatives, the combination gives rise to either Explicit or Implicit schemes. In this study, an explicit centered difference scheme (Forward time Central Space, Centered Scheme (FTCSCS)) is used to determine the solution. Discretization of time and space derivatives of 𝑐∗ in Equation (10) using FTCSCS at (𝑖, 𝑗)𝑡ℎ node is given by Finite difference method is applied to solve the dimensionless Equation (10) subject to the initial and boundary conditions in Equations (11) – (13). We discretize the domain by dividing the solution domain in the xt - plane into equal meshes with grid points, each mesh with step size ∆𝑥∗ by ∆𝑡∗. The interval [0, 𝑙] is divided into 𝑛 equal parts by the points 𝑥1 ∗, 𝑥2 ∗, … … … … … , 𝑥𝑛 + 1 ∗ with step length ∆𝑥∗= 𝑥𝑛+1 ∗ − 𝑥1∗ 𝑛 . Similarly, the time interval is divided into m equal parts by the points 𝑡1 ∗, 𝑡2 ∗, … … … … … , 𝑡𝑚 + 1 ∗ with time step ∆𝑡∗= 𝑡𝑚+1 ∗ − 𝑡1∗ 𝑚 . We compute 𝑐∗(𝑥𝑖, 𝑡𝑗) for 𝑖= 1, 2, … … . . , 𝑛+ 1; 𝑗= 1,2, … … . , 𝑚+ 1. Applying the dimensionless quantities in Equation (9) on Equation (5) yields ∗) 𝜕2𝑐∗ 𝜕𝑥∗2 −𝑃𝑒𝑓2(𝑚𝑡∗) 𝜕 𝑐∗ 𝜕 𝑥∗ (10) 𝜕𝑐∗ 𝜕𝑡∗= 𝑓1(𝑚𝑡∗) 𝜕2𝑐∗ 𝜕𝑥∗2 −𝑃𝑒𝑓2(𝑚𝑡∗) 𝜕 𝑐∗ 𝜕 𝑥∗ (10) http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology [3] ijesrt.com© International Journal of Engineering Sciences & Research Technology [3] http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technolog http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology [3] [ ] IJESRT is licensed under a Creative Commons Attribution 4.0 International License. IJESRT is licensed under a Creative Commons Attribution 4.0 International License. IJESRT is licensed under a Creative Commons Attribution 4.0 International License. ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 ISSN: 2277-9655 [Raoul et al., 12(1): January, 2023] Impact Factor: 5.164 IC™ Value: 3.00 CODEN: IJESS7 Non – dimensionalizing the initial and boundary conditions in Equations (6) – (8) yields 𝑐∗= 0; 𝑡∗= 0 (11) 𝑐∗= 1; 𝑥∗= 0 (12) 𝜕𝑐∗ 𝜕𝑥∗= 0; 𝑥∗= 𝑙 (13) 𝑐∗= 0; 𝑡∗= 0 𝑐∗= 1; 𝑥∗= 0 𝜕𝑐∗ 𝜕𝑥∗= 0; 𝑥∗= 𝑙 (12) (13) Numerical and analytical solutions of Equations (10) – (13) are determined. Numerical and analytical solutions of Equations (10) – (13) are determined. Numerical and analytical solutions of Equations (10) – (13) are determined. ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 2.2.1 Numerical Solution ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 ISSN: 2277-9655 [Raoul et al., 12(1): January, 2023] Impact Factor: 5.164 IC™ Value: 3.00 CODEN: IJESS7 SSN: 9655 [Raoul et al., 12(1): January, 2023] Impact Factor: 5.164 IC™ Value: 3.00 CODEN: IJESS7 𝑐∗ 𝑖 𝑗+1 = 𝛿𝑐∗ 𝑖 −1 𝑗 + 𝛽 𝑐∗ 𝑖 𝑗+ 𝛼𝑐∗ 𝑖 + 1 𝑗 (22) 𝑐∗ 𝑖 𝑗+1 = 𝛿𝑐∗ 𝑖 −1 𝑗 + 𝛽 𝑐∗ 𝑖 𝑗+ 𝛼𝑐∗ 𝑖 + 1 𝑗 𝛿𝑐∗ 𝑖 −1 𝑗 + 𝛽 𝑐∗ 𝑖 𝑗+ 𝛼𝑐∗ 𝑖 + 1 𝑗 (22) (22) Equation (22) is used for the interior nodes. The initial condition in Equation (11) is discretized as 𝑐∗ 𝑖 0 = 𝑐∗(𝑥∗ 𝑖, 0) = 0 𝑐∗ 𝑖 0 = 𝑐∗(𝑥∗ 𝑖, 0) = 0 (23) The discretized inlet boundary condition in Equation (12) is given by 𝑐∗ 1 𝑗+1 = 1 (24) (24) To discretize the boundary condition in Equation (13) at 𝑖= 𝑛, we introduce a ghost boundary node 𝑥𝑛+ 1 ∗ and its corresponding approximation as 𝑐 𝑛+ 1 ∗𝑗 = 𝑐∗( 𝑥𝑛+ 1 ∗ , 𝑡𝑗 ∗). Then using Equation (13) and the central difference formula in Equation (15) at the last boundary ( 𝑥𝑛 ∗), we get 𝑐∗ 𝑛 + 1 𝑗 = 𝑐∗ 𝑛−1 𝑗 (25) 𝑐∗ 𝑛 + 1 𝑗 = 𝑐∗ 𝑛−1 𝑗 (25) Rewriting Equation (22) using 𝑖= 𝑛 𝑐∗ 𝑛 𝑗+1 = 𝛿𝑐∗ 𝑛−1 𝑗 + 𝛽 𝑐∗ 𝑛 𝑗+ 𝛼𝑐∗ 𝑛 + 1 𝑗 (26) Applying Equation (25) on Equation (26) gives: 𝑐∗ 𝑛 + 1 𝑗 = 𝑐∗ 𝑛−1 𝑗 (25) Rewriting Equation (22) using 𝑖= 𝑛 Rewriting Equation (22) using 𝑖= 𝑛 𝑐∗ 𝑛 𝑗+1 = 𝛿𝑐∗ 𝑛−1 𝑗 + 𝛽 𝑐∗ 𝑛 𝑗+ 𝛼𝑐∗ 𝑛 + 1 𝑗 (26) Applying Equation (25) on Equation (26) gives: Applying Equation (25) on Equation (26) gives: 𝑐∗ 𝑛 𝑗+1 = (𝛼+ 𝛿)𝑐∗ 𝑛−1 𝑗 + 𝛽𝑐∗ 𝑛 𝑗 (27) 𝑐∗ 𝑛 𝑗+1 = (𝛼+ 𝛿)𝑐∗ 𝑛−1 𝑗 + 𝛽𝑐∗ 𝑛 𝑗 (27) (𝛼+ 𝛿)𝑐∗ 𝑛−1 𝑗 + 𝛽𝑐∗ 𝑛 𝑗 (27) 𝑐∗ 𝑛 𝑗+1 = (𝛼+ 𝛿)𝑐∗ 𝑛−1 𝑗 + 𝛽𝑐∗ 𝑛 𝑗 (27) Equation (27) is the discretized equation of boundary condition in Equation (13). 2.2.1 Numerical Solution Discretization of time and space derivatives of 𝑐∗ in Equation (10) using FTCSCS at (𝑖, 𝑗)𝑡ℎ node is given by 𝜕𝑐∗ 𝜕𝑡∗= 𝑐∗ 𝑖 𝑗+1− 𝑐∗ 𝑖 𝑗 ∆𝑡∗ (14) 𝜕𝑐∗ 𝜕𝑥∗= 𝑐∗ 𝑖+1 𝑗 − 𝑐∗ 𝑖−1 𝑗 2∆𝑥∗ (15) 𝜕2𝑐∗ 𝜕𝑥∗2 = 𝑐∗ 𝑖 −1 𝑗 − 2𝑐∗ 𝑖 𝑗 + 𝑐∗ 𝑖+1 𝑗 ∆𝑥∗2 (16) 𝜕𝑐∗ 𝜕𝑡∗= 𝑐∗ 𝑖 𝑗+1− 𝑐∗ 𝑖 𝑗 ∆𝑡∗ (14) 𝜕𝑐∗ 𝜕𝑥∗= 𝑐∗ 𝑖+1 𝑗 − 𝑐∗ 𝑖−1 𝑗 2∆𝑥∗ (15) 𝜕2𝑐∗ 𝜕𝑥∗2 = 𝑐∗ 𝑖 −1 𝑗 − 2𝑐∗ 𝑖 𝑗 + 𝑐∗ 𝑖+1 𝑗 ∆𝑥∗2 (16) (14) (15) (16) Substituting Equations (14) - (16) in Equation (10) gives: ing Equations (14) - (16) in Equation (10) gives: 𝑐∗ 𝑖 𝑗+1 = 𝑐∗ 𝑖 𝑗 +∆𝑡∗𝑓1 𝑗[ 𝑐∗ 𝑖 −1 𝑗 − 2𝑐∗ 𝑖 𝑗 + 𝑐∗ 𝑖+1 𝑗 ∆𝑥∗2 ] −∆𝑡∗𝑓2 𝑗𝑃𝑒[ 𝑐∗ 𝑖+1 𝑗 − 𝑐∗ 𝑖−1 𝑗 2∆𝑥∗ ] (17) Let 𝛾= ∆𝑡∗ ∆𝑥∗2 (18) Let 𝜗= 𝑃𝑒∆𝑡∗ ∆𝑥∗ (19) 𝑐∗ 𝑖 𝑗+1 = 𝑐∗ 𝑖 𝑗 +∆𝑡∗𝑓1 𝑗[ 𝑐∗ 𝑖 −1 𝑗 − 2𝑐∗ 𝑖 𝑗 + 𝑐∗ 𝑖+1 𝑗 ∆𝑥∗2 ] −∆𝑡∗𝑓2 𝑗𝑃𝑒[ 𝑐∗ 𝑖+1 𝑗 − 𝑐∗ 𝑖−1 𝑗 2∆𝑥∗ ] (17) Let 𝛾= ∆𝑡∗ 2 (18) 𝑐∗ 𝑖 𝑗+1 = 𝑐∗ 𝑖 𝑗 +∆𝑡∗𝑓1 𝑗[ 𝑐∗ 𝑖 −1 𝑗 − 2𝑐∗ 𝑖 𝑗 + 𝑐∗ 𝑖+1 𝑗 ∆𝑥∗2 ] −∆𝑡∗𝑓2 𝑗𝑃𝑒[ 𝑐∗ 𝑖+1 𝑗 − 𝑐∗ 𝑖−1 𝑗 2∆𝑥∗ ] (17) (17) Let 𝛾= ∆𝑡∗ ∆𝑥∗2 (18) Let 𝜗= 𝑃𝑒∆𝑡∗ ∆𝑥∗ (19) Let 𝛾= ∆𝑡∗ ∆𝑥∗2 (18) et 𝜗= 𝑃𝑒∆𝑡∗ ∆𝑥∗ 𝑃𝑒∆𝑡∗ ∆𝑥∗ (19) (19) Rewriting Equation (17) using Equations (18) and (19), then rearranging it according to the time levels on 𝑐∗ gives: 𝑐∗ 𝑖 𝑗+1 = (𝛾𝑓1 𝑗+ 𝜗𝑓2 𝑗 2 ) 𝑐∗ 𝑖 −1 𝑗 + (1 −2𝛾𝑓1 𝑗) 𝑐∗ 𝑖 𝑗+ (𝛾𝑓1 𝑗− 𝜗𝑓2 𝑗 2 ) 𝑐∗ 𝑖 + 1 𝑗 (20) Let 𝛿= 𝛾𝑓1 𝑗+ 𝜗𝑓2 𝑗 2 ; 𝛽= 1 −2𝛾𝑓1 𝑗; 𝛼= 𝛾𝑓1 𝑗− 𝜗𝑓2 𝑗 2 (21) (20) Let (21) Rewriting Equation (20) using Equation (21) gives: Rewriting Equation (20) using Equation (21) gives: quation (20) using Equation (21) gives: p: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology IJESRT is licensed under a Creative Commons Attribution 4.0 International License. 2.2.1 Numerical Solution Consider the transformation 𝑋∗= 𝑥 𝑓2(𝑚𝑡∗) 𝑓1(𝑚𝑡∗) (29) Rewriting Equation (10) using Equation (29) gives Rewriting Equation (10) using Equation (29) gives Rewriting Equation (10) using Equation (29) gives 𝑓1 𝑓22 𝜕𝑐∗ 𝜕𝑡∗= 𝜕2𝑐∗ 𝜕𝑋∗2 −𝑃𝑒 𝜕 𝑐∗ 𝜕 𝑋∗ (30) 𝑓1 𝑓22 𝜕𝑐∗ 𝜕𝑡∗= 𝜕2𝑐∗ 𝜕𝑋∗2 −𝑃𝑒 𝜕 𝑐∗ 𝜕 𝑋∗ (30) Introducing a new time variable using the transformation Introducing a new time variable using the transformation 𝑇∗= ∫ 𝑓22 𝑓1 𝑑𝜏 𝑡∗ 0 (31) 𝑇∗= ∫ 𝑓22 𝑓1 𝑑𝜏 𝑡∗ 0 (31) Rewriting Equation (30) using Equation (31) gives Rewriting Equation (30) using Equation (31) gives ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 ISSN: 2277-9655 [Raoul et al., 12(1): January, 2023] Impact Factor: 5.164 IC™ Value: 3.00 CODEN: IJESS7 ISSN: 2277 9655 [Raoul et al., 12(1): January, 2023] Impact Factor: 5.164 IC™ Value: 3.00 CODEN: IJESS7 𝜕𝑐∗ 𝜕𝑇∗= 𝜕2𝑐∗ 𝜕𝑋∗2 −𝑃𝑒 𝜕 𝑐∗ 𝜕 𝑋∗ 𝜕𝑐∗ 𝜕𝑇∗= 𝜕2𝑐∗ 𝜕𝑋∗2 −𝑃𝑒 𝜕 𝑐∗ 𝜕 𝑋∗ (32) (32) Transforming the initial and boundary conditions in Equations (11) – (13) yields 𝑐∗(𝑋∗, 0) = 0 (33) 𝑐∗(0, 𝑇∗) = 1 (34) 𝜕𝑐∗(𝐿,𝑇∗) 𝜕𝑋∗ = 0 (35) 𝑐∗(𝑋∗, 0) = 0 (33) 𝑐∗(0, 𝑇∗) = 1 (34) 𝜕𝑐∗(𝐿,𝑇∗) 𝜕𝑋∗ = 0 (35) 𝑐∗(𝑋∗, 0) = 0 𝑐∗(0, 𝑇∗) = 1 𝜕𝑐∗(𝐿,𝑇∗) 𝜕𝑋∗ = 0 (33) (35) Further application of the transformation Further application of the transformation Further application of the transformation 𝑐∗(𝑋∗, 𝑇∗) = 𝐾∗(𝑋∗, 𝑇∗) exp ( 𝑃𝑒 2 𝑋∗− 𝑃𝑒2 4 𝑇∗ ) (36) to eliminate 𝜕 𝑐∗ 𝜕 𝑋∗ in Equation (32) gives a diffusion equation 𝜕𝐾∗ 𝜕𝑇∗= 𝜕2𝐾∗ 𝜕𝑋∗2 Transforming Equations (33) – (35) using Equation (36) 𝐾∗(𝑋∗, 0) = 0 Transforming Equations (33) – (35) using Equation (36) 𝐾∗(𝑋∗, 0) = 0 𝐾∗(0, 𝑇∗) = 𝑒𝜇𝑇∗ ; 𝜇= 𝑃𝑒2 4 𝜕𝐾∗ 𝜕𝑋∗+ 𝑃𝑒 2 𝐾∗= 0 𝐾∗(0, 𝑇∗) = 𝑒𝜇𝑇∗ ; 𝜇= 𝑃𝑒2 4 𝜕𝐾∗ 𝜕𝑋∗+ 𝑃𝑒 2 𝐾∗= 0 Applying Laplace transforms to Equations (37) – (40) produces Applying Laplace transforms to Equations (37) – (40) produces 𝑑2𝐾̅∗ 𝑑𝑋∗2 −𝑝𝐾̅∗= 0 (41) 𝐾̅∗(0, 𝑝) = 1 𝑝− 𝜇 (42) 𝑑𝐾̅∗ 𝑑𝑋∗+ 𝑃𝑒 2 𝐾̅∗= 0 (43) 𝑑2𝐾̅∗ 𝑑𝑋∗2 −𝑝𝐾̅∗= 0 𝐾̅∗(0, 𝑝) = 1 𝑝− 𝜇 𝑑𝐾̅∗ 𝑑𝑋∗+ 𝑃𝑒 2 𝐾̅∗= 0 (41) (42) (43) Where 𝐾̅∗(𝑋∗, 𝑝) = 𝐿[𝐾∗(𝑋∗, 𝑇∗)] = ∫ 𝐾∗(𝑋∗, 𝑇∗)𝑒−𝑝𝑇∗𝑑𝑇∗ ∞ 0 Where 𝐾̅∗(𝑋∗, 𝑝) = 𝐿[𝐾∗(𝑋∗, 𝑇∗)] = ∫ 𝐾∗(𝑋∗, 𝑇∗)𝑒−𝑝𝑇∗𝑑𝑇∗ ∞ 0 The particular solution to Equations (41) – (43) is obtained as 𝐾̅∗(𝑋∗, 𝑝) = 1 𝑝−𝜇𝑒− √𝑝𝑋∗ (44) p 𝐾̅∗(𝑋∗, 𝑝) = 1 𝑝− 𝜇𝑒− √𝑝𝑋∗ (44) 𝑝 𝜇 Applying Laplace inverse on Equation (44) using the tables in Van and Alves (1982) gives the solution to our diffusion equation as 𝑝 𝜇 Applying Laplace inverse on Equation (44) using the tables in Van and Alves (1982) gives the solution to our diffusion equation as q 𝐾∗(𝑋∗, 𝑇∗) = 1 2 [exp ( 𝑃𝑒2𝑇∗ 4 − 𝑃𝑒𝑋∗ 2 ) 𝑒𝑟𝑓𝑐( 𝑋∗ 2√𝑇∗− 𝑃𝑒√𝑇∗ 2 ) + 𝑒𝑥𝑝( 𝑃𝑒2𝑇∗ 4 + 𝑃𝑒𝑋∗ 2 ) 𝑒𝑟𝑓𝑐( 𝑋∗ 2√𝑇∗+ 𝑃𝑒√𝑇∗ 2 )] (45) Inserting Equation (45) in Equation (36) and further applying Equations (29) and (31) gives the analytical solution to the model: , 𝑡∗) = 1 2 [𝑒𝑟𝑓𝑐( 𝑥𝑓2(𝑚𝑡∗) 2√𝑇∗𝑓1(𝑚𝑡∗) − 𝑃𝑒√𝑇∗ 2 ) + 𝑒𝑥𝑝( 𝑥 𝑓2(𝑚𝑡∗) 𝑓1(𝑚𝑡∗) 𝑃𝑒 ) 𝑒𝑟𝑓𝑐( 𝑥𝑓2(𝑚𝑡∗) 2√𝑇∗𝑓1(𝑚𝑡∗) + 𝑃𝑒√𝑇∗ 2 )] (46 𝑐∗(𝑥∗, 𝑡∗) = 1 2 [𝑒𝑟𝑓𝑐( 𝑥𝑓2(𝑚𝑡∗) 2√𝑇∗𝑓1(𝑚𝑡∗) − 𝑃𝑒√𝑇∗ 2 ) + 𝑒𝑥𝑝( 𝑥 𝑓2(𝑚𝑡∗) 𝑓1(𝑚𝑡∗) 𝑃𝑒 ) 𝑒𝑟𝑓𝑐( 𝑥𝑓2(𝑚𝑡∗) 2√𝑇∗𝑓1(𝑚𝑡∗) + 𝑃𝑒√𝑇∗ 2 )] (46) (46) 2.2.1 Numerical Solution Using Equations (24), (22) and (27), the governing equation and the boundary conditions is expressed as a system of linear equations: 𝑐∗ 1 𝑗+1 = 1 𝑐∗ 𝑖 𝑗+1 = 𝛿𝑐∗ 𝑖 −1 𝑗 + 𝛽 𝑐∗ 𝑖 𝑗+ 𝛼𝑐∗ 𝑖 + 1 𝑗 𝑓𝑜𝑟 𝑖= 2, … . . , 𝑛 𝑐∗ 𝑛 𝑗+1 = (𝛼+ 𝛿)𝑐∗ 𝑛−1 𝑗 + 𝛽𝑐∗ 𝑛 𝑗 } (28) 𝑐∗ 1 𝑗+1 = 1 (28) The solution to Equation (28) is obtained iteratively. To ensure stability of the scheme, the parameters 0 ≤𝜗 ≤ 1 𝑃𝑒 and 0 ≤ 𝛾 ≤ 1 2 (Andallah L.S. and Khatun, (2020)) are used. 𝜗 and 𝛾 are defined in Equations (18) and (19) respectively. The solution to Equation (28) is obtained iteratively. To ensure stability of the scheme, the parameters 0 ≤𝜗 ≤ 1 𝑃𝑒 and 0 ≤ 𝛾 ≤ 1 2 (Andallah L.S. and Khatun, (2020)) are used. 𝜗 and 𝛾 are defined in Equations (18) and (19) respectively. 2 2 2 A l i l S l i 2.2.2 Analytical Solution Analytical solution of Equation (10) subject to the initial and boundary conditions in Equations (11) – (13) is determined. Our solution is the dimensionless form of the solution obtained by Kumar et al (2011) for input concentration 𝐶0 = 1. ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 3. RESULTS AND DISCUSSION http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology 3. RESULTS AND DISCUSSION A comparison of numerical and analytical solutions is provided using the solutions obtained in Equations (28) and (46) respectively. A discussion of results based on the simulations of the validated results obtained in [6] [6] [6] IJESRT is licensed under a Creative Commons Attribution 4.0 International License. IJESRT is licensed under a Creative Commons Attribution 4.0 International License. IJESRT is licensed under a Creative Commons Attribution 4.0 International License. http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology [7] IJESRT is licensed under a Creative Commons Attribution 4.0 International License. ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 Equation (28) is also provided. Results are presented on the basis of effect of various Peclet numbers on pollutant concentration and effect of temporally varying velocity and dispersion coefficients with time on pollutant concentration. 3.1. Comparison of Analytical and Numerical Solutions 3.1. Comparison of Analytical and Numerical Solutions In this section, we compare the analytical and numerical solutions based on Equations (46) and (28) respectively. The simulations are obtained at a fixed time (𝑡∗= 1) using 𝑈0 = 1.14, 𝐷0 = 1.40 (𝑃𝑒 ~ 1). Both velocity and dispersion coefficients are considered as exponentially increasing with time. Figure 1: Plot of Analytical and Numerical Solutions of 𝒄∗ against 𝒙∗ when 𝒕∗= 𝟏 ure 1 shows concentration profile obtained when analytical (exact) and numerical (approximate) solutions compared. Absolute error is evaluated using concentration values from both the exact (𝑐𝑒 ∗) and approximate ) solutions. The errors obtained at different points are summarized in Table 1: Figure 1: Plot of Analytical and Numerical Solutions of 𝒄∗ against 𝒙∗ when 𝒕∗= 𝟏 Figure 1 shows concentration profile obtained when analytical (exact) and numerical (approximate) solutions are compared. Absolute error is evaluated using concentration values from both the exact (𝑐𝑒 ∗) and approximate (𝑐𝑎 ∗) solutions. The errors obtained at different points are summarized in Table 1: Figure 1 shows concentration profile obtained when analytical (exact) and numerical (approximate) solutions are compared. Absolute error is evaluated using concentration values from both the exact (𝑐𝑒 ∗) and approximate (𝑐𝑎 ∗) solutions. The errors obtained at different points are summarized in Table 1: Figure 1 shows concentration profile obtained when analytical (exact) and numerical (approximate) solutions are compared. Absolute error is evaluated using concentration values from both the exact (𝑐𝑒 ∗) and approximate (𝑐𝑎 ∗) solutions. The errors obtained at different points are summarized in Table 1: Table 1: Comparison of Exact and Approximate Solutions Table 1: Comparison of Exact and Approximate Solutions Table 1: Comparison of Exact and Approximate Solutions 𝑥∗ 𝑐𝑒 ∗ 𝑐𝑎 ∗ Absolute Error 1 0.77900 0.787800 8.8 × 10− 3 2 0.490400 0.506500 1.61 × 10−2 3 0.239900 0.255700 1.98 × 10− 2 4 0.088890 0.098870 9.98 × 10− 3 5 0.024540 0.037690 1.315 × 10−2 6 0.007038 0.007038 0.00 7 - 10 0.000000 0.000000 0.00 ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 If 𝑃𝑒 ≪1, advection term is significantly smaller than diffusion term. The simulations are obtained using initial velocity and dispersion coefficients as 𝑈0 = 0.05, 𝐷0 = 1.25 respectively. We observe from Figure 2(a) that at a fixed point near the source (𝑥∗ = 0), concentration increases with increasing time. For example at 𝑥∗ = 2, for 𝑡∗= 0.25, 𝑐∗= 0.0472; for 𝑡∗= 0.5, 𝑐∗= 0.2393; for 𝑡∗= 0.75, 𝑐∗= 0.3673 and for 𝑡∗= 1, 𝑐∗= 0.4587. Increase in concentration is as a result of increased spreading of pollutant cloud around the source. Pollutant cloud spreads in the medium faster than it is transported downstream. Concentration however becomes zero at some distance from the source. This is based on the assumption that pollutants being considered are miscible and there are no storage areas or dead zones in the river that would retain pollutants then release them after some time. We further observe that at any fixed time, concentration decreases with increasing distance. For example when 𝑡∗= 0.5, for 𝑥∗= 1, 𝑐∗= 0.5594; for 𝑥∗= 2, 𝑐∗= 0.2393; for 𝑥∗= 3, 𝑐∗= 0.07609; for 𝑥∗ = 4, 𝑐∗= 0.01765; for 𝑥∗= 5, 𝑐∗= 0.004327; for 𝑥∗= 6, 𝑐∗= 0.000551 and for 𝑥∗≥7, 𝑐∗≈0. When 𝑃𝑒 ≪1, there is minimal spreading of pollutant molecules at some far distance from the source point thus less significant effect on the concentration at far distance. When 𝑃𝑒 ~ 1, the advection and diffusion terms are not significantly different and neither process dominates over the other. The results in Figure 2(b) are obtained using initial velocity and dispersion coefficients as 𝑈0 = 1.14, 𝐷0 = 1.40 respectively. We note that at 𝑥∗ = 2, 𝑡∗= 0.25, 𝑐∗= 0.0972; for 𝑡∗= 0.5, 𝑐∗= 0.4588; for 𝑡∗= 0.75, 𝑐∗= 0.6657; and for 𝑡∗= 1, 𝑐∗= 0.7943. The high concentration levels around source point and gradual decrease of pollutant concentration thereafter is due to the fact that once a mass of pollutant is released at a single instant of time in the river, it spreads out as it moves downstream due to molecular diffusion caused by random motion of pollutant molecules, spreading caused by the variations of the microscopic velocities through the pores in the river and advection due to bulk movement of water. We also observe that for a fixed time, concentration decreases with increasing distance. For example, when 𝑡∗= 0.5: for 𝑥∗= 1, 𝑐∗= 0.7616, for 𝑥∗= 2, 𝑐∗= 0.4588; for 𝑥∗= 3, 𝑐∗= 0.2096, for 𝑥∗ = 4, 𝑐∗= 0.07062; for 𝑥∗= 5, 𝑐∗= 0.2346; for 𝑥∗= 6, 𝑐∗= 0.00379, for 𝑥∗= 7, 𝑐∗= 0.0005803 and for 𝑥∗≥8, 𝑐∗≈0. Though concentration decreases with distance as we move downstream concentration in this case at any point downstream is higher as g decreases with distance as we move downstream, concentration in this case at any point downstream is higher as compared to when 𝑃𝑒 ≪1, due to the effect of advection process during flow. g decreases with distance as we move downstream, concentration in this case at any point downstream is higher as compared to when 𝑃𝑒 ≪1, due to the effect of advection process during flow. g s with distance as we move downstream, concentration in this case at any point downstream is higher as d to when 𝑃𝑒 ≪1, due to the effect of advection process during flow. Simulations are further obtained using initial velocity and dispersion coefficients as 𝑈0 = 4, 𝐷0 = 1.05 respectively for 𝑃𝑒 ≫ 1. In this case, advection term is significantly bigger than the diffusion term. Results in Figure 2(c) show that at a fixed point near the source, concentration increases with increasing time and reaches a maximum value (𝑐∗= 1) after a given time. For example at 𝑥∗= 4, for 𝑡∗= 0.25, 𝑐∗= 0.02354; for 𝑡∗= 0.5, 𝑐∗= 0.8515; for 𝑡∗= 0.75, 𝑐∗= 0.9999; and for 𝑡∗= 1, 𝑐∗= 0.9999. When advection dominates the flow, spreading is minimal with the cloud of pollutant being simply moved along by the flow. This is because the pollutant is transported downstream very first and has less time to spread. We also observe that for a fixed time, concentration decreases with increasing distance. ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 From Table 1 above, we observe that the numerical results obtained using FTCSCS agree with the analytical results to a great degree. We conclude that our scheme is accurate. Simulations obtained in our study are based on the numerical scheme. 3.2. Effect of Velocity and Dispersion on Pollutant Concentration ff f y p Both advection and diffusion processes move pollutants from one place to another, but each accomplishes this differently. Whereas advection transports pollutant molecules downstream, diffusion transports pollutant molecules in both ways regardless of the stream direction. In order to understand the effect of each process on pollutant concentration, a comparison of advection and diffusion fluxes is performed using the ratio of their scales. This ratio is given by Peclet number (Pe). Simulations are obtained for pollutant concentration when 𝑃𝑒 ≪1, 𝑃𝑒 ~ 1 and 𝑃𝑒 ≫ 1. http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology [8] IJESRT is licensed under a Creative Commons Attribution 4.0 International License. (a) (b) (c) Figure 2: Plot of 𝒄∗(𝒙∗) for 𝒕∗= 𝟎. 𝟐𝟓, 𝟎. 𝟓, 𝟎. 𝟕𝟓 𝒂𝒏𝒅 𝟏 for (a) 𝑷𝒆 ≪𝟏 (b) 𝑷𝒆 ~ 𝟏 (c) 𝑷𝒆 ≫ 𝟏 (a) (b) (b) (a) (b) (c) (c) ( ) http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology [8] IJESRT is licensed under a Creative Commons Attribution 4.0 International License. Figure 2: Plot of 𝒄∗(𝒙∗) for 𝒕∗= 𝟎. 𝟐𝟓, 𝟎. 𝟓, 𝟎. 𝟕𝟓 𝒂𝒏𝒅 𝟏 for (a) 𝑷𝒆 ≪𝟏 (b) 𝑷𝒆 ~ 𝟏 (c) 𝑷𝒆 ≫ 𝟏 Figure 2: Plot of 𝒄∗(𝒙∗) for 𝒕∗= 𝟎. 𝟐𝟓, 𝟎. 𝟓, 𝟎. 𝟕𝟓 𝒂𝒏𝒅 𝟏 for (a) 𝑷𝒆 ≪𝟏 (b) 𝑷𝒆 ~ 𝟏 (c) 𝑷𝒆 ≫ 𝟏 [8] IJESRT is licensed under a Creative Commons Attribution 4.0 International Licens http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology For example, when 𝑡∗= 0.5: for 𝑥∗= 1, 𝑐∗= 1, for 𝑥∗= 2, 𝑐∗= 1, for 𝑥∗= 3, 𝑐∗= 0.9553, for 𝑥∗ = 4, 𝑐∗= 0.8515; for 𝑥∗= 5, 𝑐∗= 0.7009; for 𝑥∗= 6, 𝑐∗= 0.4553, for 𝑥∗= 7, 𝑐∗= 0.2273, for 𝑥∗= 8, 𝑐∗= 0.08369, for 𝑥∗= 9, 𝑐∗= 0.02209, and for ∗ 10 ∗ 0 005173 D i ifi ff f h f d i i i l l 𝑥∗= 10, 𝑐∗= 0.005173. Due to significant effect of the process of advection in transporting molecules downstream, we note that at any point, there are pollutant molecules present at downstream especially for 0.5 ≤𝑡∗≤1. 𝑥∗= 10, 𝑐∗= 0.005173. Due to significant effect of the process of advection in transporting molecules downstream, we note that at any point, there are pollutant molecules present at downstream especially for 0.5 ≤𝑡∗≤1. Comparison of pollutant concentration for different Peclet numbers across the domain 0 ≤𝑥∗≤10 at a fixed time (𝑡∗= 0.5) is provided in Table 2: Comparison of pollutant concentration for different Peclet numbers across the domain 0 ≤𝑥∗≤10 at a fixed time (𝑡∗= 0.5) is provided in Table 2: Table 2: Table Showing Pollutant Concentration for Various 𝒙∗ and Pe 𝑥∗ 𝑃𝑒 ≪1 𝑃𝑒 ~ 1 𝑃𝑒 ≫1 1 0.559400 0.761600 1.000000 2 0.239300 0.458800 1.000000 3 0.076090 0.209600 0.955300 4 0.017650 0.070620 0.851500 5 0.004327 0.023460 0.700900 6 0.000551 0.004379 0.455300 [9] REFERENCES 1. Ahmed S.G., “A Numerical Algorithm for Solving Advection Diffusion Equation with Constant and Variable Coefficients,” in Open Numerical Method Journal. Vol. 4, pp. 1 - 7, 2012. 1. Ahmed S.G., “A Numerical Algorithm for Solving Advection Diffusion Equation with Constant and Variable Coefficients,” in Open Numerical Method Journal. Vol. 4, pp. 1 - 7, 2012. 2. Alebrahem J., “Forward Time Centered Space Scheme for the Solution of Transport Equation,” i International Annals of Science. Vol. 2, No. 1, pp. 1 – 5, 2017. 3. Andallah L.S. and Khatun M.R., “Numerical Solution of Advection Diffusion Equation using Finite Difference,” in Bangladesh Journal of Industrial Research. Vol. 55, No.1, pp. 15 - 22, 2020. 3. Andallah L.S. and Khatun M.R., “Numerical Solution of Advection Diffusion Equation using Finite Difference,” in Bangladesh Journal of Industrial Research. Vol. 55, No.1, pp. 15 - 22, 2020. g pp 4. Appadu, A.R., “Numerical Solution of the 1D Advection Diffusion Equation using Standard and Non Standard Finite Difference Scheme,” in Journal of Applied Maths. ID 734374, 2013. g pp 4. Appadu, A.R., “Numerical Solution of the 1D Advection Diffusion Equation using Standard and Non Standard Finite Difference Scheme,” in Journal of Applied Maths. ID 734374, 2013. pp 5. Duffy, D.G., “Green’s Function with Applications,” in Chapman and Hall/CRC, 2001. 6. Gurarslan G, Sari M and Zeytinoglu A., “Higher Order Finite Difference Schemes for Solving the Advection Diffusion Equation,” in Mathematical Modeling and Computational Applications, Vol. 15, No.3, pp. 449 – 460, 2010. 6. Gurarslan G, Sari M and Zeytinoglu A., “Higher Order Finite Difference Schemes for Solving the Advection Diffusion Equation,” in Mathematical Modeling and Computational Applications, Vol. 15, No.3, pp. 449 – 460, 2010. pp 7. Huang K, Simunek J and Genuchten M.T.H., “A Third Order Numerical Scheme with Upwind Weighting for Solving the Solute Transport Equation” in International Journal for Numerical Methods in Engineering,” Vol. 40, pp. 1623 – 37, 1997. 7. Huang K, Simunek J and Genuchten M.T.H., “A Third Order Numerical Scheme with Upwind Weighting for Solving the Solute Transport Equation” in International Journal for Numerical Methods in Engineering,” Vol. 40, pp. 1623 – 37, 1997. g g pp 8. Johari, H., Rusli, N. and Yahya, Z. “Finite Difference Formulation for the Prediction of Water Pollution,” in IOP Conf. Series: Materials Science and Engineering. Vol. 318, 012005. 2018. g g pp 8. Johari, H., Rusli, N. and Yahya, Z. 4. CONCLUSION We have noted that concentration generally increases around the source point and gradually decreases with increasing distance from the source point. At some far point from source, the concentration values converge to very small positive constant, almost zero. This implies that if we manage to maintain low pollutant levels at source point then the water downstream will be safe for human consumption. The simulations generally show skewness in the longitudinal distribution of the concentration with the average concentration being more downstream to the original source. The skewness is as a result of imbalance between the advective and dispersive processes. When varying Peclet numbers, we note that concentration is highest for 𝑃𝑒 ≫1. Physically, 𝑃𝑒 ≫1 represents surface waters in mountain regions, where the stream/ river is characterized by large velocities which makes advection more significant while 𝑃𝑒 ≪1 represents surface waters in plain areas where advection is characterized by smaller velocities. The results can be applied in many physical situations described by advection diffusion phenomena to help in the planning and management of rivers flowing through cities. 5. ACKNOWLEDGEMENTS This work is part of the thesis done by the first author towards her doctoral studies. We acknowledge the Technical University of Kenya for the financial support in terms of tuition waiver to the first author. ISSN: 2277-9655 Impact Factor: 5.164 CODEN: IJESS7 [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 It is observed that at any fixed point, concentration is lower when both velocity and dispersion coefficients are exponentially increasing with time. For example, when 𝑥= 2, 𝑐∗= 0.09349. When both flow velocity and dispersion parameters are increasing with time, there is rapid mixing of pollutants after injection and immediate transportation of pollutants downstream occur simultaneously such that very few pollutant molecules are left around the source point in the shortest time possible. Concentration is however found to be higher when both velocity and dispersion coefficients are exponentially decreasing with time, (𝑥= 2, 𝑐∗= 0.4026) as compared to the former case. This would be attributed to accumulation of pollutant molecules within the medium since spreading and transportation processes takes place at a much slower rate. There is no significant difference in concentration obtained when an exponentially decreasing dispersion in an exponentially decelerating flow (𝑥= 2, 𝑐∗= 0.4226) is compared to that of exponentially increasing dispersion in an exponentially decelerating flow (𝑥= 2, 𝑐∗= 0.4217). It is further observed that concentration is highest (𝑥= 2; 𝑐∗= 0.4420) when we have exponentially decreasing dispersion in an exponentially accelerating flow. Again, because of less dispersion effect there tends to be increased accumulation of pollutant molecules. ISSN: 2277-9655 [Raoul et al., 12(1): January, 2023] Impact Factor: 5.164 IC™ Value: 3.00 CODEN: IJESS7 7 0.000000 0.000580 0.227300 8 0.000000 0.000000 0.083690 9 0.000000 0.000000 0.0220900 10 0.000000 0.000000 0.005173 Comparing the three cases of varying Peclet number, at a fixed time say 𝑡∗= 0.5, it is observed that at any point 𝑥∗, concentration is much higher for 𝑃𝑒 ≫1 as compared to when 𝑃𝑒 ≪1 and 𝑃𝑒 ~ 1. When flow velocity is significantly higher than diffusion, more pollutant molecules are transported downstream faster than they are spread around the source point. The effect of bulk transport of pollutant molecules is also seen at the downstream for 𝑃𝑒 ≫1 where concentration is evident unlike the two cases where 𝑃𝑒 ≪1 and 𝑃𝑒 ~ 1. 3.3. Effect of Temporally Varying Velocity and Dispersion Coefficients on Pollutant Concentration p y y g y p To study the effect of time dependent dispersion (𝐷(𝑡) = 𝐷0𝑓1(𝑚𝑡)) and velocity coefficients (𝑈(𝑡) = 𝑈0𝑓2(𝑚𝑡)) on concentration, 𝑓𝑖(𝑚𝑡)for 𝑖= 1,2 was considered to be an exponential function of time. Simulations were obtained at a fixed time (𝑡∗= 1) using 𝑃𝑒 ~ 1 for four different combinations of 𝑓𝑖(𝑚𝑡) for 𝑖= 1,2. Table 3: Summary of Different Combinations of 𝒇𝒊(𝒎𝒕); 𝒊= 𝟏, 𝟐 𝒇𝟏(𝒎𝒕) 𝒇𝟐(𝒎𝒕) Description of dispersion 𝑫(𝒕) = 𝑫𝟎𝒇𝟏(𝒎𝒕)) in a flow of velocity 𝒖(𝒕) = 𝑼𝟎𝒇𝟐(𝒎𝒕) 𝑒𝑚𝑡 𝑒𝑚𝑡 Exponentially increasing dispersion in an exponentially accelerating flow 𝑒𝑚𝑡 𝑒− 𝑚𝑡 Exponentially increasing dispersion in an exponentially decelerating flow 𝑒− 𝑚𝑡 𝑒𝑚𝑡 Exponentially decreasing dispersion in an exponentially accelerating flow 𝑒− 𝑚𝑡 𝑒− 𝑚𝑡 Exponentially decreasing dispersion in an exponentially decelerating flow Simulation obtained for various time dependent advection and diffusion coefficients given in Table 3 are Simulation obtained for various time dependent advection and diffusion coefficients given in Table 3 are provided in Figure 3: Simulation obtained for various time dependent advection and diffusion coefficients given in Table 3 are provided in Figure 3: http: // www.ijesrt.com© International Journal of Engineering Sciences & Research Technology [10] IJESRT is licensed under a Creative Commons Attribution 4.0 International License. Figure 3: Plot of 𝒄∗ against 𝒙∗ when 𝒕∗= 𝟏, for Different Time Dependent Diffusion and Velocity Coefficients Figure 3: Plot of 𝒄∗ against 𝒙∗ when 𝒕∗= 𝟏, for Different Time Dependent Diffusion and Velocity Coefficients Figure 3: Plot of 𝒄∗ against 𝒙∗ when 𝒕∗= 𝟏, for Different Time Dependent Diffusion and Velocity Coefficients [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 [11] REFERENCES “Finite Difference Formulation for the Prediction of Water Pollution,” in IOP Conf. Series: Materials Science and Engineering. Vol. 318, 012005. 2018. 9. Kumar, A., Kumar, D.J, and Kumar, R.R., “Analytical Solutions to the One - Dimensional Advection Diffusion Equation with Time Dependent Coefficients,” in Journal of Water Resources and Protection. Vol. 3, pp. 76 - 84, 2011. 9. Kumar, A., Kumar, D.J, and Kumar, R.R., “Analytical Solutions to the One - Dimensional Advection Diffusion Equation with Time Dependent Coefficients,” in Journal of Water Resources and Protection. Vol. 3, pp. 76 - 84, 2011. [11] ISSN: 2277-9655 aoul et al., 12(1): January, 2023] Impact Factor: 5.164 ™ Value: 3.00 CODEN: IJESS7 10. Kaya, B. and Gharehbaghi, “Implicit Solutions of Advection Diffusion Equation by Various Numerical Methods,” in Australian Journal of Basic and Applied Sciences. Vol. 8, No.1, pp. 381 - 391, 2014. [Raoul et al., 12(1): January, 2023] IC™ Value: 3.00 10. Kaya, B. and Gharehbaghi, “Implicit Solutions of Advection Diffusion Equation by Various Numerical Methods,” in Australian Journal of Basic and Applied Sciences. Vol. 8, No.1, pp. 381 - 391, 2014. 11. Manitcharoen, N. and Pimpunchat, B., “Analytical and Numerical Solutions of Pollution Concentration with Uniformly and Exponentially Increasing Forms of Sources,” in Journal of Applied Mathematics. 9 pages. Article ID 95, 2020. 12. Van Genuchten, M. Th., and Alves, W.J., “Analytical Solutions of the One Dimensional Convective – Dispersive Solute Transport Equation,” in U.S. Department of Agriculture, Technical Bulletin. No. 1661, 1982. 13. Yip, B.F., Alias, N.A.F and Kasiman, E.H., “Numerical Modeling of Pollutant Transport in a Straight Narrow Channel using Upwind Finite Difference Method,” in IOP Conf. Series: Materials Science and Engineering. 1153,012003, 2021. [12]
https://openalex.org/W1874745064	http://seer.faccat.br/index.php/coloquio/article/download/103/pdf_39	Portuguese	null	Um modelo aplicado à melhoria dos processos de planejamento estratégico e autoavaliação em Instituições de Ensino Superior Privadas	Colóquio	2,014	cc-by-sa	8,885	Abstract This paper presents a methodological framework developed to improve the processes of planning and self-assessment applied to the management of Private Higher Education Institutions - IESP. Therefore, we analyzed literature which resulted in a Model of Strategic Planning articulated institutional self-evaluation, regulatory requirements for the regulation process of Higher Education, established by Law nº 10.861, dated april 14, 2004. Keywords: Private Institutions of Higher Education. Self-assessment. Strategic planning. Resumo Este artigo apresenta um modelo metodológico desenvolvido para melhoria dos processos de planejamento e autoavaliação aplicado à gestão de Instituições de Ensino Superior Privadas - IESP. Para tanto, foi realizada uma análise bibliográfica, que resultou em um Modelo de Planejamento Estratégico articulado à autoavaliação institucional, requisito normativo para o processo de regulação do Ensino Superior, instituída pela Lei nº 10.861, de 14 de abril de 2004. Palavras-chave: Instituições de Ensino Superior Privadas. Autoavaliação. Panejamento estratégico. j @g 2 Pós-Doutor em Engenharia de Produção. Professor do Mestrado em Desenvolvimento Regional da Faccat. carlosfernandojung@gmail.com Um modelo aplicado à melhoria dos processos de planejamento estratégico e autoavaliação em Instituições de Ensino Superior Privadas Josias Ezequiel Julierme Mazzurana1 Carlos Fernando Jung2 p josiasmazzurana@gmail.com 2 1 Especialista em Gestão Educacional: Supervisão e Orientação Educacional. 1 Introdução A intensidade e diversidade das mudanças contemporâneas e a expansão das instituições de ensino, resultado das políticas públicas de democratização do acesso à educação superior, configuram novos desafios aos Gestores de Instituições de Ensino Superior Privadas (IESP). No cenário brasileiro, das 2.378 jos as a u a a@g a co 2 Pós-Doutor em Engenharia de Produção. Professor do Mestrado em Desenvolvimento Regional da Faccat. carlosfernandojung@gmail.com 168 instituições de ensino superior, 88,3% são privadas; destas, 85,2% são faculdades; em relação às Instituições Federais, no período de 2001 a 2010, estas apresentam um aumento de 140,5% relativamente ao número de ingressantes, definindo maior competitividade às Instituições de pequeno porte (INEP, 2010). Neste contexto, o planejamento Estratégico, como ferramenta de gestão, adquire nova importância como resposta ao mercado competitivo, representando o comprometimento com o futuro da IES, e somente terá sentido com a sua implementação (MEYER JÚNIOR; SERMANN; MANGOLIM, 2004). Para isso, torna-se necessário o comprometimento integrado dos sujeitos (gestores, comunidades acadêmicas e civis organizadas), para a constituição de objetivos e estratégias, de acordo com a realidade institucional (VIANNA, 2004). A ação inicial para a implementação do Planejamento Estratégico é a análise do ambiente organizacional, configurada pela identificação do contexto em que a IES está inserida e sua situação estratégica (MAXIMIANO, 2004). Desta forma, a pesquisa proposta tem por objetivo a reflexão sobre o processo de autoavaliação, instituído pela Lei nº 10.861, de 14 de abril de 2004, que possui como finalidade o desenvolvimento de conhecimentos sobre a própria IES, como componente do planejamento estratégico. Por meio da revisão bibliográfica, buscou-se contemplar os aspectos fundamentais do tema e, como resultado, é apresentada uma proposição de Modelo de Planejamento Estratégico para Instituições de Ensino Superior Privadas - IESP, a partir do Modelo de Rojo (2001), com o objetivo de subsidiar pesquisas futuras, relacionadas ao Planejamento Estratégico articulado com a autoavaliação. O artigo estrutura-se da seguinte forma: na seção 2, é apresentada a fundamentação teórica; na seção 3, os procedimentos metodológicos, e a seção 4 traz as considerações finais. ÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 201 ÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 201 2.1 Concepções sobre Planejamento Estratégico Devido a mudanças sociais, políticas e econômicas que afetam a sociedade contemporânea, a Gestão do Ensino Superior, nos últimos anos, adquire novo e 169 importante papel. O cenário mais competitivo, relacionado à expansão das Instituições de Ensino, principalmente a partir da década de 1990, exige reflexões sobre as questões estratégicas (DOURADO; CATANI; MANCEBO; OLIVEIRA, 2003; FILHO 2009; LIMA; TOMIELLO; SILVEIRA, 2004). Nesse contexto, o Planejamento estratégico torna-se fundamental para o desenvolvimento organizacional, uma vez que fomenta reflexões em torno dos princípios básicos que definem a instituição, a missão, a visão, as políticas, os objetivos (LIMA; TOMIELLO; SILVEIRA, 2004). Para Oliveira (2009), a natureza que constitui o planejamento estratégico também o configura como complexo, por considerar, em processo contínuo, o futuro organizacional. Para isso, deve ponderar o contexto no qual a organização está inserida, pois o planejamento, ao projetar à frente os objetivos, está afeto à conjuntura em que a organização se encontra. Como componentes principais do processo de elaboração do planejamento estratégico, formalizados a partir de análises e decisões, Maximiano (2004) define: i) análise da situação estratégica; ii) análise do ambiente externo; iii) análise dos pontos fortes e fracos; iv) definição de objetivos e estratégias; v) estratégias funcionais e operacionais; vi) execução e avaliação. Na sequência, apresenta-se o detalhamento de tais etapas: i) Análise da situação estratégica: seu objetivo é o diagnóstico da circunstância estratégica em que se encontra a organização, tendo por principais pontos a missão, o desempenho, as vantagens competitivas e as estratégias vigentes; ii) Análise do ambiente: representa um dos pontos essenciais do planejamento. Considerando que a maior competitividade que configura o cenário contemporâneo afeta o desempenho da organização, a análise do contexto torna-se imprescindível para o desenvolvimento institucional. Como elementos fundamentais a serem considerados para a análise, destacam-se ramos de negócio, mudanças tecnológicas, ação e controle do governo, conjuntura econômica e tendências sociais; iii) Análise dos pontos fortes e fracos: compreende o meio de identificação dos processos da organização, sua capacidade e limitações, podendo ser identificados a partir da avaliação de desempenho, incidindo na 170 elaboração das estratégias. A análise dos pontos fortes e fracos deve considerar a análise das áreas funcionais e os projetos benchmarking3. elaboração das estratégias. A análise dos pontos fortes e fracos deve considerar a análise das áreas funcionais e os projetos benchmarking3. elaboração das estratégias. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2 3 Técnica pela qual uma organização compara seu desempenho com outra (MAXIMIANO, 2004). 2.1 Concepções sobre Planejamento Estratégico A análise dos pontos fortes e fracos deve considerar a análise das áreas funcionais e os projetos benchmarking3. iv) Definição de objetivos e estratégias: como resultado da identificação das ameaças, oportunidades, fraquezas e potencialidades, esta etapa compreende ações para a efetivação do Planejamento, estratégias que visam cumprir os objetivos definidos a partir do alinhamento com a situação estratégica, a missão e a visão. v) Estratégias funcionais e operacionais: configuram o curso das ações, como o objetivo de realização da missão e objetivos; a organização define as estratégias, que consistem em determinar ações sistematizadas, permitindo a efetividade do Plano Estratégico; e vi) Execução e Avaliação: a execução do planejamento dá-se pela efetivação dos planos táticos e operacionais; sua avaliação deve permitir o monitoramento do desenvolvimento do Plano Estratégico, possibilitando reflexões e aprimoramentos dos objetivos e das estratégias institucionais. Maximiano (2004) conceitua, a partir dessas etapas, o planejamento como ferramenta de reflexões, que deve subsidiar decisões comprometidas com o futuro da organização. Bateman e Snell (1998) definem o conceito de planejamento estratégico como processo contínuo e racional para a tomada de decisões. Em seu modelo de processo de administração estratégica, trata das principais etapas do Planejamento Estratégico, constituído por: i) estabelecimento de uma missão e uma visão; ii) análise ambiental; iii) avaliação interna; iv) formulação de estratégias; v) implementação de estratégias e vi) controle estratégico. As etapas são, a seguir, descritas: i) Estabelecimento da missão e visão: constituída com o objetivo de definir a razão da existência da IES em seu contexto, a missão institucional estabelece a finalidade e a razão de ser da organização. No entanto, a visão Institucional perpassa a missão, pois promove a perspectiva e o direcionamento da organização, configurando-a por meio de seus objetivos e almejos, traduzindo as intenções e aspirações futuras; Técnica pela qual uma organização compara seu desempenho com outra (MAXIMIANO, 2004). 171 ii) Análise ambiental: configura-se como um dos fatores críticos para a elaboração do Planejamento Estratégico, constituída pelo exame da organização e do ambiente em que está inserida; trata-se da avaliação interna (pontos fortes e fracos) e externa (oportunidades e ameaças), que poderá ser desenvolvida através da análise SWOT4 (BATEMAN; SNELL, 1998; SCHERMERHORN, 2006; MACHADO, 2008; MAXIMIANO, 2004); iii) Avaliação interna: configura-se pelo panorama das competências e recursos da organização. 4 Ferramenta utilizada para a análise ambiental, sendo subsídio para a Gestão e o Planejamento Estratégico. O termo deriva de quadro palavras em inglês: Strengths (Forças), Weaknesses (Fraquezas), Opportunities (Oportunidades) e Threats (Ameaças). COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./ju COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 4 Ferramenta utilizada para a análise ambiental, sendo subsídio para a Gestão e o Planejamento Estratégico. O termo deriva de quadro palavras em inglês: Strengths (Forças), Weaknesses (Fraquezas), Opportunities (Oportunidades) e Threats (Ameaças). UIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 2.1 Concepções sobre Planejamento Estratégico Como componentes básicos, destacam-se análise financeira, de recursos humanos, de marketing, de produção e operações; iv) Formulação de estratégias: decorrentes do diagnóstico da análise ambiental, que descreve a realidade da organização, devem formular caminhos que estabeleçam a direção de ações em decorrência dos objetivos; v) Implementação de estratégias: configura-se como desafio ao gestor, devendo considerar, além das análises dos fatores externos e internos, a estrutura, a tecnologia, os recursos humanos, a liderança e a cultura da instituição, além do comprometimento dos sujeitos, este, fator imprescindível para o sucesso de execução das estratégias. Rojo (2001), ao considerar a dinâmica da sociedade contemporânea, que define o cenário de maior competitividade das instituições de Ensino Superior, apresenta um modelo de Planejamento Estratégico voltado para Instituições de Ensino Superior Privadas. Ver Figura 1: 172 Figura 1 - Modelo de Planejamento Estratégico para IES privadas, segundo Rojo (2001) Figura 1 - Modelo de Planejamento Estratégico para IES privadas, segundo Rojo (2001) Fonte: Elaborado pelos autores com base em Rojo (2001). 1.1 Ambiente Interno 1.2 Ambiente Externo 1.1.1 Pontos Fortes 1.1.2 Pontos Fracos 1.2.1 Ameaças 1.2.2 Oportunidades 2 Determinação da Missão e Objetivos 2.1 Missão: criar ou revisar 2.2 Objetivos qualificados 2.3 Objetivos quantitativos 2.4 Metas 3 Avaliação das opções de Estratégias 4 Implementação 5 Monitoramento 1 Análise do Ambiente 6 Resultados 1.2 Ambiente Externo 1.2.1 Ameaças 1.2.2 Oportunidades 2 Determinação da Missão e Objetivos 2.1 Missão: criar ou revisar 2.2 Objetivos qualificados Fonte: Elaborado pelos autores com base em Rojo (2001). 1. Análise do Ambiente: permite obter informações do contexto em que a Instituição de Ensino Superior (IES) está inserida, constituindo-se do diagnóstico dos ambientes interno e externo. Para o ambiente interno (1.1), sua análise deve permitir identificar dados que caracterizam a IES, considerando sua estrutura, corpo docente, discente e técnico administrativo; o grau de satisfação dos alunos, entre outros elementos que possam permitir a análise de pontos Forte (1.1.1) e Fracos (1.1.2) da instituição. Em relação ao ambiente externo (1.2), sua avaliação deve considerar fatores que afetam o desempenho da IES, resultado do 173 contexto histórico em que se encontra, sendo elementos incontroláveis pela instituição. Assim, a análise do contexto externo deve ser constituída pela identificação das ameaças (1.2.1) e oportunidades (1.2.2); contexto histórico em que se encontra, sendo elementos incontroláveis pela instituição. Assim, a análise do contexto externo deve ser constituída pela identificação das ameaças (1.2.1) e oportunidades (1.2.2); contexto histórico em que se encontra, sendo elementos incontroláveis pela instituição. Assim, a análise do contexto externo deve ser constituída pela identificação das ameaças (1.2.1) e oportunidades (1.2.2); 2. Determinação da Missão e Objetivos: a missão (2.1), considerando o contexto identificado pelas análises dos ambientes interno e externo, deve passar por um processo de reflexão, permitindo rever a razão de ser da IES. Os objetivos são enquadrados em qualitativos (2.2), que permitam a mensuração, e quantitativos (2.3). As metas (2.4) estão associadas aos objetivos, considerando o tempo de realização e as prioridades; 3. Avaliação das opções de Estratégias: deve considerar todas as implicações levantadas pela análise ambiental, considerando o cenário, a missão e as metas, a fim de que se possa optar pela estratégia mais assertiva para atingir os objetivos; 4. Implementação: com o foco nos objetivos, em consonância com a missão, a implementação representa a efetivação das estratégias. 5. ÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 201 2. 2 Autoavaliação das Instituições de Ensino Superior - Fundamentos Legais 1, jan./jun. 2014 Art. 3º A avaliação das instituições de educação superior terá por objetivo identificar o seu perfil e o significado de sua atuação, por meio de suas atividades, cursos, programas, projetos e setores, considerando as diferentes dimensões institucionais, dentre elas obrigatoriamente as seguintes: I - a missão e o plano de desenvolvimento institucional; II - a política para o ensino, a pesquisa, a pós-graduação, a extensão e as respectivas formas de operacionalização, incluídos os procedimentos para estímulo à produção acadêmica, as bolsas de pesquisa, de monitoria e demais modalidades; III - a responsabilidade social da instituição, considerada especialmente no que se refere à sua contribuição em relação à inclusão social, ao desenvolvimento econômico e social, à defesa do meio ambiente, da memória cultural, da produção artística e do patrimônio cultural; IV - a comunicação com a sociedade; V - as políticas de pessoal, as carreiras do corpo docente e do corpo técnico-administrativo, seu aperfeiçoamento, desenvolvimento profissional e suas condições de trabalho; VI - organização e gestão da instituição, especialmente o funcionamento e representatividade dos colegiados, sua independência e autonomia na relação com a mantenedora, e a participação dos segmentos da comunidade universitária nos processos decisórios; VII - infraestrutura física, especialmente a de ensino e de pesquisa, biblioteca, recursos de informação e comunicação. Art. 3º A avaliação das instituições de educação superior terá por objetivo identificar o seu perfil e o significado de sua atuação, por meio de suas atividades, cursos, programas, projetos e setores, considerando as diferentes dimensões institucionais, dentre elas obrigatoriamente as seguintes: Art. Figura 1 - Modelo de Planejamento Estratégico para IES privadas, segundo Rojo (2001) Monitoramento: incide no acompanhamento da execução das estratégias e das implicações em relação à implementação; 6. Resultados: consiste na análise de dados, incidindo na revisão de todos os componentes do processo. Segundo Rojo (2001), o Planejamento Estratégico, por considerar a organização como um todo e analisar uma gama complexa de fatores, amplia o conceito de planejar, configura-se pela formalização de um processo e pelo estabelecimento de estratégias que possibilitem cumprir a missão e a visão institucional. Como importante ferramenta de gestão, o planejamento estratégico permite o equilíbrio das tomadas de decisões, considerando o contexto em que a organização está inserida, pois oportuniza a reflexão dos fatores ambientais, analisando variáveis internas e externas. Para Maximiano (1995), o planejamento estratégico torna-se essencial para a) enfrentar fatos que certamente ocorrerão; b) criar um futuro desejável; c) coordenar fatos entre si. Desta forma, considerando as mudanças do cenário nacional procedentes do processo de globalização, o planejamento estratégico permite olhar, de forma sistemática, para o futuro, com o objetivo de assegurar o desenvolvimento da organização. 174 UIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 Art. 3º A avaliação das instituições de educação superior terá por objetivo identificar o seu perfil e o significado de sua atuação, por meio de suas atividades, cursos, programas, projetos e setores, considerando as diferentes dimensões institucionais, dentre elas obrigatoriamente as seguintes: 2. 2 Autoavaliação das Instituições de Ensino Superior - Fundamentos Legais 2. 2 Autoavaliação das Instituições de Ensino Superior - Fundamentos Legais Entre maio e abril de 2003, o Secretário de Educação Superior do Ministério da Educação – SESu/MEC, Carlos Roberto Antunes dos Santos, emite as Portarias nº 11 e nº 19, que instituem a Comissão Especial da Avaliação da Educação Superior (CEA), presidida pelo professor José Dias Sobrinho (Universidade Estadual de Campinas - Unicamp), com a finalidade de rever processos, políticas e instrumentos de Avaliação da Educação Superior (INEP, 2009). Data não coincide Em abril de 2004, é sancionada a Lei º 10.861, consolidando uma nova dinâmica de avaliação do ensino superior, integrando os processos de avaliação das instituições, cursos e estudantes. O Sistema Nacional de Avaliação da Educação Superior (SINAES) busca, de forma integrada, a avaliação de Instituições, cursos e estudantes, tendo por objetivo a melhoria da qualidade do ensino superior e sua expansão, o aumento permanente da sua eficácia institucional e a efetividade acadêmica. Por meio da consolidação da autonomia institucional, tende a afirmação do respeito à diversidade, e o efetivo aprofundamento da responsabilidade social das IES (Lei nº 10.861/2004). Para isso, com base no Artigo 3º, da Lei 10.861/2004, o SINAES estrutura-se em dez dimensões: Entre maio e abril de 2003, o Secretário de Educação Superior do Ministério da Educação – SESu/MEC, Carlos Roberto Antunes dos Santos, emite as Portarias nº 11 e nº 19, que instituem a Comissão Especial da Avaliação da Educação Superior (CEA), presidida pelo professor José Dias Sobrinho (Universidade Estadual de Campinas - Unicamp), com a finalidade de rever processos, políticas e instrumentos de Avaliação da Educação Superior (INEP, 2009). Data não coincide Em abril de 2004, é sancionada a Lei º 10.861, consolidando uma nova dinâmica de avaliação do ensino superior, integrando os processos de avaliação das instituições, cursos e estudantes. O Sistema Nacional de Avaliação da Educação Superior (SINAES) busca, de forma integrada, a avaliação de Instituições, cursos e estudantes, tendo por objetivo a melhoria da qualidade do ensino superior e sua expansão, o aumento permanente da sua eficácia institucional e a efetividade acadêmica. Por meio da consolidação da autonomia institucional, tende a afirmação do respeito à diversidade, e o efetivo aprofundamento da responsabilidade social das IES (Lei nº 10.861/2004). Para isso, com base no Artigo 3º, da Lei 10.861/2004, o SINAES estrutura-se em dez dimensões: COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 2. 2 Autoavaliação das Instituições de Ensino Superior - Fundamentos Legais 3º A avaliação das instituições de educação superior terá por objetivo identificar o seu perfil e o significado de sua atuação, por meio de suas atividades, cursos, programas, projetos e setores, considerando as diferentes dimensões institucionais, dentre elas obrigatoriamente as seguintes: p ; II - a política para o ensino, a pesquisa, a pós-graduação, a extensão e as respectivas formas de operacionalização, incluídos os procedimentos para estímulo à produção acadêmica, as bolsas de pesquisa, de monitoria e demais modalidades; p ; II - a política para o ensino, a pesquisa, a pós-graduação, a extensão e as respectivas formas de operacionalização, incluídos os procedimentos para estímulo à produção acadêmica, as bolsas de pesquisa, de monitoria e demais modalidades; ; III - a responsabilidade social da instituição, considerada especialmente no que se refere à sua contribuição em relação à inclusão social, ao desenvolvimento econômico e social, à defesa do meio ambiente, da memória cultural, da produção artística e do patrimônio cultural; IV - a comunicação com a sociedade; V - as políticas de pessoal, as carreiras do corpo docente e do corpo técnico-administrativo, seu aperfeiçoamento, desenvolvimento profissional e suas condições de trabalho; VI - organização e gestão da instituição, especialmente o funcionamento e representatividade dos colegiados, sua independência e autonomia na relação com a mantenedora, e a participação dos segmentos da comunidade universitária nos processos decisórios; p VII - infraestrutura física, especialmente a de ensino e de pesquisa, biblioteca, recursos de informação e comunicação. ÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 201 175 Prosseguindo: Prosseguindo: VIII - planejamento e avaliação, especialmente os processos, resultados e eficácia da autoavaliação institucional; IX - políticas de atendimento aos estudantes; X - sustentabilidade financeira, tendo em vista o significado social da continuidade dos compromissos na oferta da educação superior(Lei nº 10.861/2004). VIII - planejamento e avaliação, especialmente os processos, resultados e eficácia da autoavaliação institucional; A Avaliação do ensino superior, atendendo ao SINAES, divide-se em três modalidades: Avaliação Institucional (interna e externa), Avaliação de Curso e o Exame Nacional de Avaliação do Desempenho dos Estudantes – ENADE (BRASIL, 2012). Às IES cabe a responsabilidade pela autoavaliação institucional, segundo a Lei nº 10.861/2004, Art. 5 Revogada em 2010, após a republicação da Portaria Normativa MEC nº 40/2007. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 5 Revogada em 2010, após a republicação da Portaria Normativa MEC nº 40/2007. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 5 Revogada em 2010, após a republicação da Portaria Normativa MEC nº 40/2007. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./ju COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 6 Republicada em 29/12/2010, Seção 1, p. 23, por ter saído no DOU nº 239, de 13/12/2007, Seção 1, p. 39, com incorreção no original. 2. 2 Autoavaliação das Instituições de Ensino Superior - Fundamentos Legais 11 Cada instituição de ensino superior, pública ou privada, constituirá Comissão Própria de Avaliação - CPA, no prazo de 60 (sessenta) dias, a contar da publicação desta Lei, com as atribuições de condução dos processos de avaliação internos da instituição, de sistematização e de prestação das informações solicitadas pelo INEP, obedecidas as seguintes diretrizes: I – constituição por ato do dirigente máximo da instituição de ensino superior, ou por previsão no seu próprio estatuto ou regimento, assegurada a participação de todos os segmentos da comunidade universitária e da sociedade civil organizada, e vedada a composição que privilegie a maioria absoluta de um dos segmentos; I – constituição por ato do dirigente máximo da instituição de ensino superior, ou por previsão no seu próprio estatuto ou regimento, assegurada a participação de todos os segmentos da comunidade universitária e da sociedade civil organizada, e vedada a composição que privilegie a maioria absoluta de um dos segmentos; g II – atuação autônoma em relação a conselhos e demais órgãos colegiados existentes na instituição de educação superior. Em julho de 2004, é publicada, no Diário Oficial da União, a Portaria nº 2.0515, do Ministério da Educação – MEC, que regulamenta os procedimentos de avaliação do Sistema Nacional de Avaliação da Educação Superior (SINAES), e, em relação à autoavaliação, delibera: Art. 7º As Comissões Próprias de Avaliação (CPAs), previstas no Art. 11 da Lei nº 10.861, de 14 de abril de 2004, e constituídas no âmbito de cada instituição de educação superior, terão por atribuição a coordenação dos processos internos de avaliação da instituição, de sistematização e de prestação das informações solicitadas pelo INEP. Este artigo dá outras informações: 176 § 1º As CPAs atuarão com autonomia em relação a conselhos e demais órgãos colegiados existentes na instituição de educação superior; § 2º A forma de composição, a duração do mandato de seus membros, a dinâmica de funcionamento e a especificação de atribuições da CPA deverão ser objeto de regulamentação própria, a ser aprovada pelo órgão colegiado máximo de cada instituição de educação superior, observando-se as seguintes diretrizes: I - necessária participação de todos os segmentos da comunidade acadêmica (docente, discente e técnico-administrativo) e de representantes da sociedade civil organizada, ficando vedada a existência de maioria absoluta por parte de qualquer um dos segmentos representados; II - ampla divulgação de sua composição e de todas as suas atividades. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./ju 2. 2 Autoavaliação das Instituições de Ensino Superior - Fundamentos Legais § 1º As CPAs atuarão com autonomia em relação a conselhos e demais órgãos colegiados existentes na instituição de educação superior; § 1º As CPAs atuarão com autonomia em relação a conselhos e demais órgãos colegiados existentes na instituição de educação superior; g g ç ç p § 2º A forma de composição, a duração do mandato de seus membros, a dinâmica de funcionamento e a especificação de atribuições da CPA deverão ser objeto de regulamentação própria, a ser aprovada pelo órgão colegiado máximo de cada instituição de educação superior, observando-se as seguintes diretrizes: g I - necessária participação de todos os segmentos da comunidade acadêmica (docente, discente e técnico-administrativo) e de representantes da sociedade civil organizada, ficando vedada a existência de maioria absoluta por parte de qualquer um dos segmentos representados; II - ampla divulgação de sua composição e de todas as suas atividades. Vinculada ao processo de regulação, a autoavaliação é instrumento essencial e obrigatório para que a IES se integre formalmente ao sistema regular da Educação Superior (INEP, 2009). Para isso, a Portaria Normativa MEC nº 40, de 12 de dezembro de 20076, dispõe: nculada ao processo de regulação, a autoavaliação é instrumento essencial Art. 61-D Será mantido no cadastro e-MEC, junto ao registro da instituição, campo para inserção de relatório de auto-avaliação, validado pela CPA, a ser apresentado até o final de março de cada ano, em versão parcial ou integral, conforme se trate de ano intermediário ou final do ciclo avaliativo. Os relatórios devem apresentar dados relacionados a ações de caráter pedagógico e administrativo resultantes do processo de autoavaliação, e indicadores necessários para melhorias das carências identificadas, devendo subsidiar os processos de recredenciamento de IES e de autorização, reconhecimento e renovação de reconhecimento de cursos. (Portaria Normativa MEC nº 40/2007) O processo de autoavaliação com a participação expressiva da comunidade, a qual está afeta direta ou indiretamente às ações da IES, torna o processo mais significativo e democrático. Dessa forma, a autoavaliação configura-se pela participação plural da comunidade acadêmica e externa, em um processo social de reflexão e construção de conhecimentos sobre a Instituição que possibilitem transformações (INEP, 2009). As organizações sociais, assim como as Instituições de Ensino Superior - IES, COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 7 Revogada pela Resolução CNE/CES n.º 11, de 10 de julho de 2006. No entanto, o PDI permanece como requisito básico para os processos de Regulação, Supervisão e Avaliação, segundo o Decreto Executivo nº 5.773/2006. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2 2.3 Instituições de Ensino Superior, Planejamento Estratégico e Autoavaliação 2.3 Instituições de Ensino Superior, Planejamento Estratégico e Autoavaliação As organizações sociais, assim como as Instituições de Ensino Superior - IES, 177 são partes integrantes de um novo contexto, no qual a sociedade contemporânea, de características mais dinâmicas, relacionadas a fatores econômicos, políticos, culturais e tecnológicos, além de um cenário mais competitivo, exige o posicionamento estratégico por parte da Gestão (MEYER JÚNIOR; SERMANN; MANGOLIM, 2004). O Planejamento Estratégico, neste contexto, busca preparar a Instituição às mudanças do ambiente e à competitividade, representando importante ferramenta gerencial, com métodos do processo administrativo, que permitem a idealização do futuro, com o dever de cumprir a missão e visão institucional (LIMA; TOMIELLO; SILVEIRA, 2004; PAPA FILHO, 2009). Dessa forma, o planejamento estratégico configura-se pelo compromisso com o futuro da organização; quando formalizado, permite, através de suas etapas de elaboração, o conhecimento da realidade, do cenário, dos recursos, das potencialidades e limitações, fomentando o desenvolvimento de ações que possibilitem maior racionalidade administrativa. (PAPA FILHO, 2009). Por vez, consciente da importância do Planejamento, não somente para a estabilidade da Instituição, como para o desenvolvimento do ensino de qualidade e afirmação de seu compromisso social, o Estado, desde março de 2002, com a publicação da Resolução nº 107, do Conselho Nacional da Educação – CNE e Secretaria de Educação Superior - SESu, torna obrigatória a elaboração do Plano de Desenvolvimento Institucional – PDI, como requisito básico para o pleno funcionamento das Instituições de Ensino Superior, públicas e privadas. A implementação do Plano de Desenvolvimento Institucional possibilita aos Gestores a reflexão sobre as principais questões atinentes à instituição (MEYER JÚNIOR; SERMANN; MANGOLIM, 2004; CARDOSO, 2006). Com o objetivo de permitir a expansão do ensino superior, em 2003, acirram- se os debates frente à Reforma Universitária, com o compromisso com a qualidade e inclusão, discutindo-se a necessidade de políticas públicas que possibilitem aferir as condições do sistema nacional de ensino superior (ANDRIOLA, 2009). Em abril de 2004, é instituído o Sistema Nacional de Avaliação da Educação 178 Superior (SINAES), a partir da Lei nº 10.861. Esta estabeleceu a autoavalição institucional como requisito para o processo de regulação do Ensino Superior, ao mesmo tempo em que torna compulsório às Instituições de Ensino Superior (IES) a constituição de uma Comissão Própria de Avaliação (CPA), responsável pela condução deste processo, que conta com a participação da comunidade acadêmica8 e civil organizada. ÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 201 8 A expressão comunidade acadêmica engloba o corpo docente, discente e técnico-administrativo. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 2.3 Instituições de Ensino Superior, Planejamento Estratégico e Autoavaliação Destaca-se a autovaliação como um processo que visa à melhoria da qualidade do ensino, diante de um processo de participação coletiva, através de princípios democráticos, que possibilitem a visão multilateral sobre IES, permitindo a reflexão por parte das diferentes percepções, seja docente, discente, administrativa e social. Uma vez efetiva, a autoavaliação representa uma ferramenta imprescindível para a Gestão do Ensino Superior, sendo, também, subsídio para o aperfeiçoamento do Plano de Desenvolvimento Institucional e Projetos Pedagógicos de cursos (ANDRIOLA, 2009; GALDINO, 2011). Porém, para que autoavaliação se consolide como subsídio para a Gestão, é essencial que as etapas da avaliação interna ocorram contemplando a participação plural dos sujeitos, a transparência e o planejamento do processo, permitindo a criação e permanência da cultura avaliativa (GALDINO, 2011). A autoavaliação, componente para a regulação do ensino superior, possibilita o diagnóstico da realidade interna institucional, subsidiando a gestão estratégica, com vista ao alcance dos objetivos institucionais. Desta forma, a autoavaliação amplia seu significado estratégico, quando da participação da comunidade acadêmica e externa, que fazem da avaliação um instrumento para melhoria contínua da Gestão (MEYER JÚNIOR; SERMANN; MANGOLIM, 2004). A implementação do Planejamento Estratégico em Instituições de Ensino Superior representa um grande desafio à gestão, diante dos aspectos organizacionais, culturais e financeiros, pois o Planejar compreende a totalidade Institucional, soma-se à complexidade de sua natureza, ou seja, seu produto é o conhecimento. Associado ao processo de autoavaliação, o desafio para a gestão está em integrar todos os segmentos da comunidade comprometidos com a IES, de forma responsável e crítica, considerando as características institucionais. Desta forma, planejar estrategicamente, diante da modernidade e, consequentemente, da competitividade, 179 representa uma exigência às Instituições de Ensino (VIANNA, 2004). representa uma exigência às Instituições de Ensino (VIANNA, 2004). ÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 201 3.1 Cenário 3.1 Cenário Segundo os dados do Censo da Educação Superior, do ano de 2010, mensurados pelo Instituto Nacional de Estudos e Pesquisa Educacionais Anísio Teixeira (INEP), o total de instituições no Brasil passa de 1.391, em 2001, para 2.378, em 2010 (INEP, 2012). Esses totais, considerado o ano de realização do Censo, representam 88,3% de instituições privadas; 4,5%, estaduais; 4,2%, federais e 3,0%, municipais. Destas, 85,2% são faculdades; 8,0% são universidades; 5,3% são centros universitários e 1,6% são institutos federais de educação, ciência e tecnologia – IFs - e centros federais de educação tecnológica - Cefets (INEP, 2012). Segundo os dados do INEP, o setor público apresenta significativa expansão, resultado das políticas públicas de democratização do acesso à educação superior, ao mesmo tempo em que o setor privado apresenta certa estabilização. De 2001 a 2010, as instituições federais tiveram um aumento de 85,9% em suas matrículas, e as estaduais, de 66,7% (INEP, 2012). Em relação aos ingressantes nos cursos de graduação, em 2010, presencial e a distância, entraram 2.182.229 alunos, representando um aumento de 140,5% em instituições federais e 115,4% em instituições privadas (INEP, 2012). Indagando o índice de Inadimplência no setor de Educação Superior, a pesquisa realizada pela assessoria econômica do Sindicato das Entidades Mantenedoras de Estabelecimentos de Ensino Superior no Estado de São Paulo (Semesp) -, por meio do Sistema de Informações do Ensino Superior Particular – Sindata - demonstra que o índice de inadimplência no ensino superior privado, no ano de 2010, chegou a ser 68% superior à inadimplência de todos os setores da economia consolidados. Em 2010, a inadimplência total no país foi de 5,7%, enquanto que o setor do ensino superior privado registrou um índice de 9,58% (SINDICATO, 2011). 180 Considerando as informações acima elencadas, pode-se compreender alguns desafios aos Gestores de Instituições de Ensino Superior, especialmente para instituições privadas de pequeno porte, configuradas como Faculdades. As instituições de ensino superior privadas, segundo o relatório do INEP, no ano de 2010, corresponderam a 83,3% das 2.378 instituições, sendo 85,2% credenciadas como faculdades (INEP, 2012). A concorrência não está restrita somente a este perfil de IES, acrescenta-se à expansão, de 2001 a 2010, das instituições federais, que tiveram um aumento de 85,9%, em suas matrículas e 140,5% no número de ingressantes, em cursos presenciais e a distância (INEP, 2012). 3.1 Cenário Quanto ao índice de inadimplência, o atraso acima de 90 dias nas mensalidades das instituições de pequeno porte9 atingiu 11,63%, em 2010 (SINDICATO, 2011). Outros indicadores que caracterizam o cenário nacional devem ser considerados, a exemplo da taxa de desemprego medida pelo Instituto Brasileiro de Geografia e Estatística (IBGE), nas seis maiores regiões metropolitanas10, registrada em agosto de 2012, sendo de 5,3 % (IBGE, 2012) Desta forma, o cenário contemporâneo exige das Instituições de Ensino Superior, especialmente das Faculdades Privadas, o posicionamento estratégico. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 9 A pesquisa do Semesp considerou como instituições de pesqueno porte, aquelas com até dois mil alunos. 10 Regiões metropolitanas de Recife, Salvador, Belo Horizonte, Rio de Janeiro, São Paulo e Porto Alegre. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./ju 3.2 Metodologia Os procedimentos para execução da pesquisa têm por princípios metodológicos uma abordagem qualitativa baseada em uma revisão bibliográfica que, segundo Miles e Huberman (1994), consiste em um processo investigativo no qual o pesquisador, gradualmente, compreende o sentido de um fenômeno social ao constatar, comparar, reproduzir, catalogar e classificar o objeto de estudo. Desta forma, a revisão bibliográfica tem como finalidade contemplar os aspectos fundamentais do tema, por meio da análise, que estabelece a correlação entre Planejamento Estratégico, Autoavaliação e Gestão, viabilizando a descoberta de novos conhecimentos e permitindo a proposição de um modelo aplicado à melhoria dos processos de gestão em Instituições de Ensino Superior Privadas, a 181 partir do Modelo de Planejamento Estratégico de Rojo (2001), para Instituições de Ensino Superior Privadas. partir do Modelo de Planejamento Estratégico de Rojo (2001), para Instituições de Ensino Superior Privadas. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 3.3 Modelo proposto O modelo proposto tem por objetivo integrar a autoavaliação institucional ao Planejamento Estratégico de Instituições de Ensino Superior Privadas. O Planejamento configura-se como importante ferramenta de gestão, definindo estratégias com o objetivo de cumprir a visão e a missão institucional, fomentando o sucesso competitivo das IES no atual cenário de mudanças constantes (SCHERMERHOR, 2006). É, portanto, essencial para o desenvolvimento organizacional da Instituição, frente ao mercado competitivo. Articulada com o processo de Gestão, a autoavaliação, requisito para o processo de regulação do Ensino Superior, estabelecida pela Lei nº 10.861, de 14 de abril de 2004, quando constituída com a finalidade de integrar o Planejamento Estratégico, subsidia a análise ambiental, através do diagnóstico interno da IES. Na Figura 2, apresentada na próxima página, é apresentado o Modelo de Planejamento Estratégico para Instituições de Ensino Superior Privadas, em forma de diagrama, em seguida, é feita a descrição de suas etapas. 182 Figura 2 - Modelo Proposto de Planejamento Estratégico para IES privadas COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 Figura 2 - Modelo Proposto de Planejamento Estratégico para IES privadas Fonte: Elaborado pelos autores. 1.2 Ambiente Interno 1.1 Ambiente Externo 1.2.2.2 Pontos Fortes 1.2.2.3 Pontos Fracos 1.1.2.2 Ameaças 1.1.2..3 Oportunidades 2 Determinação da Missão e Visão 2.1 Missão 2.2 Visão 2.3 Objetivos 2.4 Metas 3 Avaliação das opções de Estratégias 4 Implementação 5 Monitoramento 1 Análise do Ambiente 6 Resultados 7 Avaliação 1.2.1 Preparação – Autoavaliação 1.2.1.2 Constituição da CPA 1.2.1. 3 Elaboração do Projeto 1.2.1.4 Sensibilização 1.2.2 Execução 1.2.2.1 Avaliação 1.3 Consolidação 1.3.1 Análise Swot 1.3.3 Balanço Crítico 1.1.3 Relatórios 1.2.4 Relatórios 8 Tomadas de Decisões : implementação de Melhorias 1.1.1.2 Constituição de Comissão 1.1.1 Preparação 1.1.1.3 Elaboração de Projeto para levantamento de dados 1.1.2 Execução 1.1.2.1Avaliação 1.3.4 Identificação da Situação Estratégica feedback - Modelo Proposto de Planejamento Estratégico para IES privadas Fonte: Elaborado pelos autores. UIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 183 Etapa 1. Análise do Ambiente: compreende um dos pontos-chave do planejamento, permitindo a identificação da Situação Estratégica da Instituição. Seu resultado é o diagnóstico da IES, composto pelo exame da instituição e do ambiente em que está inserida. A avaliação ambiental deve permitir melhor adequação das estratégias aos objetivos, constituindo-se da análise do ambiente externo (1.1) e interno (1.2). ÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 201 3.3 Modelo proposto 1.1 Ambiente Externo: representa elementos incontroláveis por parte da IES, considerando o contexto no qual a Instituição se encontra, e que interferem em seu desempenho. Por sua amplitude, o ambiente externo caracteriza-se como o componente mais complexo dos segmentos ambientais. O arranjo deste processo prevê o acontecimento de etapas distintas, para que seja possível identificar as ameaças e oportunidades com precisão; sua preparação (1.1.1) caracteriza-se pela definição de ações e responsáveis. Primeiramente definem-se os integrantes da Comissão de Desenvolvimento do Planejamento (1.1.1.2), responsáveis pela condução do processo, com representantes dos segmentos administrativos e da gestão, com atribuições para definir estratégias, através da elaboração do Projeto, para o levantamento de dados. No Projeto (1.1.1.2), definem-se diretrizes, metodologias, indicadores e instrumentos, observando fatores que definem o contexto: socioeconômicos, políticos, culturais, tecnológicos e jurídicos, considerando os objetivos institucionais e alocação de recursos necessários para sua execução (1.1.1.2). Concluída a execução, inicia-se o processo de avaliação (1.1.2.1) das informações coletadas, mensurando as informações em um processo de identificação das ameaças (1.1.2.2) e oportunidades (1.1.2.3), possibilitando a elaboração de relatórios (1.1.3) que definem a situação do ambiente externo. 1.2 Análise do Ambiente Interno: tem por objetivo evidenciar a situação em que se encontra a IES, identificando limitações e potencialidades. A organização deste processo prevê a ocorrência de diferentes etapas, sua preparação (1.2.1) caracteriza-se pelo planejamento das ações, de forma que permita sistematizar os procedimentos e seus responsáveis, fatores necessários para a implementação da avaliação interna. Inicialmente, 184 define-se a composição da Comissão Própria de Avaliação – CPA (1.2.1.2), regulamentada pela Lei nº 10.861/2004, que deve contar com representantes da comunidade acadêmica e da sociedade civil organizada, responsáveis por todo o processo de autoavaliação, e em articulação com a administração da IES para alocação de recursos necessários para a execução da avaliação. A elaboração do Projeto de Autoavaliação (1.2.1.3) tem por finalidade a definição dos objetivos, estratégias, procedimentos, prazos, entre outros elementos fundamentais ao processo, além da elaboração de instrumentos e indicadores, considerando a missão e a visão institucional. A sensibilização (1.2.1.4) deve permitir a participação, através de palestras e seminários, da comunidade acadêmica na elaboração da proposta avaliativa. Tais processos subsidiam a execução (1.2.2) da autoavaliação e avaliação, uma vez que o sujeito se autoavalia e avalia o objeto da avaliação, observando as ações previstas no projeto, nos instrumentos, em coerência com a implementação. ÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 201 3.3 Modelo proposto Concluída esta etapa, inicia-se a avaliação (1.2.2.1) das informações coletadas, com o foco na identificação dos pontos fortes (1.2.2.2) e fracos (1.2.2.3), resultando na elaboração dos relatórios (1.2.3). Finalizados os relatórios da análise ambiental externa (1.1) e interna (1.2), a comissão responsável pelo planejamento, configurada na análise ambiental externa, assume as demais etapas, entre elas o processo de consolidação das informações coletadas (1.3), composto pela análise Swot (1.3.1), articulando as oportunidades, as ameaças, os pontos fortes e fracos ao prazo, ao grau de impacto e às consequências sobre a IES. Em seguida, relacionam-se as oportunidades aos pontos fortes e as ameaças, aos pontos fracos, subsidiando o Balanço Crítico (1.3.3), permitindo identificar a situação estratégica da Instituição (1.3.4). Etapa 2. Determinação da missão e visão: considerando a situação estratégica identificada através da análise ambiental (1), consolidada pelo Balanço Crítico (1.3.3), a missão (2.1), razão de ser da Instituição, e a visão (2.2), projeção futura da IES, devem passar por um processo de reflexão que pode resultar na redefinição 185 destes valores. Os objetivos (2.3), alinhados à missão, visão e situação estratégica consistem em almejos (qualitativos) que a instituição tem a alcançar. As metas (2.4) correspondem à quantificação destes objetivos, que incidem em estratégias que se caracterizam por ações que permitam atingir tanto os objetivos, quanto as metas; Etapa 3. Avaliação das opções de Estratégias: deve possibilitar a identificação das estratégias apropriadas ao contexto da IES, considerando as implicações apontadas pela análise ambiental (1), com a finalidade de cumprir com os objetivos e metas, resultando em planos de ações. Estes se configuram em plano tático, que compreende o detalhamento de ações a partir das estratégias, por áreas e setores, e plano operacional, que concebe a efetividade das ações propostas no plano tático. Etapa 4. Implementação: representa a efetivação do planejamento, sendo necessário considerar os recursos financeiros e infraestrutura, além do comprometimento dos sujeitos. Neste processo, observam-se as ações previstas e a coerência com o Plano de ação. Etapa 5. Monitoramento: assegura, através do processo de acompanhamento do desenvolvimento do Plano Estratégico, o alinhamento entre ações, objetivos e metas. Etapa 6. Resultados: consiste no desfecho das ações implementadas, em dados qualitativos dos objetivos e quantitativos das metas alcançadas, constituindo um dos elementos do diagnóstico do Planejamento, identificado na avaliação do desenvolvimento. Etapa 7. ÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 201 3.3 Modelo proposto Avaliação: caracteriza-se pela análise do processo desenvolvido, dos resultados alcançados, das estratégias utilizadas, dos obstáculos e dos avanços, permitindo o planejamento de ações futuras, subsidiando a tomada de decisões. Etapa 8. Tomadas de Decisões - implementação de melhorias: compreende um dos pontos de maior importância do Planejamento Estratégico, configurando-se pela materialização das ações, produzindo significado à participação dos sujeitos envolvidos e ao processo, tornando a IES mais competitiva no mercado, retroalimentando o sistema, uma vez que, ocorrendo mudanças, altera-se a Situação 186 Estratégica da IES. Estratégica da IES. O Modelo de Planejamento Estratégico, aqui apresentado, pretende destacar a importância do processo de autoavaliação, compreendido como diagnóstico da realidade interna da IES, como subsídio para a elaboração do Planejamento Estratégico em Instituições de Ensino Superior Privadas em contribuição com a Gestão Estratégica. UIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 4 Considerações finais A sociedade contemporânea caracteriza-se pela dinâmica das relações políticas, econômicas, sociais, culturais e tecnológicas. Neste contexto de mudanças, a Gestão do Ensino Superior passa a enfrentar novos desafios, tornando necessárias reflexões no âmbito estratégico. Neste mesmo cenário, os conceitos tradicionais de avaliação tornam-se incapazes de oferecer resultados satisfatórios. Diante desta concepção, os processos avaliativos estendem-se a vários domínios do Estado, principalmente nas políticas públicas voltadas à Educação. A diversificação e ampliação histórica das práticas avaliativas destacam sua importância política. Em 2004, é instituído o Sistema Nacional de Avaliação da Educação Superior (SINAES), compreendendo a autoavaliação como requisito normativo para o processo de regulação do Ensino Superior. A autoavaliação representa a reflexão sobre a própria IES, gera conhecimentos fundamentais para a melhoria dos procedimentos internos e, quando constituída com a finalidade de integrar o Planejamento, permite subsidiar a elaboração do diagnóstico institucional, por meio da análise do ambiente interno, componente da análise ambiental que oportuniza identificar a situação estratégica da IES, fator fundamental para o desenvolvimento do Planejamento. O Planejamento Estratégico representa o comprometimento dos Gestores com o futuro da Instituição, compreendendo-a em sua globalidade, a partir da reflexão de sua situação estratégica, de seus objetivos e metas, alinhados à missão e visão institucional. Com a finalidade de assegurar o desenvolvimento institucional, através de melhorias dos processos, das atividades administrativas, pedagógicas e da 187 infraestrutura, assim como a autoavaliação, o Planejamento Estratégico deve contar com a participação da comunidade acadêmica e da sociedade. A autoavaliação, pela dificuldade de se atribuir significado e identidade ao processo, assim como a participação, esta principalmente em relação ao Planejamento Estratégico, quando definido em moldes tradicionais, representam desafios à Gestão. O Modelo de Planejamento Estratégico proposto deverá permitir que esses desafios sejam minimizados, quando considerada a autoavaliação institucional como componente do Planejamento. Esta integração, além de tornar o processo mais participativo, torna-o coerente com o histórico social da IES, além de possibilitar mudanças na cultura organizacional da Instituição, fomentando a cultura avaliativa e o comprometimento dos sujeitos, a partir da implementação de melhorias, resultado da efetivação do Planejamento. A presente pesquisa teve por objetivo articular a autoavaliação institucional ao Planejamento Estratégico, desenvolvida através da revisão bibliográfica, que resultou na proposição de um modelo de Planejamento Estratégico para Instituições de Ensino Superior Privadas. É importante destacar que a pesquisa não teve por finalidade esgotar as reflexões sobre o tema, mas subsidiar pesquisas futuras. Referências ANDRIOLA, Wagner Bandeira. Planejamento Estratégico e Gestão Universitária como atividades oriundas da auto-avaliação de instituições de ensino superior (IES): o exemplo da universidade federal do ceará (ufc). Revista Iberoamericana de Evaluación Educativa 2009. vl. 2, n 2. p. 82-103. BATEMAN, Thomas S; SNELL, Scott A. Administração: construindo vantagem competitiva. São Paulo, Atlas, 1998. BRASIL. Executivo. Lei no 10.861, de 14 de abril de 2004. Institui o Sistema Nacional de Avaliação da Educação Superior – SINAES e dá outras providências. Disponível em: <planalto.gov.br >.Acesso em: 10 ago. 2012b BRASIL. Executivo. Lei no 10.861, de 14 de abril de 2004. Institui o Sistema Nacional de Avaliação da Educação Superior – SINAES e dá outras providências. Disponível em: <planalto.gov.br >.Acesso em: 10 ago. 2012b ______. Instituto de Estudos e Pesquisas Educacionais Anísio Teixeira. SINAES – Sistema Nacional de Avaliação da Educação Superior: da concepção à regulamentação. 5 ed. Brasília; 2009. ______. Instituto de Estudos e Pesquisas Educacionais Anísio Teixeira. SINAES – Sistema Nacional de Avaliação da Educação Superior: da concepção à regulamentação. 5 ed. Brasília; 2009. ______. Ministério da Educação. Comissão Nacional de Avaliação da Educação Superior (CONAES). Diretrizes para a Avaliação das Instituições de Educação ______. Ministério da Educação. Comissão Nacional de Avaliação da Educação Superior (CONAES). Diretrizes para a Avaliação das Instituições de Educação UIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 188 Superior. Brasília, 2004. ______. ______. Comissão Nacional de Avaliação da Educação Superior (CONAES). Documento orientador das comissões de avaliação in loco. Brasília, 2012a. ______. ______. Comissão Nacional de Avaliação da Educação Superior (CONAES). Instituto Nacional de Estudos e Pesquisas Educacionais (INEP). Orientações Gerais para o Roteiro da Auto-Avaliação das Instituições. 2004. Disponível em: <inep.gov.br>. Acesso em: 10 agosto. 2012. ______. ______. Portaria Normativa Nº 40, de 12 de dezembro de 2007. Institui o e- MEC, sistema eletrônico de fluxo de trabalho e gerenciamento de informações relativas aos processos de regulação, avaliação e supervisão da educação superior no sistema federal de educação, e o Cadastro e-MEC de Instituições e Cursos Superiores e consolida disposições sobre indicadores de qualidade, banco de avaliadores (Basis) e o Exame Nacional de Desempenho de Estudantes (ENADE) e outras disposições. Republicada em 29/12/2010, Seção 1, p. 23, por ter saído no Diário Oficial da União nº 239, de 13/12/2007, Seção 1, p. 39, com incorreção no original. ______. ______. Secretaria de Educação Superior – SESu. Referências Portaria nº 11, de 28 de abril de 2003. Institui Comissão Especial com a finalidade de analisar, oferecer subsídios, fazer recomendações, propor critérios e estratégias para a reformulação dos processos e políticas de avaliação do ensino superior e elaborar a revisão crítica dos seus instrumentos, metodologias e critérios utilizados. Publicada no Diário Oficial da União em 30/04/2003, Seção 1, p. 19. ______. ______. Secretaria de Educação Superior – SESu. Portaria nº 19, de 27 de maio de 2003. Designa professores para integrarem a Comissão Especial, instituída pela Portaria MEC/SESu, n° 11, de 28 de abril de 2003, publicada no DOU de 30 de abril de 2003, seção 2, p. 19. Publicada no Diário Oficial da União em 28/05/2003 , Seção 1 p. 11 CARDOSO, Wille Muriel. O Impacto do Plano de Desenvolvimento Institucional na Profissionalização das Instituições Privadas de Ensino Superior. Dissertação (Mestrado em Administração) - Faculdades Integradas de Pedro Leopoldo, Belo Horizonte, 2006. DOURADO, Luiz Fenandes; CATANI, Afrânio Mendes; MANCEBO, Deise; OLIVEIRA, João Ferreira de (Org.) Políticas e Gestão da Educação Superior: transformações recentes e debates atuais. São Paulo: Xamã, 2003. GALDINO, Mary Neuza Dias. A Autoavaliação Institucional no Ensino Superior como Instrumento de Gestão. Fundação CESGRANRIO/ Universidade do Grande Rio. 2011. XXV Simpósio Brasileiro II Congresso IBERO-AMERICANO de Políticas e Administração da Educação. Jubileu de Ouro da ANPAE, no período de 26 a 29 de abril de 2011. UIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 189 INSTITUTO Brasileiro de Geografia Estatística – IBGE. Pesquisa Mensal de Emprego. Disponível em: INSTITUTO Brasileiro de Geografia Estatística IBGE. Pesquisa Mensal de Emprego. Disponível em: <ttp://www.ibge.gov.br/home/presidencia/noticias/noticia_visualiza.php?id_noticia=22 20&id_pagina=1. Acesso em: 11 out. 2012. <ttp://www.ibge.gov.br/home/presidencia/noticias/noticia_visualiza.php?id_noticia=22 20&id_pagina=1. Acesso em: 11 out. 2012. INSTITUTO Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira INEP. Censo da educação superior: 2010 – resumo técnico. Brasília: Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira, 2012. LIMA, Arnaldo José de; TOMIELLO, Naira; SILVEIRA, Rosana Rosa. Metodologias de Planejamento Estratégico: uma discussão preliminar para IES. IV Colóquio Internacional sobre Gestão Universitária na América do Sul. Florianópolis, 8 a 10 de dezembro de 2004. Disponível em: <http://www.inpeau.ufsc.br/coloquio04/area1.htm>. Acesso em: 24 set. 2012. LIMA, Arnaldo José de; TOMIELLO, Naira; SILVEIRA, Rosana Rosa. Metodologias de Planejamento Estratégico: uma discussão preliminar para IES. IV Colóquio Internacional sobre Gestão Universitária na América do Sul. Florianópolis, 8 a 10 de dezembro de 2004. Disponível em: <http://www.inpeau.ufsc.br/coloquio04/area1.htm>. Referências Acesso em: 24 set. 2012. MACHADO, Luiz Eduardo. Gestão Estratégica para Instituições de Ensino Superior. Rio de Janeiro: FGV, 2008, MAXIMIANO, Antonio César Amaru. Introdução à Administração. 4. ed. São Paulo: Atlas, 1995. MAXIMIANO, Antonio César Amaru. Introdução à Administração. 4. ed. São Paulo: Atlas, 1995. _. Introdução à Administração. 6. ed. São Paulo: Atlas, 2004. . Introdução à Administração. 6. ed. São Paulo: Atlas, 2004. MEYER JÚNIOR; Victor; SERMANN, Lúcia I. C; MANGOLIM Lúcia. Planejamento e Gestão Estratégica: viabilidade nas IES. IV Colóquio Internacional sobre Gestão Universitária na América do Sul. Florianópolis, 8 a 10 de dezembro de 2004. Disponível em: <http://www.inpeau.ufsc.br/coloquio04/area1.htm>. Acesso em: 24 set. 2012. MEYER JÚNIOR; Victor; SERMANN, Lúcia I. C; MANGOLIM Lúcia. Planejamento e Gestão Estratégica: viabilidade nas IES. IV Colóquio Internacional sobre Gestão Universitária na América do Sul. Florianópolis, 8 a 10 de dezembro de 2004. Disponível em: <http://www.inpeau.ufsc.br/coloquio04/area1.htm>. Acesso em: 24 set. 2012. MILES, M. B.; HUBERMAN, A. M. Qualitative da analysis: a sourcebook of new methods (2nded.). Newbury Park, CA: Sage. 1994. In: CRESWELL, John W. Projetos de pesquisa: métodos qualitativos, quantitativos e misto. 2. ed. Porto Alegre: Artmed, 2007. MILES, M. B.; HUBERMAN, A. M. Qualitative da analysis: a sourcebook of new methods (2nded.). Newbury Park, CA: Sage. 1994. In: CRESWELL, John W. Projetos de pesquisa: métodos qualitativos, quantitativos e misto. 2. ed. Porto Alegre: Artmed, 2007. OLIVEIRA, Djalma de Pinho Rebouças de. Planejamento Estratégico: conceitos, metodologias e práticas. 26. ed. São Paulo, Atlas, 2009; OLIVEIRA, Djalma de Pinho Rebouças de. Planejamento Estratégico: conceitos, metodologias e práticas. 26. ed. São Paulo, Atlas, 2009; PAPA FILHO, Sudário. Planejamento Estratégico em Instituições de Ensino Superior. In: GOULART, Íris Barbosa; PAPA FILHO, Sudário (Coord.). Gestão de Instituições de Ensino Superior. Curitiba: Juruá, 2009; PAPA FILHO, Sudário. Planejamento Estratégico em Instituições de Ensino Superior. In: GOULART, Íris Barbosa; PAPA FILHO, Sudário (Coord.). Gestão de Instituições de Ensino Superior. Curitiba: Juruá, 2009; ROJO, Claudio Antonio. Diagnóstico Ambiental - uma etapa do Planejamento estratégico para Instituições de ensino superior da iniciativa privada: O caso da faculdade de Ciências Sociais Aplicadas de Cascavel – UNIVEL. Dissertação (Mestrado em Engenharia de Produção), Universidade Federal de Santa Catarina, Florianópolis, 2001 ÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 201 190 SCHERMERHORN, Johm R. Administração: conceitos fundamentais. Rio de Janeiro: LTC, 2006. SCHERMERHORN, Johm R. Administração: conceitos fundamentais. Rio de Janeiro: LTC, 2006. SINDICATO das Entidades Mantenedoras de Estabelecimentos de Ensino Superior no Estado de São Paulo – Semesp. Revista Ensino Superior. Para não perder de vista. Disponível em: VIANNA; Ilca Oliveira de Almeida. Planejamento Estratégico e Participativo: elaboração, fatores facilitadores e dificultadores de sua implantação na universidade. IV Colóquio Internacional sobre Gestão Universitária na América do Sul. Florianópolis, 8 a 10 de dezembro de 2004. Disponível em: <http://revistaensinosuperior.uol.com.br/imprime.asp?codigo=12806>. COLÓQUIO – Revista do Desenvolvimento Regional - Faccat - Taquara/RS - v. 11, n. 1, jan./jun. 2014 . Introdução à Administração. 6. ed. São Paulo: Atlas, 2004. Acesso em: 24 set. 2012.
https://openalex.org/W2573118780	https://www.nature.com/articles/srep40279.pdf	English	null	Exocyst subunit SEC3A marks the germination site and is essential for pollen germination in Arabidopsis thaliana	Scientific reports	2,017	cc-by	9,356	Exocyst subunit SEC3A marks the germination site and is essential for pollen germination in Arabidopsis thaliana received: 12 September 2016 accepted: 05 December 2016 Published: 11 January 2017 Yan Li, Xiaoyun Tan, Mengru Wang, Bingxuan Li, Yanxue Zhao, Chengyun Wu, Qingchen Rui, Junxia Wang, Zhongyuan Liu & Yiqun Bao Yan Li, Xiaoyun Tan, Mengru Wang, Bingxuan Li, Yanxue Zhao, Chengyun Wu, Qingchen Ru Junxia Wang, Zhongyuan Liu & Yiqun Bao Arabidopsis exocyst subunit SEC3A has been reported to participate in embryo development. Here we report that SEC3A is involved during pollen germination. A T-DNA insertion in SEC3A leads to an absolute, male-specific transmission defect that can be complemented by the expression of SEC3A coding sequence from the LAT52 promoter or SEC3A genomic DNA. No obvious abnormalities in the microgametogenesis are observed in the sec3a/SEC3A mutant, however, in vitro and in vivo pollen germination are defective. Further studies reveal that the callose, pectin, and cellulose are apparently not deposited at the germination site during pollen germination. SEC3A is expressed ubiquitously, including in pollen grains and pollen tubes. Notably, SEC3A-GFP fusion proteins are specifically recruited to the future pollen germination site. This particular localization pattern is independent of phosphatidylinositol 4,5-bisphosphate (PI-4,5P2), although SEC3-HIS fusion proteins are able to bind to several phosphoinositols in vitro. These results suggest that SEC3A plays an important role in the establishment of the polar site for pollen germination. Pollen germination is a very important event among a series of pollination processes by which pollen tube deliv- ers the sperm cells into the ovule to complete fertilization. In Arabidopsis, once a desiccated, pollen grain contacts a papilla cell on the stigmatic surface, it became hydrated within a short period of time. The presence of a Ca2+ gradient beneath the potential germination site1,2, the reorganization of F-actin cytoskeleton3,4, and the massive deposition of callose, pectin, and cellulose at the germination plaque5–7 are key events taking place before tube emergence. Until now, it is not yet clear how the the germination site is established. g y g In order to satisfy pollen germination and the rapid pollen tube tip growth, cell wall material, proteins and other membrane components, are transported to the growing tip of the pollen tube via the vesicle trafficking system8,9. Compared to intensive researches on pollen tube growth, limited number of players functioning from the onset of pollen germination have been identified. These included proteins involved in cell wall material synthesis and modification. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China. Correspondence and requests for materials should be addressed to Y.B. (email: baoyiqun@njau.edu.cn) Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 Exocyst subunit SEC3A marks the germination site and is essential for pollen germination in Arabidopsis thaliana For example, BUP encodes a novel Golgi-located glycosyltransferase, the bup is affected during pollen germination and pollen tube growth by affecting pectin synthesis or delivery7. Pollen grains from homozy- gous plants mutated in PECTIN METHYLESTERASE48 gene developed multiple germinating sites due to the presence of more abundant highly methylesterified pectin in the intine wall10. A T-DNA insertion line mutated in the Callose Synthase 9 gene produced pollen grains able to in situ precociously germinate inside the anther6, and mutations in CSLD1 and CSLD4 caused a significant reduction in cellulose deposition and an alteration of the cell wall organization leading to defective pollen germination and tube growth11. These data suggested that tight regulation of cell wall synthesis and modification is crucial for the germination site establishment and pollen tube emergence. Additionally, components involved in vesicle trafficking, an intimately related process for cell wall material delivery, have also been implicated during pollen germination12. For example, a mutation in AtSYT2, a homolog of mammalian Synaptotagmins implicated in regulating membrane fusion during exo/endocytosis, affected pollen germination and pollen tube elongation13–15. Pollen-specific GNL2 was shown to be essential for pollen germination and pollen tube tip growth based on its necessary role in polar recycling. GNL2 is localized to the germination site and pollen tube tip, and absolutely no pollen germination was observed in gnl2 mutants16,17. Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 1 www.nature.com/scientificreports/ Furthermore, proteins involved in intracellular signaling18, and dynamic actin regulation3,4 have also been shown to play a role during pollen germination.t p y g p g Vesicle tethering is required after vesicle delivery but preceding the SNARE-mediated docking/fusion steps at the target membrane19. The exocyst, composed of SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84 subunits, is an evolutionally conserved octameric protein complex that tethers secretory vesicles to specific domains of the plasma membrane20. Spatial regulation of membrane trafficking by the exocyst complex is fun- damental to epithelial cell polarization, neuronal synaptogenesis and the polar growth of budding yeast21, which suggested a role of exocyst in polar exocytosis. Several Arabidopsis exocyst subunits have been implicated in biological processes that rely on regulated vesicle trafficking. For example, mutations in SEC5, SEC6, SEC8, and SEC15A dramatically reduced pollen germination and pollen tube growth which led to a male-specific transmis- sion defect22,23. Mutations in SEC8 and EXO70A1 locus reduced pectin deposition in the seed coat24. Results Th The expression pattern of SEC3A and SEC3B genes. The time and location of gene expression often implies its function. To investigate the expression pattern of SEC3A in detail, a 1024 bp promoter region upstream from the ATG start codon was fused with a GUS reporter gene and introduced into wild type Arabidopsis plants. Fifteen independent pSEC3A:GUS transgenic plants were obtained and six of them were chosen randomly and examined further. Strong GUS activities were detected in flowers (Fig. 1A), mature pollen (Fig. 1B) and pollen tubes (Fig. 1C). Notably, SEC3A started its expression only in pollen from the bicellular stage (Fig. 1D,F), which corresponded to the stage 11 flower onwards29 (Fig. 1E,F). In addition, SEC3A expression was detected in seed- lings, root columella cells, vascular bundles of root maturation zone, and embryos (Supplementary Fig. S1), sug- gesting a role of SEC3A in sporophytic development as well. In Arabidopsis, SEC3B shares 96.6% sequence identity to SEC3A in the coding region, and the two genes are arranged in tandem. It is therefore very important to explore the expression pattern of SEC3B to predict whether they are functionally redundant in a particular tissue. In four pSEC3B:GUS lines obtained, the GUS staining of SEC3B looked much weaker than that of SEC3A which is consistent with the RT-PCR results generated with gene-specific primers (Supplementary Fig. S2, Supplementary Table S1). SEC3B expression was not detected in the pollen and pollen tube, although it was noticed in tissues such as the cotyledon, the root vascular bundles, and the mature leaves (Supplementary Fig. S3). Furthermore, homozygotes obtained from the progeny of two sec3b/ SEC3B mutants (SALK_071060, SALK_124458) showed no observable phenotype. These results indicate that SEC3A is the major SEC3 paralog in pollen. A mutation in SEC3A gene causes male sterility. SEC3A gene contains 25 exons and 24 introns. One T-DNA line of the SEC3A gene (sec3a/SEC3A, GK_652H12) was obtained where the insertion was in the last exon (Fig. 1G). PCR-based genotyping revealed that no homozygous sec3a mutant plants could be identified (n > 180) (Fig. 1H). The progeny from the self-pollinated sec3a/SEC3A plants segregated in a ratio of roughly 1:1 (n = 243) instead of the expected 3:1 (Table 1), indicating a disorder in gametophytic transmission rather than a zygotic lethality of the mutation. Exocyst subunit SEC3A marks the germination site and is essential for pollen germination in Arabidopsis thaliana exo70a1, exo84b, and pollen rescued sec6 mutants (PRsec6) display cytokinesis defects25,26. In maize roothairless1 where SEC3 was mutated, root hairs could not elongate properly27. g p p y Previous investigations of the sec3a mutation (SALK_145185) indicated that the deletion of SEC3A gene resulted in embryo-lethality. The authors showed that in the interphase cells, SEC3A-GFP is present in the cytosol and at the plasma membrane where it accumulates as immobile punctate structures over the cell surface of the root hairs and the root epidermal cells, furthermore its recruitment to the plasma membrane is not mediated by the conventional secretory pathway28. y y In this report, with another T-DNA insertion mutant of the SEC3A gene (GK_652H12), we provided genetic, molecular, and cellular evidence that SEC3A is crucial for male gametophytic transmission. The mutation in SEC3A gene led to the lack of pollen germination along with perturbations in the deposition of cell wall material. SEC3A-GFP fusion protein was found to accumulate at the future site of pollen germination, which is independ- ent on PI-4,5P2 content. These results suggest that the polar localization of SEC3A at the bulge of the germinating pollen grain is essential for pollen germination. Results Th GKLB, P1, P2 are the primers used in the PCR assays. (H) Genotyping of sec3a/SEC3A by PCR analysis. TUB8 was used as an internal control. Bars = 0.2 mm for (A,F), 10 μm for (B,C), 0.4 mm for (D), and 5 μm for (E). Figure 1. SEC3A expression pattern in pollen and the genotyping of sec3a/SEC3A mutant. (A) The flower, (B) Mature pollen grains, and (C) Pollen tube showed strong SEC3A expression. (D) Flowers at stage 9, 11, 12 were used for anther dissection. (E) DAPI staining showed that pollen from stage 9, 11, 12 flowers was at unicellular, bicellular, and the tricellular stages, respectively. (F) GUS staining of anthers from flowers of (D). (G) A schematic representation of SEC3A transcript and the site of T-DNA insertion. The T-DNA insert is located in the last Exon. The black boxes represent exons, lines in between represent introns. GKLB, P1, P2 are the primers used in the PCR assays. (H) Genotyping of sec3a/SEC3A by PCR analysis. TUB8 was used as an internal control. Bars = 0.2 mm for (A,F), 10 μm for (B,C), 0.4 mm for (D), and 5 μm for (E). Figure 1. SEC3A expression pattern in pollen and the genotyping of sec3a/SEC3A mutant. (A) The flower, (B) Mature pollen grains, and (C) Pollen tube showed strong SEC3A expression. (D) Flowers at stage 9, 11, 12 were used for anther dissection. (E) DAPI staining showed that pollen from stage 9, 11, 12 flowers was at unicellular, bicellular, and the tricellular stages, respectively. (F) GUS staining of anthers from flowers of (D). (G) A schematic representation of SEC3A transcript and the site of T-DNA insertion. The T-DNA insert is located in the last Exon. The black boxes represent exons, lines in between represent introns. GKLB, P1, P2 are the primers used in the PCR assays. (H) Genotyping of sec3a/SEC3A by PCR analysis. TUB8 was used as an internal control. Bars = 0.2 mm for (A,F), 10 μm for (B,C), 0.4 mm for (D), and 5 μm for (E). Pollen germination in vitro and in vivo were carried out with quartets of sec3a/SEC3A qrt1/qrt1 (Fig. 2). Due to the 2:2 ratio of the wild type versus mutant within a quartet from a heterozygote, germination of three or four pollen grains in the quartet requires the germination of one or both mutant pollen grains. Results Th To investigate if the mutation in SEC3A affected male or female gametophytic development, the sec3a/SEC3A plants were used as male or female donors to cross with the wild type. When the sec3a/SEC3A plants were crossed as female parents, approximately 49% (n = 271) of the resulting F1 progenies were heterozygotes as expected for normal transmission. In contrast, when pollen grains from the sec3a/SEC3A were pollinated to wild type plants, none (n = 224) of the F1 seedlings were heterozygous plants (Table 1). These results indicated that genetic transmission of sec3a mutation through the male was abolished in the mutant, while female gametophytic transmission was normal. The sec3a mutant is defective during pollen germination. Male gametophytic lethal might be due to two possible defects. One is that haploid cells inherited the mutant allele after meiosis do not develop into mature pollen, or they developed normally, but are defective during pollen germination and/or tube growth, or later stages of double fertilization. To address these two possibilities, sec3a was introgressed into the quartet 1 (qrt1) mutant background where four microspores from a microsporocyte fail to separate after meiosis, but their functions are virtually unaffected30,31. Within a quartet, two microspores are mutant (sec3a) and the other two are wild type (SEC3A). Pollen grain development was examined using the quartets from sec3a/SEC3A qrt1/qrt1 plants. No difference in nuclear composition was observed in quartets at the unicellular, bicellular and tricellular stages using DAPI staining (Fig. 2A–C). In addition, sec3a pollen grains were morphologically comparable to wild type under the scanning electron microscope (Fig. 2D,E), and they are viable as revealed by the Alexander stain- ing (Fig. 2F,G). Together, these results indicated that the mutation in SEC3A did not affect pollen development. Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 2 www.nature.com/scientificreports/ ntificreports/ Figure 1. SEC3A expression pattern in pollen and the genotyping of sec3a/SEC3A mutant. (A) The flower, (B) Mature pollen grains, and (C) Pollen tube showed strong SEC3A expression. (D) Flowers at stage 9, 11, 12 were used for anther dissection. (E) DAPI staining showed that pollen from stage 9, 11, 12 flowers was at unicellular, bicellular, and the tricellular stages, respectively. (F) GUS staining of anthers from flowers of (D). (G) A schematic representation of SEC3A transcript and the site of T-DNA insertion. The T-DNA insert is located in the last Exon. The black boxes represent exons, lines in between represent introns. Results Th Therefore, a relative low percentage of quartets with three or four grains germinated would indicate a pollen germination defect. When cultured on a solid medium, approximately 31.7% (n = 600) of quartets from qrt1/qrt1 plants had three or four pollen grains germinated (Fig. 2H,J), while no quartet from sec3a/SEC3A qrt1/qrt1 (n = 600) plants could do the same (Fig. 2I,J). In vivo pollination assay was carried out using male-sterile plants (ms1) as pollen recipient to avoid the potentially complicating effects of stigma maturity and emasculation stresses32. While approximately 47% (n = 108) of qrt1/qrt1 quartet could germinate three or four pollen tubes into the pistil as revealed by aniline blue staining (Fig. 2K,L), none of the sec3a/SEC3A qrt1/qrt1 (n = 60) quartets was able to do that (Fig. 2M,N). These results suggested that sec3a mutation significantly inhibited pollen germination in vitro and in vivo. Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 3 www.nature.com/scientificreports/ No. of Progeny Genotypes of Progeny χ2 P SEC3A/SEC3A sec3a/SEC3A sec3a/sec3a Self-cross 25 (%) 50 (%) 25 (%) Expected 243 48 52 0 125.7 <0.001a Out-cross (♀ × ♂) 50 (%) 50 (%) 0 (%) Expected WT × sec3a/SEC3A 224 100 0 0 224 <0.001a sec3a/SEC3A × WT 271 51 49 0 0.1328 NS Table 1. Genotyping of the sec3a/SEC3A mutant. P, values were calculated using the χ2 test. aSignificant difference between the observed and the expected ratios. NS, Not significantly different. No. of Progeny Genotypes of Progeny χ2 P SEC3A/SEC3A sec3a/SEC3A sec3a/sec3a Self-cross 25 (%) 50 (%) 25 (%) Expected 243 48 52 0 125.7 <0.001a Out-cross (♀ × ♂) 50 (%) 50 (%) 0 (%) Expected WT × sec3a/SEC3A 224 100 0 0 224 <0.001a sec3a/SEC3A × WT 271 51 49 0 0.1328 NS Table 1. Genotyping of the sec3a/SEC3A mutant. P, values were calculated using the χ2 test. aSignificant difference between the observed and the expected ratios. NS, Not significantly different. Table 1. Genotyping of the sec3a/SEC3A mutant. P, values were calculated using the χ2 test. aSignificant difference between the observed and the expected ratios. NS, Not significantly different. Table 1. Genotyping of the sec3a/SEC3A mutant. P, values were calculated using the χ2 test. aSignificant difference between the observed and the expected ratios. NS, Not significantly different. Phenotype of sec3a/SEC3A is rescued by pLAT52:SEC3A, pLAT52:SEC3A-GFP and gSEC3A transgenes, respectively. Results Th To demonstrate that the mutant phenotype is caused by the disruption of SEC3A gene, sec3a/SEC3A plants were transformed with SEC3A coding sequence driven by the pollen-specific LAT52 promoter33 in a vector that confers resistance to hygromycin. Transgenic plants hemizygous for pLAT52:SEC3A (20 lines) and pLAT52:SEC3A-GFP (12 lines) loci in sec3a/SEC3A mutant were obtained, and several lines of each genotype were chosen randomly for further characterization. The male transmission defects were com- plemented, with sec3a/sec3a, sec3a/SEC3A and wild type progeny appearing at the expected Mendelian ratio (Table 2). Moreover, sec3a homozygotes, named as PRsec3a (Pollen-Rescued sec3a: sec3a/sec3a pLAT52:SEC3A/ pLAT52:SEC3A), were identified using PCR-based genotyping with progeny lines in which all seedlings showed hygromycin resistant (pLAT52:SEC3A transgene selection marker) (Fig. 3A). RT-PCR analysis of PRsec3a mutants indicated that the transcripts spanning (P5 + P7) or behind (P6 + P7) the T-DNA insertion sites were not expressed, while the transcript (P3 + P4) before the insertion was detected (Figs 1A and 3B). However, it should be unstable due to the lack of Poly(A) tail. Similarly, PRsec3a-GFP lines (sec3a/sec3a pLAT52:SEC3A-GFP/ pLAT52:SEC3A-GFP) were obtained (Table 2, Supplementary Fig. S4). Furthermore, twelve sec3a/SEC3A lines bearing hemizygous SEC3A genomic DNA were generated (Supplementary Fig. S4). In two randomly selected lines, the male transmission efficiency increased to approximately 2:1, indicating a complete complementation (Supplementary Table S2). pp y In vitro pollen germination of two randomly selected PRsec3a lines were evaluated on the solid media. Under the same conditions, 78% of wild type pollen grains (n = 672) and 41% (n = 638) of that from sec3a/SEC3A plants were able to germinate (Fig. 3C,D). Remarkably, in PRsec3a mutant, the ratio was restored to that of the wild type (73%, n = 683) (Fig. 3C,D). These date demonstrated that the male gametophytic defects were indeed due to T-DNA insertion in the SEC3A gene. SEC3A-GFP decorates the pollen germination site, and the localization is independent on PI-4, 5P2. To analyze the role of SEC3A more precisely, the dynamic localization of SEC3A in the germinating pol- len grains were explored. Our studies showed that the male transmission was restored to normal in sec3a/ SEC3A mutant with hemizygous pLAT52:SEC3A-GFP (Table 2), and PRsec3a-GFP lines could be identified (Supplementary Fig. S4). These results indicated that the SEC3A-GFP protein is functional in pollen, and there- fore should display its correct subcellular localization. Results Th In pLAT52:SEC3A-GFP transgenic plants, SEC3A proteins appeared from the binucleated pollen stage onwards (Supplementary Fig. S5), consistent with the GUS analy- sis results (Fig. 1). In mature pollen prior to activation, SEC3A-GFP was dispersed in the cytoplasm (Fig. 4A). Interestingly, after being placed in liquid germination medium for 30 min, SEC3A-GFP was shift to the cortex of the future germination site before any visible change of pollen morphology was noticed (Fig. 4B, Supplementary Video. S1), and the signals oscillated at this position throughout the tube emerging process (Supplementary Videos S1 and S2). ) In yeast, Sec3p directly interacted with PI-4,5P2 and marked the exocytic site at the bud34. To test whether SEC3A could bind to PIPs directly in vitro, SEC3A was fused to HIS and expressed in E. Coli. Purified proteins were used in PIPs strip overlay assays. As shown in Fig. 4D, SEC3A bound to several membrane phosphoinositides including PI-4,5P2, while the control with BSA alone could not (Fig. 4D). In plants, PI-4,5P2 could be generated through the phosphorylation of phosphatidylinositol-4-phosphate (PI4P) by phosphatidylinositol-4-phosphate 5-kinases (PIP5K)35,36. In pip5k4 mutant, the production of PI-4,5P2 and the membrane recycling was decreased in pollen tubes37,38. pLAT52:SEC3A-GFP was introduced into the pip5k4 homozygous mutant (SALK_001138, Supplementary Fig. S6), and its localization was examined. Interestingly, SEC3A proteins still accumulated at the bulge of the germinating pip5k4 pollen grain with bright fluorescent labeling (Fig. 4C), just as in the wild type (Fig. 4B), indicating that the localization of SEC3A in pollen was not dependent on PI-4,5P2. Polar accumulation of cell wall material was not observed in the germinating sec3a pollen grains. Pollen germination started with hydration, which might act as a triggering signal. Following hydration, plaque formation starts and is completed within 1 h, by then the pollen tube has emerged39. Polar deposition of callose, cellulose and pectin is observed in the incipient bulge of germinating pollen grains5,7,40. The phenotypes of sec3 mutant and the localization pattern of SEC3A prompted us to look into the cell wall deposition at the germination site. Pollen grains from sec3a mutant hemizygous for pLAT52:SEC3A-GFP were used since mutant pollen (sec3a) can easily distinguished from the complemented one (sec3a pLAT52:SEC3A-GFP) by the absence or presence of the GFP signal. Calcofluor white which labels cellulose and callose41, appeared at the germination site in the complemented pollen grain, colocalized with the SEC3A-GFP signals (Fig. 5A). Results Th Similarly, antibodies against Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 4 www.nature.com/scientificreports/ Figure 2. sec3a pollen is defective during pollen germination. Quartets from sec3a/SEC3A qrt1/qrt1 plants was stained with DAPI at the unicellular (A), bicellular (B) and tricellular (C) stages. Scanning electron microscopy (SEM) analysis of qrt1/qrt1 (D) and sec3a/SEC3A qrt1/qrt1 quartets (E). Alexander staining of quartets from qrt1/qrt1 (F) and sec3a/SEC3A qrt1/qrt1 (G). No abnormality was observed for sec3a/SEC3A qrt1/qrt1 quartets in above assays. In vitro germination of quartets of qrt1/qrt1 (H) and sec3a/SEC3A qrt1/qrt1 (I) plants on solid germination medium. (J) Statistic analysis of qrt1/qrt1 and sec3a/SEC3A qrt1/qrt1 quartets showing 0, 1 + 2, 3 + 4 pollen tube(s) 6 h after germination. Values represent the means ± SD. Means P < 0.05 by Student’s t test (n = 600 pollen grains for each genotype). In vivo germination of pollen grains from qrt1/ qrt1 (K,L) and sec3a/SEC3A qrt1/qrt1 (M,N) quartets. (L,N) are corresponding bright-field images of (K,M), respectively. Arrowheads in (I) indicated non-germinated pollen grains. Arrows in (K,M) indicated pollen tubes. Bars = 10 μm for (A–G), 20 μm for (H,I,K,M). Figure 2. sec3a pollen is defective during pollen germination. Quartets from sec3a/SEC3A qrt1/qrt1 plants was stained with DAPI at the unicellular (A), bicellular (B) and tricellular (C) stages. Scanning electron microscopy (SEM) analysis of qrt1/qrt1 (D) and sec3a/SEC3A qrt1/qrt1 quartets (E). Alexander staining of quartets from qrt1/qrt1 (F) and sec3a/SEC3A qrt1/qrt1 (G). No abnormality was observed for sec3a/SEC3A qrt1/qrt1 quartets in above assays. In vitro germination of quartets of qrt1/qrt1 (H) and sec3a/SEC3A qrt1/qrt1 (I) plants on solid germination medium. (J) Statistic analysis of qrt1/qrt1 and sec3a/SEC3A qrt1/qrt1 quartets showing 0, 1 + 2, 3 + 4 pollen tube(s) 6 h after germination. Values represent the means ± SD. Means P < 0.05 by Student’s t test (n = 600 pollen grains for each genotype). In vivo germination of pollen grains from qrt1/ qrt1 (K,L) and sec3a/SEC3A qrt1/qrt1 (M,N) quartets. (L,N) are corresponding bright-field images of (K,M), respectively. Arrowheads in (I) indicated non-germinated pollen grains. Arrows in (K,M) indicated pollen tubes. Bars = 10 μm for (A–G), 20 μm for (H,I,K,M). Complemented T1 No. Results Th of Progeny Genotypes of Progeny χ2 P SEC3A/SEC3A sec3a/SEC3A sec3a/sec3a 33.3 (%) 50 (%) 16.7 (%) Expected pLAT52:SEC3A 1# 249 35.3 42.1 22.6 8.42 0.0148 pLAT52:SEC3A 2# 194 29.4 54.1 16.5 1.56 NS pLAT52:SEC3A 3# 201 27.9 48.8 23.3 7.31 0.0258 pLAT52:SEC3A-GFP 1# 204 37.7 50.5 11.8 2.34 NS pLAT52:SEC3A-GFP 2# 166 32.5 52.4 15 0.08 NS Table 2. Complementation analysis of sec3a/SEC3A mutants. pLAT52:SEC3A represents pLAT52:SEC3A hemizygous transgene in sec3a/SEC3A mutant background. pLAT52:SEC3A-GFP represents pLAT52:SEC3A- GFP hemizygous transgene in sec3a/SEC3A mutant background. P, values were calculated using the χ2 test. NS NS, Not significantly different. Complemented T1 No. of Progeny Genotypes of Progeny χ2 P SEC3A/SEC3A sec3a/SEC3A sec3a/sec3a 33.3 (%) 50 (%) 16.7 (%) Expected pLAT52:SEC3A 1# 249 35.3 42.1 22.6 8.42 0.0148 pLAT52:SEC3A 2# 194 29.4 54.1 16.5 1.56 NS pLAT52:SEC3A 3# 201 27.9 48.8 23.3 7.31 0.0258 pLAT52:SEC3A-GFP 1# 204 37.7 50.5 11.8 2.34 NS pLAT52:SEC3A-GFP 2# 166 32.5 52.4 15 0.08 NS Table 2. Complementation analysis of sec3a/SEC3A mutants. pLAT52:SEC3A represents pLAT52:SEC3A hemizygous transgene in sec3a/SEC3A mutant background. pLAT52:SEC3A-GFP represents pLAT52:SEC3A- GFP hemizygous transgene in sec3a/SEC3A mutant background. P, values were calculated using the χ2 test. NS NS, Not significantly different. callose (anti-callose, Fig. 5C), low methylestified pectins (JIM 5, Fig. 5E), and high methylestified pectins (JIM7, Fig. 5G) labeled the germination plaques in the complemented pollen grains, respectively. In contrast, mutant pollen exhibited an even staining of the cell wall with no sign of germination (Fig. 5B,D,F,H). In addition, qrt1/ qrt1 and sec3a/SEC3A qrt1/qrt1 quartets were stained with ruthenium red, which labels a broad range of methy- lesterified pectins. Ruthenium red strongly labeled the germination plaques of all four pollen grains of qrt1/qrt1 (Fig. 5I) while only two pollen grains of sec3a/SEC3A qrt1/qrt1 quartet could be labeled (Fig. 5J). Even after 6 h of germination when a quartet from qrt1/qrt1 plant produced four pollen tubes (Fig. 5K), sec3a/SEC3A qrt1/qrt1 quartet still had two non-germinated pollen grains that show no sign of polar pectin deposition (Fig. 5L). Thus pectin distribution revealed by ruthenium red is consistent with the JIM5 and JIM7 staining (Fig. 5E,F,G,H). Together, these data demonstrated that polar accumulation of several types of cell wall materials was not observed in the germinating sec3a pollen. Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 5 ntificreports/ Figure 3. Complementation of sec3a mutants with pLAT52:SEC3A transgene. (A) Genotyping of PRsec3a. Results Th (B) RNAs were extracted from true leaves, and P3 + P4, P5 + P7 or P6 + P7 primer pairs were used to analyze different SEC3A transcripts. (C) In vitro germination of pollen grains from wild type, sec3a/SEC3A, and PRsec3a plants after 6 h on the solid medium. Arrowheads indicated ungerminated pollen. (D) Statistic analysis showed that pLAT52:SEC3A transgene restored in vitro germination rate of sec3a/SEC3A pollen to normal. Values represent the means ± SD. Means P < 0.05 by Student’s t test. Bars = 20 μm. www.nature.com/scientificreports/ Figure 3. Complementation of sec3a mutants with pLAT52:SEC3A transgene. (A) Genotyping of PRsec3a. (B) RNAs were extracted from true leaves, and P3 + P4, P5 + P7 or P6 + P7 primer pairs were used to analyze different SEC3A transcripts. (C) In vitro germination of pollen grains from wild type, sec3a/SEC3A, and PRsec3a plants after 6 h on the solid medium. Arrowheads indicated ungerminated pollen. (D) Statistic analysis showed that pLAT52:SEC3A transgene restored in vitro germination rate of sec3a/SEC3A pollen to normal. Values represent the means ± SD. Means P < 0.05 by Student’s t test. Bars = 20 μm. Figure 3. Complementation of sec3a mutants with pLAT52:SEC3A transgene. (A) Genotyping of PRsec3a. (B) RNAs were extracted from true leaves, and P3 + P4, P5 + P7 or P6 + P7 primer pairs were used to analyze different SEC3A transcripts. (C) In vitro germination of pollen grains from wild type, sec3a/SEC3A, and PRsec3a plants after 6 h on the solid medium. Arrowheads indicated ungerminated pollen. (D) Statistic analysis showed that pLAT52:SEC3A transgene restored in vitro germination rate of sec3a/SEC3A pollen to normal. Values represent the means ± SD. *Means P < 0.05 by Student’s t test. Bars = 20 μm. Discussion (I–L) Ruthenium red staining of tetrads from qrt1/qrt1 (I,K) and sec3a/SEC3A qrt1/qrt1 (J,L) plants. Bars = 10 μm. Figure 5. Accumulation of Cell wall materials are not observed in sec3a germinating pollen. (A,B) Calcofluor white staining. (C,D) Pollen grains labeled with anti-callose monoclonal antibody. (E,F) Pollen grains labeled with JIM5 monoclonal antibody. (G,H) Pollen grains labeled with JIM7 monoclonal antibody. sec3a pLAT52:SEC3A-GFP (A,C,E,G) and sec3a (B,D,F,H) pollen were used for the assays. (I–L) Ruthenium red staining of tetrads from qrt1/qrt1 (I,K) and sec3a/SEC3A qrt1/qrt1 (J,L) plants. Bars = 10 μm. Figure 5. Accumulation of Cell wall materials are not observed in sec3a germinating pollen. (A,B) Calcofluor white staining. (C,D) Pollen grains labeled with anti-callose monoclonal antibody. (E,F) Pollen grains labeled with JIM5 monoclonal antibody. (G,H) Pollen grains labeled with JIM7 monoclonal antibody. sec3a pLAT52:SEC3A-GFP (A,C,E,G) and sec3a (B,D,F,H) pollen were used for the assays. (I–L) Ruthenium red staining of tetrads from qrt1/qrt1 (I,K) and sec3a/SEC3A qrt1/qrt1 (J,L) plants. Bars = 10 μm. zero with four pollen germinated in an in vitro assay; Among 73 sec3a heterozygous tetrads, only one with three pollen, and zero with four pollen germinated in an in vivo assay. Therefore, we believed that pollen germination defect, rather than pollen tube growth, is the major physiological aberrance revealed by the sec3a/SEC3A mutant, and our data is consistent with the report43. Given the fact that mutations in SEC6, SEC8, SEC15A, and SEC5 dramatically affect pollen germination (1% to 7% germination rate in respective mutants) as well22,23, it is most likely that SEC3A acts together with other exocyst subunits, but as a more crucial player, in tethering secretory vesicles containing newly synthesized pectin, callose or cellulose synthase to establish and consolidate the ger- mination aperture. It is therefore interesting to explore in the future the dynamic localization of other exocyst subunits at the germination aperture. However, other possibilities cannot be totally excluded. For example, the failure of pollen germination in sec3a/SEC3A mutant might be caused by defects in synthesis of some cell wall material or by an abnormal rapid degradation of cell wall materials. SEC3A appeared in a dynamic cone-shaped area and at the extreme plasma membrane at the tip of elongating pollen tube (Supplementary Video S3 and Supplementary Fig. S8) confirming recent findings43 and implying a role of SEC3A in pollen tube growth as well. Discussion In this report, we demonstrated that a mutation in SEC3A led to the failure of pollen germination with no polar accumulation of cell wall materials. SEC3A is expressed in pollen and pollen tube, and SEC3A marked the pollen germination site. Thus, SEC3A is a key player during pollen germination, and its recruitment to the germination site is not dependent on PI4,5-P2. p 2 Mutations in distinct exocyst subunit caused polar growth defects in processes such as root hair elongation, hypocotyl elongation, and pollen tube growth22,23,27,42. In this study, we found that in sec3a mutant (GK_652H12), germination of the pollen grains was hindered, resulting in an absolute male-specific transmission defect which could be rescued by pLAT52:SEC3A, pLAT52:SEC3A-GFP or genomic SEC3A transgene (Figs 2 and 3, Table 2, Supplementary Fig. S4, Supplementary Table S2). The sec3a pollen grains developed normally, however, no pollen germination was observed in in vitro and in vivo germination assays (Fig. 2), let alone pollen tube growth. In a recent paper43, the authors showed that among 1769 sec3a heterozygous tetrads, only one with three pollen, and Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 6 www.nature.com/scientificreports/ Figure 4. SEC3A proteins marks the pollen germination site, and this localization is independ PI4, 5-P2. pLAT52:SEC3A-GFP was transformed into wild type (Col-0) (A,B), and pip5k4 mutant (C), respectively, and pollen grains were collected and germinated in liquid medium. (A) SEC3A-G localization before pollen germination. (B) SEC3A-GFP localization during pollen germination. ( GFP localization during pollen germination in pip5k4 mutant background. (D) SEC3A protein lip binding assay. Bars = 10 μm. Figure 4. SEC3A proteins marks the pollen germination site, and this localization is independent on PI4, 5-P2. pLAT52:SEC3A-GFP was transformed into wild type (Col-0) (A,B), and pip5k4 mutant plants (C), respectively, and pollen grains were collected and germinated in liquid medium. (A) SEC3A-GFP localization before pollen germination. (B) SEC3A-GFP localization during pollen germination. (C) SEC3A- GFP localization during pollen germination in pip5k4 mutant background. (D) SEC3A protein lipid overlay binding assay. Bars = 10 μm. Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Accumulation of Cell wall materials are not observed in sec3a germinating pollen. (A,B) Calcofluor white staining. (C,D) Pollen grains labeled with anti-callose monoclonal antibody. (E,F) Pollen grains labeled with JIM5 monoclonal antibody. (G,H) Pollen grains labeled with JIM7 monoclonal antibody. sec3a pLAT52:SEC3A-GFP (A,C,E,G) and sec3a (B,D,F,H) pollen were used for the assays. Discussion This localization is however in contrast with an earlier report of SEC3A being localized to immobile puncta at the plasma membrane of root hairs28.h p The expression pattern of SEC3A (Supplementary Fig. S1) is suggestive of a possible role of SEC3A in embryo development. Previous investigations of sec3a mutant line (SALK_145185) indicated that disruption of the SEC3A gene caused embryo lethality28. However, in our hand, PCR-based genotyping (Supplementary Table S1) of the SALK_145185 line did not yield any T-DNA specific band. Therefore we could not confirm the reported embryo lethality of this particular line. PRsec3a generated in our study represented sporophytic sec3a homozygotes according to the genotyping results (Fig. 3), but its embryo development was normal (Supplementary Fig. S7). Our results was supported by the recent report where sec3a mutant was shown to have a male transmission defect43. How pollen germination site is established and pollen tube elongation initiated are not well known. In S. cerevisiae, bud tip localized Sec3p is thought to be a spatial landmark for polarized exocytosis and for the 8 Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 www.nature.com/scientificreports/ recruitment of other exocyst subunits to the exocytic sites44,45. The polarized localization of Sec3p is depended on its interaction with PI4,5-P2 and the Rho family of small GTPases34,46,47. In this study, SEC3A has been shown to be recruited from the cytosol to the cell cortex which marks the future germination site (Fig. 4, Supplementary Videos S1 and S2). There are two scenarios under which SEC3A could be recruited to this particular site. In plant, Rac/Rop small GTPases accumulate specifically at the plasma membrane of the tip of elongating pollen tubes and are key regulators of polar cell expansion48. Interestingly, Rop1 GTPase effector ICR1/RIP1, an interacting protein of SEC3A49, has been shown to localize and oscillate at the germination site50 in a similar manner to that of SEC3A (Supplementary Videos S1 and S2). Moreover, the icr1 mutant were reported to be partially male sterile, although the details of pollen abnormality remained to be studied49. Under this scenario, ROP GTPase might be involved in the recruitment of exocyst to the site of pollen germination through the interaction between SEC3A and ICR1. PI4,5-P2 has been shown to accumulate exclusively at the apex of elongating pollen tube to control polar secretion by modulating actin organization and membrane traffic51. Materials and Methods Plant materials and growth conditions. Arabidopsis thaliana Columbia ecotype (Col-0) was used as the wild type. T-DNA insertion lines of SEC3A (GK_652H12) and PIP5K4 (SALK_001138) were obtained from the Nottingham Arabidopsis Stock Center (NASC) and Arabidopsis Biological Resource Center (ABRC), respec- tively. Seeds were surface sterilized and plated on half-strength Murashige and Skoog medium with 0.8% agar, imbibed at 4 °C for 3 days, and then placed in a growth chamber at 22 °C with a 16-h-light/8-h-dark cycle. Seven- day-old seedlings were then transferred to soil and maintained under the same condition. GUS staining. For GUS staining, different tissues of transgenic Arabidopsis plants were vacuum infil- trated for 15 min in GUS staining solution of 100 mM phosphate buffer (pH 7.0), 0.1% Triton X-100, 0.5 mM [K3Fe(CN)6], 0.5 mM [K4Fe(CN)6], 10 mM EDTA and 0.5 mg ml−1 bromochloroindoyl-β-glucuronide (X-Gluc). Samples were then incubated overnight at 37 °C and cleared in acetic acid:ethanol (1:3 v/v). Phenotypic analysis of mutants. To determine different pollen development stages, pollen grains were stained in a DAPI solution (0.1 M phosphate buffer solution pH 7.0, 1 mM EDTA, 0.1% Triton X-100 and 1 μg ml−1 DAPI) for 15 min before observation. The viability of pollen grains was assessed using Alexander staining52. For SEM, mature pollen grains were coated directly with gold particles (EIKO IB-3) and observed on HITACHI S-3000N scanning electron microscope. g p In vitro pollen germination was conducted essentially according to described previously53. Pollen harvested from newly fully opened flowers was placed onto pollen germination medium (PGM) consisting of 1 mM CaCl2, 1 mM Ca(NO3)2, 1 mM MgSO4, 0.01% (w/v) H3BO3, 18% (w/v) sucrose, pH 7.0, which was solidified with 0.8% (w/v) agar, and grown in a growth chamber in the dark at 22 °C. g g g For in vivo pollen germination assay, mature pistils of the male-sterile mutant ms132 were pollinated with a limited number (3–5) of quartets from sec3a/SEC3A qrt1/qrt1 and qrt1/qrt1 plants. The pollen tubes in the pistils were stained with aniline blue and viewed with a confocal microscope Zeiss LSM710 system. Complementation experiments. For the pollen-rescue experiment, the LAT52 promoter and the coding sequence (CDS) of SEC3A were amplified by PCR using the primer pairs LAT52-S (SacI)/LAT52-A (KpnI) and SEC3A1-S (BamHI)/SEC3A1-A (NheI), respectively (Supplementary Table S1). The resulting DNA fragments were cloned into the pCAMBIA1300 vector (CAMBIA, http://www.cambia.org) and sequencing validated. Discussion In pip5k4 mutant where PI4,5-P2 level was reduced, but the position of SEC3A at the germination pore is unaffected (Fig. 4). Hence, the alternative scenario that SEC3A binds directly to PI4,5-P2 to achieve its polar localization is not true. Given the fact that SEC3A labeled pollen germination site (Fig. 4), mutant pollen from sec3a/SEC3A plant could not germinate, and partial complementation of sec3a resulted in multiple germination sites43, we suggest that SEC3A is required for germination site selection and/or establishment. Materials and Methods For genomic DNA complementation experiment, about 1.1 kb promoter region along with 8230 bp genomic sequence of SEC3A was PCR-amplified using the primer pair SEC3A2-S/SEC3A2-A (Supplementary Table S1). The ampli- fied fragment was cloned into the vector pCAMBIA1300221 (CAMBIA, http://www.cambia.org) using in-fusion HD cloning kit according to the manufacturer’s instructions (Clontech). The above constructs were introduced into the sec3a/SEC3A plants using the Agrobacterium (strain EHA105)-mediated infiltration method54, followed by hygromycin selection. Confocal microscopy. Confocal images of pollen grains or pollen tubes were collected by UltraView spinning-disc confocal scanner unit (Perkin Elmer). The wavelength was 488 nm for GFP excitation and 505– 530 nm for detection. The excitation and detection wavelengths for DAPI and Calcofluor white were 359 nm and 385–405 nm for excitation, 461 nm and 437–445 nm for detection, PIP strip overlay binding assay. For the PIP strip binding assays, phospholipid membranes (Echelon) were blocked with 3% bovine serum albumin (BSA) in PBS-T (0.1% v/v Tween-20) for 1 h at room temperature. After that, membranes were incubated in a buffer with or without SEC3A-HIS fusion proteins (0.5 μg ml−1) in 3% BSA/PBS-T for another hour. Unbound protein was washed away with PBST for three times, 10 min each. The membranes were then incubated with anti-HIS antibodies to detect the bound proteins. Immunofluorescence and cytochemical staining. Pollen grains were adhered to poly-L-lysine-covered glass slides after germination in liquid PGM for 30 min. Samples were fixed in 4% (w/v) polyformaldehyde in PIPES buffer (50 mM PIPES, 1 mM EGTA, 5 mM MgSO4, 0.5 mM CaCl2, 0.1% TritonX-100, pH 7) for 1 h. After washing with PBS (100 mM potassium phosphate, 138 mM NaCl, and 2.7 mM KCl, pH 7.3), samples were Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 9 www.nature.com/scientificreports/ incubated in the blocking buffer (0.8% BSA, 0.1% gelatin, and 2 mM NaN3 in PBS) at room temperature for 30 min and then incubated with the primary antibodies (1:200 diluted in blocking buffer) for 1 h. Pectins with low and high degrees of methylesterification were labeled with JIM5 and JIM755,56, respectively (Plant Probes). Callose was labeled with anti-callose57 (Biosupplies Australia Pty Ltd.). The pollen grains were washed three times with PBS and incubated for 30 min with Alexa Fluor 594 conjugated secondary antibodies (1:100 dilution in blocking buffer). The samples were washed for five times with PBS before analysis using an UltraView spinning-disc con- focal scanner unit (Perkin Elmer). References Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall. The Plant journal 15, 79–88 (1998). 31. Rhee, S. Y. & Somerville, C. R. Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persisten pectic polysaccharides of the pollen mother cell wall. The Plant journal 15, 79–88 (1998).i h 32. Zinkl, G. M., Zwiebel, B. I., Grier, D. G. & Preuss, D. 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Acknowledgementsh g This research was supported by grant 31200236 from the National Science Foundation of China (NSFC); Grants KYTZ201402 and KJQN201534 from the Fundamental Research Funds for the Central Universities in China; Grant 2014ZX0800925B from the Ministry of Agriculture of China for Transgenic Research; A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. g This research was supported by grant 31200236 from the National Science Foundation of China (NSFC); Grants KYTZ201402 and KJQN201534 from the Fundamental Research Funds for the Central Universities in China; Grant 2014ZX0800925B from the Ministr of Agriculture of China for Transgenic Research A Project Funded b g This research was supported by grant 31200236 from the National Science Foundation of China (NSFC); Grants g This research was supported by grant 31200236 from the National Science Foundation of China (NSFC); Grants KYTZ201402 and KJQN201534 from the Fundamental Research Funds for the Central Universities in China; Grant 2014ZX0800925B from the Ministry of Agriculture of China for Transgenic Research; A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Grant 2014ZX0800925B from the Ministry of Agriculture of China for Transgenic Research; A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are required for pollen germination and tube growth in Arabidopsis. Plant Cell 16, 229–240 (2004). 39. Shi.D. Q. & Yang.W. C. Pollen Germination and Tube Growth. Plant Developmental Biology – Biotechnological Perspectives 1 (2010). 40 Lalanne E et al SETH1 and SETH2 two components of the glycosylphosphatidylinositol anchor biosynthetic pathway are required 39. Shi.D. Q. & Yang.W. C. Pollen Germination and Tube Growth. Plant Developmental Biology – Biotechnological Perspectives 1 (2010). 40. Lalanne, E. et al. SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are required Q g p gy g p ( 40. Lalanne, E. et al. SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are req for pollen germination and tube growth in Arabidopsis. Plant Cell 16, 229–240 (2004). Scientific Reports \| 7:40279 \| DOI: 10.1038/srep40279 10 www.nature.com/scientificreports/ 1. Wood, P. J. & Fulcher, R. G. Dye interactions. A basis for specific detection and histochemistry of polysaccharides. The journal o histochemistry and cytochemistry 31, 823–826 (1983).i y y y 2. Synek, L. et al. AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development. The Plant journal 48, 54–72 (2006).i p g p ph j , ( ) 43. Bloch, D. et al. Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth. Plant Physiol (2016). 3. Bloch, D. et al. Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth. Plant Physio (2016). 4 Finger F P Hughes T E & Novick P Sec3p is a spatial landmark for polarized secretion in budding yeast Cell 92 559–571 (1998) Hughes, T. E. & Novick, P. Sec3p is a spatial landmark for polarized Finger, F. P., Hughes, T. E. & Novick, P. Sec3p is a spatial landmark fo 45. Boyd, C., Hughes, T., Pypaert, M. & Novick, P. Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. Journal of Cell Biology 167, 889–901 (2004). Guo, W., Tamanoi, F. & Novick, P. Spatial regulation of the exocyst c 46. Guo, W., Tamanoi, F. & Novick, P. Spatial regulation of the exocyst complex by Rho1 GTPase. Nature cell biology 3, 353–360 (2001). 47. Yamashita, M. et al. Author Contributions Y.L. and X.Y.T. performed most of the experiments. Y.L. and Y.Q.B. wrote, and revised this manuscript. M.R.W., B.X.L., Y.X.Z., C.Y.W. and Q.C.R. helped analyze data. J.X.W. and Z.Y.L. contributed to manuscript modification. All authors have participated in this research and approved the final manuscript. References Structural basis for the Rho- and phosphoinositide-dependent localization of the exocyst subunit Sec3. Nature structural & molecular biology 17, 180–186 (2010). gy , ( ) 48. Zheng, Z. L. & Yang, Z. The Rrop GTPase switch turns on polar growth in pollen. Trends Plant Sci 5, 298–303 (2000).ffi 48. Zheng, Z. L. & Yang, Z. The Rrop GTPase switch turns on polar 49. Lavy, M. et al. A Novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking. Current b CB 17, 947–952 (2007). , ( ) 50. Li, S., Gu, Y., Yan, A., Lord, E. & Yang, Z. B. RIP1 (ROP Interactive Partner 1)/ICR1 marks pollen germination sites and may a the ROP1 pathway in the control of polarized pollen growth. Molecular plant 1, 1021–1035 (2008). 51. Kost, B. et al. Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth. The Journal of cell biology 145, 317–330 (1999).f p p gh f gy 52. Alexander, M. P. Differential staining of aborted and nonaborted pollen. Stain technology 44, 117–122 (1969). l b d f d h f f bl d l d h ll b l 53. Ye, J. et al. Arabidopsis formin3 directs the formation of actin cables and polarized growth in pollen tubes. Plant Cell 21, 3868–3884 (2009).ih 54. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant journal 16, 735–743 (1998).i j 5. Willats, W. G. et al. Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls I li ti f ti th l t ti t i ti d ll dh i J Bi l Ch 276 19404 19413 (2001) 5. Willats, W. G. et al. Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls Implications for pectin methyl esterase action, matrix properties, and cell adhesion. J Biol Chem 276, 19404–19413 (2001). 56. Dardelle, F. et al. Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall. Plant Physiol 153, 1563–1576 (2010).h 57. Chebli, Y., Kaneda, M., Zerzour, R. & Geitmann, A. The cell wall of the Arabidopsis pollen tube spatial distribution, recycling, and network formation of polysaccharides. Plant Physiol 160, 1940–1955 (2012). Additional Information upplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Li, Y. et al. Exocyst subunit SEC3A marks the germination site and is essential for pollen germination in Arabidopsis thaliana. Sci. Rep. 7, 40279; doi: 10.1038/srep40279 (2017). How to cite this article: Li, Y. et al. Exocyst subunit SEC3A marks the germination site and is essential for pollen germination in Arabidopsis thaliana. Sci. Rep. 7, 40279; doi: 10.1038/srep40279 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. 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https://openalex.org/W2510025839	https://seer.ufrgs.br/index.php/EmQuestao/article/download/59514/37953	Portuguese	null	Descoberta de conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação	Em Questão	2,016	cc-by	9,684	E-ISSN 1808-5245 E-ISSN 1808-5245 E-ISSN 1808-5245 Descoberta de conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Palavras-chave: Descoberta de conhecimento. Correlação. Associação. Informações não estruturadas. Temporalidade. Descoberta de conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio Doutoranda; Universidade Federal de Santa Catarina (UFSC), Florianópolis, SC, Brasil; marinacarradore@egc.ufsc.br Thales do Nascimento da Silva Doutorando; Universidade Federal de Santa Catarina (UFSC), Florianópolis, SC, Brasil; thales788@gmail.com Alexandre Leopoldo Gonçalves Doutor; Universidade Federal de Santa Catarina (UFSC), Florianópolis, SC, Brasil; a.l.goncalves@ufsc.br Resumo: O atual momento da tecnologia vem promovendo meios para o aumento exponencial no volume de informações disponíveis na internet ou em organizações. Considerando que grande parte desta informação encontra-se em formato textual, este fato representa um desafio para as áreas de coleta, armazenamento, recuperação e análise de informações visando à explicitação de conhecimento. Este trabalho tem como objetivo apresentar um modelo para Descoberta de Conhecimento com base nas técnicas de correlação e associação temporal a partir de grandes coleções de documentos. Os procedimentos metodológicos utilizados compreenderam uma pesquisa descritiva e exploratória, envolvendo artigos coletados da base de dados Science Direct® como uma ferramenta para a coleta e a análise dos dados. Através deste tipo de informação é possível extrair regras, padrões, tendências e redes, capazes de auxiliar no processo de tomada de decisão nas organizações a fim de gerar vantagem competitiva. Como principal contribuição destaca-se a proposição de um modelo voltado ao entendimento de aspectos temporais, considerando relacionamentos factuais (através de correlações) ou não (através de associação) entre termos de um domínio. Palavras-chave: Descoberta de conhecimento. Correlação. Associação. Informações não estruturadas. Temporalidade. \| 87 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 E-ISSN 1808-5245 1 Introdução A Correlação é responsável por determinar o grau de relacionamento entre duas Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 variáveis, enquanto que a Associação se encarrega de evidenciar relacionamentos indiretos, buscando explicitar conexões potencialmente úteis entre os termos. Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 variáveis, enquanto que a Associação se encarrega de evidenciar Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação variáveis, enquanto que a Associação se encarrega de evidenciar relacionamentos indiretos, buscando explicitar conexões potencialmente úteis entre os termos. Afim de revelar padrões latentes em grandes coleções de documentos que estejam disponíveis no meio da web ou em organizações ao mesmo passo que envolvidos em um determinado período temporal, o desenvolvimento desta pesquisa se dá por meio da motivação para prover soluções para os desafios de produzir conhecimento útil ao processo de tomada de decisão pelas organizações. Metodologias, modelos, técnicas e algoritmos provenientes de diferentes áreas que promovam suporte à Descoberta de Conhecimento são fundamentais para o desenvolvimento de sistemas capazes de lidar com tais demandas. Os sistemas atuais de descoberta de conhecimento em bases de dados não estruturados aplicam métodos de correlação/associação objetivando extrair conhecimento, porém não fornecem todos os elementos dos processos de armazenamento, pré-processamento e recuperação do conteúdo textual considerando a dimensão temporal. Deste modo, o presente artigo visa propor um modelo de descoberta de conhecimento aplicado a bases de documentos textuais por meio de técnicas de correlação e associação de maneira temporal com suporte da computação distribuída. As demais seções do artigo são estruturadas de modo que a seção 2 apresente os principais referenciais teóricos envolvidos na proposição deste artigo, enquanto a seção 3 apresenta o modelo proposto e na seção 4 é explanado sobre a metodologia de pesquisa utilizada para o desenvolvimento deste trabalho. 1 Introdução A discussão dos resultados ocorre ao longo da seção 5, até que finalmente sejam detalhadas as considerações finais e os trabalhos futuros na última seção. 1 Introdução As evoluções dos meios computacionais juntamente com o aumento da capacidade de processamento, armazenamento e conectividade, estão provocando um crescimento exponencial no volume de informação (FLEUREN; ALKEMA, 2015). Pesquisas realizadas por Hilbert e López (2011) concluíram que até 2007 haviam sido produzidos 295 exabytes de informações, e segundo Wu et al. (2014) todos os dias 2,5 quintilhões de bytes de dados são criados, sendo que 90% dos dados produzidos no mundo foram gerados nos últimos anos. Estima-se que até 2020 o volume de informação, a nível mundial, cresça em 35 trilhões de gigabytes (GANTZ; REINSEL, 2010). Aproximadamente 80% destas informações se encontram em formato textual (SOMASUNDARAM; SHRIVASTAVA, 2011; RÊGO, 2013). Este cenário promove desafios quanto à coleta, armazenamento, recuperação e análise de informação não estruturada a ponto de gerar conhecimento, com o intuito de servir como uma fonte de vantagem competitiva para as organizações. Aproximadamente 80% destas informações se encontram em formato textual (SOMASUNDARAM; SHRIVASTAVA, 2011; RÊGO, 2013). Este cenário promove desafios quanto à coleta, armazenamento, recuperação e análise de informação não estruturada a ponto de gerar conhecimento, com o intuito de servir como uma fonte de vantagem competitiva para as organizações. Para lidar com tais desafios tornam-se necessários modelos, processos, metodologias, entre outros, para identificar e reaproveitar conhecimentos. Entre estes se encontra o processo de Descoberta de Conhecimento em Texto (do inglês, Knowledge Discovery in Text - KDT) entendido como uma versão da Descoberta de Conhecimento em Bases de Dados (do inglês, Knowledge Discovery in Database - KDD) voltada à manipulação de informação não estruturada. Considerando o extenso volume de documentos disposto em linguagem natural, o processo de KDT tornou-se o foco de diversos estudos (HASHIMI; HAFEZ; MATHKOUR, 2015). Este processo tem como objetivo desvendar padrões e tendências, classificando e comparando os mais variados documentos. Em razão de sua potencialidade, torna-se de suma importância o desenvolvimento de modelos embasados em técnicas que possibilitem simplificar o processo de descoberta de padrões em bases dessa natureza. Dentre as técnicas apresentadas na literatura encontram-se a Correlação e Associação. 2.1 Descoberta de conhecimento em bases de dados O processo de Descoberta de Conhecimento em Bases de Dados tem por objetivo identificar e desvendar relacionamentos implícitos entre as informações armazenadas nas bases de dados organizacionais (SILVA; ROVER, 2011). O KDD surgiu como uma alternativa para solucionar o problema da sobrecarga de dados na era da informação digital (SHABBIR et al., 2014). Para Zhu et al. (2013), o processo de KDD se constitui na análise e na exploração automática ou semiautomática de grandes volumes de dados, objetivando desvendar regras e padrões significativos. Os padrões, após descobertos, são empregados no auxílio à tomada de decisão em determinado contexto (CAO et al., 2010). Desta forma, o processo visa a descoberta de conhecimento interessante e útil (VASHISHTHA; KUMAR; RATNOO, 2012), e o KDD destina-se a facilitar e acelerar a extração de conhecimento a partir de fontes de dados persistentes (NOACK; SCHMITT, 2013). O processo de descoberta de conhecimento em bases de dados compreende as etapas de seleção dos dados, o pré-processamento que adequa os dados aos algoritmos, a mineração efetiva dos dados que compreendem o uso de técnicas geralmente baseadas na Inteligência Artificial ou Estatística (MAIA; SOUZA, 2010), a validação dos resultados e a análise e interpretação dos resultados para aquisição do conhecimento. O principal objetivo deste processo é a tradução de dados brutos em informações relevantes para posterior utilização e descoberta (ZHU et al., 2013). 2 Descoberta de conhecimento Os processos de Descoberta de Conhecimento se destinam à análise de grandes conjuntos de dados (FENG, 2010), buscando padrões que resultem em \| 89 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 conhecimento útil e que tenham surgido como uma solução fundamental para a Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Descoberta de Conhecimento a partir de informaçõe não estruturadas por meio de técnicas de correlação associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves ç Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves conhecimento útil e que tenham surgido como uma solução fundamental para a compreensão do valor real dos dados coletados, objetivando auxiliar o processo de tomada de decisão nas organizações. conhecimento útil e que tenham surgido como uma solução fundamental para a compreensão do valor real dos dados coletados, objetivando auxiliar o processo de tomada de decisão nas organizações. 2.3 Estrutura de apresentação da informação A estrutura de apresentação da informação pode ser dividida em estruturada, semiestruturada e não estruturada. A informação estruturada é representada normalmente em tabelas, gerenciadas por softwares de banco de dados (RAMOS; BRÄSCHER, 2009). A informação semiestruturada, por sua vez, é normalmente apresentada entre marcadores (tags), tais como documentos XML e HTML (CHEN et al., 2009), onde a estrutura de apresentação possibilita o entendimento por parte dos meios computacionais. Por outro lado, a informação não estruturada é disposta em linguagem natural e não segue um padrão de apresentação (LIM; LIU; LEE, 2009), ou seja, não contém estrutura tabular e nem marcação. É o caso de exemplos como e-mails, artigos, comentários em redes sociais e documentos na Web. 2.2 Descoberta de conhecimento em textos O processo de Descoberta de Conhecimento em Textos (KDT) assemelha-se ao \| 90 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação KDD, porém é voltado ao tratamento de documentos textuais (HASHIMI; HAFEZ; MATHKOUR, 2015). Tanto o KDT quanto o KDD referem-se ao processo de extração de padrões não triviais e de conhecimento útil (ZHU et al., 2013; HASHIMI; HAFEZ; MATHKOUR, 2015). Entretanto, a área que envolve o KDT torna-se mais complexa devido à falta de estruturação da informação descrita em linguagem natural (ZHU et al., 2013). KDD, porém é voltado ao tratamento de documentos textuais (HASHIMI; HAFEZ; MATHKOUR, 2015). Tanto o KDT quanto o KDD referem-se ao processo de extração de padrões não triviais e de conhecimento útil (ZHU et al., 2013; HASHIMI; HAFEZ; MATHKOUR, 2015). Entretanto, a área que envolve o KDT torna-se mais complexa devido à falta de estruturação da informação descrita em linguagem natural (ZHU et al., 2013). Documentos textuais possuem uma estrutura que necessita da aplicação de técnicas especializadas para serem analisados por sistemas computacionais, devido ao significado implícito atribuído a cada palavra na linguagem humana (SABOL et al., 2009). Segundo Hashimi, Hafez e Mathkour (2015), grande parte do conhecimento existente está disposto no formato textual, e em função deste motivo, tal conhecimento precisa ser identificado, representado e manipulado de modo a tornar-se potencialmente útil para as organizações. Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 2.4 Modelos baseados em coocorrência No processo de Descoberta de Conhecimento e considerando fontes de \| 91 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação ç Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves informação não estruturadas, torna-se necessário o emprego de técnicas para realizar a agregação da informação. Os modelos baseados em coocorrência entre termos1 permitem evidenciar a combinação destes considerando um conjunto de dados. O termo “correlação” significa literalmente “correlacionamento”, sendo possível evidenciar o grau de relacionamento entre duas variáveis. O grau de correlação entre os termos contidos nos documentos textuais pode ser representado em cálculos oriundos da estatística. A finalidade do cálculo de correlação é a determinação da força do relacionamento entre dois elementos em análise (BARALIS et al., 2013). Entre os modelos utilizados para determinar a correlação encontram-se a Frequência Conjunta, Média e Variância, Teste T (MANNING; SCHÜTZE, 1999), o Chi-square (CHURCH; MERCER, 1993), o Phi-squared (CHURCH; GALE, 1991; CONRAD; UTT, 1994) e a Informação Mútua (CHURCH; HANKS, 1990). Neste trabalho o cálculo utilizado foi o Phi-squared, que segundo Church e Gale (1991) é definido como: A aplicação do Phi-squared utiliza uma tabela 2*2 (tabela de contingência), conforme observado no Quadro 1. Quadro 1 - Tabela de contingência. Fonte: Sérgio (2013) Fonte: Sérgio (2013) Fonte: Sérgio (2013) Sendo que a representa a frequência em que os termos e ocorrem de forma conjunta, b representa as ocorrências do termo onde não há a presença de , c representa as ocorrências de sem a presença de , e d é o tamanho da coleção de documentos menos o número de documentos que não contenham e/ou , sendo d=N-a-b-c, onde N é o tamanho da base. \| 92 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação 2.5 Associação entre termos 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Cosseno definida da maneira como se segue: Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Cosseno definida da maneira como se segue: Cosseno definida da maneira como se segue: Onde wqi e wqk representam os pesos dos ith e kth termos do vetor q, e woi e woj representam os pesos dos ith e jth termos do vetor o. 2.5 Associação entre termos O tópico anterior apresenta meios para o estabelecimento de relacionamentos diretos entre termos. Ainda que isto possa promover uma visão inicial do contexto em que os termos estejam inseridos, relacionamentos diretos não são capazes de capturar a dinâmica de associação entre os termos que podem promover um entendimento mais detalhado sobre determinado domínio de análise. Neste sentido, o processo de associação é responsável por evidenciar relacionamentos indiretos, com o objetivo de explicitar conexões potencialmente úteis entre os termos. A área biomédica e da bioinformática vem provocando grandes avanços envolvendo a associação entre termos, tendo em vista revelar novos conhecimentos (WOSZEZENKI; GONÇALVES, 2013). Na base destas pesquisas encontram-se os trabalhos relativos à área de Descoberta Baseada em Literatura (DBL – do inglês Literature-Based Discovery), proposta inicialmente por um cientista norte americano, Don R. Swanson, que efetuou pesquisas envolvendo a área biomédica e a descoberta de relacionamento implícito entre padrões (SWANSON, 1986). O objetivo principal da Descoberta Baseada em Literatura é desvendar relacionamentos implícitos em bases científicas, com o intuito de originar potenciais proposições para novas descobertas (SMALHEISER, 2012). O modelo vetorial no contexto de associação indireta visa determinar o coeficiente de semelhança entre um conjunto de termos. Cada termo a ser analisado possui o seu vetor de contexto determinado pelas relações estabelecidas através de correlação. O vetor de contexto é responsável por descrever determinado termo em que cada posição é preenchida com um termo relacionado e o seu grau de correlação. Para que se obtenha a similaridade desejada são utilizadas algumas medidas, sendo que medidas como o índice Jaccard, o índice Dice, a medida Overlap (máxima e mínima), a medida do Cosseno e a medida do Pseudo-cosseno (JONES; FURNAS, 1987; EGGHE; MICHEL, 2002) recebem destaque. Neste trabalho considerou-se a equação do \| 93 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 2.6 Computação distribuída como suporte ao extenso volume de dados Entre os anos de 1945 e 1985, computadores ocupavam um grande espaço e tinham um custo elevado, e, de modo geral, estes computadores trabalhavam de forma independente devido à inexistência de uma forma de interligá-los (TANENBAUM; STEEN, 2008). Com a evolução das redes de computadores, tornou-se possível a conexão de computadores, e progressivamente a velocidade atingida nestas conexões tornava-se cada vez maior. A partir do cenário descrito e uma necessidade de maior capacidade de processamento, a computação distribuída destaca-se como uma alternativa viável a esta demanda. O conceito de “computação distribuída” pode ser definido como um sistema composto por vários componentes de hardware ou software que se comunicam, compartilham recursos e coordenam suas ações por meio da troca de mensagens (COULOURIS et al., 2013). Tanenbaum e Steen (2008) citam algumas vantagens de sistemas distribuídos quando comparados a sistemas centralizados: a) maior poder de processamento: um sistema distribuído pode ter mais capacidade de processamento em relação a servidores centralizados; b) crescimento incremental: o poder computacional pode crescer incrementalmente; c) compartilhamento de dados e recursos: tornam-se possíveis c) compartilhamento de dados e recursos: tornam-se possíveis \| 94 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 aplicações envolverem máquinas separadas geograficamente; d) maior confiabilidade: o sistema pode continuar funcionando mesmo perdendo alguns componentes; e) menor custo/benefício: os sistemas distribuídos têm melhor custo/beneficio em relação aos sistemas centralizados. Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 m Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 oi: http://dx.doi.org/10.19132/1808-5245222.87-113 3 Modelo proposto Aliada ao fácil acesso aos meios de comunicação, a rápida evolução dos meios de armazenamento levou a um veloz aumento no volume de informação. Grande parte da informação produzida encontra-se em formatos não estruturados, como textos em geral. E este tipo de informação, por não possuir uma estrutura formal, torna-se difícil de ser analisada. Com a utilização do processo de Descoberta de Conhecimento em Textos (KDT), é possível extrair conhecimento desta fonte de informação. Apesar disso, a aplicação do KDT não é trivial, principalmente devido ao grande volume e ao fator de ambiguidade existente na informação. Outro aspecto de fundamental importância é o fator temporal, característica que permite descobrir comportamentos que descrevam fatos que já ocorreram ou que podem vir a ocorrer. O fator temporal é apontado como uma limitação adicional das abordagens existentes, pois estes trabalhos tentam determinar conexões implícitas significativas, considerando a distribuição dos termos ou conceitos de um corpus em um único ponto do tempo (COHEN; SCHVANEVELDT, 2010). A detecção de associações presentes em um conjunto de análise num espaço temporal pode ser vista como a previsão de futuras ligações explícitas (COHEN; SCHVANEVELDT; WIDDOWS, 2010; YETISGEN-YILDIZ; PRATT, 2009). Mudanças no grau de associação ao longo do tempo seriam importantes para prever conexões explícitas no futuro. O presente artigo propõe um modelo computacional utilizando como base as técnicas de Correlação e Associação entre termos e a computação distribuída para a descoberta de conhecimento, com destaque para a dimensão \| 95 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 temporal. A Figura 1 ilustra as etapas que compõem o modelo proposto. Tais etapas possibilitam a interconexão do conteúdo textual representado por conceitos em um domínio de análise, cujo objetivo é prover suporte ao processo de descoberta de conhecimento. Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 3 Modelo proposto Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação temporal. A Figura 1 ilustra as etapas que compõem o modelo proposto. Tais etapas possibilitam a interconexão do conteúdo textual representado por conceitos em um domínio de análise, cujo objetivo é prover suporte ao processo de descoberta de conhecimento. \| 96 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Figura 1 - Modelo proposto de descoberta de conhecimento com base na correlação e associação de termos. Fonte: Sérgio (2013) Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Figura 1 - Modelo proposto de descoberta de conhecimento com base na correlação e associação de termos. Fonte: Sérgio (2013) Fonte: Sérgio (2013) 3.1 Etapa de correlação A etapa 1 é detalhada a partir da Figura 2. O primeiro passo consiste na definição de um dicionário que contenha os termos necessários para uma determinada análise, juntamente com a indexação de todos os documentos coletados para compor a base de dados. Neste ponto torna-se fundamental a intervenção de um especialista de domínio, pois o mesmo é responsável por definir e registar na base de dados os termos que serão utilizados no processo de correlação, e posteriormente no processo de associação. \| 97 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Figura 2 – Detalhamento da etapa de correlação. Fonte: Sérgio (2013) Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Figura 2 – Detalhamento da etapa de correlação. Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Figura 2 – Detalhamento da etapa de correlação. Fonte: Sérgio (2013) Fonte: Sérgio (2013) No passo 2, a aplicação requisita uma lista de termos, sendo que estes foram inseridos na etapa anterior. No passo seguinte, levando em consideração cada termo a constar na lista, através de um serviço de consulta verifica-se em quais documentos o termo é mencionado. Ao final deste passo, obtém-se uma lista de termos e suas respectivas frequências individuais representando o número de documentos que contenham o termo pesquisado. No passo 4, monta-se uma estrutura que seja capaz de prover todos os dados, de modo que o cálculo de correlação possa ser executado distribuidamente. Para que as tarefas fossem executadas de forma distribuída, foi utilizado o framework/middleware GridGain®. O GridGain® possibilita o desenvolvimento de aplicações distribuídas de alto desempenho e escalabilidade (IVANOV; DMITRIY, 2012). Já no passo 5, cada nodo que integra a rede distribuída é responsável por obter a frequência conjunta dos termos. Em outras palavras, é requisitado ao servidor de consulta a quantidade de documentos em que dois termos quaisquer aparecem conjuntamente. Q , g , , , p , / g doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Q , g , , , p , / g doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 3.1 Etapa de correlação Para realizar este passo, todo nodo possui um termo origem e uma lista de termos destino. Sendo assim, o nodo calcula a frequência conjunta do termo origem com cada termo destino que compõe a lista. Já tendo obtido os valores da frequência individual e da frequência conjunta, a partir do passo 6 é possível calcular o coeficiente de \| 98 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação correlação que representa a força de correlação entre dois termos. O cálculo utilizado foi apresentado anteriormente no item sobre Modelos baseados em coocorrência. Finalmente, ao chegar no último passo, cada nodo da rede é responsável por persistir os coeficientes de correlação que calculou. A quantidade de nodos criados é igual o tamanho da lista de termos menos um. Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 3.2 Etapa de associação O primeiro passo desta etapa é a requisição dos termos sem correlação. Sendo assim, a etapa é realizada apenas com termos que não possuem correlação direta, uma vez que a partir dos dados gerados objetivam-se análises onde exista uma tendência de aumento no coeficiente de associação ao longo do tempo, no entanto antes que estes passem a coocorrerem em um mesmo documento. A Figura 3 detalha a etapa de associação. Figura 3 – Detalhamento da etapa de associação. Fonte: Sérgio (2013) Fonte: Sérgio (2013) Em seguida realiza-se a divisão dos nodos da rede distribuída que serão responsáveis por calcular o coeficiente de associação. Esta divisão tem como objetivo verificar a associação de uma lista de termos em diferentes datas, de modo que seja possível que se aplique uma análise de associação temporal. É no \| 99 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação ç Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves passo seguinte que o coeficiente de associação é calculado por meio da utilização do modelo vetorial apresentado anteriormente, de modo que no último processo relativo à esta etapa, cada nodo realize a inserção do resultado do cálculo de associação na base de dados. 4 Metodologia da pesquisa Os procedimentos metodológicos utilizados neste trabalho são de natureza descritiva e exploratória, utilizando a base de dados Science Direct® como fonte de coleta dos artigos para a análise dos dados. 3.3 Etapa de descoberta de conhecimento Após as etapas de associação e correlação serem concluídas pelos nodos da rede, obtém-se o grau de similaridade entre os termos da pesquisa. Neste momento é realizada a descoberta de conhecimento subsidiada pelos passos anteriores. A base de dados resultante permite que seu conteúdo seja explorado, visando à obtenção de tendências e padrões (HASHIMI; HAFEZ; MATHKOUR, 2015) que auxiliem na descoberta de conhecimento relevante e útil para o apoio a tomada de decisão. Este conhecimento pode ser exposto através de gráficos de correlação e associação, histogramas, e mesmo mapas de tópicos, temporais ou não. Dentre as possibilidades citadas, a característica da análise temporal possibilita um acompanhamento da possível evolução do grau de associação entre dois termos. A Figura 4 ilustra o processo de descoberta de conhecimento. Figura 4 – Detalhamento da etapa de descoberta de conhecimento. Fonte: Sérgio (2013) Figura 4 – Detalhamento da etapa de descoberta de conhecimento. \| 100 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação é Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 4.1 Detalhamento do cenário de pesquisa A construção do cenário de aplicação envolveu a coleta de artigos na base de dados Science Direct®, com o objetivo de evidenciar relações temporais existentes entre termos de determinado domínio. Critérios como a abrangência de áreas, assim como a confiabilidade e credibilidade, o volume de artigos publicados (aproximadamente 12 milhões) e os filtros de pesquisa se fizeram decisivos para escolha da base de coleta. Abaixo, o Quadro 2 expõe os elementos da pesquisa para criação da base de dados. Na primeira e segunda coluna são apresentados os termos pesquisados e o foco da análise. Na terceira coluna, é apontado o período de realização da coleta e na última coluna, o momento em que ocorre a coocorrência entre os termos em pelo menos um documento. Quadro 2 - Elementos de pesquisa para montagem da base de dados. Termo de Pesquisa 1 Termo de Pesquisa 2 Período de realização da pesquisa Momento em que ocorre a coocorrência Biotechnology Genetic Engineering 1993 a 2002 2003 Nanotechnology Medicine 1984 a 1993 1994 Fonte: Sérgio (2013) Quadro 2 - Elementos de pesquisa para montagem da base de dados. O período de coleta e extração dos dados para compor a base de dados compreendeu o ano de 2013. Foram coletados 313 artigos para o primeiro estudo de caso e 238 artigos para o segundo, conferindo um total de 551 documentos. No primeiro estudo de caso, os termos de consulta foram: \| 101 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação não estruturadas por meio de técnicas de correlação associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves ç Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves (“Biotechnology” and “Genetic Engineering”), enquanto que no segundo os termos analisados foram (“Nanotechnology” and “Medicine”). A escolha dos termos foi motivada pela presença de termos relacionados à área da saúde em pesquisas envolvendo modelos de Descoberta de Conhecimento. A pesquisa foi realizada considerando a presença do termo no documento como um todo. Para o primeiro estudo de caso (“Biotechnology” and “Genetic Engineering”), compreendendo apenas o período de associação – ou seja, quando não existe coocorrência –, foram coletados 138 documentos. 4.1 Detalhamento do cenário de pesquisa No período em que passa a existir coocorrência e a determinação da correlação se torna possível, foram coletados 175. Para o segundo estudo de caso (Nanotechnology and Medicine), coletou-se 185 documentos para o período de associação, e 53 para o período de correlação. Para criação da base de dados foi necessário extrair as meta-informações dos artigos e estruturá-los na forma de documentos XML – processo que foi realizado manualmente. E em razão do processo de coleta manual, optou-se por selecionar apenas uma quantidade limitada de documentos por ano. O documento XML criado é composto por um identificador sequencial, o título, o ano, o nome do(s) autor(es) com sua(s) respectiva(s) organização(ões) e as palavras-chave. Caso as palavras-chave não existissem o documento era lido e as palavras-chave elencadas no arquivo XML correspondente. Na etapa seguinte os arquivos no formato XML e os documentos completos em formato PDF foram indexados via um servidor de indexação visando permitir as consultas para a identificação das frequências individuais e conjuntas dos termos. Considerando as palavras-chave dos artigos obteve-se 710 termos que foram utilizados no primeiro estudo envolvendo Biotechnology e Genetic Engineering, e 506 termos utilizados no segundo estudo envolvendo Nanotechnology e Medicine. 5 Discussão dos resultados \| 102 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação ç Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves O modelo apresentado neste artigo promove suporte à evolução temporal dos relacionamentos entre termos. Por meio de gráficos e mapas de tópicos, com o objetivo de explicitar conhecimento em bases textuais e consequentemente auxiliar na tomada de decisão, se busca demonstrar a evolução dos relacionamentos entre termos. Baseado nas frequências tanto individuais quanto conjuntas dos termos pesquisados é possível aplicar o cálculo da equação Phi-squared para que se obtenha o grau de correlação. Posteriormente, a partir da correlação aplica-se o cálculo do modelo vetorial para a obtenção do grau de associação entre dois termos. Ao se utilizar os resultados como base, torna-se possível gerar análises sobre os dados observados. Tais análises podem indicar tendências associativas e apontar possíveis correlações passíveis de investigação em determinado período. O Gráfico 1 apresenta o momento em que ocorre a associação entre os elementos em análise e o momento posterior, a correlação. Neste caso, pode-se observar que o grau de associação entre Biotechnology e Genetic Engineering evolui até 2002, ou seja, compartilham termos presentes na representação vetorial, porém não coocorrem em um mesmo documento. E a partir de 2003, os termos passam a ser mencionados conjuntamente. Gráfico 1 - Grau de associação entre Biotechnology e Genetic engineering Fonte: Sérgio (2013) Gráfico 1 - Grau de associação entre Biotechnology e Genetic engineering Fonte: Sérgio (2013) Fonte: Sérgio (2013) \| 103 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 5 Discussão dos resultados 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação O gráfico acima apresenta uma evolução do comportamento associativo entre dois termos quaisquer de interesse em uma análise. Deste modo, o aumento da tendência pode disparar alertas, visando uma análise mais detalhada nas fontes de informação. Cabe mencionar que a evolução da associação não garante que coocorrências irão acontecer de fato, mas criam indícios que podem auxiliar na tomada de decisão. Este tipo de gráfico tem impacto nos mais variados domínios, sejam científicos ou mesmo análises de contexto social. Por outro lado, a evolução do grau de associação entre os elementos pode não ocorrer de maneira incremental. Entretanto, tal comportamento não indica que os termos não possam ser mencionados conjuntamente, visto que a determinação da coocorrência pode acontecer ao acaso. No Gráfico 2 pode-se observar um cenário em que a associação não é crescente, mas conduz a coocorrência mesmo assim. Gráfico 2 – Evolução temporal entre os termos Nanotechnology e Medicine Fonte: Sérgio (2013) Gráfico 2 – Evolução temporal entre os termos Nanotechnology e Medicine Fonte: Sérgio (2013) Os resultados obtidos na segunda análise, ainda que modestos, apontam um incremento nos valores, que vão de 0,193 para 0,329, o que equivale a aproximadamente 70% na evolução do grau de associação. Além do comportamento apresentando nas análises acima, a associação entre dois termos pode sofrer decréscimos ao longo do tempo, indicando um afastamento dos mesmos. Este afastamento poderia, por exemplo, ser explicado pela evolução de determinada área em que um termo ou passa a ter menos \| 104 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 influência ou se altera para termos correlatos, uma vez que a designação original não representava adequadamente o conceito. 5 Discussão dos resultados Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 i fl ê i lt t l t d i ã i i l Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação influência ou se altera para termos correlatos, uma vez que a designação original não representava adequadamente o conceito. Os dados obtidos contribuem ainda para a geração de novos conhecimentos, possibilitando a exploração de conteúdo centrado na obtenção de padrões e tendências que possam conduzir a descoberta de conhecimento através de ferramentas que considerem os aspectos visuais de como os termos se interconectam. Os mapas de tópicos estão entre as ferramentas que possibilitam este tipo de exploração, objetivando auxiliar no entendimento de determinado domínio de análise. A escolha de mapas de tópicos como meio de representação foi motivada pel a sua utilidade quanto à representação e descrição da informação, e bem como a estrutura conceitual de determinado domínio (AHMED; MOORE, 2005). A elaboração dos mapas de tópicos é conduzida recursivamente, selecionando em cada nível do mapa os cinco termos mais relacionados a determinado termo de interesse – neste caso, Biotechnology, Genetic Engineering, Nanotechnology e Medicine. A expansão de cada mapa considerou dois níveis a partir do termo central. A Figura 5 apresenta o mapa de tópicos gerado a partir do termo Biotechnology. Figura 5 – Mapa de tópicos referente ao termo Biotechnology Figura 5 – Mapa de tópicos referente ao termo Biotechnology Fonte: Sérgio (2013) As ligações entre os termos não possuem direção. Como mencionado, cada termo a partir da origem (termo de interesse) se conecta aos demais termos \| 105 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação é em que possui a maior correlação. A cor vermelha representa o termo de análise, a cor amarela representa o primeiro nível, e a cor azul o segundo nível. A Figura 6 apresenta o mapa de tópicos gerado a partir do termo Genetic Engineering. em que possui a maior correlação. Figura 7 – Mapa de tópicos referente ao termo Nanotechnology 5 Discussão dos resultados A cor vermelha representa o termo de análise, a cor amarela representa o primeiro nível, e a cor azul o segundo nível. A Figura 6 apresenta o mapa de tópicos gerado a partir do termo Genetic Engineering. Figura 6 – Mapa de tópicos referente ao termo Genetic Engineering Fonte: Sérgio (2013) de tópicos referente ao termo Genetic Engineering Nos dois mapas pode-se verificar que os termos Transgenic, Molecules e Cell promovem a conexão entre os termos Biotechnology e Genetic Engineering. A Figura 7 e a Figura 8 apresentam cada uma mapas de tópicos obtidos a partir dos termos Nanotechnology e Medicine. Como é possível observar, os termos Engineering, Technology e Data promovem a conexão entre Nanotechnology e Medicine. Figura 7 – Mapa de tópicos referente ao termo Nanotechnology \| 106 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 5 Discussão dos resultados 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Fonte: Sérgio (2013) Figura 8 – Mapa de tópicos referente ao termo Medicine Fonte: Sérgio (2013) A base de dados gerada para a condução dos estudos possibilita, ao nível de mapas de tópicos, análises temporais como as apresentadas no Gráficos 1 e no Gráfico 2 que forneçam uma visão estrutural de determinado domínio em Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Fonte: Sérgio (2013) Descoberta de Conhecimento a partir de informaçõ não estruturadas por meio de técnicas de correlaçã associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Fonte: Sérgio (2013) Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação não estruturadas por meio de técnicas de correlação associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves Fonte: Sérgio (2013) Figura 8 – Mapa de tópicos referente ao termo Medicine Fonte: Sérgio (2013) A base de dados gerada para a condução dos estudos possibilita, ao nível de mapas de tópicos, análises temporais como as apresentadas no Gráficos 1 e no Gráfico 2 que forneçam uma visão estrutural de determinado domínio em função do tempo. Mapas de tópicos temporais podem promover indícios importantes sobre a evolução ou a retração de determinado domínio do \| 107 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 conhecimento visando à condução, por exemplo, de investimentos em pesquisa, desenvolvimento e inovação. Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Descoberta de Conhecimento a partir de informaçõe não estruturadas por meio de técnicas de correlação associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves conhecimento visando à condução, por exemplo, de investimentos em pesquisa, desenvolvimento e inovação. Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 6 Considerações finais 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação é associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 auxiliar no desenvolvimento e crescimento destas organizações, com o intuito de gerar vantagens competitivas e posicionamentos estratégicos. Referências AHMED, K.; MOORE, G. An introduction to topic maps. The Architecture Journal, [S.l.], v. 5, n. 5, jul. 2005. Disponível em: <http://msdn.microsoft.com/en-us/library/aa480048.aspx>. Acesso em: 31 ago. 2015. BARALIS, E. et al. GraphSum: discovering correlations among multiple terms for graph-based summarization. Information Sciences, New York, v. 249, p. 96-109, nov. 2013. BARALIS, E. et al. GraphSum: discovering correlations among multiple terms for graph-based summarization. Information Sciences, New York, v. 249, p. 96-109, nov. 2013. 6 Considerações finais A fim de desenvolver um modelo que possibilitasse a aplicação de técnicas de correlação e associação que considerasse a dimensão temporal e permitisse lidar com grandes volumes de dados não estruturados, o emprego da computação distribuída se mostrou essencial, ao passo que também demonstrou flexibilidade e escalabilidade. Seguindo por esta linha, buscou-se então a descoberta de relacionamentos entre termos que descrevessem determinado domínio de aplicação, e que fossem de caráter indireto e temporal. Os resultados apresentados foram responsáveis por estabelecer a base para a geração das análises sobre o domínio pesquisado, sendo as mesmas explanadas por meio de gráficos temporais que tornam evidente a existência de padrões comportamentais entre os termos em questão. E com o intuito de melhorar o entendimento do domínio, foram gerados mapas de tópicos a partir dos vetores de contexto envolvendo determinado termo de análise. Devido à aplicação do protótipo em um conjunto restrito de tempos e documentos – uma vez que não foi possível acessar completamente a base de dados Science Direct® – percebe-se a construção do cenário como uma das limitações existentes. Em função disso, a base de dados foi desenvolvida por meio de um especialista de domínio. A partir da aplicação do processo de Descoberta de Conhecimento, padrões e tendências podem ser evidenciados. No âmbito de trabalhos futuros, vislumbra-se a evolução do software desenvolvido, gerando novas informações no processo e novas formas de visualização da informação, visando a descoberta de conhecimento por meio destes. Quanto a possíveis análises, destacam-se cenários onde haja a necessidade e a demanda por parte de organizações para compreender a competitividade ou a identificação de tendência de mercados, com dados oriundos da Web. Desta maneira, o conhecimento obtido pode \| 108 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. CAO, L. et al. Flexible Frameworks for Actionable Knowledge Discovery. IEEE Transactions on Knowledge and Data Engineering, [S.l.], v. 22, n. 9, p. 1299-1312, set. 2010. CAO, L. et al. Flexible Frameworks for Actionable Knowledge Discovery. IEEE Transactions on Knowledge and Data Engineering, [S.l.], v. 22, n. 9, p. 1299-1312, set. 2010. CHEN, Y. et al. Keyword search on structured and semi-structured data. In: ACM SIGMOD INTERNATIONAL CONFERENCE ON MANAGEMENT OF DATA, 1., 2009, Providence. Proceedings… Providence: ACM, 2009. CHURCH, K. W.; GALE, W. A. Concordances for Parallel Text. In: ANNUAL CONFERENCE OF THE UW CENTRE FOR THE NEW OED AND TEXT RESEARCH, 8. 1991, Oxford. Proceedings… Oxford: [s.n], 1991. p. 40-62. CHURCH, K. W.; HANKS, P. Word association norms, mutual information, and lexicography. Computational Linguistics, Cambridge, v. 16, n. 1, p. 22-29, mar. 1990. CHURCH, K. W.; MERCER, R. L. Introduction to the Special Issue on Computational Linguistics Using Large Corpora. Computational Linguistics, Cambridge, v. 19, n. 1, p. 1-24, mar. 1993. COHEN, T.; SCHVANEVELDT, R. W. The trajectory of scientific discovery: concept co-occurrence and converging semantic distance. Studies in Health Technology and Informatics, Amsterdam, v. 160, p. 661-665, 2010. Disponível em: <http://ebooks.iospress.nl/publication/13521>. Acesso em: 22 abr. 2016. COHEN, T.; SCHVANEVELDT, R. W. The trajectory of scientific discovery: concept co-occurrence and converging semantic distance. Studies in Health T h l d I f i A d 160 661 665 2010 Technology and Informatics, Amsterdam, v. 160, p. 661-665, 2010. Disponível em: <http://ebooks.iospress.nl/publication/13521>. Acesso em: 22 abr. 2016. COHEN, T.; SCHVANEVELDT, R.; WIDDOWS, D. Reflective Random Indexing and Indirect Inference: A Scalable Method for Discovery of Implicit Connections. Journal of Biomedical Informatics, San Diego, v. 43, n. 2, p. 240-256, abr. 2010. \| 109 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 CONRAD, J. G.; UTT, M. H. A system for discovering relationships by feature extraction from text databases. 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Perspectivas em Ciência da \| 110 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Informação, Belo Horizonte, v. 15, n. 1, p.154-172, jan./abr. 2010. Disponível em: <http://portaldeperiodicos.eci.ufmg.br/index.php/pci/article/view/875/717>. Acesso em: 22 abr. 2016. Informação, Belo Horizonte, v. 15, n. 1, p.154-172, jan./abr. 2010. Disponível em: <http://portaldeperiodicos.eci.ufmg.br/index.php/pci/article/view/875/717>. Acesso em: 22 abr. 2016. Informação, Belo Horizonte, v. 15, n. 1, p.154-172, jan./abr. 2010. 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Journal of biomedical informatics, San Diego, v. 46, n. 2, p. 200-211, abr. 2013. Disponível em: \| 112 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113 E-ISSN 1808-5245 Descoberta de Conhecimento a partir de informações não estruturadas por meio de técnicas de correlação e associação Marina Carradore Sérgio, Thales do Nascimento da Silva e Alexandre Leopoldo Gonçalves Knowledge Discovery from Unstructured Information through Correlation and Association Techniques Abstract: Nowadays, technology’s current status seeks means to support the exponential increase of information available around the Internet or in organizations, and regarding that most of said information comes in a textual form, this is a challenge to the areas of crawling, storage, retrieval and analysis of information. This article aims to provide a Knowledge Discovery model based on the temporal correlation and association from large document collections. The methodology set for this process involve descriptive and explorative researches using papers taken right from the Science Direct® database as a tool for data collection and analysis. Through this kind of information is possible to extract rules, patterns, trends, and networks, all of them being usufel to the process of making decisions within organizations in order to generate competitive advantage. Thus, the main contribution of this paper relies on the proposition of a model towards the understanding of temporal aspects, considering factual relationships (through correlations) or not (through associations) between terms in a domain. Keywords: Knowledge discovery. Correlation. Association. Unstructured information. Temporality. Recebido: 22/10/2015 Aceito: 11/04/2016 1 Por “termos” compreendem-se palavras simples e/ou compostas que podem ser nomeadas/classificadas (chamados de entidades) ou não. \| 113 Em Questão, Porto Alegre, v. 22, n. 2, p. 87-113, mai/ago. 2016 doi: http://dx.doi.org/10.19132/1808-5245222.87-113
https://openalex.org/W3166270316	https://link.springer.com/content/pdf/10.1007/s10640-021-00561-1.pdf	English	null	Adoption Gaps of Environmental Adaptation Technologies with Public Effects	Social Science Research Network	2,019	cc-by	16,683	Environmental and Resource Economics (2022) 83:313–339 https://doi.org/10.1007/s10640-021-00561-1 Environmental and Resource Economics (2022) 83:313–339 https://doi.org/10.1007/s10640-021-00561-1 * Angelo Antoci antoci@uniss.it Angelo Antoci1 · Simone Borghesi2,3 · Giulio Galdi3 · Sergio Vergalli4,5 Angelo Antoci1 · Simone Borghesi2,3 · Giulio Galdi3 · Sergio Vergalli4,5 Accepted: 12 April 2021 / Published online: 2 June 2021 © The Author(s) 2021 The authors would like to express their sincere appreciation to two anonymous reviewers for their very useful suggestions. 1 Department of Economic and Business Sciences, University of Sassari, Sassari, Italy 2 Department of Political and International Sciences, University of Siena, Siena, Italy 3 Florence School of Regulation, European University Institute, Florence, Italy 4 Department of Economics and Management, University of Brescia, Brescia, Italy 5 Fondazione Eni Enrico Mattei (FEEM), Milan, Italy 1 Introduction Climate change and environmental hazards are exerting pressure on our societies at an increasing pace, requiring rapid and swift change in our economic systems in order to have a chance to stay below a 2◦C degrees global temperature increase (Rogelj et al. 2018). We had to forgo the comforting idea that “natural systems fluctuate within an unchanging enve- lope of variability”(Milly et al. 2008). The scale of the challenge requires immediate action to cope with our rapidly changing climate. The IPCC and the UNEP1 characterise the pos- sible responses to environmental and climatic hazards dichotomously, as either adapta- tion or mitigation strategies (UNEP 2019; IPCC 2014), with the implementation of one not excluding the other’s. On the one hand, there are mitigation strategies at our disposal, which tackle the problem at its source and combat the causes of increased environmental risks. Efficient water management, restoration of soil, substitution of fossil fuels with agri- cultural by-products are all examples of mitigation techniques for the agricultural sector (Smith et al. 2007). Mitigation strategies not only reduce the environmental hazards for the adopter, but for all agents, thus generating a positive externality to other agents. On the other hand, adverse effects of climate change and environmental degradation can already be felt, prompting the development of solutions to deviate or alleviate the damages, i.e. adaptation strategies. Such strategies allow to cope with a changing climate, reducing the exposure of adopters to the ensuing harm. This includes responding to abnormally hot or cold temperatures, adopting new agricultural techniques to cope with the impoverishment of soil, creating artificial snow in ski resorts, and much more (for a broad review on many other forms of adaptation, see Tompkins et al. 2010). With respect to mitigation strate- gies, adaptation does not aim to reduce the problem, but rather to avoid at least part of its adverse affects. At times, this is done at the expense of other agents, i.e. adaptation strate- gies may generate negative externalities. An adaptation technology that exemplifies this dynamics and is recently gaining salience is solar geoengineering, whose aim is to reduce solar radiation on Earth. There are many techniques with which this could be attained, but all of them seem to carry their own flaw which could endanger vulnerable areas or com- munities (Reynolds 2019; Wagner and Weitzman 2015; Zhang et al. 2015). 1 United Nations Environment Programme. Abstract As the Intergovernmental Panel on Climate Change (2018) testifies, the world is a long way from halting climate change, let alone reverting it. The existence of adaptation and mitigation technologies did not prove sufficient, their adoption being respectively faulted or hindered by the presence of externalities. In this work, we study how externalities, whether positive or negative, lead the system away from Pareto-dominant (social optimum) states, towards Pareto-dominated ones. We show that adoption gaps, i.e. differences between socially optimum vs current adoption shares, of both (mal)adaptation and mitigation tech- nologies are caused by the externalities emitted. In particular, over-adoption may occur for maladaptive technologies, whereas under-adoption may occur in case of mitigation. We employ a model with two regions at different stages of development and also derive rel- evant considerations on possible counterproductive effects of green policies and environ- mental dumping. Keywords Maladaptation · Mitigation · Externalities · Environmental dumping JEL Classification C70 · D62 · O13 · O40 · Q20 The authors would like to express their sincere appreciation to two anonymous reviewers for their very useful suggestions. 1 Department of Economic and Business Sciences, University of Sassari, Sassari, Italy 2 Department of Political and International Sciences, University of Siena, Siena, Italy 3 Florence School of Regulation, European University Institute, Florence, Italy 4 Department of Economics and Management, University of Brescia, Brescia, Italy 5 Fondazione Eni Enrico Mattei (FEEM), Milan, Italy A. Antoci et al. 314 1 Introduction Indeed, the IPCC (2018) remarks that, according to extant knowledge on solar engineering, there are concerns of potential conflicts between the various techniques and sustainable development goals. Another notorious instance of maladaptation in the scientific literature are cooling and heating systems: by improving domestic temperature for the user, it increases the risk of energy shortages and ultimately worsens the problem of climate change (Lundgren and Kjellstrom 2013). These two notable examples and, in general, all strategies that shift envi- ronmental hazards onto others, postpone them for future generations, or disproportionally affect the most vulnerable, are defined by the literature as maladaptation, rather than sim- ply as adaptation (Antoci et al. 2020; UNEP 2019; Barnett and O’Neill 2010). It should be noted that the UNEP (2019) stresses that every adaptation strategy that increases the opportunity cost of moving to a more sustainable alternative is maladaptation, as it has detrimental effects on long term sustainability. In this work we study the dynamics of miti- gation and maladaptation, showing how global externalities lead to under-adoption of strat- egies of the former type and over-adoption of the latter 1 3 Adoption Gaps of Environmental Adaptation Technologies with… 315 Since mitigation strategies actually reduce environmental hazards instead of (temporar- ily) avoiding its effects, it is usually considered to be the most desirable strategy (IPCC 2014). However, there are reasons why humans did not respond with enthusiasm to the emergence of mitigation solutions in the face of environmental hazards. Firstly, many mitigation strategies require long-term investments to pay off, with a time scale that may exceed the average life expectancy of a person before they become effective (Hallegatte 2009). The incapacity of humans to make long-term investments and their preference for the present are additional threats to our capacity to make long-term commitments to stop environmental degradation (Warburton 2018), leading to issues of intergenerational equity (Glotzbach and Baumgartner 2012), which is a characterising feature of maladapta- tion (UNEP 2019). We remark that the existence of mitigation solutions is not a sufficient condition for the abatement of environmental damage. The literature has uncovered sev- eral ways in which externalities of any type, either negative or positive (as is the case for mitigation strategies) may undermine the achievement of the social optimum. On the one hand, whenever an agent may transfer her cost to protect against environmental hazards onto others, i.e. 2 In an experimental setting, Hasson et al. (2010) show that agents rarely contribute to the mitigation solution and that their contributions to a common mitigation policy are not sensitive to the likelihood of extremely adverse events. In a somewhat similar experiment, Milinski et al. (2006) show that reputation effects may nudge agents to contribute to a public good in an environmental framing. 1 Introduction she emits negative externalities, in a way that has no consequences on her- self, she has little incentive to adopt an existing mitigation strategy. For instance, an agent might prefer to install a substantially cheaper air conditioning system instead of investing to enhance house insulation. On the other hand, if a strategy actually reduces environmen- tal risks not only for the adopter, but also for other agents, i.e. it has a positive externality, then it may happen that all agents wait for the others to tackle environmental degradation for everyone, but none is willing to pay the cost for the benefit of others2. This is but an instance of the well known free rider problem, which emerges from the non-excludability of agents from the benefits of a public good (Heller and Starrett 1976). Scholars studying these shiftable externalities highlighted that policy tools hindering maladaptive strategies and promoting mitigation ones are desirable, e.g. a tax on negative externalities or a sub- sidy on positive ones (Bird 1987; Shaw and Shaw 1991; Shogren and Crocker 1991; Geaun 1993). In this work, we focus on the dichotomy between maladaptation and mitigation, studying the adoption dynamics of the related strategies. We assume that individuals from two regions at different stages of development have the possibility to adopt a technology which provides benefits for the adopters, but also generates externalities on other agents. In particular, each region has a local environmental indicator which is affected by the adoption dynamics of both regions, so that the externalities have a global effect. These externalities may be either nega- tive, in case of a maladaptation technology, or positive, in case of a mitigation technology. We highlight what is the underlying mechanism that leads to over-adoption of maladaptation strategies and under-adoption of mitigation ones. In addition, we show what are the effects on the less developed region if the maladaptation technology is such that it disproportionally burdens its population with respect to the agents from the more developed region. Finally, we briefly analyse the case of a green policy in the developed region (abating local externalities) which has negative impacts on the environment in the less developed region (environmental dumping). We show that in this context the green policy may actually reduce the well-being of agents in both regions. The adoption dynamics of the technologies is modelled by a two 1 3 3 A. Antoci et al. 2 The Model Let us consider two regions j = N, S . At any given time t, the well-being of agents from both regions depends on the quality of an environmental indicator, Ej t , that is subject to be degraded or enhanced by other agents, i.e. by their externalities. Agents may decide to adopt an adapta- tion/mitigation strategy A to improve the personal benefit they derive from Ej t , while being aware that they are in turn affected by the public effect, i.e. the externality, from the adapta- tion/mitigation strategies of other agents from the same region. One might think of neighbour- ing farmlands, where the land of one farmer can be threatened by the actions of others (e.g. volatile pesticides crossing over) or, vice versa, enhanced by them (e.g. neighbour’s beehives increasing own crop fertility). Using pesticides or keeping a beehive are both actions that pro- vide a private benefit to the adopter, while having an effect on the neighbouring farmland. In our model, this private effect is denoted by pj and is always positive. If agents decide not to use the adaptation/maladaptation technology, i.e. they choose strategy NA , they cannot enjoy the private effect. Independently from their chosen strategy, agents are also subject to public effect Pj t , which is the summation of the externalities generated by agents adopting strategy A and is defined later in (2). Finally, the environmental quality of region j when all agents in both countries adopt strategy NA is equal to E j . The overall environmental quality for agents in j = N, S adopting strategy i = A, NA is thus described as follows: Ej t = { E j + Pj t if i = NA E j + Pj t + pj if i = A Ej t = { E j + Pj t if i = NA E j + Pj t + pj if i = A where Pj can take either sign. We define the well-being of an agent from region j as the natural logarithm of Ej minus the adoption cost Cj for agents choosing strategy A: (1) Πj i ∶= { ln(E j + Pj t) if i = NA ln(E j + Pj t + pj) −Cj if i = A (1) where the adoption cost Cj is strictly positive. 1 Introduction 316 population evolutionary game which employs replicator equations, so that all agents may imi- tate their peers in the region, if the well-being of the latter is greater. Our analysis leads to three major conclusions: (1) when only a maladaptive technology is available, either all agents adopt it or none, depending on the initial distribution of strategies; (2) when only a mitigation technology is available, the system typically reaches a state in which a part of the population adopts the technology while the rest does not; no path dependency arises (3) if the more devel- oped region dumps negative externalities onto the less developed one, it might happen the the well-being of all agents decreases. In Sect. 2 we illustrate our model. We then employ it to analyse the adoption dynamics of a maladaptive technology (Sect. 3) and of a mitigation one (Sect. 4). Finally, in Sect. 5 we elaborate on the results, draw some policy implications and sketch future research directions. 2 The Model We now define the public effect Pj t , which depends on the shares of agents xt, zt ∈[0, 1] adopting strategy A at time t in regions N and S, respectively. We also differentiate between domestic and foreign effects of the adap- tation/mitigation technology, respectively defined as dj and f j . The former describes the impact on a local environmental indicator of same-region adopters, whereas the latter 1 3 3 Adoption Gaps of Environmental Adaptation Technologies with… Adoption Gaps of Environmental Adaptation Technologies with… 317 describes the impact of cross-region adopters. For the sake of simplicity, we assume that the public effects are determined by linear functions: describes the impact of cross-region adopters. For the sake of simplicity, we assume that the public effects are determined by linear functions: (2a) PN t ∶= −dN ⋅xt −f N ⋅zt (2b) PS t ∶= −f S ⋅xt −dS ⋅zt (2a) PN t ∶= −dN ⋅xt −f N ⋅zt (2a) (2b) PS t ∶= −f S ⋅xt −dS ⋅zt (2b) where parameters dj and f j measure the domestic and the foreign public effects, respec- tively, for region j = N, S . They represent the public impact of adoption by all agents on the local environmental indicator of region j. In other terms, dj and f j represent the externali- ties embedded in adopting strategy A . These externalities might either depend on techno- logical factors (as in solar engineering) and/or on policies regulating them (e.g. emissions standards). Since the analysis of these factors are beyond the scope of the present paper, we assume them to be exogenously determined. Domestic effects dj are caused by agents in region j and worsen the quality of their own local environmental indicator, whereas foreign effects f j affect the local environmental indicator of region j but are caused by agents in the other region. We do not apply any sign restriction on the public effects, so that externalities of adoption of the environmental adaptation technology may take either sign. When a pub- lic effect Pj is positive, adoption of strategy A by an agent carries part of its benefits over to other agents. This case qualifies as a mitigation case, in which an agent is working for the cooperative improvement of environmental quality, or equivalently towards the abatement of pollution. 2 The Model By contrast, when the public effect is negative, an agent adopting strategy A is actually benefiting herself by worsening environmental quality for others. From the concavity of (1), we may add that a negative public effect affects relatively more (reduces well-being by a higher amount) the agents who are not adopting the environment enhanc- ing strategy A . By the definition provided by Barnett and O’Neill (2010), this is a case of maladaptation. In order to study the dynamics of this system, we now describe the way in which the share of agents adopting strategy A in either country varies. We assume that if the differ- ence in well-being ΔΠj = Πj A −Πj NA between strategy A and strategy NA is positive for region j, then the share of agents adopting the technology (either a maladaptive or a mitiga- tion one) in that region will increase, since it provides higher payoffs. The opposite holds if the payoff difference is negative. Finally, if the payoff difference equals zero, economic agents are indifferent between adopting or not adopting the technology, so that the popula- tion shares of agents adopting the technology keeps constant over time. Therefore, we have that: (3) ΔΠN(xt, zt) ⋛0 ⇒̇x ⋛0 ΔΠS(xt, zt) ⋛0 ⇒̇z ⋛0 (3) where ̇x and ̇z are the time derivatives of xt and zt , respectively. Hence, in each region the payoff difference ΔΠj(xt, zt) in N and ΔΠS(xt, zt) in S has the same sign as the time deriva- tive of the population share that adopts the environmental adaptation technology in that region. Referring to the well-being definition (1), we may derive the payoff difference ΔΠj: (4) ΔΠj(xt, zt) = Πj A(xt, zt) −Πj NA(xt, zt) = ln E j + pj + Pj t E j + Pj t −Cj (4) We assume that the dynamics of xt and zt is given by the so-called “replicator dynamics”(see e.g. Weibull 1995): We assume that the dynamics of xt and zt is given by the so-called “replicator dynamics”(see e.g. Weibull 1995): A. Antoci et al. 318 (5) { ̇x = x(1 −x)ΔΠN(x, z) ̇z = z(1 −z)ΔΠS(x, z) (5) where we omitted the temporal subscript t to improve readability. Dynamics (5) describes an adaptive process based on an imitation mechanism: every period t, a (very) small frac- tion of the population changes its strategy adopting the more remunerative one. 2.1 Basic Mathematical Results As the shares of agents adopting strategy A are defined in the interval [0, 1], the dynamic system (5) is defined in the square Q: Q = {(x, z) ∶0 ≤x ≤1, 0 ≤z ≤1}. We will henceforth denote with Qx=0 the side of Q along which x = 0 , and with Qx=1 the side along which x = 1 . Similar interpretations apply to Qz=0 and Qz=1 . All sides of this square are invariant; in other terms, if the pair (x, z) initially lies on one of the sides, then the whole correspondent trajectory also lies on that side. Note that the states {(x, z) = (0, 0), (0, 1), (1, 0), (1, 1)} are always stationary states of the dynamic system (5). In such states, only one strategy (either A or NA ) is played in each region. Other stationary states are the points of intersection between the interior of the sides Qx=0 , Qx=1 (where ̇x = 0 ) and the locus ΔΠS(x, z) = 0 (where ̇z = 0 ) and the points of intersection between the interior of sides Qz=0 , Qz=1 (where ̇z = 0 ) and the locus ΔΠN(x, z) = 0 (where ̇x = 0 ). In such stationary states, there is a region (either S or N) where both available strategies are played by a positive share of agents, while in the other region all agents choose the same strategy. In addition, the point in the interior of Q where the loci ΔΠN(x, z) = 0 and ΔΠS(x, z) = 0 meet are other possible stationary states. In such points, both strategies are adopted by a positive share of agents in both regions.i Finally, we find that the loci ̇x = 0 and ̇z = 0 in the interior of Q are respectively repre- sented by the lines: (6a) z = E N f N − pN f N(eCN −1) −dN f N x (6b) z = E S dS − pS dS(eCS −1) −f S dS x (6a) (6b) z = E S dS − pS dS(eCS −1) −f S dS x (6b) where we recall that eCj −1 > 0 since the adoption cost Cj is strictly positive. The above lines are obtained by substituting the public effects (2) into the well-being differential (4) and setting it equal to zero. 2 The Model Differently from the “classical” contexts where replicator dynamics are introduced (in which economic agents are pairwise randomly matched), here the well-being of each agent depends on the technological choice by all agents, in both regions, and at the same instant; that is, we ana- lyse a population game. Replicator dynamics may be generated by several learning mecha- nisms in a random matching context (see e.g. Börgers and Sarin 1997; Schlag 1998); how- ever, rationales for such dynamics can be found also in our context (see e.g. Sacco 1994). Sethi and Somanathan (1996) propose an application of replicator equations in a context similar to ours. 2.1 Basic Mathematical Results Other relevant cases could be investigated, yet we restrain the analysis to these two cases for the sake of parsimony. 3 In the unlikely circumstance that lines (6a) and (6b) have the same slope and the same intercept, the two lines completely overlap and all their points in the interior of Q are stationary states. 2.1 Basic Mathematical Results Note that the slope of (6a) and (6b) is negative if the domestic 1 3 Adoption Gaps of Environmental Adaptation Technologies with… 319 effect dj and the foreign effect f j have the same sign, for j = N, S respectively. Further- more, the slope of (6a) is greater than the slope of (6b) if dN f N < f S dS . Finally, we note that ΔΠN(x, z) is positive (i.e. ̇x > 0 ) above (6a) if f N > 0 (vice versa if f N < 0 ) and that ΔΠS(x, z) is positive (i.e. ̇z > 0 ) above (6b) if dS > 0 (vice versa if dS < 0 ). Since both (6a) and (6b) are straight lines, there generally3 exists at most one stationary state in the interior of each side of Q and at most one in the interior of Q . Consequently, by recalling that all vertices are stationary states, as well, the highest number of stationary states that can be generally observed is nine (four vertices, four points on the sides, and an internal point). Let us now outline the possible scenarios the system may reach when the adaptation/ mitigation technology is characterised by: (1) Negative public effects towards all agents. In such a context, the technology is mala- daptive, i.e. it is such that it lowers the environmental quality for all. Formally, this maladaptation technology has both a domestic and a foreign negative public effect. One common example of such a technology in the literature is air conditioning: it provides the person with an improvement of her environmental quality at the cost of a small deterioration of the environmental quality (and energy security) for all other people (Deschênes and Greenstone 2011; Lundgren and Kjellstrom 2013). From an analytical perspective, this translates into all public effect parameters being strictly positive: dN , dS , f S , f N > 0.f (2) Positive public effects towards all agents. In such a context, the technology increases the environmental quality for all (mitigation). This mitigation technology has both a domestic and a foreign positive public effect, and this translates into all public effect parameters being strictly negative: dN , dS, f S , f N < 0. Other relevant cases could be investigated, yet we restrain the analysis to these two cases for the sake of parsimony. rspective, this , f S , f N > 0.f Proof See “Mathematical appendix A”. ◻ 3.2 Stability Properties of the Vertices The analysis in the Mathematical appendix B allows us to illustrate the stability properties of the stationary states (0, 0), (0, 1), (1, 0), (1, 1). The analysis in the Mathematical appendix B allows us to illustrate the stability properties of the stationary states (0, 0), (0, 1), (1, 0), (1, 1). 3 Technologies with Negative Public Effects In this section, we analyse scenario (1), that is the case of a maladaptation technology. In this context, the following inequalities hold: (7a) 𝜕ΔΠN(x, z) 𝜕x = pNdN ( E N + PN )( E N + pN + PN ) > 0 (7b) 𝜕ΔΠS(x, z) 𝜕z = pSdS ( E S + PS )( E S + pS + PS ) > 0 (7a) (7b) Inequalities (7a) and (7b) indicate that the well-being differential of adopting strategy A increases with the share of adopters. The higher is the proportion of agents adopting it in 3 In the unlikely circumstance that lines (6a) and (6b) have the same slope and the same intercept, the two lines completely overlap and all their points in the interior of Q are stationary states. A. Antoci et al. 320 either group, the higher is the incentive for others to do the same. To fix ideas, we can think to air conditioning. As the additional production of energy demanded by the air condition- ing systems of other agents increases the heat in the region, even more agents are incen- tivised to adopt the maladaptive technology. In this scenario, strategy A yields the highest payoffs when the majority of agents adopts it. As we will see, such a context favours the emergence of self-reinforcing processes that lead agents to adopt only one strategy, in each region S and N. Proposition 1 Under the assumption that dN , dS , f S , f N > 0 , the system (5) has the follow- ing features: (a) Every trajectory of the system approaches a stationary state. (b) Only the vertices of Q , i.e. the stationary states (0, 0), (0, 1), (1, 0), (1, 1), can be attrac- tive. Proof See “Mathematical appendix A”. Proof See “Mathematical appendix A”. 3.1 Dynamic Regimes First of all, we note that if dN , dS , f S , f N > 0 , then both lines (6a) and (6b), along which ̇x = 0 and ̇z = 0 , respectively, have negative slope. Above these lines, we have that the share of agents adopting strategy A increases. In particular, ̇x > 0 above line (6a) and ̇z > 0 above line (6b), whereas the reverse occurs below these lines. This is very informative with respect to the behaviour of agents: for a higher value of x, z must be lower in order for agents in either region to be indifferent to the maladaptation technology, or else they would prefer to adopt strategy A . From another perspective, for a given point (x, z) which lies on either line (6a) or (6b), a translation (i.e. shift) to the right would make A the preferred strategy in region N or S (or both), respectively. Finally, we note that lines (6a) and (6b) move downwards if the autonomous environmental quality for region N or S is lower. For sufficiently low values of E N and E S , we have that ̇x > 0 and ̇z > 0 , respectively, for all points in Q . The reverse applies when E N and E S are sufficiently high. fi The following proposition characterises the dynamics of the system when dN , dS , f S , f N > 0. Proposition 1 Under the assumption that dN , dS , f S , f N > 0 , the system (5) has the follow- ing features: 3.2.1 Stability of the Stationary State (0, 0) In this stationary state, no agent adopts the technology. In order for this non-adoption scenario to be attractive, it must be individually convenient to adopt strategy NA in both regions. In order for this to hold, the following condition must be satisfied: 3 1 Adoption Gaps of Environmental Adaptation Technologies with… Adoption Gaps of Environmental Adaptation Technologies with… 321 (8) E j > pj eCj −1 with j = N, S (8) with j = N, S (8) To the right hand side of this inequality we have the ratio of the positive private effect of the technology over a measure of its cost of adoption, so that we may interpret the ratio as the efficiency of the adaptation technology in region j. We note that the denominator is strictly positive since Cj > 0 . To the left hand side we have the autonomous environ- mental quality in j, which also coincides with the overall environmental quality since no agent is adopting strategy A ( x = 0, z = 0 ). Condition (8) thus requires that in both regions the efficiency of the technology is lower than the environmental quality. To continue with the example proposed above, mild temperatures (high level of autonomous environmental quality) and costly air conditioning systems hinder their diffusion in the population. 3.2.2 Stability of the Stationary State (0, 1) This stationary state represents the S-adoption scenario, in which all agents in S adopt the technology, while no agent does so in N. It is locally attractive if the following conditions hold: (9) E N −f N > pN eCN −1 (10) E S −dS < pS eCS −1 (9) (10) E S −dS < pS eCS −1 (10) We note that condition (9) is similar to condition (8) however, in this case, the autonomous environmental quality E N is adjusted by the negative public effect ( −f N < 0 ) of the agents in S adopting strategy A (since z = 1 ). In other terms, in order for the agents in N to be more convenient not to adopt the technology, its efficiency needs to be lower than the over- all environmental quality, which includes the public effects of agents in S. f According to condition (10), the adoption of the technology by agents in S requires that its efficiency is greater than the environmental quality, which includes the negative domes- tic public effect −dS. 3.2.3 Stability of the Stationary State (1, 0) This stationary state represents the N-adoption scenario and is specular to the previous one, with all agents in N adopting the technology and no agent adopting it in S. It is locally attractive if the following conditions hold: (11) E N −dN < pN eCN −1 (12) E S −f S > pS eCS −1 (11) (12) E S −f S > pS eCS −1 (12) According to condition (11), in region N the efficiency of strategy A must be greater than the environmental quality adjusted by the domestic (negative) public effect −dN . A. Antoci et al. 322 Analogously, according to condition (12), in region S the efficiency of strategy A must be lower than environmental quality, adjusted by the foreign (negative) public effect −f S. Fig. 1 All nine stationary states exist: the vertices are attractors, the ones on the sides are saddles and the internal one is a repeller Fig. 1 All nine stationary states exist: the vertices are attractors, the ones on the sides are saddles and the internal one is a repeller Analogously, according to condition (12), in region S the efficiency of strategy A must be lower than environmental quality, adjusted by the foreign (negative) public effect −f S. Analogously, according to condition (12), in region S the efficiency of strategy A must be lower than environmental quality, adjusted by the foreign (negative) public effect −f S. 3.2.4 Stability of the Stationary State (1, 1) Finally, the stationary state (1, 1) represents a full adoption scenario, in which all agents from both regions adopt the technology. As discussed more in detail later in the paper, this may represent an undesirable outcome. This stationary state is locally attractive if the fol- lowing conditions hold: (13) E j −(dj + f j) < pj eCj −1 with j = N, S (13) On the left hand side of condition (13) we see that now the environmental quality is affected by both domestic and foreign public effects, since all agents are adopting A . The condition requires the efficiency of the technology for both regions to be greater than the environmental quality. A cheap (or a very efficient) technology is easily diffused in a popu- lation living with a highly degraded environment. Finally, we remark that the vertices of Q can be simultaneously attractive, which occurs when the following condition holds: (14) pj eCj −1 + f j < E j < pj eCj −1 + dj with j = N, S (14) We note that in order for condition (14) to hold, it is necessary that f j < dj for j = N, S . By checking their definitions in (2), we can see that this implies that foreign public effects must be lower than domestic public effects, in both N and S. If foreign public effects were stronger than domestic ones in at least one region, then the stationary states (1, 0) and (0, 1) (respectively, N-adoption and S-adoption) could not be simultaneously attractive. Indeed, it would not be otherwise convenient for an agent not to adopt strategy A when all agents in the other region are doing so unless foreign public effects were neglectable with respect to domestic ones. We note that in order for condition (14) to hold, it is necessary that f j < dj for j = N, S . By checking their definitions in (2), we can see that this implies that foreign public effects must be lower than domestic public effects, in both N and S. If foreign public effects were stronger than domestic ones in at least one region, then the stationary states (1, 0) and (0, 1) (respectively, N-adoption and S-adoption) could not be simultaneously attractive. Fig. 2 In this case, there are three attractors: (0, 0), (0, 1), (1, 1), whereas the other station- ary states are either repellers or saddles Fig. 3 In this case only the sta- tionary states (0, 0) and (1, 0) are attractors. The stationary state in the interior of the top side of Q is a repeller whereas the one lying in the interior of the botom side is a saddle Fig. 4 There are two attractors, corresponding to the full adop- tion (1, 1) and the non-adoption (0, 0) scenarios. There are also a saddle point on the right hand side and a repeller in the asym- metric state (0, 1) 3.2.4 Stability of the Stationary State (1, 1) Indeed, it would not be otherwise convenient for an agent not to adopt strategy A when all agents in the other region are doing so unless foreign public effects were neglectable with respect to domestic ones. 1 3 Adoption Gaps of Environmental Adaptation Technologies with… Adoption Gaps of Environmental Adaptation Technologies with… Adoption Gaps of Environmental Adaptation Technologies with… 323 1 3 Fig. 3 In this case only the sta- tionary states (0, 0) and (1, 0) are attractors. The stationary state in the interior of the top side of Q is a repeller whereas the one lying in the interior of the botom side is a saddle Fig. 4 There are two attractors, corresponding to the full adop- tion (1, 1) and the non-adoption (0, 0) scenarios. There are also a saddle point on the right hand side and a repeller in the asym- metric state (0, 1) 1 3 A. Antoci et al. 324 Fig. 5 In this case, the vertices (0, 1) and (1, 1) are attractors, whereas a repeller lies on the interior of the bottom side of Q Fig. 5 In this case, the vertices (0, 1) and (1, 1) are attractors, whereas a repeller lies on the interior of the bottom side of Q Fig. 5 In this case, the vertices (0, 1) and (1, 1) are attractors, whereas a repeller lies on the interior of the bottom side of Q Some examples of multistability are shown in Figs. 1, 2, 3, 4 and 5, where attractive stationary states are represented by full dots ∙ , repulsive ones by open dots ◦ , and saddles by squares ◻ . In all cases graphically represented, agents in each region coordinate on one of the two strategies. The most interesting dynamics of this kind is the one represented in Fig. 1, where condition (14) is satisfied. In this case all vertices of Q are attractive, whereas all other stationary states along the sides of Q are saddle points and the stationary state inside Q is a source. As Fig. 1 shows, almost every trajectory4 will lead to a vertex of Q , where each region ends up choosing a single strategy (either adopting the environmental maladaptation technology or not). The basins of attraction of the vertices are delimited by the stable manifolds of the saddle point in the interior of the sides of Q. 4 The stable branches of the saddles are exceptions, as they lead the system toward the saddle points. 3.3 Well‑Being Analysis We now examine the average level of well-being in the two regions when all public effects are negative, i.e. the coefficients are positive: dN , dS , f S , f N > 0 . The average level of well- being in N and in S is equal to the weighted average of the well-being of agents adopting strategy A and the well-being of agents adopting NA , where the weights are given by share of adopters in the region. Formally, we have that: (15) ̃ΠN(x, z) ∶= x ⋅ΠN A(x, z) + (1 −x) ⋅ΠN NA(x, z) (16) ̃ΠS(x, z) ∶= z ⋅ΠS A(x, z) + (1 −z) ⋅ΠS NA(x, z) (15) (16) ̃ΠS(x, z) ∶= z ⋅ΠS A(x, z) + (1 −z) ⋅ΠS NA(x, z) (16) so that ̃ΠN(0, z) = ΠN NA(0, z) represents the average well-being in N when no agent is adopt- ing A in this region, whereas ̃ΠN(1, z) = ΠN A(1, z) represents the opposite case. The inter- pretation is analogous for region S. The following proposition applies: 1 3 Adoption Gaps of Environmental Adaptation Technologies with… Adoption Gaps of Environmental Adaptation Technologies with… 325 Fig. 6 There exist a global attrac- tor, corresponding to the full adoption scenario (1, 1). There is also a repeller in the interior of the bottom side of Q Fig. 6 There exist a global attrac- tor, corresponding to the full adoption scenario (1, 1). There is also a repeller in the interior of the bottom side of Q Proposition 2 If dN , dS , f S , f N > 0 , then: Proposition 2 If dN , dS , f S , f N > 0 , then: (a) for agents in N, the non-adoption stationary state (0, 0) Pareto-dominates all other stationary states in Q , when they exist, with 0 ≤x < 1 and 0 ≤z ≤1 . Equivalently, ΠN(0, 0) > ΠN(x, z) for every (x, z) ≠(0, 0) with x and z such that 0 ≤x < 1 and 0 ≤z ≤1. (b) for agents in S, the non-adoption stationary state (0, 0) Pareto-dominates all other stationary states in Q , when they exist, with 0 ≤x ≤1 and 0 ≤z < 1 . Equivalently, ΠS(0, 0) > ΠS(x, z) for every (x, z) ≠(0, 0) with x and z such that 0 ≤x ≤1 and 0 ≤z < 1. 3.3 Well‑Being Analysis (c) for agents in both regions N and S, the non-adaptation stationary state (0, 0) Pareto- dominates all the other stationary states (among these, also the full adoption station- ary state (1, 1)), when the efficiency of strategy A , net of domestic public effects, is lower than local autonomous environmental quality. Equivalently, Πj(0, 0) > Πj(x, z) for every (x, z) ≠(0, 0) when E j > pj−dj eCj −1 , with j = N, S. Fig. 6 There exist a global attrac- tor, corresponding to the full adoption scenario (1, 1). There is also a repeller in the interior of the bottom side of Q 3.4 Environmental Dumping At the centre of debates of both environmental and development economists, environmen- tal dumping is the phenomenon for which an economic activity in a more developed coun- try results in the disproportionate degradation of the environment of a less developed coun- try. Some scholars even argued that policies targeted to improve environmental quality in more developed countries lead to increased pollution in less developed ones. For instance, scholars investigating the Pollution Haven Effect5 maintain that carbon taxes and stricter environmental regulation are a push factor for firms, which offshore to less developed countries with laxer environmental regulation. Opponents of this theory argue that interna- tional trade and offshoring incentivise less developed countries to raise their environmental standards and thus help tackling the problem of environmental degradation. The analysis of the North American Free Trade Agreement performed by Gallagher (2000) seems to partly support both claims: Mexican firms reduced their emission intensity following the agree- ment, yet overall emissions increased due to the relatively lower Mexican standards with respect to the US ones. Since CO2 emissions are a public bad (their negative effects affect the whole world), this increased pollution might have damaged more developed countries, as well. We here investigate this hypothesis, for which shifting environmental burden from one country to the other might worsen the well-being of all agents. More precisely, our model allows to study the adoption dynamics of an environmental maladaptation technology with negative public effects and which asymmetrically degrades the environmental quality indi- cator of one of the two regions. We here discuss what happens when an exogenous factor (e.g. a new policy) decreases domestic effect dN in N, whereas it raises the foreign effect f S in S. This is the case of a green tax or policy in the more developed region which decreases the domestic effect on the local resource but increases the foreign effect on the resource of the other region, further degrading it. By recalling the payoff differentials (4), we may per- form a simple comparative dynamics analysis, noting that a smaller value of dN improves the environmental quality in N and decreases the well-being differential of adopters of the maladaptation technology. This leads to a reduction in the share of adopters in N and the ensuing reduction in environmental degradation. Proof See Mathematical appendix A ◻ By the above proposition and by virtue of Sect. 3.2, it is easy to check that when the stationary state (0, 0) is locally attractive, then it Pareto-dominates all others. Further- more, the non-adoption stationary state (0, 0) may Pareto-dominate the stationary state (1, 1) (in both regions) even if (1, 1) is the only attractive stationary state (see Fig. 6), provided that E N and E S are sufficiently high. In such case, the adoption of maladap- tation technologies in both regions reduces the well-being of agents as system moves from the repulsive non-adoption state (0, 0) to the attractive full adoption state (1, 1). One could also check that if (0, 0) does not Pareto-dominate all other stationary states (in both N and S), then the dynamics (5) is trivial, i.e. ̇x and ̇z are always positive in Q . In such case, the stationary state (1, 1) is globally attractive and Pareto-dominates any other possible state (x, z) in N and S. 1 3 A. Antoci et al. 326 Remark From the well-being analysis above, in the context represented in Fig. 1, every agent, from each region, achieves its highest level of well-being in (0, 0). Therefore, only one of the four attractive vertices yields the maximum level of well-being. Furthermore, the lowest level of well-being is achieved in (1, 1), whereas intermediate levels are reached in (0, 1) and (1, 0). 5 See Copeland and Scott Taylor (2004) for a definition of the concept and its differences with the slightly similar Pollution Haven Hypothesis. 3.4 Environmental Dumping However, an increase in the foreign effect f S on the local environmental indicator of S increases the payoff differential for adopters, incentivising more agents to adopt the maladaptation technology. A higher share of adop- ters in S, i.e. a greater z, would then increase the public effect for agents in N. The overall well-being effects for agents in N cannot be assessed a priori. If the reduction in the domes- tic effect dN is sufficiently large with respect to the increase in f S , it might counterbalance the additional degradation deriving from more adopters in the S region, who emit the for- eign effect f N affecting the environmental quality in region N. Vice versa, if the domestic 1 3 Adoption Gaps of Environmental Adaptation Technologies with… 327 effect is weaker with respect to the increased adoption induced in the foreign region, then the well-being of N decreases as a consequence of the exogenous change.i effect is weaker with respect to the increased adoption induced in the foreign region, then the well-being of N decreases as a consequence of the exogenous change.i A graphical illustration is provided by Figs. 3 and 4. In the former figure, only the Pareto-dominant non adoption state (0, 0) and the N-adoption state (1, 0) are attractive. However, a green policy affecting the value of dN and f S may cause the stationary state (1, 0) to lose attractiveness and the Pareto-dominated state (1, 1) to become attractive (see Sect. 3.2), as represented in the latter figure. This implies that, depending on the initial condition, a green policy as previously described may push the system towards a Pareto- dominated state. This analysis highlights that environmental policies in a more developed region may have either a positive or a negative effect for its agents, depending on the feed- back effects from agents in the less developed region. 4 Technologies with Positive Public Effects We now study the case in which all public effects of the environmental adaptation tech- nology are positive, that is: dN , dS , f S , f N < 0 . This case describes the adoption dynam- ics of a mitigation technology, which thus improves environmental quality for all agents. An instance relating to household mitigation strategies is home insulation, as it reduces the energy demand of the residents thus also stimulating lower (polluting) production (see Gupta and Gregg 2012; Hallegatte 2009, for other instances of adaptation technolo- gies with mitigation features). If we think of the agents as firms, instances of such tech- nologies might be the installation of a water treatment plant on a common water basin or, equivalently, of a technology which reduces emissions or waste water usage. Other exam- ples might draw from businesses dealing with the management of common environmental resources, such as fisheries or forestries (Olson 1965). i Analytically, the opposite of inequalities (7a) and (7b) hold, that is: (17a) 𝜕ΔΠN(x, z) 𝜕x = pNdN ( E N + PN )( E N + pN + PN ) < 0 (17b) 𝜕ΔΠS(x, z) 𝜕z = pSdS ( E S + PS )( E S + pS + PS ) < 0 (17b) 𝜕ΔΠS(x, z) 𝜕z = pSdS ( E S + PS )( E S + pS + PS ) < 0 (17b) Inequalities (17a) and (17b) describe payoff configurations of strategies A and NA in N and S, respectively, similar to those of the “elitist” narratives in (Antoci et al. 2018). Since the well-being differential of adopting strategy A decreases with the share of adopters, strategy A yields the highest payoffs when only a minority of agents adopts it. As strategy A diffuses, so the incentive to adopt it decreases, to the point that agents become indifferent toward the technology. As we will see, the presence of such a property in a region is neces- sary in order to have coexistence of strategies in such a region. 4.1 Dynamic Regimes In the common water basin example, install- ing (or contributing to the installation of) a new water treatment plant is less useful for a firm if the quality of the water is already guaranteed by the presence of treatment plants funded by other firms. In addition, we remark that if the autonomous environmental quality E is sufficiently high in N and S, then the well-being differential is always negative, i.e. ̇x < 0 and ̇z < 0 , leading agents to drop the mitigation technology and shift from A to NA . In this case, the autonomous level of environmental quality is so high that no agent finds it convenient to increase it further by an amount equal to the private effect pj , with j = 0, 1 . This might also be due to the inefficiency of the mitigation technology (a low value of pj eCj −1 ). In formal terms, we may say that lines (6a) and (6b) move downwards if the autono- mous environmental quality for region N or S is higher. For sufficiently high values of E N and E S or for sufficiently low values of pN and pS , we have that ̇x < 0 and ̇z < 0 , respec- tively, hold for every value of x and z. The reverse applies when E N and E S are sufficiently low or pN and pS sufficiently high.i N in this case ̇x > 0 below line (6a), whereas ̇x < 0 above it. Analogously, ̇z > 0 below line (6b), whereas ̇z < 0 above it. In contrast to the previous case, now the adoption dynamics is not self-reinforcing: more specifically, the incentive to adopt the environmental mitigation technology decreases if the share of agents adopting the technology in either group increases. This is the well known free riding problem, for which agents are not willing to contribute to a public good and would rather benefit from the contributions of others with- out paying the cost of their own contribution. In addition, the concavity of the well-being function with respect to the environmental quality accentuates the effect, as it makes any further improvement of the environment less desirable. Since the returns from the mitiga- tion technology decrease with the share of adopters while the cost is constant, we may see why this context favours coexistence between strategies A and NA . 4.1 Dynamic Regimes This might also be due to the inefficiency of the mitigation technology (a low value of pj eCj −1 ). In formal terms, we may say that lines (6a) and (6b) move downwards if the autono- mous environmental quality for region N or S is higher. For sufficiently high values of E N and E S or for sufficiently low values of pN and pS , we have that ̇x < 0 and ̇z < 0 , respec- tively, hold for every value of x and z. The reverse applies when E N and E S are sufficiently low or pN and pS sufficiently high. Fig. 7 The non-adoption station- ary state (0, 0) is globally attrac- tive, whereas the full adoption one (1, 1) is repulsive A. Antoci et al. 328 Fig. 7 The non-adoption station- ary state (0, 0) is globally attrac- tive, whereas the full adoption one (1, 1) is repulsive Fig. 7 The non-adoption station- ary state (0, 0) is globally attrac- tive, whereas the full adoption one (1, 1) is repulsive in this case ̇x > 0 below line (6a), whereas ̇x < 0 above it. Analogously, ̇z > 0 below line (6b), whereas ̇z < 0 above it. In contrast to the previous case, now the adoption dynamics is not self-reinforcing: more specifically, the incentive to adopt the environmental mitigation technology decreases if the share of agents adopting the technology in either group increases. This is the well known free riding problem, for which agents are not willing to contribute to a public good and would rather benefit from the contributions of others with- out paying the cost of their own contribution. In addition, the concavity of the well-being function with respect to the environmental quality accentuates the effect, as it makes any further improvement of the environment less desirable. Since the returns from the mitiga- tion technology decrease with the share of adopters while the cost is constant, we may see why this context favours coexistence between strategies A and NA . Indeed, as more and more agents adopt the mitigation technology, the well-being differential of such strategy falls to zero, allowing for a stationary state in which in the same region there are agents adopting strategy A and agents adopting NA . Fig. 7 The non-adoption station- ary state (0, 0) is globally attrac- tive, whereas the full adoption one (1, 1) is repulsive 4.1 Dynamic Regimes We first note that if dN , dS , f S , f N < 0 , both the straight lines (6a) (where ̇x = 0 ) and (6b) (where ̇z = 0 ) have negative slope. Differently from the case with negative public effects, 1 3 328 A. Antoci et al. in this case ̇x > 0 below line (6a), whereas ̇x < 0 above it. Analogously, ̇z > 0 below line (6b), whereas ̇z < 0 above it. In contrast to the previous case, now the adoption dynamics is not self-reinforcing: more specifically, the incentive to adopt the environmental mitigation technology decreases if the share of agents adopting the technology in either group increases. This is the well known free riding problem, for which agents are not willing to contribute to a public good and would rather benefit from the contributions of others with- out paying the cost of their own contribution. In addition, the concavity of the well-being function with respect to the environmental quality accentuates the effect, as it makes any further improvement of the environment less desirable. Since the returns from the mitiga- tion technology decrease with the share of adopters while the cost is constant, we may see why this context favours coexistence between strategies A and NA . Indeed, as more and more agents adopt the mitigation technology, the well-being differential of such strategy falls to zero, allowing for a stationary state in which in the same region there are agents adopting strategy A and agents adopting NA . In the common water basin example, install- ing (or contributing to the installation of) a new water treatment plant is less useful for a firm if the quality of the water is already guaranteed by the presence of treatment plants funded by other firms. In addition, we remark that if the autonomous environmental quality E is sufficiently high in N and S, then the well-being differential is always negative, i.e. ̇x < 0 and ̇z < 0 , leading agents to drop the mitigation technology and shift from A to NA . In this case, the autonomous level of environmental quality is so high that no agent finds it convenient to increase it further by an amount equal to the private effect pj , with j = 0, 1 . 4.1 Dynamic Regimes Indeed, as more and more agents adopt the mitigation technology, the well-being differential of such strategy falls to zero, allowing for a stationary state in which in the same region there are agents adopting strategy A and agents adopting NA . In the common water basin example, install- ing (or contributing to the installation of) a new water treatment plant is less useful for a firm if the quality of the water is already guaranteed by the presence of treatment plants funded by other firms. In addition, we remark that if the autonomous environmental quality E is sufficiently high in N and S, then the well-being differential is always negative, i.e. ̇x < 0 and ̇z < 0 , leading agents to drop the mitigation technology and shift from A to NA . In this case, the autonomous level of environmental quality is so high that no agent finds it convenient to increase it further by an amount equal to the private effect pj , with j = 0, 1 . This might also be due to the inefficiency of the mitigation technology (a low value of pj eCj −1 ). In formal terms, we may say that lines (6a) and (6b) move downwards if the autono- mous environmental quality for region N or S is higher. For sufficiently high values of E N and E S or for sufficiently low values of pN and pS , we have that ̇x < 0 and ̇z < 0 , respec- tively, hold for every value of x and z. The reverse applies when E N and E S are sufficiently low or pN and pS sufficiently high.i fi We find that the following proposition characterises the adoption dynamics when: dN , dS f S f N < 0 fi We find that the following proposition characterises the adoption dynamics when: dN , dS , f S , f N < 0. 1 3 Adoption Gaps of Environmental Adaptation Technologies with… 329 Proposition 3 Under the assumption that dN , dS , f S , f N < 0, the system (5) has the follow- ing features: Proposition 3 Under the assumption that dN , dS , f S , f N < 0, the system (5) has the follow- ing features: (a) Every trajectory of the system approaches a stationary state. Fig. 8 The full adoption station- ary state (1, 1) is globally attrac- tive, whereas the non-adoption one (0, 0) is repulsive Proof See Mathematical appendix A Fig. 10 The internal stationary state is a saddle, whereas the asymmetric states (0, 1) and (1, 0) are attractors. The non- adoption state (0, 0) and the full adoption one (1, 1) are repellers Fig. 9 The internal stationary state is an attractor. There are also three saddles on the sides and three repellers on the vertices (0, 0), (1, 0), (1, 1) 4.1 Dynamic Regimes (b) When the stationary state (0, 0) is attractive (see Sect. 3.2), then it is globally attractive, i.e. there is no other attractive stationary state (see Fig.7). (c) When the stationary state (1, 1) is attractive (see Sect. 3.2), then it is globally attractive (see Fig. 8). (d) If there is no stationary state in the interior of Q , then there exists only one attractive stationary state in the boundary of Q ; it may either be one of the vertices or lie on the interior of the edges of Q.f (e) If dNdS −f Nf S > 0 , i.e. the domestic effects are larger than the foreign ones, the sta- tionary state in the interior of Q (in which both strategies are played in both regions) is globally attractive, when it exists (see Fig. 9).f (f) If dNdS −f Nf S < 0, i.e. the domestic effects are smaller than the foreign ones, the stationary state in the interior of Q is a saddle point, when it exists. In addition, there exist two attractive stationary states lying in the edges of Q : they may be the vertices (0, 1) and (1, 0) or lie in the interior of the edges Q (see Figs.10, 11, 12 and 13).f (g) If pN = pS = 0 (i.e. the private effect of strategy A is 0 in both regions), then non-adop- tion is individually convenient for all agents: ΠN NA > ΠN A and ΠS NA > ΠS A , whatever the values of x and z are. This implies that ̇x < 0 and ̇z < 0 always hold and consequently (0, 0) is globally attractive (the classical free-riding problem arises for public goods provision). Proof See Mathematical appendix A ◻ ◻ 1 3 Fig. 8 The full adoption station- ary state (1, 1) is globally attrac- tive, whereas the non-adoption one (0, 0) is repulsive 1 3 Fig. 8 The full adoption station- ary state (1, 1) is globally attrac- tive, whereas the non-adoption one (0, 0) is repulsive 3 3 A. Antoci et al. 330 Fig. 9 The internal stationary state is an attractor. There are also three saddles on the sides and three repellers on the vertices (0, 0), (1, 0), (1, 1) Fig. 10 The internal stationary state is a saddle, whereas the asymmetric states (0, 1) and (1, 0) are attractors. 4.1 Dynamic Regimes The non- adoption state (0, 0) and the full adoption one (1, 1) are repellers Fig. 10 The internal stationary state is a saddle, whereas the asymmetric states (0, 1) and (1, 0) are attractors. The non- adoption state (0, 0) and the full adoption one (1, 1) are repellers Fig. 11 The internal staationary state is a saddle and both the non-adoption (0, 0) and the full adoption (1, 1) states are repel- lers. Two attractors lie on the interiors of the bottom side and of the top side of Q 1 3 Adoption Gaps of Environmental Adaptation Technologies with… 331 Fig. 12 The internal station- ary state is a saddle and both the non-adoption (0, 0) and the full adoption (1, 1) states are repellers. Two attractors lie on the interiors of the side to the left and of the side to the right of Q Fig. 12 The internal station- ary state is a saddle and both the non-adoption (0, 0) and the full adoption (1, 1) states are repellers. Two attractors lie on the interiors of the side to the left and of the side to the right of Q 7 This occurs, for instance, when pN eCN −1 < E N < pN−dN−f N eCN −1 and pS eCS −1 < E S < pS−dS−f S eCS −1 hold. Indeed, in this case (0, 0) is attractive but is Pareto-dominated by (1, 1). 4.2 Well‑Being in the Context with Positive Externalities We now examine the average level of well-being in the two regions when all public effects are positive: dN , dS , f S , f N < 0 (see (15) and (16) in the previous section for a comparison). The following proposition applies: Proposition 4 Assume dN , dS , f S , f N < 0 . In such context, it holds: Proposition 4 Assume dN , dS , f S , f N < 0 . In such context, it holds: Proposition 4 Assume dN , dS , f S , f N < 0 . In such context, it holds: (a) The stationary state (0, 0) is Pareto-dominated (in both regions) by any attractive stationary state with x > 0 and/or z > 0 . When (0, 0) is attractive6, it may be Pareto- dominated by other stationary states.7 (a) The stationary state (0, 0) is Pareto-dominated (in both regions) by any attractive stationary state with x > 0 and/or z > 0 . When (0, 0) is attractive6, it may be Pareto- dominated by other stationary states.7 (a) The stationary state (0, 0) is Pareto-dominated (in both regions) by any attractive stationary state with x > 0 and/or z > 0 . When (0, 0) is attractive6, it may be Pareto- dominated by other stationary states.7 (b) The stationary state (1, 1) Pareto dominates (in both regions) any other stationary state when it is attractive (remember that, in such case, no other stationary state can be attractive). Furthermore, even if it is unstable, it Pareto dominates the stationary state in the interior of Q , when it exists. (b) The stationary state (1, 1) Pareto dominates (in both regions) any other stationary state when it is attractive (remember that, in such case, no other stationary state can be attractive). Furthermore, even if it is unstable, it Pareto dominates the stationary state in the interior of Q , when it exists. 6 As stated in Proposition 3, point (b), in this case (0, 0) is globally attractive. N S 6 As stated in Proposition 3, point (b), in this case (0, 0) is globally attractive. Proof See “Mathematical Appendix A” ◻ Remark From the well-being analysis above, in the context in which the stationary state (x, z) in the interior of Q is attractive, we have that (x, z) Pareto-dominates (0, 0) but is Pareto-dominated by (1, 1); however, the latter stationary state cannot be reached because it is not attractive. These results are reversed with respect to the case with negative public effects. Indeed, in the previous case (0, 0) Pareto-dominates all stationary states in most cases, although it is not attractive. The selfish nature of the maladaptation technology leads agents towards p p g y 7 This occurs, for instance, when pN eCN −1 < E N < pN−dN−f N eCN −1 and pS eCS −1 < E S < pS−dS−f S eCS −1 hold. Indeed, in this case (0, 0) is attractive but is Pareto-dominated by (1, 1). 3 A. Antoci et al. 332 adoption, although it results in a lower level of well-being for all. The technology is thus over-adopted with respect to the Pareto-optimum. With positive public effects, we have that (0, 0) is Pareto-dominated by all other stationary states whereas (1, 1) Pareto-dominates them when it is attractive. All agents benefit from the mitigation technology adopted by others, but they are less willing to pay its cost as they do not internalise the well-being of others. In this case, the technology is under-adopted, as the full adoption scenario would be the Pareto optimum. This last result is in line with the results by Shogren and Crocker (1991). Fig. 13 In this case, one attractor is the asymmetric state (0, 1) and another lies on the interior of the right-hand of square Q . The internal stationary state is a saddle and both the non-adoption (0, 0) and the full adoption (1, 1) states are repellers Fig. 13 In this case, one attractor is the asymmetric state (0, 1) and another lies on the interior of the right-hand of square Q . The internal stationary state is a saddle and both the non-adoption (0, 0) and the full adoption (1, 1) states are repellers adoption, although it results in a lower level of well-being for all. The technology is thus over-adopted with respect to the Pareto-optimum. With positive public effects, we have that (0, 0) is Pareto-dominated by all other stationary states whereas (1, 1) Pareto-dominates them when it is attractive. Proof See “Mathematical Appendix A” All agents benefit from the mitigation technology adopted by others, but they are less willing to pay its cost as they do not internalise the well-being of others. In this case, the technology is under-adopted, as the full adoption scenario would be the Pareto optimum. This last result is in line with the results by Shogren and Crocker (1991). adoption, although it results in a lower level of well-being for all. The technology is thus over-adopted with respect to the Pareto-optimum. With positive public effects, we have that (0, 0) is Pareto-dominated by all other stationary states whereas (1, 1) Pareto-dominates them when it is attractive. All agents benefit from the mitigation technology adopted by others, but they are less willing to pay its cost as they do not internalise the well-being of others. In this case, the technology is under-adopted, as the full adoption scenario would be the Pareto optimum. This last result is in line with the results by Shogren and Crocker (1991). Fig. 13 In this case, one attractor is the asymmetric state (0, 1) and another lies on the interior of the right-hand of square Q . The internal stationary state is a saddle and both the non-adoption (0, 0) and the full adoption (1, 1) states are repellers 5 Discussion and Conclusions In this work, we studied the case of two regions whose agents may adopt an environmental adaptation technology which yields a private benefit to the adopter, while also transferring a negative or positive externality both to agents in the same region and to agents in the other one. We defined same-region externalities as domestic public effects and cross-region externalities as foreign public effects. We excluded altruistic consideration on the part of agents towards either same-region and cross-region agents. In other terms, we assumed that the actions of agents are only driven by self-interest considerations. In addition, we posed no restriction on the initial distribution of the system, so that it might initially lie on a point which is characterised by a positive share of adopters in both regions, even if in that point the dynamics of the system converges to the non-adoption state (0, 0). We preferred not to make any assumption on the initial distribution, which could be affected for instance by technological shocks or by early adoption of agents during a learning phase in which the payoff of each strategy is not already known. The model here proposed is very broad, so that a complete analysis of all possible specifications is beyond the scope of this paper. Instead, we focused on two salient characterisations. On the one hand, we analysed the case of a maladaptation technology, whose domestic and foreign public effects are both nega- tive. In this case, an adopter shifts the environmental load to agents from both regions. A common example of this kind of technologies is air conditioning (Lundgren and Kjellstrom 2013). On the other hand, we analysed the case of a mitigation technology, whose domestic 1 3 3 1 Adoption Gaps of Environmental Adaptation Technologies with… 333 and foreign public effects are both positive. In this case, each adopter is improving the well-being of agents from both regions. In analogy with the previous example, we may think of home insulation, as it allows each household to reduce both heating and air condi- tioning, benefiting the environment on a global scale. Our results show that for the malad- aptation technology the social optimum is represented by the non-adoption scenario, unless the efficiency of the technology is extremely high (greater than the autonomous level of environmental quality). However, Pareto-dominated states may be reached, because agents do not internalise the externalities of the technology. 5 Discussion and Conclusions In this case, we talk of over-adoption of the maladaptation technology. The reverse occurs with a mitigation technology, which would have a full adoption scenario as its Pareto-optimum. However, an intermediate state (in which only some agents are adopters) is typically reached, since the returns on adop- tion decrease for each additional adopter. Also in this case, agents do not take into account the (positive) externalities of adoption on other agents, this time leading to under-adoption of the technology with respect to the Pareto-dominant state. Finally, under the hypothesis of a maladaptation technology, with negative public effects, we analysed the effects of a green policy which results in environmental dumping. We represented a green policy as one reducing domestic emissions in the more developed region while increasing emis- sions toward the less developed region. Although it is intuitive that the agents from the less developed region would be worse off in this case, we showed that the implications for agents from the more developed region are not straightforward. Indeed, according to the relative magnitude of the domestic and the foreign effect of the policy, the well-being of agents from the more developed region could decrease as well, with the system reaching a Pareto-dominated state that was not previously attractive.i This last result is particularly interesting, although its plausibility should be verified by further research. Indeed, instances of such negative feedbacks could provide greater insight on the cost-benefit analysis of many maladaptive strategies or policies available to the more developed regions. In addition, further research should try to map the speci- fications which are not illustrated in this work. Interesting dynamics could arise, for example, if the public effects had different signs according to whether they are domestic or foreign. In particular, a case in which all domestic public effects are null or positive, while all foreign effects are negative would depict a situation in which all adopters shift the environmental burden to foreign agents, although they increase the well-being of same-region individuals. In this case, it is not intuitive which state the system would reach. Another relevant case would be represented by technological differences between the two regions allowing the agents from the more developed region to adopt a mitiga- tion technology, whereas agents in the less developed region could only adopt a mal- adaptation technology. Appendix A: Proofs of the Propositions in Text Proof of Proposition 1 The proof of point (b) is straightforward and follows immediately from the local stability analysis (which can be found in Mathematical appendix B). To prove point (a) we have to show that limit cycles cannot exist (see e.g. Lefschetz 1963, pp. 230 ff). This is obviously the case when the internal stationary state (x, z) , with 0 < x, z < 1 , does not exist or, if it does, is a saddle point. If (x, z) is a source, then dNdS −f Sf N > 0 (see (19b) in appendix B), that is the straight line (6a) (where ̇x = 0 ) crosses from above the straight line (6b) (where ̇z = 0 ). In such case, it is easy to see that the regions in Q where ̇x and ̇z have the same sign are positively invariant, so that no oscillatory behaviour of tra- jectories can occur. This implies, by the Poincaré-Bendixson Theorem, that any trajectory starting in Q approaches a stationary state. This concludes the proof of the proposition. Proof of Proposition 2 To prove point (a) of Proposition 2, we have to show that the average payoff in N, evaluated at (0, 0), is higher than at any point (̄x, ̄z) along the line ΔΠN(x, z) = 0 (where ̇x = 0 ) and along the side Qx=0 . The average level of well-being in (0, 0) is: ̃ΠN(0, 0) = ΠN NA(0, 0) = ln E N ̃ΠN(0, 0) = ΠN NA(0, 0) = ln E N Let us now take a point (̄x, ̄z) ∈{ΔΠN(x, z) = 0} . We have that both strategies yield the same level of well-being: ΠN A(̄x, ̄z) = ΠN NA(̄x, ̄z) , which implies: Let us now take a point (̄x, ̄z) ∈{ΔΠN(x, z) = 0} . We have that both strategies yield the same level of well-being: ΠN A(̄x, ̄z) = ΠN NA(̄x, ̄z) , which implies: ΠN(̄x, ̄z) = ΠN NA( _x, ̄z) = ln ( E N −dN ⋅ _x −f N ⋅̄z ) Therefore, if ̄x and/or ̄z > 0 , it follows that: ΠN(0, 0) > ΠN(̄x, ̄z) . 5 Discussion and Conclusions Well-being analysis could highlight which region is relatively more affected by the negative externalities and which state is more likely to be reached. All similar research directions, focusing on translating real phenomena and dynamics into the model, would provide a fine extension to this work and a contribution to the understanding of the relationship between regions and countries at different stages of development and their environmental quality. 3 1 A. Antoci et al. 334 Appendix A: Proofs of the Propositions in Text This means that the aver- age well-being in the non-adoption state (0, 0) is higher than in any stationary state in the interior of Q and in any stationary state in the interior of the sides Qz=h ( h = 1, 2 ). Further- more, it is easy to check that (0, 0) always Pareto-dominates any stationary state with z > 0 in the side Qx=0 . The proof of point (b) can be obtained applying the same arguments. In order to prove point (c), we now show that, for agents in region N, (0, 0) Pareto-dom- inates any stationary state in the side Qx=1 if E N > pN−dN eCN −1 . It can be easily verified that (1, 0) always Pareto-dominates any other stationary state in the side Qx=1 . Therefore, we simply have to compare well-being in (0, 0) with the one in (1, 0). By very simple computations, we obtain that, if E N > pN−dN eCN −1 , then (0, 0) Pareto-dominates (1, 0). With similar arguments, it is easy to check that (1, 1) is Pareto-dominated by all the other stationary states when E N > pN−dN eCN −1 . To prove that analogous results hold for the well-being of region S, it suffices to apply the same arguments. Proof of Proposition 3 The proof of point (b) is straightforward and follows immediately from graphical analysis: if (0, 0) is attractive, then it must lie above the straight lines (6a) and (6b). Consequently, in the interior of Q , it holds ̇x < 0 and ̇z < 0 , which implies the global attractiveness of (0, 0). With similar arguments, point (c) can be proved. In order to prove point (e), it suffices to check that when dN∕f N > f S∕dS , the internal stationary state is locally attractive (see Proposition 6). Graphical analysis then allows to see that no other attractive stationary state can exist. It remains to show that limit cycles cannot exist. To do so, we note that the straight line (6a), along which ̇x = 0 , crosses the straight line (6b), along which ̇z = 0 , from above. Appendix A: Proofs of the Propositions in Text In such case, the regions of Q where ̇x and ̇z have opposite signs are positively invariant; this implies that no oscillatory behaviour of trajec- tories may occur and consequently that the internal stationary state is globally attractive by 1 Adoption Gaps of Environmental Adaptation Technologies with… 335 the Poincaré-Bendixson Theorem. We now prove point (f): if dN∕f N < f S∕dS , the internal stationary state is a saddle point (see Sect. 3.2); consequently, no limit cycle may exist. Furthermore, we note that the straight line (6a) crosses the straight line (6b) from below. In such case, the regions of Q where ̇x and ̇z have opposite sign are positively invariant and, in each of these regions, the trajectories approach a stationary state lying on the boundary of Q . Finally, the proof of points (a), (d) and (g) is straightforward. Proof of Proposition 4 To prove point (a) of the proposition, we first consider the average well-being in N, which in (0, 0) is equal to: Proof of Proposition 4 To prove point (a) of the proposition, we first consider the average well-being in N, which in (0, 0) is equal to: ̃ΠN(0, 0) = ΠN NA(0, 0) = ln E N Let us now consider a point (̄x, ̄z) ∈Q . If (̄x, ̄z) is a stationary state belonging to the curve ΔΠN(x, z) = 0 , then it holds that ΠN A(̄x, ̄z) = ΠN NA(̄x, ̄z) , and consequently we have: Let us now consider a point (̄x, ̄z) ∈Q . If (̄x, ̄z) is a stationary state belonging to the curve ΔΠN(x, z) = 0 , then it holds that ΠN A(̄x, ̄z) = ΠN NA(̄x, ̄z) , and consequently we have: ΠN(̄x, ̄z) = ΠN NA(̄x, ̄z) = ln ( E N −dN ⋅̄x −f N ⋅̄z ) Therefore, since dN , dS , f S , f N < 0 , if either ̄x or ̄z > 0 , we have that: ΠN(0, 0) < ΠN(̄x, ̄z) . Thus, average payoff in (0, 0) is lower than in any stationary state in the interior of Q and in any stationary state in the interior of the sides Qz=k ( k = 1, 2 ). Furthermore, it is easy to check that (0, 0) is always Pareto-dominated by any stationary state in the side Qx=0 . Appendix A: Proofs of the Propositions in Text It remains to prove that (0, 0) is Pareto-dominated by any attractive stationary state in the side Qx=1 . Easy algebraic manipulations show that ΠN(0, 0) < ΠN(1, 1) if and only if E N < pN−dN−f N eCN −1 . The latter condition is always satisfied if (1, 1) is attractive (see Sect. 3.2). In the same way, it can be checked that ΠN(0, 0) < ΠN(1, 0) when (1, 0) is attractive. Finally, it is left to prove that (0, 0) is Pareto-dominated by any attractive stationary state (1, z) lying in the interior of Qx=1 . As already seen above, the well-being in (0, 0) is lower than in any stationary state, so that ΠN(0, 0) = ΠN NA(0, 0) < ΠN NA(1, ̄z) . Furthermore, we note that if (1, z) is attractive, then the curve ΔΠN(x, z) = 0 must lie on the right of it (see Proposition 5); consequently, on the left of ΔΠN(x, z) = 0 , it holds that ΔΠN(x, z) > 0 . This implies that ΠN NA(1, ̄z) < ΠN A(1, ̄z) . Therefore, ΠN(0, 0) < ΠN(1, ̄z) , being ΠN(1, ̄z) = ΠN A(1, ̄z) . The corre- sponding results for S can be proved following the same steps. To check the remaining part of point (a), we simply have to solve the inequality ΠN(0, 0) < ΠN(1, 1) and draw from the stability results in Sect. 3.2 about the stationary state (0, 0). The proof of point (b) follows very similar steps. Therefore, since dN , dS , f S , f N < 0 , if either ̄x or ̄z > 0 , we have that: ΠN(0, 0) < ΠN(̄x, ̄z) . Thus, average payoff in (0, 0) is lower than in any stationary state in the interior of Q and in any stationary state in the interior of the sides Qz=k ( k = 1, 2 ). Furthermore, it is easy to check that (0, 0) is always Pareto-dominated by any stationary state in the side Qx=0 . It remains to prove that (0, 0) is Pareto-dominated by any attractive stationary state in Appendix B: Stability Properties of the Stationary States We here study the stability of the stationary states, in order to understand toward which the system may converge. Indeed, the attractive states are of particular interest, as they are the only limit sets that can actually be reached by the system. We recall that the condition for a 3 1 A. Antoci et al. 336 stationary state to be attractive is that both the eigenvalues of the Jacobian matrix evaluated on it are negative.8. stationary state to be attractive is that both the eigenvalues of the Jacobian matrix evaluated on it are negative.8. 8 If the eigenvalues are both positive, then the state is repulsive and cannot be reached by system (unless it coincides with its initial condition). If they have opposite signs, instead, the state is a saddle and can only be reached if the initial condition of the system lies on its stable branch. Appendix B.1: Stability Analysis of the Vertices In order to assess the stability properties of the vertices of Q , we derive the Jacobian matrix of the system (5) evaluated at the stationary state (x, z) = (i, k) , i = 0, 1 and k = 0, 1: (18) ( (1 −2i)ΔΠN(i, k) 0 0 (1 −2k)ΔΠS(i, k) ) (18) which has the eigenvalues (1 −2i)ΔΠN(i, k) and (1 −2k)ΔΠS(i, k) , in the direction of Qz=0 and Qx=0 , respectively. The analysis of the sign of the eigenvalues allows us to illustrate the stability properties of the stationary states (0, 0), (0, 1), (1, 0), (1, 1). It is easy to check that: In (0, 0) both the eigenvalues are strictly negative if: In (0, 0) both the eigenvalues are strictly negative if: E j > pj eCj −1 with j = N, S In (0, 1), the eigenvalue in direction of Qz=1 is strictly negative if: In (0, 1), the eigenvalue in direction of Qz=1 is strictly negative if: E N −f N > pN eCN −1 whereas the eigenvalue in direction of Qx=0 is strictly negative if: whereas the eigenvalue in direction of Qx=0 is strictly negative if: E S −dS < pS eCS −1 In (1, 0), the eigenvalue in direction of Qz=0 is strictly negative if In (1, 0), the eigenvalue in direction of Qz=0 is strictly negative if: E N −dN < pN eCN −1 E N −dN < pN eCN −1 while the eigenvalue in direction of Qx=1 is strictly negative if: while the eigenvalue in direction of Qx=1 is strictly negative if: E S −f S > pS eCS −1 Finally, in (1, 1) the eigenvalues in direction of Qz=1 and Qx=1 are strictly negative if: E j −(dj + f j) < pj eCj −1 with j = N, S E j −(dj + f j) < pj eCj −1 with j = N, S 8 If the eigenvalues are both positive, then the state is repulsive and cannot be reached by system (unless it coincides with its initial condition). If they have opposite signs, instead, the state is a saddle and can only be reached if the initial condition of the system lies on its stable branch. 1 3 Adoption Gaps of Environmental Adaptation Technologies with… 337 Appendix B.2: Stability Properties of the Stationary States in the Interior of the Edges of Q Note that the attractiveness conditions of the stationary states belonging to the interior extreme of Qh=k which are the vertices of Q , have positive eigenvalues in direction of Qh=k. Note that the attractiveness conditions of the stationary states belonging to the interior of the edges of the square Q require that: Note that the attractiveness conditions of the stationary states belonging to the interior of the edges of the square Q require that: 𝜕ΔΠN(x, i) 𝜕x < 0 𝜕ΔΠS(i, z) 𝜕z < 0 Such conditions are never (always) satisfied in the context of technologies with negative (positive) public effects (see formulas (17a) and (17b)). So, in the context where technolo- gies have a negative public effect, the stationary states internal to the edges Qh=k , h = x, z and k = 0, 1 , are never attractive (they may be either saddle points or sources). Appendix B.2: Stability Properties of the Stationary States in the Interior of the Edges of Q Let us now consider the stability properties of the stationary states belonging to the interior of the edges of the square Q , i.e. those where both adoption choices coexist in N while all agents in S play the same strategy or vice versa. Proposition 5 The Jacobian matrix of the system (5) evaluated at the stationary states in the interior of the edges Qx=h (h = 0 , 1) is: (19) ( (1 −2h)ΔΠN(h, z) 0 z(1 −z) 휕ΔΠS(h,z) 휕x z(1 −z) 휕ΔΠS(h,z) 휕z ) (19) where z is the value of z at the stationary state, and has the eigenvalues: z(1 −z) 휕ΔΠS(h,z) 휕z (in direction of Qx=h) and (1 −2h)ΔΠN(h, z) (in direction of the interior of Q). The Jacobian matrix of the system (5) evaluated at the stationary states in the interior of the edges Qz=h (h = 0 , 1) is: where z is the value of z at the stationary state, and has the eigenvalues: z(1 −z) 휕ΔΠS(h,z) 휕z (in direction of Qx=h) and (1 −2h)ΔΠN(h, z) (in direction of the interior of Q). The Jacobian matrix of the system (5) evaluated at the stationary states in the interior of the edges Qz=h (h = 0 , 1) is: (20) ( x(1 −x) 휕ΔΠN(x,h) 휕x x(1 −x) 휕ΔΠN(x,h) 휕z 0 (1 −2h)ΔΠS(x, h) ) (20) where x is the value of x at the stationary state, and has the eigenvalues: x(1 −x) 휕ΔΠN(x,h) 휕x (in direction of Qz=h) and (1 −2h)ΔΠS(x, h) (in direction of the interior of Q). We remark that, given a stationary state in an edge Qh=k , h = x, z and k = 0, 1 , the sign of its eigenvalue in direction of Qh=k is negative if and only if the stationary states at the extreme of Qh=k which are the vertices of Q , have positive eigenvalues in direction of Qh=k. Note that the attractiveness conditions of the stationary states belonging to the interior of the edges of the square Q require that: We remark that, given a stationary state in an edge Qh=k , h = x, z and k = 0, 1 , the sign of its eigenvalue in direction of Qh=k is negative if and only if the stationary states at the extreme of Qh=k which are the vertices of Q , have positive eigenvalues in direction of Qh=k. Appendix B.3: Stability Properties of Stationary States in the Interior of Q Giulio Galdi gratefully acknowledges the scholarship received from the Department of International and Political Sciences of the University of Siena for the research activities conducted under the supervision of Simone Borghesi (PSR2017). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Appendix B.3: Stability Properties of Stationary States in the Interior of Q In this subsection we analyse the stability of stationary states in the interior of the square Q , in which a positive share of agents adopts each strategy in both regions. Proposition 6 The Jacobian matrix of the system (5) evaluated at a stationary state (x, z) in the interior of Q (i.e. 0 < x, z < 1) is: 3 338 A. Antoci et al. A. Antoci et al. 338 (21a) ( x(1 −x) 휕ΔΠN(x,z) 휕x x(1 −x) 휕ΔΠN(x,z) 휕z z(1 −z) 휕ΔΠS(x,z) 휕x z(1 −z) 휕ΔΠS(x,z) 휕z ) (21a) ( x(1 −x) 휕ΔΠN(x,z) 휕x x(1 −x) 휕ΔΠN(x,z) 휕z z(1 −z) 휕ΔΠS(x,z) 휕x z(1 −z) 휕ΔΠS(x,z) 휕z ) where the sign of the determinant of (21a) is equal to the sign of the expression: (21b) dNdS −f Sf N (21b) and the trace of (21a) is equal to: (21c) x(1 −x) 휕ΔΠN(x,z) 휕x + z(1 −z) 휕ΔΠS(x,z) 휕z = = x(1 −x) pNdN eCN ( E N+PN )2 + z(1 −z) pSdS eCS ( E S+PS )2 (21c) If expression (21b) is strictly negative, then the internal stationary state is a saddle (i.e. it is unstable). If it is positive, then the stationary state may be a repeller or an attractor. In the context in which expression (21b) is strictly positive, the condition dN , dS > 0 (< 0 ) is a sufficient condition for the repulsiveness (attractiveness) of the internal stationary state (see formula (21c)). Therefore, in the context where technologies have a negative public effect, the internal stationary state is never attractive (it may be either a saddle point or a source). Funding Open access funding provided by Università degli Studi di Sassari within the CRUI-CARE Agree- ment. The research of Angelo Antoci was supported by the University of Sassari (“Finanziamento straordi- nario una tantum per la ricerca 2020”). Giulio Galdi gratefully acknowledges the scholarship received from the Department of International and Political Sciences of the University of Siena for the research activities conducted under the supervision of Simone Borghesi (PSR2017). Funding Open access funding provided by Università degli Studi di Sassari within the CRUI-CARE Agree- ment. The research of Angelo Antoci was supported by the University of Sassari (“Finanziamento straordi- nario una tantum per la ricerca 2020”). Antoci A, Bellanca N, Galdi G (2018) At the relational crossroads: narrative selection, contamination, bio- diversity in trans-local contexts. J Econ Behav Organ 150:98–113 Antoci A, Russu P, Ticci E (2020) Modeling maladaptation in the inequality–environment nexus. J Econ Interact Coord, in press. https://doi.org/10.1007/s11403-020-00301-6 Barnett J, O’Neill S (2010) Maladaptation. Global Environ Change 2:211–213 Bird PJWN (1987) The transferability and depletability of externalities. J Environ Econ Manage 14:54–57 Börgers T, Sarin R (1997) Learning through reinforcement and replicator dynamics. J Econ Theory 77:1–14 Copeland BR, Scott Taylor M (2004) Trade, growth, and the environment. 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https://openalex.org/W2400962464	https://europepmc.org/articles/pmc5100731?pdf=render	English	null	A comparison of healthy infants and adults with respect to indirect microparticle activity and the parameters of thrombin generation test	Turkish journal of hematology	2,016	cc-by	1,334	Sağlıklı Süt Çocukları ve Erişkinlerin İndirekt Mikropartikül Aktivitesi ve Trombin Jenerasyon Parametrelerine Göre Karşılaştırılması Filiz Şimşek Orhon1, Nejat Akar2, Yonca Eğin2, Betül Ulukol1, Sevgi Başkan1 1Ankara University Faculty of Medicine, Department of Pediatrics, Divisions of Social Pediatrics, Ankara, Turkey 2Ankara University Faculty of Medicine, Department of Pediatrics, Divisions of Pediatric Molecular Genetics, Ankara, Turkey Physiologic concentrations of coagulation proteins gradually increase after birth [4]. Karlaftis et al. showed that procoagulant phospholipid activity was increased in neonates and decreased in children aged 1-16 years [5]. We show that the levels of indirect microparticle activity are increased in healthy adults as compared to healthy infants. This may suggest that aging is correlated to an increase in the indirect microparticle activity, and also possibly to its procoagulant and proinflammatory features. To the Editor, Microparticles express phospholipids and support thrombin generation, which increases with age [1,2]. In a recently published study, we showed age-dependent changes in thrombin generation parameters in a healthy infant population aged 1-24 months [3]. The aim of this present study was to compare the levels of both indirect microparticle activity and thrombin generation parameters of healthy infants from our recent study to those of a healthy adult population. The adult population consisted of medical students of the Ankara University School of Medicine. Blood was collected into tubes containing 1 mL of 0.109 M trisodium citrate. For indirect microparticle activity, plasma samples were studied using the STA-PROCOAG- PPL Kit (Diagnostica Stago Inc., Asnières sur Seine, France). Plasma samples were measured using thrombin generation kits, including a Thrombin Calibrator, PPP-Reagent 5 pM, and the FluCa-Kit (Diagnostica Stago). Thrombin generation curves were calculated using Thrombinoscope software (Thrombinoscope BV, Maastricht, the Netherlands). The following parameters were derived from the curves: lag time (LT, min), time to initiation of thrombin generation; endogenous thrombin potential (ETP, nmol/L/min), area under the thrombin generation curve; peak thrombin activity (peak, nmol/L); and time to peak thrombin generated (TTP, min). Statistical analysis was performed using Statistical Package for the Social Sciences 16. Thrombin generation is influenced by different variables like age, sex, body mass index, genetic factors, and acquired conditions [6,7]. In a previous study, the ETP values of children were found to be lower than those of adults [8]. Positive correlations were found for age versus thrombin generation parameters in calibrated automated thrombography in two recent studies [9,10]. We showed that ETP and peak levels were higher in adults as compared to infants. Thus, we suggest that ETP and peak levels, the main parameters of thrombin generation, increase gradually from infancy to adulthood. As for limitations, our adult group was not adequate for representing all ages of the adult population and there was a difference between the groups Table 1. Data on indirect microparticle activity and thrombin generation parameters of the study groups. Healthy Infants (n=85)* Healthy Adults (n=58)* p** Microparticle release time (s) 31.7±7.5 39.7±9.8 0.001 Lag time (min) 3.2±0.8 3.1±0.7 0.357 ETP (nmol/L/min) 1363.6±262.2 1691.5±378.1 0.001 Peak (nmol/L) 256.5±79.7 358.4±79.9 0.001 TTP (min) 6.7±1.7 5.4±0.9 0.001 Values are presented as mean ± standard deviation, *t-test. ETP: Endogenous thrombin potential, peak: peak thrombin activity, TTP: time to peak thrombin generated. Table 1. LETTERS TO EDITOR Turk J Hematol 2016;33:163-166 in terms of sex ratios. However, we may conclude that plasma from adults may be more procoagulant than that of infants. Our findings may confirm the presence of a regulation mechanism in the coagulation parameters throughout the course of life. Authorship Contributions Study Conception and Design: Nejat Akar, Filiz Şimşek Orhon; Acquisition and Blood Collection: Filiz Şimşek Orhon, Sevgi Başkan; Laboratory Analysis: Yonca Eğin; Interpretation of Data: Nejat Akar, Filiz Şimşek Orhon; Literature Search: Filiz Şimşek Orhon, Betül Ulukol; Drafting and Writing: Filiz Şimşek Orhon. 5. Karlaftis V, Attard C, Summerhayes R, Monagle P, Ignjatovic V. The microparticle-specific procoagulant phospholipid activity changes with age. Int J Lab Hem 2014;36:e41-e43. 6. Castoldi E, Rosing J. Thrombin generation tests. Thromb Res 2011;127(Suppl 3):S21-S25. 7. Butenas S, van’t Veer C, Mann KG. “Normal” thrombin generation. Blood 1999;94:2169-2178. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included. 8. Haidl H, Cimenti C, Leschnik B, Zach D, Muntean W. Age-dependency of thrombin generation measured by means of calibrated automated thrombography (CAT). Thromb Haemost 2006;95:772-775. 9. Schneider T, Siegemund T, Siegemund R, Petros S. Thrombin generation and rotational thromboelastometry in the healthy adult population. Hamostaseologie 2015;35:181-186. Financial Disclosure: This study was supported in part by the Ankara University Research Fund. 10. Wu J, Zhao HR, Zhang HY, Ge YL, Qiu S, Zhao J, Song Y, Zhao JZ, Lu SS. Thrombin generation increasing with age and decreasing with use of heparin indicated by calibrated automated thrombogram conducted in Chinese. Biomed Environ Sci 2014;27:378-384. Received/Geliş tarihi: September 29, 2015 Accepted/Kabul tarihi: December 11, 2015 Received/Geliş tarihi: September 29, 2015 Accepted/Kabul tarihi: December 11, 2015 Address for Correspondence/Yazışma Adresi: Filiz ŞİMŞEK ORHON, M.D., Ankara University Faculty of Medicine, Department of Pediatrics, Divisions of Social Pediatrics, Ankara, Turkey Phone : +90 312 595 72 02 Address for Correspondence/Yazışma Adresi: Filiz ŞİMŞEK ORHON, M.D., Ankara University Faculty of Medicine, Department of Pediatrics, Divisions of Social Pediatrics, Ankara, Turkey Phone : +90 312 595 72 02 DOI: 10.4274/tjh.2015.0341 References 1. Chironi GN, Boulanger CM, Simon A, Dignat-George F, Freyssinet JM, Tedgui A. Endothelial microparticles in diseases. Cell Tissue Res 2009;335:143-151. 1. Chironi GN, Boulanger CM, Simon A, Dignat-George F, Freyssinet JM, Tedgui A. Endothelial microparticles in diseases. Cell Tissue Res 2009;335:143-151. 2. Brummel-Ziedins KE, Everse SJ, Mann KG, Orfeo T. Modeling thrombin generation: plasma composition based approach. J Thromb Thrombolysis 2014;37:32-44. Keywords: Infant, Adult, Microparticle, Thrombin 3. Orhon FS, Egin Y, Ulukol B, Baskan S, Akar N. Evaluation of indirect microparticle activity and parameters of thrombin generation test in healthy infants. Thromb Res 2014;133:281-284. Anahtar Sözcükler: Süt çocuğu, Erişkin, Mikropartikül, Trombin Anahtar Sözcükler: Süt çocuğu, Erişkin, Mikropartikül, Trombin Authorship Contributions 4. Kenet G, Krumpel A, Nowak-Gottl U. Bleeding issues in neonates, infants and young children. Thromb Res 2009;123(Suppl 2):S35-S37. Yorum: Cevap Olarak “Geriletilmiş Lenfoma: Diffüz Büyük B-Hücreli Lenfoma Olduğu Bilinen Bir Olguda B-Kronik Lenfositik Lösemi - De Novo Oluşum veya Dönüşüm” Burak Uz, Kadir Acar Burak Uz, Kadir Acar University Faculty of Medicine, Department of Internal Medicine, Division of Adult Hematology, Ankara, Turkey radiotherapy. He was well for nearly 5 years, but subsequently his disease locally relapsed. Unfortunately, a planned intensive salvage regimen could not be given because the patient was lost to follow-up. In 2010, despite not being given any treatment modality, he presented with small lymphocytic lymphoma. Finally, 22 months thereafter, he was diagnosed with Rai stage IV chronic lymphocytic leukemia and 6 cycles of fludarabine, Comment: In Response to “Downgraded Lymphoma: B-Chronic Lymphocytic Leukemia in a Known Case of Diffuse Large B-Cell Lymphoma - De Novo Occurrence or Transformation” Yorum: Cevap Olarak “Geriletilmiş Lenfoma: Diffüz Büyük B-Hücreli Lenfoma Olduğu Bilinen Bir Olguda B-Kronik Lenfositik Lösemi - De Novo Oluşum veya Dönüşüm” To the Editor, Data on indirect microparticle activity and thrombin generation parameters of the study groups. Table 1. Data on indirect microparticle activity and thrombin generation parameters of the study groups. A total of 58 healthy adults (23 males and 35 females; mean age: 23.2±0.4 years) were admitted to the study. In our recent study, 85 healthy infants (51 males and 34 females; mean age: 12.6±8.3 months) were studied. The indirect microparticle activity in the infant group was significantly lower than that of the adult group (p<0.001). The ETP and peak levels in the infant group were significantly lower than those of adults. Furthermore, the TTP levels of the adult group were lower than those of infants (p=0.001) (Table 1). 163 LETTERS TO EDITOR To the Editor, We read the letter submitted by Gajendra et al. with deep interest [1]. The authors described a patient diagnosed with diffuse large B-cell lymphoma (DLBCL) non-germinal center B-cell type in 2002 who received 6 cycles of cyclophosphamide, adriamycin, vincristine, and prednisolone (CHOP) followed by 164
https://openalex.org/W2210427063	https://europepmc.org/articles/pmc4689448?pdf=render	English	null	Analyses of the Erosive Effect of Dietary Substances and Medications on Deciduous Teeth	PloS one	2,015	cc-by	9,697	Analyses of the Erosive Effect of Dietary Substances and Medications on Deciduous Teeth Adrian Lussi, Thiago Saads Carvalho Department of Preventive, Restorative and Pediatric Dentistry, University of Bern, Bern, Switzerland Adrian Lussi, Thiago Saads Carvalho Department of Preventive, Restorative and Pediatric Dentistry, University of Bern, Bern, Switzerland Adrian Lussi, Thiago Saads Carvalho Department of Preventive, Restorative and Pediatric Dentistry, University of Bern, Bern, Switzerland adrian.lussi@zmk.unibe.ch OPEN ACCESS Citation: Lussi A, Carvalho TS (2015) Analyses of the Erosive Effect of Dietary Substances and Medications on Deciduous Teeth. PLoS ONE 10(12): e0143957. doi:10.1371/journal.pone.0143957 Citation: Lussi A, Carvalho TS (2015) Analyses of the Erosive Effect of Dietary Substances and Medications on Deciduous Teeth. PLoS ONE 10(12): e0143957. doi:10.1371/journal.pone.0143957 Editor: Brian Lee Beatty, New York Institute of Technology College of Osteopathic Medicine, UNITED STATES Received: April 22, 2015 Accepted: November 11, 2015 Published: December 23, 2015 Copyright: © 2015 Lussi, Carvalho. 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: April 22, 2015 Accepted: November 11, 2015 Published: December 23, 2015 Copyright: © 2015 Lussi, Carvalho. 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. Copyright: © 2015 Lussi, Carvalho. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The data underlying the findings are fully available and all relevant data are presented within the manuscript. Funding: This study was funded by the Deutsche Gesellschaft für Zahnerhaltung (DGPZM- Wissenschaftsfonds) to AL and TSC. Funding: This study was funded by the Deutsche Gesellschaft für Zahnerhaltung (DGPZM- Wissenschaftsfonds) to AL and TSC. RESEARCH ARTICLE Abstract This study aimed at analysing the erosive potential of 30 substances (drinks, candies, and medicaments) on deciduous enamel, and analyse the associated chemical factors with enamel dissolution. We analysed the initial pH, titratable acidity (TA) to pH 5.5, calcium (Ca), inorganic phosphate (Pi), and fluoride (F) concentration, and degree of saturation ((pK -pI)HAP, (pK -pI)FAP, and (pK−pI)CaF2) of all substances. Then, we randomly distributed 300 specimens of human deciduous enamel into 30 groups (n = 10 for each of the substances tested. We also prepared 20 specimens of permanent enamel for the sake of comparison between the two types of teeth, and we tested them in mineral water and Coca-Cola1. In all specimens, we measured surface hardness (VHN: Vickers hardness numbers) and sur- face reflection intensity (SRI) at baseline (SHbaseline and SRIbaseline), after a total of 2 min (SH2min) and after 4 min (SH4min and SRI4min) erosive challenges (60 ml of substance for 6 enamel samples; 30°C, under constant agitation at 95 rpm). There was no significant differ- ence in SHbaseline between deciduous and permanent enamel. Comparing both teeth, we observed that after the first erosive challenge with Coca-Cola1, a significantly greater hard- ness loss was seen in deciduous (−90.2±11.3 VHN) than in permanent enamel (−44.3±12.2 VHN; p = 0.007), but no differences between the two types of teeth were observed after two challenges (SH4min). After both erosive challenges, all substances except for mineral water caused a significant loss in relative surface reflectivity intensity, and most substances caused a significant loss in surface hardness. Multiple regression analyses showed that pH, TA and Ca concentration play a significant role in initial erosion of deciduous enamel. We conclude that drinks, foodstuffs and medications commonly consumed by children can cause erosion of deciduous teeth and erosion is mainly associated with pH, titratable acidity and calcium concentration in the solution. Introduction Dental erosion is the acid dissolution of dental hard tissues caused by multiple factors. One of these factors are acidic substances in the diet (nutrition-related factors) [1]. Erosion can occur Competing Interests: The authors have declared that no competing interests exist. PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 1 / 15 Erosive Effect of Different Substances on Deciduous Teeth in both deciduous and permanent teeth [2–5]. It starts with a softening of the tooth surface (enamel) and progresses to extensive loss of tooth substance when contact with the acids con- tinues [6–8]. Various dietary substances and medicaments have been associated with dental erosion [9–15], and many studies have investigated which chemical factors are most signifi- cantly associated with enamel dissolution [16–23]. However, many studies have focused on permanent teeth, and more detailed investigations should be carried out to find out the effect of different dietary substances on deciduous enamel, and which chemical factors will play a role on erosive demineralization of these teeth. Deciduous enamel is histologically different to permanent enamel. Basically, prism arrange- ments in deciduous and permanent enamel are similar [24], but the prisms in deciduous enamel are smaller, with more complete boundaries, and are more widely spread than those in permanent enamel [25]. Also, the prisms in deciduous enamel are more gently curved, and have slightly less pronounced Hunter-Schreger bands [25]. Deciduous enamel is considerably less mineralized [26], has greater total carbonate content [27], and a higher organic content [28] than permanent enamel. These histological differences could also lead to different erosion patterns in deciduous and permanent enamel, so it is important to fully investigate the effect of different dietary substances on deciduous enamel. Moreover, in a study by Ganss et al. [29], children who initially presented with erosive lesions in deciduous teeth had a significantly greater risk (3.9-fold) of having erosive lesions in their permanent teeth. Similar results were also reported by Harding et al. [30], who showed that 5-year-old children who present with severe erosive tooth wear in deciduous teeth are 5 times more likely to present erosive tooth wear in permanent teeth at the age of 12 years. Introduction It is, thus, suggested that tooth wear in deciduous teeth ought to be regarded as a predictive factor for wear in permanent teeth, and health professionals should be fully aware of the erosive effect of different dietary substances on deciduous enamel in order to be able to give children and parents the best oral health recommendations. Consequently, the aim of this study was to analyse the potential of different substances to cause erosion of deciduous enamel, and to deter- mine which chemical factors are most strongly associated with enamel dissolution in deciduous teeth. PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 Chemical analysis of the substances For the chemical analyses [22], we used 10 g of each solution at 30°C to measure the initial pH and the titratable acidity to pH 5.5 (total amount of base needed to raise the pH of the sub- stance to 5.5). An automatic titrator (Toledo DL 53, Mettler Toledo, Electrode DG 101-SC, Software: LabX pro, Schwerzenbach, Switzerland) established the initial pH of the solutions, which were then individually titrated with 0.5 mol/l NaOH in steps of 0.02 ml [23]. Titratable acidity was calculated as the amount of base (mmol/L of sample) required to raise the pH to 5.5. Calcium (Ca) concentration was measured with the standard atomic absorption method, using an atomic absorption spectrometer with an air/acetylene flame. Lanthanum was added to all the products and standards (final end concentration 0.2%) to suppress interference from inorganic phosphates (Pi). Total Pi concentration was analysed by the ammonium molybdate method of Chen et al. (1956) [32]. Fluoride (F) concentration was determined using an F ion- specific electrode (Orion 960900, Boston, MA, USA). Before F measurement, we added total ionic strength adjustment buffer (TISAB) to all products and standard solutions (1:1 ratio), without previously neutralizing the substances. The concentrations of Ca and Pi are expressed in mmol/l and those of F in ppm. The degree of saturation (pK−pI) with respect to hydroxyap- atite (HAP), fluorapatite (FAP), and calcium fluoride (CaF2) was calculated from the pH and the concentrations of Ca, Pi and F using a computer program [33]. This program assumes a solubility product for HAP of 10−58.5 and for FAP of 10−59.6 [34, 35]. The concentrations of Ca, Pi and F, the pH, and the titratable acidity were measured in duplicate. Substances tested In the present study, we tested 30 substances, ranging from drinks, candies, and medicaments frequently used by children and young adolescents (Table 1). For the experiment, all carbon- ated drinks, candies, and medicaments were pre-treated as follows. The carbonated drinks were degassed by stirring at room temperature (10 min). The candy was dissolved in deionized water (5.2 g candy / 10 ml water), under constant mixing at 45°C; the resulting candy solution was then cooled and used at 30°C for the experiments. The medicaments and concentrated drinks were all prepared with deionized water according to the manufacturer’s instructions. The chewing gum was ground for 5 min (2 g chewing gum in 10 ml of deionized water) using a mortar and pestle, and the resulting solution was used in the experiment. The fruits were squeezed/crushed and the juice was then passed through a sieve (1.0 x 1.0 mm). Preparation of enamel specimens From a pool of extracted teeth, we randomly selected 150 caries-free human deciduous molars and 20 (permanent) premolars. The teeth were extracted by dental practitioners in Switzerland. Before the extraction, the patients and their parents were informed about the use of their teeth for research purposes and their oral consent was obtained. Because we are using teeth from a pooled bio-bank, the local ethics committee categorized the samples as “irreversibly anon- ymised”, and no previous approval was necessary. The crowns of all teeth were separated from the roots, and cut in two halves (into buccal and lingual surfaces). The enamel slabs were embedded in acrylic resin blocks (Paladur1, Bad Homburg, Germany) using two planar paral- lel moulds of 8 mm and 0.2 mm. The latter mould was removed and the blocks were then seri- ally ground (LaboPol-21 rotating polishing machine, Struers, Ballerup, Denmark) with silicon carbide paper discs (grade 18 μm for 30 s, 8 μm grade for 30 s, 5 μm grade for 1 min, 3 μm dia- mond abrasive paste for 1 min), removing 200 μm of enamel from each specimen. After each polishing step, the resin blocks were rinsed and sonicated for 2 min in tap water and all speci- mens were then stored in a saturated mineral solution (1.5 mM CaCl2, 1.0 mM KH2PO4, 50 mM NaCl, pH 7.0 [31]) until the time of the experiment. The 300 deciduous enamel samples PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 2 / 15 Erosive Effect of Different Substances on Deciduous Teeth were randomly distributed into 30 groups (n = 10 for each of the substances tested). The per- manent enamel samples were divided into two groups (n = 10). PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 Surface hardness measurement The present method describes hardness measurements using nanoindentations. Surface hard- ness (SH) of each enamel specimen was determined with a Vickers diamond under a pressure of 50 mN for 15 s (Fischerscope HM 2000 XYp; Helmut Fischer, Hünenberg, Switzerland). A total of six baseline indentations were made at intervals of 50 μm. Further indentations next to the previous indentations were made following the experimental procedure. Vickers hardness was automatically calculated from the depth of the indentations by the computer program. The load resolution was 0.04 mN and the indentation depth was 600 nm for sound enamel and < 1000 nm for most softened specimens. The device allowed fully automatic measure- ments using a programmable x, y stage. The WIN-HCU software calculated SH. The SH value for each enamel slab was determined by calculating the average of six indentations. 3 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 Table 1. Basic information on the substances tested and their chemical parameters: pH, titratable acidity to pH 5.5 (mmol OH−/l to pH 5.5), calcium [Ca], inorganic phosphate [Pi], and fluoride [F] concentrations, degree of saturation with respect to hydroxyapatite ((pK−pI)HAP), with respect to fluorapatite ((pK−pI)FAP), and with respect to calcium fluoride ((pK−pI)CaF2). Substance Brand name/ producer Flavour Erosion- related ingredients* pH mmol OH−/l to pH 5.5 [Ca] (mmol/l) [Pi] (mmol/l) [F] (ppm) (pK−pI)HAP (pK−pI)FAP (pK−pI)CaF2 MINERAL WATER Mineral water Valser1, Coca- Cola Company – – 6.53 – 10.57 < 0.01 0.58 −0.35 3.47 –0.82 SOFT DRINKS Coca-Cola1 Coca-Cola1, Coca-Cola Company Cola Phosphoric acid, carbonic acid, 2.55 9.32 0.53 5.39 0.05 −20.59 −14.31 –5.45 Pepsi Cola1 Pepsi Cola1, PepsiCo Cola Phosphoric acid, citric acid, carbonic acid, and ﬂavours 2.51 8.30 0.22 5.38 <0.05 −22.83 −17.09 −7.00 Fanta1 Regular Fanta1, Coca −Cola Company Orange Orange fruit, citric acid, carbonic acid, and ﬂavours 2.59 36.19 0.56 0.14 <0.05 −24.76 −18.65 −5.64 Sprite1 Sprite1, Coca −Cola Company Lemon Carbonic acid, citric acid, acidity regulator, and ﬂavours 2.57 31.56 0.47 < 0.01 <0.05 −34.71 −28.78 −6.12 Guaraná Antártica1 Antártica Guaraná Citric acid and carbonic acid 2.62 15.55 0.03 < 0.01 <0.05 −36.96 −31.02 −7.21 Rivella1 Red Rivella NA Milk serum, carbonic acid, citric acid, and ﬂavours 3.28 32.88 2.95 2.72 0.07 −12.52 −6.41 −3.61 Ice tea NA, Coop (supermarket in Switzerland) NA Black tea extract, citric and ascorbic acids 2.43 24.36 0.03 0.06 0.88 −33.58 −26.06 −4.45 Ice tea peach Lipton, Unilever Peach Black tea extract and peach juice 2.65 25.15 0.08 0.13 0.55 −28.39 −21.12 −4.01 FRUITS, JUICES AND SMOOTHIES Kiwi (fruit) NA NA NA 3.24 159.81 1.06 3.40 <0.05 −14.53 −9.93 −7.12 Orange (fruit) NA NA NA 3.93 71.93 1.50 1.18 <0.05 −10.22 −5.22 −4.77 Orange juice Hohes C, Eckes AG Orange Orange juice 3.63 83.56 2.11 1.58 <0.05 −11.32 −5.89 −4.38 Apple juice Ramseier Premium, Ramseier Suisse AG Apple Apple juice and pear juice 3.24 70.30 1.17 1.62 <0.05 −15.23 −9.44 −4.68 Apple juice for babies Nestlè Apple and pear Apple juice, pear juice, vitamin C 3.59 48.19 2.55 1.96 0.17 −10.98 −4.70 −2.68 Ribena1 Lucozade Ribena Suntory Blackcurrant Blackcurrant juice concentrate, citric acid, and vitamin C 2.51 27.94 0.36 0.17 0.01 −26.06 −20.42 −6.93 (Continued) Erosive Effect of Different Substances on Deciduous Teeth Erosive Effect of Different Substances on Deciduous Teeth stances tested and their chemical parameters: pH, titratable acidity to pH 5.5 (mmol OH−/l to pH 5.5), calcium oride [F] concentrations, degree of saturation with respect to hydroxyapatite ((pK−pI)HAP), with respect to pect to calcium fluoride ((pK−pI)CaF2). avour Erosion- related ingredients* pH mmol OH−/l to pH 5.5 [Ca] (mmol/l) [Pi] (mmol/l) [F] (ppm) (pK−pI)HAP (pK−pI)FAP (pK−pI)CaF2 – 6.53 – 10.57 < 0.01 0.58 −0.35 3.47 –0.82 ola Phosphoric acid, carbonic acid, 2.55 9.32 0.53 5.39 0.05 −20.59 −14.31 –5.45 ola Phosphoric acid, citric acid, carbonic acid, and ﬂavours 2.51 8.30 0.22 5.38 <0.05 −22.83 −17.09 −7.00 range Orange fruit, citric acid, carbonic acid, and ﬂavours 2.59 36.19 0.56 0.14 <0.05 −24.76 −18.65 −5.64 emon Carbonic acid, citric acid, acidity regulator, and ﬂavours 2.57 31.56 0.47 < 0.01 <0.05 −34.71 −28.78 −6.12 uaraná Citric acid and carbonic acid 2.62 15.55 0.03 < 0.01 <0.05 −36.96 −31.02 −7.21 A Milk serum, carbonic acid, citric acid, and ﬂavours 3.28 32.88 2.95 2.72 0.07 −12.52 −6.41 −3.61 A Black tea extract, citric and ascorbic acids 2.43 24.36 0.03 0.06 0.88 −33.58 −26.06 −4.45 each Black tea extract and peach juice 2.65 25.15 0.08 0.13 0.55 −28.39 −21.12 −4.01 A NA 3.24 159.81 1.06 3.40 <0.05 −14.53 −9.93 −7.12 A NA 3.93 71.93 1.50 1.18 <0.05 −10.22 −5.22 −4.77 range Orange juice 3.63 83.56 2.11 1.58 <0.05 −11.32 −5.89 −4.38 pple Apple juice and pear juice 3.24 70.30 1.17 1.62 <0.05 −15.23 −9.44 −4.68 pple and ear Apple juice, pear juice, vitamin C 3.59 48.19 2.55 1.96 0.17 −10.98 −4.70 −2.68 ackcurrant Blackcurrant juice concentrate, citric acid, and vitamin C 2.51 27.94 0.36 0.17 0.01 −26.06 −20.42 −6.93 (Continued) Table 1. Basic information on the substances tested and their chemical parameters: pH, titratable acidity to pH 5.5 (mmol OH−/l to pH 5.5), calcium [Ca], inorganic phosphate [Pi], and fluoride [F] concentrations, degree of saturation with respect to hydroxyapatite ((pK−pI)HAP), with respect to fluorapatite ((pK−pI)FAP), and with respect to calcium fluoride ((pK−pI)CaF2). Substance Brand name/ producer Flavour Erosion- related ingredients* pH mmol OH−/l to pH 5.5 [Ca] (mmol/l) [Pi] (mmol/l) [F] (ppm) (pK−pI)HAP (pK−pI)FAP (pK−pI)CaF2 MINERAL WATER Mineral water Valser1, Coca- Cola Company – – 6.53 – 10.57 < 0.01 0.58 −0.35 3.47 –0.82 SOFT DRINKS Coca-Cola1 Coca-Cola1, Coca-Cola Company Cola Phosphoric acid, carbonic acid, 2.55 9.32 0.53 5.39 0.05 −20.59 −14.31 –5.45 Pepsi Cola1 Pepsi Cola1, PepsiCo Cola Phosphoric acid, citric acid, carbonic acid, and ﬂavours 2.51 8.30 0.22 5.38 <0.05 −22.83 −17.09 −7.00 Fanta1 Regular Fanta1, Coca −Cola Company Orange Orange fruit, citric acid, carbonic acid, and ﬂavours 2.59 36.19 0.56 0.14 <0.05 −24.76 −18.65 −5.64 Sprite1 Sprite1, Coca −Cola Company Lemon Carbonic acid, citric acid, acidity regulator, and ﬂavours 2.57 31.56 0.47 < 0.01 <0.05 −34.71 −28.78 −6.12 Guaraná Antártica1 Antártica Guaraná Citric acid and carbonic acid 2.62 15.55 0.03 < 0.01 <0.05 −36.96 −31.02 −7.21 Rivella1 Red Rivella NA Milk serum, carbonic acid, citric acid, and ﬂavours 3.28 32.88 2.95 2.72 0.07 −12.52 −6.41 −3.61 Ice tea NA, Coop (supermarket in Switzerland) NA Black tea extract, citric and ascorbic acids 2.43 24.36 0.03 0.06 0.88 −33.58 −26.06 −4.45 Ice tea peach Lipton, Unilever Peach Black tea extract and peach juice 2.65 25.15 0.08 0.13 0.55 −28.39 −21.12 −4.01 FRUITS, JUICES Table 1. Basic information on the substances tested and their chemical parameters: pH, titratable acidity to pH 5.5 (mmol OH/l to pH 5.5), calcium [Ca], inorganic phosphate [Pi], and fluoride [F] concentrations, degree of saturation with respect to hydroxyapatite ((pK−pI)HAP), with respect to fluorapatite ((pK−pI)FAP), and with respect to calcium fluoride ((pK−pI)CaF2). Table 1. Basic information on the substances tested and their chemical parameters: pH, titratable acidity to [Ca], inorganic phosphate [Pi], and fluoride [F] concentrations, degree of saturation with respect to hydroxy fluorapatite ((pK−pI)FAP), and with respect to calcium fluoride ((pK−pI)CaF2). (Continued) PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 4 / 15 Erosive Effect of Different Substances on Deciduous Teeth Table 1. (Continued) Substance Brand name/ producer Flavour Erosion- related ingredients* pH mmol OH−/l to pH 5.5 [Ca] (mmol/l) [Pi] (mmol/l) [F] (ppm) (pK−pI)HAP (pK−pI)FAP (pK−pI)CaF2 Fruit smoothie innocent Kiwi, apple and limes Apple juice, grape juice, kiwi juice, lime juice, and pineapple juice 3.27 82.44 2.10 0.27 <0.05 −16.13 −10.62 −4.94 YOGHURT Forest berries yoghurt NA, Migros (Supermarket in Switzerland) Berries Forest Berries; 4.13 62.86 37.39 10.72 <0.05 −0.55 4.63 −2.86 SOUR CANDIES Candy spray Mega Mouth1 Candy Spray, Bazooka Candy Brands International Ltd NA Citric acid 2.14 441.75 0.12 0.16 <0.05 −31.67 −26.76 −9.65 Sour candy Haribo1 Pommes, Haribo GmbH & Co.,Germany Apple Citric, malic, and tartaric acids 2.46 88.10 0.07 0.12 <0.05 −30.57 −24.64 −7.18 Sour chewing gum Trident1 Senses, Modelez Mega Mystery Citric acid, malic acid 2.74 22.57 0.37 0.03 <0.05 −26.56 −21.57 −7.76 SPORTS AND ENERGY DRINKS Monster Energy Drink1 Monster Energy Drink1, Vertrieb Spar GmbH, Austria NA Citric, sorbic, carbonic, and benzoic acids, vitamin B, taurine 3.35 62.39 0.07 0.03 <0.05 −25.05 −19.38 −5.82 Red Bull1 Energy Drink Red Bull1, Red Bull GmbH, Austria NA Sodium citrate, carbonic acid, taurine, vitamin B 3.35 67.76 1.41 < 0.01 0.13 −25.72 −19.38 −3.27 Gatorade1 Gatorade1, PepsiCo NA Citric acid, ﬂavours 2.89 37.38 0.05 2.98 0.05 −23.94 −17.74 −5.97 MEDICAMENTS Dafalgan1 syrup for children Bristol−Myers Squibb NA NA 5.26 7.91 0.07 < 0.01 <0.05 −15.16 −11.65 −6.37 Mucosolvon1 for children Boehringer Ingelheim NA Benzoic acid 3.13 14.43 0.01 0.01 <0.05 −31.47 −26.41 −8.21 Fluimucil1 Effervescent Zambon Schweiz NA NA 4.48 14.04 0.01 < 0.01 <0.05 −29.35 −25.55 −8.26 Tossamin1 sugar free syrup Novartis Consumer Health Schweiz NA Sorbic acid 4.43 19.46 0.01 1.46 <0.05 −16.42 −12.59 −8.12 Ventolin1 syrup Glaxo Smith Kline NA NA 3.19 56.08 0.02 < 0.01 <0.05 −36.98 −32.35 −8.85 Claritine1 syrup MSD Merk Sharp & Dohme AG NA Peach aroma 2.98 74.34 0.07 < 0.01 <0.05 −37.13 −32.23 −8.74 (Continued) (Continued) PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 5 / 15 Surface reflection intensity For the surface reflection intensity (SRI) measurements, we used a recently developed table-top reflection device [36–38]. The device was connected to a computer running a specific software that registers the point of highest reflection intensity, which is expressed as a SRI value. We measured SRI initially (SRIbaseline) and after the second challenge (SRI4min), and from these SRI values, we calculated the relative percentage decrease in reflection intensity (rSRI) using the formula rSRIi = (100×(SRI4min−SRIbaseline)) / SRIbaseline. In practical terms, more negative rSRI values represent greater decrease in reflection intensity, which, in turn, represent more erosion of the enamel surface. Erosive Effect of Different Substances on Deciduous Teeth Table 1. (Continued) Substance Brand name/ producer Flavour Erosion- related ingredients* pH mmol OH−/l to pH 5.5 [Ca] (mmol/l) [Pi] (mmol/l) [F] (ppm) (pK−pI)HAP (pK−pI)FAP (pK−pI)CaF2 Maltofer1 syrup Vifor (International) AG NA NA 4.90 5.48 0.12 < 0.01 <0.05 −20.68 −17.47 −7.45 * Erosion-related ingredients are those listed on the packaging of each substance. NA = not available. When [Pi] values were <0.01mmol/l, exact values of 0.0001 mmol/l were used in the (pK−pI) calculations. doi:10.1371/journal.pone.0143957.t001 NA not available. When [Pi] values were <0.01mmol/l, exact values of 0.0001 mmol/l were used in the (pKpI) calculations. When [Pi] values were <0.01mmol/l, exact values of 0.0001 mmol/l were used in the (pKpI) calculations. doi:10.1371/journal.pone.0143957.t001 PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 Statistical analyses Wilcoxon’s signed rank tests were used to compare the SH and SRI values before and after immersion in the respective drink or solution. Changes in SH (ΔSH) were calculated as follows: for the first 2-min erosive challenge ΔSH2–0 = SH2min −SHbaseline; for the second 2-min erosive challenge ΔSH4–2 = SH4min −SH2min; and for the total 4-min erosive challenge ΔSH4–0 = SH4min−SHbaseline. Associations between the changes in SRI (rSRI, denoted as dependent vari- able), ΔSH (denoted as the dependent variable) and pH, titratable acidity, and Ca, Pi and F con- centrations, HAP saturation, FAP saturation, CaF2 saturation (independent variables) were investigated using Spearman’s Correlation Coefficients. Since HAP and FAP saturation are not independent of pH, titratable acidity, and Ca, Pi and F concentrations, care was taken not to include them in the regression analyses. Multiple linear regression analyses were carried out to verify the association of ΔSH2−0 and ΔSH4−0 with pH, titratable acidity, Ca, Pi and F concentra- tions. Association between ΔSH4−0 and rSRI were investigated using spearman’s correlation coefficient and linear regression analysis. Furthermore, additional differences between decid- uous and permanent enamel were verified using the Mann-Whitney U test. The significance level was set at 0.05 for all analyses. Study design Immediately prior to the experimental procedures, the resin blocks were further polished with 1 μm diamond abrasive for 1 min (LaboPol-6, DP-Mol Polishing, DP-Stick HQ; Struers, Copenhagen, Denmark) to ensure the removal of possible remnants from storage. Initially, the samples were incubated in freshly collected human saliva (20 ml / 6 enamel samples, 3 h, 37°C, under constant shaking). For that, stimulated saliva was collected from one healthy adult donor (stimulated salivary flow rate 2.32 ml/min) by chewing on a piece of paraffin pellets (Fluka; Sigma-Aldrich Chemie GmbH, Munich, Germany) for 30 min. An approval from the institutional review board is not necessary for collecting saliva samples, so the local Ethical Committee (Kantonale Ethikkommission) waived the need for ethical approval. In the eyes of the Ethical Committee, when collecting saliva samples, we are only required to obtain the con- sent from the saliva expeditor, which can be done verbally. In our study, the saliva donor gave a verbal consent, since written consent was not required. The saliva was collected in an ice- cooled tube at least 1 h after the donor had consumed any food or drink [39, 40]. The samples were then carefully rinsed with tap water (50 s) and with deionized water (10 s), then dried with oil-free air (5 s). All enamel samples had their baseline SH and SRI individually measured (SHbaseline and SRIbaseline), after which they were subjected to two consecutive erosive chal- lenges. Each erosive challenge consisted of individually immersing the specimens into the respective test substance (10 ml / sample) for 2 min at 30°C, under constant agitation (95 rpm). The samples were then taken out of the solution, washed (10 s) and dried (5 s), and a second SH measurement was performed (SH2min). Subsequently, the samples were submitted to another erosive challenge (2 min), rinsed, dried, and a final SH and SRI measurement was carried out (SH4min and SRI4min). A total of 10 deciduous enamel specimens were tested per substance (5 buccal and 5 lingual surfaces randomly chosen). In addition, the two groups PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 6 / 15 Erosive Effect of Different Substances on Deciduous Teeth containing the permanent enamel samples were also submitted to the same experimental pro- tocol, and were treated with mineral water (n = 10) or Coca-Cola1 (n = 10). PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 Erosive Effect of Different Substances on Deciduous Teeth Table 2. Mean and standard error of the mean (SEM) for surface hardness at baseline (SHbaseline), the difference in surface hardness between baseline and the first erosive challenge (ΔSH2−0), the difference in surface hardness between the first and the second erosive challenges (ΔSH4−2), and the relative difference in surface reflection intensity between baseline and the second erosive challenge (rSRI4−0). Table 2. Mean and standard error of the mean (SEM) for surface hardness at baseline (SHbaseline), the difference in surface hardness between baseline and the first erosive challenge (ΔSH2−0), the difference in surface hardness between the first and the second erosive challenges (ΔSH4−2), and the relative difference in surface reflection intensity between baseline and the second erosive challenge (rSRI4−0). Table 2. Mean and standard error of the mean (SEM) for surface hardness at baseline (SHbaseline), the difference in surface hardness between baseline and the first erosive challenge (ΔSH2−0), the difference in surface hardness between the first and the second erosive challenges (ΔSH4−2), and the relative difference in surface reflection intensity between baseline and the second erosive challenge (rSRI4−0). Table 2. Mean and standard error of the mean (SEM) for surface hardness at baseline (SHbaseline), the difference in surface hardness between baseline and the first erosive challenge (ΔSH2−0), the difference in surface hardness between the first and the second erosive challenges (ΔSH4−2), and the relative difference in surface reflection intensity between baseline and the second erosive challenge (rSRI4−0). lower pH values and Ca concentration, and higher titratable acidity values are significantly related to more loss of SH during erosion. Comparing permanent enamel with deciduous enamel treated with the same substances (Table 5), we observed no significant differences in initial hardness between the two kinds of teeth. However, a significant difference was observed in the change in SH when the samples were immersed in Coca-Cola1. After the first erosive challenge (ΔSH2−0), deciduous enamel and the relative difference in surface reflection intensity between baseline and the second erosive challenge (rSRI40). SHbaseline ΔSH2−0 ΔSH4−2 rSRI4−0 Mean SEM Mean SEM p-value Mean SEM p-value Mean SEM p-value MINERAL WATER Mineral water 509.5 19.6 −5.0 7.7 0.695 −6.1 5.5 0.375 15.6 11.0 0.301 SOFT DRINKS Coca−Cola1 501.0 12.7 −90.2 11.3 0.002 −79.1 10.3 0.002 −83.0 2.0 0.002 Pepsi−Cola1 497.6 10.3 −60.7 7.6 0.002 −86.4 8.8 0.002 −87.7 1.2 0.004 Fanta1 Regular 491.2 10.9 −100.6 9.8 0.002 −105.1 14.8 0.002 −85.8 1.5 0.002 Sprite1 511.0 13.0 −124.4 4.7 0.002 −134.1 8.2 0.002 −85.3 1.4 0.002 Guaraná Antártica1 502.5 14.4 −32.3 8.6 0.014 −58.5 7.8 0.002 −77.1 2.1 0.002 Rivella1 Red 491.1 14.2 −44.8 15.0 0.002 −112.9 11.9 0.002 −78.1 2.3 0.002 Ice tea 500.8 10.5 −63.7 5.4 0.002 −84.1 8.4 0.002 −82.8 2.5 0.004 Ice tea Peach 483.6 10.0 −25.5 11.2 0.106 −101.2 9.8 0.002 −82.2 1.6 0.004 FRUITS, JUICES AND SMOOTHIES Kiwi (fruit) 498.9 10.3 −60.8 15.9 0.004 −142.3 13.5 0.002 −94.3 1.9 0.002 Orange (fruit) 502.0 11.5 −16.2 5.1 0.014 −43.4 7.3 0.002 −60.7 2.1 0.004 Orange juice 499.4 13.3 −19.2 5.2 0.006 −30.0 5.6 0.002 −72.4 6.1 0.002 Apple juice 480.2 7.6 −37.5 13.6 0.027 −107.4 17.7 0.004 −93.4 1.1 0.002 Apple juice for babies 494.6 9.8 −15.4 7.6 0.065 −48.6 6.0 0.002 −71.0 3.7 0.004 Ribena1 506.8 11.3 −50.1 7.0 0.002 −91.4 14.8 0.004 −84.6 2.1 0.002 Fruit smoothie 532.6 15.5 −38.8 10.8 0.006 −77.2 5.3 0.002 −71.6 3.2 0.002 YOGHURT Forest berries yoghurt 494.5 6.2 24.7 11.4 0.037 1.6 12.8 0.922 −23.9 7.6 0.006 SOUR CANDIES Candy spray 509.9 13.1 −301.7 11.3 0.002 −110.7 12.5 0.002 −97.2 2.4 0.004 Sour candy 525.7 9.0 −74.1 14.3 0.002 −110.7 15.1 0.002 −84.0 2.3 0.002 Sour chewing gum 490.3 13.6 −53.9 7.0 0.002 −81.5 6.7 0.002 −80.7 1.6 0.002 SPORTS AND ENERGY DRINKS Monster Energy Drink1 509.9 14.9 −51.6 6.7 0.002 −77.1 14.0 0.004 −75.4 2.1 0.002 Red Bull1 Energy Drink 515.5 15.3 −52.6 9.3 0.004 −92.2 8.7 0.002 −74.9 2.4 0.002 Gatorade1 541.8 18.4 −115.4 20.2 0.002 −89.1 9.2 0.002 −71.7 3.0 0.002 MEDICAMENTS Dafalgan syrup 478.9 13.1 17.1 8.4 0.049 18.7 15.6 0.232 −20.6 6.1 0.006 Mucosolvon cough syrup 520.1 13.7 −7.8 9.2 0.625 −41.6 7.5 0.002 −69.9 3.8 0.002 Fluimucil effervescent 496.4 6.8 −11.9 4.7 0.020 −36.1 2.6 0.002 −46.4 4.3 0.006 Tossamin sugar free syrup 510.5 10.3 15.8 14.0 0.492 −13.5 10.6 0.193 −49.5 6.7 0.004 Ventolin syrup 512.9 9.4 −54.2 5.9 0.002 −85.3 6.7 0.002 −74.0 3.7 0.004 Claritine syrup 527.9 15.9 −10.8 5.8 0.106 −13.8 6.4 0.065 −40.8 3.5 0.002 Maltofer syrup 501.7 8.2 9.9 5.7 0.131 −5.4 6.1 0.432 −19.8 4.3 0.002 doi:10.1371/journal.pone.0143957.t002 PLOS ONE \| DOI:10 1371/journal pone 0143957 December 23 2015 8 / 15 lower pH values and Ca concentration, and higher titratable acidity values are significantly related to more loss of SH during erosion. Results Table 1 presents the 30 substances and their chemical parameters. The SH values at baseline (SHbaseline), the mean SH loss (ΔSH) after the first (ΔSH2−0) and second (ΔSH4−2) erosive chal- lenges, as well as the relative surface reflection intensity, are presented in Table 2. Most of the substances caused a significant decrease in SH after the first erosive challenge (p<0.05), with the exception of mineral water (negative control), ice tea peach, apple juice for babies, and some medicaments. Interestingly, during the second erosive challenge, only mineral water, yogurt and some medicaments caused no further loss of SH. After both erosive challenges, all substances caused significant loss in relative surface reflectivity intensity, except for mineral water (Table 2). There was a significant correlation (p < 0.001; ρ = 0.66) between loss in surface hardness (ΔSH4−0) and relative percentage decrease in reflection intensity (rSRI; Fig 1), with regression Eq (1) fitting the data: rSRI ¼ 46:9 þ 0:18 DSH40 ð1Þ ð1Þ rSRI ¼ 46:9 þ 0:18 DSH40 By far the most erosive substance was candy spray, which caused a loss of SH of more than 300 Vickers Hardness Numbers after the first erosive challenge, and caused the greatest relative change in SRI with a decrease of more than 95% in the SRI of the samples. Kiwi fruit caused the greatest decrease in SH during the second erosive challenge. Regarding the chemical parameters, we see that candy spray had the lowest pH and the highest titratable acidity, whereas kiwi exhibited the second-highest titratable acidity. Analysing the effect of the different chemical properties of the drinks on dental erosion in deciduous enamel, we see that pH showed a moderate positive correlation with ΔSH and rSRI, whereas all other parameters showed a weak correlation (Table 3). This was also shown by the results of the multivariate linear regression analyses (Table 4), where, despite the weak correla- tion values observed in Table 3, not only pH, but also titratable acidity, Ca concentration, and, to a lesser extent, Pi concentration all play a role in initial enamel erosion. Table 4 shows that 7 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 Comparing permanent enamel with deciduous enamel treated with the same substances (Table 5), we observed no significant differences in initial hardness between the two kinds of teeth. However, a significant difference was observed in the change in SH when the samples were immersed in Coca-Cola1. After the first erosive challenge (ΔSH2−0), deciduous enamel PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 8 / 15 Erosive Effect of Different Substances on Deciduous Teeth Fig 1. Association between relative surface reflection intensity (rSRI) and change in surface hardness (ΔSH4−0). The solid line represents the regression line (Eq 1). Fig 1. Association between relative surface reflection intensity (rSRI) and change in surface hardness (ΔSH4−0). The solid line represents the regression line (Eq 1) tive surface reflection intensity (rSRI) and change in surface hardness (ΔSH4−0). The solid line represents the doi:10.1371/journal.pone.0143957.g001 exhibited significantly greater hardness loss (−90.2 ± 11.3 VHN) than permanent enamel (−44.3 ± 12.2 VHN; p = 0.007). However, no differences between the two types of teeth were observed in the total change in SH after both challenges (ΔSH4−0), or in the surface reflection intensity. exhibited significantly greater hardness loss (−90.2 ± 11.3 VHN) than permanent enamel (−44.3 ± 12.2 VHN; p = 0.007). However, no differences between the two types of teeth were observed in the total change in SH after both challenges (ΔSH4−0), or in the surface reflection intensity. PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 Discussion Despite the great number of studies on dental erosion, there is still a lack of information regarding the erosive dissolution of deciduous teeth. In the present study, we show the erosive effect of various substances on deciduous enamel. Moreover, we analysed the effect of different chemical factors on the initial erosion process in deciduous teeth. In line with the previous studies, we observed that several soft drinks, fruit juices and smoothies, sour candies, and medicaments can cause significant erosion. This is not surprising given their degree of satura- tion with respect to HAP and FAP. Dental enamel is mostly made up of calcium (Ca2+), phosphate (PO4 3+), hydroxide (OH−), and, to a lesser extent, fluoride (F−) ions [41]. In the oral cavity, the teeth are surrounded by 9 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 Erosive Effect of Different Substances on Deciduous Teeth Table 3. Spearman’s correlation coefficients between the chemical properties of the substances and the difference in surface hardness between baseline and the first erosive challenge (ΔSH2−0), the total difference in surface hardness after all erosive challenges (ΔSH4−0), and the relative dif- ference in surface reflection intensity between baseline and the second erosive challenge (rSRI4−0). Table 3. Spearman’s correlation coefficients between the chemical properties of the substances and the difference in surface hardness between baseline and the first erosive challenge (ΔSH2−0), the total difference in surface hardness after all erosive challenges (ΔSH4−0), and the relative dif- ference in surface reflection intensity between baseline and the second erosive challenge (rSRI4−0). ΔSH2−0 ΔSH4−0 rSRI4−0 pH 0.635* 0.667* 0.644* Titratable acidity −0.197* −0.275* −0.256* [Ca] −0.018 −0.094 −0.155 [Pi] −0.094 −0.158 −0.273* [F] −0.165 −0.232*** −0.126* (pK−pI)HAP ± 0.306* 0.268* 0.153* (pK−pI)FAP † 0.289* 0.245* 0.119* (pK−pI)CaF2 ‡ 0.029 −0.033 −0.061 [Ca], [Pi], [F]: calcium, phosphate and ﬂuoride concentrations, respectively; [Ca], [Pi], [F]: calcium, phosphate and ﬂuoride concentrations, respectively; * signiﬁcant at p<0.05; *** signiﬁcant at p<0.001; ± Degree of saturation with respect to hydroxyapatite; † Degree of saturation with respect to ﬂuorapatite. ‡ Degree of saturation with respect to CaF2. saliva, and the enamel crystals are in a constant equilibrium with the saliva. In other words, there is a continuous exchange of Ca2+, PO4 3+, OH−, and F−between saliva and enamel. doi:10.1371/journal.pone.0143957.t003 p g y g ( S ) p Intercept pH Titratable acidity [Ca] [Pi] ΔSH β p β p β p β p β p ΔSH2−0 −135.70 <0.001 34.45 <0.001 −0.46 <0.001 2244.0 <0.001 ns ns ΔSH4−0 −314.70 <0.001 69.32 <0.001 −0.53 <0.001 3885.0 <0.001 −5457.0 0.023 β-estimates and p-values are listed only for variables with a signiﬁcant impact on ΔSH; [Ca] and [Pi]: calcium and phosphate concentrations, respectively; ns = not signiﬁcant. doi:10.1371/journal.pone.0143957.t004 Erosive Effect of Different Substances on Deciduous Teeth ess at baseline (SHbaseline), difference in surface hardness after the first (ΔSH2 surface reflectivity (rSRI4−0), for deciduous and permanent enamel samples. Table 5. Mean ± SEM (standard error of the mean) for surface hardness at baseline (SHbaseline), difference in surface hardness after the first (ΔSH2 −0) and both (ΔSH4−0) erosive challenges, and the relative change in surface reflectivity (rSRI4−0), for deciduous and permanent enamel samples. Table 5. Mean ± SEM (standard error of the mean) for surface hardness at baseline (SHbaseline), difference in surface hardness after the first (ΔSH2 −0) and both (ΔSH4−0) erosive challenges, and the relative change in surface reflectivity (rSRI4−0), for deciduous and permanent enamel samples. Substance Deciduous Permanent p-value Mineral water (negative control) SHBaseline 509.5±19.6 517.7±11.7 0.280 ΔSH2−0 −5.0±7.7 27.8±13.1 0.089 ΔSH4−0 −11.1±12.0 19.1±12.6 0.089 rSRI4−0 −15.6±11.0 1.6±5.2 0.436 Coca-Cola1 SHBaseline 501.0±12.7 514.8±13.3 0.579 ΔSH2−0 −90.2±11.3 −44.3±12.2 0.007* ΔSH4−0 −169.3±11.2 −139.8±10.7 0.075 rSRI4−0 −83.0±2.0 −86.6±1.4 0.143 * Signiﬁcant difference between deciduous and permanent enamel; SHbaseline: surface hardness at baseline; ΔSH2−0: surface hardness decrease between baseline and the ﬁrst erosive challenge; ΔSH4−0: surface hardness decrease between baseline and the second erosive challenge; rSRI4−0: relative difference in surface reﬂection intensity between baseline and the second erosive challenge. doi:10 1371/journal pone 0143957 t005 doi:10.1371/journal.pone.0143957.t005 values (varying from 2.14 to 6.70) and negative (pK−pI)HAP and (pK−pI)FAP values, which prompted enamel to demineralize. Although the (pK−pI)HAP and (pK−pI)FAP values are good indicators of whether enamel demineralization occurs, they are calculated based on the ionic composition of HAP and FAP of permanent enamel. Deciduous enamel, however, has a slightly different histological compo- sition, so the (pK−pI)HAP and (pK−pI)FAP values presented in Table 1 can only serve as a guide to deciduous enamel dissolution. We therefore carried out the multiple regression analyses to verify which specific variables play a significant role in erosive demineralization of deciduous enamel. Our results suggest that pH, titratable acidity, Ca2+ concentration and, to a lesser extent, Pi concentration in the substances can significantly influence erosion in deciduous enamel. Many studies have demonstrated how Ca concentrations in erosive solutions can modulate enamel demineralization [1, 12, 17, 44]. Higher Ca concentration in a given solution will increase its degree of saturation, thus lessening its erosive effect [45]. This is in line with our results, which showed that higher concentrations of Ca in the tested substances prompted significantly less erosive demineralization. PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 Discussion When the teeth are exposed to substances that have a low concentration of these ions, there is a ten- dency for enamel to release more of these ions to the environment in order to attain a new state of equilibrium [41]. Acidic substances with low pH values can exacerbate this process and lead to further demineralization. Therefore, the solubility of enamel is highly dependent on the pH of the surrounding substance, as well as the substance’s Ca2+, PO4 3+, and (to a lesser extent) F− concentrations [12, 16, 17, 42–44]. These parameters are, therefore, used to calculate the degree of saturation (pK−pI) of the substances with respect to hydroxyapatite (HAP) and fluorapatite (FAP) [33]. The degree of saturation values essentially indicate whether a substance is more or less likely to cause dissolution of enamel. When a substance has (pK−pI)HAP and (pK−pI)FAP values below zero, it is said that the substance is undersaturated with respect to HAP and FAP, and this will cause enamel to dissolve until equilibrium is reached. However, if the substance has positive (pK−pI)HAP and (pK−pI)FAP values, it is considered supersaturated with respect to HAP and FAP, and will cause ions to deposit on the tooth mineral until a new equilibrium is reached [41]. Interestingly, in the present study, the vast majority of the substances had low pH Table 4. Multiple linear regression analysis of the changes in surface hardness (ΔSH) of all specimens after immersion in all substances. Intercept pH Titratable acidity [Ca] [Pi] ΔSH β p β p β p β p β p ΔSH2−0 −135.70 <0.001 34.45 <0.001 −0.46 <0.001 2244.0 <0.001 ns ns ΔSH4−0 −314.70 <0.001 69.32 <0.001 −0.53 <0.001 3885.0 <0.001 −5457.0 0.023 β-estimates and p-values are listed only for variables with a signiﬁcant impact on ΔSH; [Ca] and [Pi]: calcium and phosphate concentrations, respectively; ns = not signiﬁcant. ple linear regression analysis of the changes in surface hardness (ΔSH) of all specimens after immersion in nalysis of the changes in surface hardness (ΔSH) of all specimens after immersion in all substances. Table 4. Multiple linear regression analysis of the changes in surface hardness (ΔSH) of all specimens after PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 10 / 15 Once there, this species then dissociates acting as a proton (H+) carrier into the enamel mineral, and it maintains the acidic (undersaturated) condition that promotes further dissolution [23, 47]. So, higher titratable acidity values are strong indicators of higher concentrations of the undissociated species of the acid, which, in turn, lead to more enamel erosion. Besides the effect of specific chemical factors associated with erosion in deciduous enamel, we also compared the effect of two substances (mineral water and Coca-Cola1) on both per- manent and deciduous teeth. Our results showed no significant differences between the two types of teeth when the specimens were treated with mineral water. Treatment with Coca- Cola1, however, caused a significantly greater loss of SH in deciduous enamel than in perma- nent enamel within the first 2 min (ΔSH2−0), but no differences were observed in the total loss of SH after two erosive challenges (ΔSH4−0). We, therefore, suggest that the initial erosive pro- cess may start differently in the two kinds of teeth, but also the lack of difference after the sec- ond erosive challenge could be due to the small sample size in the present study. In any case, conflicting results have been reported from studies on the dissolution pattern of deciduous and permanent enamel [4, 5, 48–52], so these differences should be further investigated. In the present study, we show that various soft drinks, sour candies, sports drinks and energy drinks, and some fruits and fruit juices are able to cause enamel erosion. Thus, the excessive consumption of such substances can lead to substantial dental erosion, which may compromise patients’ dentition for their entire lifetime [5]. It is important to note that, although the enamel samples were kept in saliva for 3 h to allow the formation of the salivary pellicle, all erosion challenges were made without saliva. More specifically, the sour candy and sour chewing gum were both diluted in water, and the tests on the erosive effect of these substances did not take into account the buffering effect of saliva. In a preliminary experiment carried out in our laboratory, we also dissolved sour chewing gum in human saliva and did the erosive challenge following the methods used in the present study. Dissolving the substance in 10 ml saliva caused no loss in enamel SH after 2 min or 4 min ero- sive challenge. Pi concentration, on the other hand, was not significant during the first erosive challenge (ΔSH2−0), but only became significant after 4 min immersion in the sub- stances (ΔSH4−0). Similar results were also observed by Hemingway, Parker (46], who sug- gested that calcium ions are dissolved from the hydroxyapatite before phosphate ions, thus explaining the relationship between calcium concentration and erosion, and the lack of associa- tion between phosphate concentration and erosion. In addition, Lussi, Megert (22] argue that there are four species of Pi (H3PO4, H2PO4 −, HPO4 2−and PO4 3−) that could be present in a solution, but their concentrations are strongly influenced by the pH of the solution. At acidic pH, most Pi species are in the form of H2PO4 −, and only a minute fraction is in the form of PO4 3-, which is the only species of importance in the ion activity of enamel [22, 46]. Therefore, at low pH, extremely high amounts of Pi would be necessary to increase the degree of satura- tion of a given solution to a level at which it would effectively hinder enamel demineralization [22]. In contrast to what was expected, the multivariate analysis in the present study shows that higher Pi concentrations are associated with a greater loss of SH. This is probably because, within the substances we have tested, the highest [Pi] values were measured in the highly PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 11 / 15 Erosive Effect of Different Substances on Deciduous Teeth erosive substances, such as Coca-Cola1, Pepsi1, Rivella1 Red, kiwi fruit and Gatorade1, and this may be an expression that in some of these substances, like Coca-Cola1 and Pepsi1, there is a high phosphoric acid content, and, consequently, high Pi concentrations. It is, therefore, possible to conclude that (similarly to permanent enamel) Pi concentration does not play a sig- nificant role in erosive dissolution of deciduous enamel. Dissolution of deciduous enamel is, thus, strongly influenced by the Ca concentration, pH and titratable acidity of the substance. Titratable acidity is a measure of the buffering of a solution, and it is directly related to the concentration of the undissociated form of the acid in a given substance [41]. The undissoci- ated form of the acid is of considerable importance because this species has no charge and it is able to diffuse more readily into the near-surface layer of enamel. PLOS ONE \| DOI:10.1371/journal.pone.0143957 December 23, 2015 Acknowledgments The present study was supported by the Deutsche Gesellschaft für Zahnerhaltung (DGPZM- Wissenschaftsfonds). The authors show their gratitude towards G. Fischer and Prof. Häusler, Institute of Mathematical Statistics, University of Bern, for the statistical analyses, and to Bri- gitte Megert for her appreciated efforts in the laboratory. We also thank Dr. R.P. Shellis for his valuable comments on this manuscript. However, Lagerlof and Dawes [53] showed that the maximum volume of saliva in the mouth before swallowing is 1.19 ml or 0.96 ml for males and females, respectively. So, when the sour chewing gum was dissolved in only 2 ml saliva in the preliminary experiment, we observed that even one drop of the solution was able to considerably decrease enamel SH after 2 min and 4 min challenge, which was probably related to the low pH (3.47) of the solu- tion (unpublished results). In this experiment, we used two parameters to measure enamel erosion: change in surface hardness (ΔSH) and surface reflection intenstiy (rSRI). Previous studies have shown that SRI is a viable additional method to measure the erosive demineralization of permanent enamel [37, 38, 54], because it highly correlates with Knoop surface microhardness, calcium release, and surface roughness [36]. In the present study, we were able to further demonstrate that SRI is significantly associated with surface hardness measured with Vickers nanoindentations. 12 / 15 Erosive Effect of Different Substances on Deciduous Teeth Moreover, we also show that SRI is a suitable viable option to measure erosive demineralization on deciduous enamel. In conclusion, we were able to corroborate the erosive potential of a broad range of drinks, foodstuffs and medications commonly consumed/used by children and young adolescents, and we show that erosive dissolution of deciduous enamel is significantly associated with pH, titrat- able acidity and calcium concentration in the solution. This study is an extensive overview, and it can be used to judge the erosive potential of many dietary substances and medications used by children. Author Contributions Conceived and designed the experiments: TSC AL. Performed the experiments: TSC. Analyzed the data: TSC AL. 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https://openalex.org/W2535165121	https://www.hal.inserm.fr/inserm-01382904/file/12931_2016_Article_449.pdf	English	null	Neutrophil elastase cleaves epithelial cadherin in acutely injured lung epithelium	Respiratory research	2,016	cc-by	12,066	To cite this version: Rachel Boxio, Julien Wartelle, Béatrice Nawrocki-Raby, Brice Lagrange, Laurette Malleret, et al.. Neutrophil elastase cleaves epithelial cadherin in acutely injured lung epithelium. Respiratory Re- search, 2016, 17 (1), pp.129. 10.1186/s12931-016-0449-x. inserm-01382904 Neutrophil elastase cleaves epithelial cadherin in acutely injured lung epithelium Rachel Boxio, Julien Wartelle, Béatrice Nawrocki-Raby, Brice Lagrange, Laurette Malleret, Timothee Hirche, Clifford Taggart, Yves Pacheco, Gilles Devouassoux, Abderrazzaq Bentaher © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: azzak.bentaher@inserm.fr 1Inflammation and Immunity of the Respiratory Epithelium Group, Faculté de Médecine Lyon Sud, EA 7426, UCBL 1, Inserm U-1111, Pierre Benite - Lyon Sud, France Full list of author information is available at the end of the article Neutrophil elastase cleaves epithelial cadherin in acutely injured lung epithelium Rachel Boxio1, Julien Wartelle1, Béatrice Nawrocki-Raby2, Brice Lagrange1, Laurette Malleret1, Timothee Hirche3, Clifford Taggart4, Yves Pacheco1, Gilles Devouassoux1,5 and Abderrazzaq Bentaher1* Abstract Background: In acutely injured lungs, massively recruited polymorphonuclear neutrophils (PMNs) secrete abnormally neutrophil elastase (NE). Active NE creates a localized proteolytic environment where various host molecules are degraded leading to impairment of tissue homeostasis. Among the hallmarks of neutrophil-rich pathologies is a disrupted epithelium characterized by the loss of cell-cell adhesion and integrity. Epithelial-cadherin (E-cad) represents one of the most important intercellular junction proteins. E-cad exhibits various functions including its role in maintenance of tissue integrity. While much interest has focused on the expression and role of E-cad in different physio- and physiopathological states, proteolytic degradation of this structural molecule and ensuing potential consequences on host lung tissue injury are not completely understood. Methods: NE capacity to cleave E-cad was determined in cell-free and lung epithelial cell culture systems. The impact of such cleavage on epithelial monolayer integrity was then investigated. Using mice deficient in NE in a clinically relevant experimental model of acute pneumonia, we examined whether degraded E-cad is associated with lung inflammation and injury and whether NE contributes to E-cad cleavage. Finally, we checked for the presence of both degraded E-cad and NE in bronchoalveolar lavage samples obtained from patients with exacerbated COPD, a clinical manifestation characterised by a neutrophilic inflammatory response. Results: We show that NE is capable of degrading E-cad in vitro and in cultured cells. NE-mediated degradation of E-cad was accompanied with loss of epithelial monolayer integrity. Our in vivo findings provide evidence that NE contributes to E-cad cleavage that is concomitant with lung inflammation and injury. Importantly, we observed that the presence of degraded E-cad coincided with the detection of NE in diseased human lungs. Conclusions: Active NE has the capacity to cleave E-cad and interfere with its cell-cell adhesion function. These data suggest a mechanism by which unchecked NE participates potentially to the pathogenesis of neutrophil-rich lung inflammatory and tissue-destructive diseases. Keywords: E-cadherin, Neutrophil elastase, Epithelium disruption, Lung inflammation and injury * Correspondence: azzak.bentaher@inserm.fr HAL Id: inserm-01382904 https://inserm.hal.science/inserm-01382904v1 Submitted on 17 Oct 2016 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. Boxio et al. Respiratory Research (2016) 17:129 DOI 10.1186/s12931-016-0449-x Background NE, a potent proteolytic enzyme, is stored in an active form at high concentration in pri- mary granules (~4 μg/106 cells) making it a major com- ponent of neutrophils [6]. The enzyme is rapidly discharged into the phagolysosome following bacterial uptake by neutrophils. Previously, we and others have shown that NE is required for maximal neutrophil killing of invading pathogens [7–10]. In the setting of overwhelming inflammatory condi- tions, activated neutrophils secrete abnormally NE in the extracellular space [11]. Active NE creates a localized proteolytic environment where a wide range of host soluble and insoluble molecules could be degraded. As a consequence, NE has been incriminated in the patho- genesis of different acute and chronic tissue-destructive diseases including acute lung injury/acute respiratory distress syndrome (ALI/ARDS), cystic fibrosis (CF) and chronic obstructive pulmonary diseases (COPD) (e.g., emphysema) [12, 13]. Among the common hallmarks of these pathologies is a disrupted epithelium characterized by the loss of its integrity and sloughing of epithelial cells [14]. The likelihood that NE is associated with these diseased situations is supported by at least two lines of evidence. First, NE is potent proteolytic enzyme with a large substrate repertoire comprising extracellular matrix proteins [15]. Second, various human and animal studies reported the presence of free active NE in lung tissues and fluids overwhelming the anti-NE screen [16–20]. Whether NE has the capacity to alter the tight epithelial cell-cell adhesion contributing to lung tissue destruction is not completely addressed particu- larly in vivo. The goals of these studies were several folds. We sought to determine if NE could cleave E-cad and alter its ability to maintain a stable lung epithelial cell mono- layer. Next, using mice deficient in NE in a clinically relevant experimental model of acute pneumonia, we wanted to determine whether degraded E-cad is associ- ated with lung inflammation and injury and whether NE contributes to E-cad cleavage. Finally, in order to deter- mine the potential relevance of our findings to human lung diseases, we examined whether degraded E-cad coin- cides with the presence of active NE in bronchoalveolar lavage samples obtained from patients with exacerbated COPD, a clinical condition characterized by a neutrophilic inflammatory response. E-cad represents one of the most important and ubi- quitous adherens junction protein. E-cad, a member of the cadherin superfamily, is a glycoprotein that crosses the membrane only once [21]. Background physicochemical barrier for efficient defence [1]. Indeed, various structural molecules are known to establish tight epithelial cell-cell adhesion and along with their connec- tion to the internal cytoskeleton provide the lung with an intact and impermeable epithelium [2]. To protect itself against various infectious or toxic agents, the lung relies on different mechanisms. Among these latter, the epithelium and resident macrophages are considered as the first lines of lung tissue protection. With respect to the epithelium, this cell lining partici- pates in mounting an appropriate inflammatory response against insulting agents but acts primarily as a When these “sentinel” lines are breached, neutrophils are called in. The primary purpose of this neutrophilic infiltration is phagocytosis of foreign particles and/or contribution to the resolution of associated inflamma- tion [3]. To this end, two systems categorized as oxygen- dependent and -independent have been described in neutrophils [4]. The non-oxidative system comprises the Boxio et al. Respiratory Research (2016) 17:129 Page 2 of 15 Page 2 of 15 processes including tissue/organ development, morpho- genesis, and cytoskeletal organization [23]. Much inter- est has focused on the expression and role of E-cad in different physio- and physiopathological states. Limited studies have, however, dealt with the proteolytic degrad- ation of this structural molecule by NE and ensuing con- sequences on host tissue architecture (e.g., contribution to lung tissue injury). In a rat model of pancreatitis, Mayerle J. et al. reported that neutrophil-derived NE, rather than pancreatic elastase, degraded E-Cad [24]. Evans SM and his colleagues observed released E-cad into the BAL fluids in a mouse model challenged with human NE [25]. Work by Downey G. and his colleagues identified a mechanism whereby NE-mediated cleavage of E-cad induced β-catenin signalling, which appears to play a critical role in reepithelialisation of denuded epi- thelium in a mouse lung inflammation model [26]. The ability of NE to degrade other cell-cell junction proteins prior to E-cad may not be ruled out [27]. Of interest as well, it has been reported that NE mediates degradation of vascular E-cad (VE-cad) that could compromise the endothelial vascular integrity contributing to micro- vascular injury and increased permeability and intersti- tial oedema [28]. readily active serine proteinase, neutrophil elastase (NE), among other polypeptides. NE is structurally related to its family members, cathepsin G and proteinase 3, and share the conserved charge-relay triad, His57-Asp102- Ser195, where Ser is the active residue (chymotrypsino- gen numbering) [5]. Background The extracellular portion of E-cad consists of a domain of five repeats that are highly homologous to each other and commonly desig- nated as EC1-EC5 (EC1 being the beginning of E-cad N-terminus). Each repeat is comprised of approximately 110 amino acid residues. This extracellular domain en- sures homophilic recognition and Ca2 + −dependent cell-cell adhesion. Intracellularly, E-cad cytoplasmic do- main is linked to actin filaments through interactions with catenin proteins reinforcing, as mentioned above, the stability of the epithelium [22]. Other functions of E-cad include its role as a signalling molecule and regu- lator of the epithelium permeability and polarity [23]. The protein is implicated in a number of biological Human samples, mice, cells, and bacteria Human samples, mice, cells, and bacteria Bronchoalveolar lavage (BAL) specimens. Remainders of BAL fluids were obtained in conjunction with a prior study of patients with COPD exacerbation [29, 30]. Mice. NE-deficient mice (NE−/−) were generated by targeted mutagenesis [8]. NE−/−and wild type (WT) mice (C57Bl/6 J, 8–10 weeks old) were housed in a pathogen-free facility with 12 h light/dark cycle and pro- vided with food and water ad libitum. Cells. Human bronchial epithelial cell line 16HBE (kindly provided by Dr. Gruenert, University of California at San Francisco, San Francisco, CA) and mouse alveolar cell line MLE15 (kindly provided by J. Wittset, University of Cincinati, Cincinati, OH) were cultured to confluence in DMEM (for 16HBE) or RPMI 1640 (for MLE15) culture media as previously described [31, 32]. Prior to any treatment, cells were washed three times with sterile phosphate-buffered saline (PBS) and cultured for 1 h in the absence of FCS. Methods Reagents g Purified human NE, CG and PR3 and NE corresponding polyclonal rabbit antibody were from Elastin Products Company (Owensville, MO, USA). The purity and activity of each enzyme were confirmed by sodium-dodecyl- sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and spectrophotometrically using specific substrates according to the manufacturer’s recommendations. The chromogenic peptide substrates were N-methoxysuccinyl- Al-Al-Pro-Val-pNA, N-Boc-Ala-ONp, and N-succinyl-Al- Al-Pro-Phe-pNA for NE, PR3 and CG, respectively. Chromogenic peptide substrates were obtained from Elastin Products Company. Recombinant human secretory leukocyte proteinase inhibitor (SLPI) and polyclonal rabbit Boxio et al. Respiratory Research (2016) 17:129 Page 3 of 15 Page 3 of 15 antibody for mouse extracellular E-Cadherin domain were from R&D Systems Europe. Monoclonal mouse antibodies for human extracellular and polyclonal rabbit antibody against human cytoplasmic E-Cadherin domain were from Cell Signalling Technology (Ozyme) and Takara (Cambrex Bio Science). Monoclonal mouse antibody against human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was from Chemicon International (Abcys). Secondary FITC- labelled or HRP conjugated polyclonal goat antibody against mouse immunoglobulin and secondary FITC- labelled or HRP conjugated polyclonal swine antibody against rabbit immunoglobulin were from Dakocytomation (Trappe’s). All tissue culture reagents and other chemicals were reagent grade and purchased from Invitrogen or Sigma-Aldrich. using RIPA buffer and quantified as previously described [34]. Next, equal protein aliquots (10 μg) were incubated alone or in the presence of varying concentrations of NE, CG or PR3 at 37 °C for designated periods of time. The reactions were carried out in a 20 μl volume in PBS at pH 7.4, which should approximate the pH in the extracellular milieu of the lung and corresponds to pH optimum of NE. In parallel experiments, NE (50 nM, highest concentration) was preincubated with SLPI (100 nM) at 37 °C for 5 min prior to addition to cell protein extracts. Immunofluorescence microscopy py MLE-15 epithelial cells were grown on cover slips to confluence. Next, cell monolayers were cultured alone or in the presence of designated concentration of puri- fied NE for a defined period of time. In parallel experi- ments, mouse PMNs were added to MLE-15 epithelial cells at a ratio 1:10 (epithelial cell:PMN). Prior to addition to epithelial cells, neutrophils were first primed and stimulated by addition of LPS (10 μg/ml) and formyl-methionyl-leucyl-phenylalanine (fMLP, 1 μM) [34]. NE release from activated cells was examined using NE specific chromogenic peptide substrate. Under these experimental conditions, over 80 % of neutrophils were still alive upon addition to epithelial cells, as judged by trypan blue dye exclusion. However, their viability was compromised 6 h after co-culture since we could barely detect their nuclei by staining with TO-PRO-3 iodide. Polymorphonuclear neutrophils (PMNs) were isolated from mouse WT femur and tibia tips-derived bone mar- row as previously described [33]. A Percoll gradient was employed to separate morphologically mature neutro- phils, which were used immediately. Purified neutrophils represented >95 % of the cell population and >98 % were viable as judged by differential counting and trypan blue dye exclusion respectively. Bacteria. In this work, we used Pseudomonas aerugi- nosa H103 (kindly provided by R. Hancock, University of British Columbia, Vancouver, Canada). Bacteria were grown aerobically to late exponential phase (3 h), washed twice, and resuspended in 1 ml of PBS (pH 7.4). The optical density (OD) of bacterial culture was deter- mined at 600 nm (OD) (OD 1 ≈109 bacteria/ml). Six hours post-treatment, cover slips were processed for immunofluorescence microscopy. Briefly, cells were fixed for 10 min in 3 % (w/v) paraformaldehyde. Nonspecific binding was blocked with 3 % bovine serum albumin in PBS for 30 min, and cells were incubated with polyclonal antibody specific for mouse extracellular E-cadherin domain (dilution 1:750) for 1 h at room temperature. After washing, samples were FITC-labelled secondary antibody-immunostained (dilution 1:2000) for fluorescence microscopy [34]. The nuclei were stained with TO-PRO-3 iodide (Molecular Probes) for 10 min. Exposure of epithelial cell protein extracts to NE Media of cell cultures were removed and confluent cells were scraped. Proteins were extracted from cell pellet Exposure of epithelial cells to NE Confluent 16HBE epithelial cell monolayers were cul- tured alone or in the presence of designated concentra- tion of purified NE for a defined period of time [35]. In parallel, NE was preincubated with SLPI at 37 °C for 5 min prior to addition to cells. At the end of treatment time, culture supernatants were collected, centrifuged to remove cell debris, and acetone-concentrated. Cells were scraped and proteins were extracted and quantified as described above. Equal aliquots of culture supernatants or lysate proteins (10 μg) were resuspended in PBS, and subjected to SDS-PAGE and Western blotting as described below. Exposure of epithelial cell protein extracts to NE p p p Media of cell cultures were removed and confluent cells were scraped. Proteins were extracted from cell pellet Page 4 of 15 Boxio et al. Respiratory Research (2016) 17:129 Boxio et al. Respiratory Research (2016) 17:129 and snap-frozen until use. Mouse lungs were processed for histologic analyses as previously described [35]. Following a final gentle wash, cells were mounted in Vectashield (Vector Laboratories) on slides using Secure Seal imaging spacers (Sigma). Samples were examined using a LSM 510 Meta laser scanner microscope (Carl Zeiss Inc., Thornwood, New York, USA). Equivalent concentrations of preimmune serum were used as a negative control. Fluorescence intensity was estimated by determination of the average area of stained regions [35, 36]. Briefly, 15 randomly digitized images represen- tative of each condition of the experiments were cap- tured in a blinded manner (A.B.) and analysed using the image analysis software ImageJ (NIH, Bethesda, MD). Western blotting Equal proteins aliquots derived from cell cultures and murine or human BAL fluids were resolved on 12 % SDS-polyacrylamide gels and transferred to polyvinyli- dene difluoride membranes (Millipore Corp., Bedford, MA). Of note, unlike murine BAL fluids, human BAL (5 ml) were concentrated by lyophilisation prior to determination of protein concentration [35]. Blots were incubated with the indicated antisera specific to extracel- lular or cytoplasmic E-cad domains or NE (dilution for E-cad antibodies 1:1000 and dilution for NE 1:2500) [35]. Membranes were subsequently incubated with an appropriate dilution of HRP-linked secondary IgG (dilution 1:2000) in blocking buffer. Immunoreactive fragments were visualized by enhanced chemilumines- cence (ECL, Amersham Biosciences). Labelled pro- teins or fragments were detected with the Molecular Imager ChemiDoc XRS System and quantified using Quantity One 1-D Analysis Software (Biorad, Marnes- la-Coquette, France). Mouse model of pneumonia Immunostaining on lung tissue sections was performed as previously described [34]. Sections were incubated with antibodies specific for mouse E-cad (dilution, 1:750) at 4 °C overnight. Next, samples were incubated for 20 min with biotinylated secondary antibody and labelled with HRP-conjugated streptavidin. Immune complexes were visualized using 3’3-diaminobenzidine (DAB) (Biocare Medical) as substrates for HRP and counterstained with Mayer’s hematoxylin. Slides were examined using a LSM 510 Meta laser scanner microscope. Mice were intranasally challenged with P. aeruginosa bacteria or sterile saline. Briefly, mice were anesthetized followed by intranasal administration of 50 μl of sterile PBS or containing a sublethal dose of bacteria (4×106 colony forming units (CFUs)/per mouse, which we have previously shown to injure acutely mouse lungs [34]. Groups of mice (n = 4/genotype/time point) were sacrificed at designated time points, and their lungs were lavaged in situ using Hank’s balanced salt solution (pH 7.4), cycled in three times. Identical recoveries of lavages (700 ml/mouse) were obtained for each of the experimental groups. Cell counts from BAL fluids were immediately performed by hemocytometer and aliquots of BAL fluids were cytospun and Wright-stained for differential counting (Thermo Shandon and Fisher Scientific). Next, the remaining BAL samples were cen- trifuged for 10 min at 4 °C to remove cells, aliquoted Time-lapse videomicroscopy First, confluent monolayers of 16HBE epithelial cells were first subjected to immunofluorescence staining as described above. Cells were incubated with human anti- E-Cad specific antibody against the extracellular domain (dilution 1:750) followed by FITC-labelled secondary antibody (dilution 1:2000). Next, cell monolayers were incubated alone or in the presence of NE (200 nM). In parallel experiments, cells were exposed to NE that was pretreated with SLPI (400 nM) or SLPI alone. Cell culture plates were transferred immediately to Zeiss IM35 inverted microscope (Zeiss, Oberkochen, Germany) equipped with an incubation chamber main- tained at 37 °C with 5 % CO2 in a wet atmosphere. Video recording was performed using a Panasonic WVCD51 digital camera (Osaka, Japan) controlled by a Sparc 2 Sun workstation (Sun Microsystems, Mountain View, CA) with a video board (Parallax Graphics, Santa Clara, CA) [31]. Images of untreated or NE-treated cells were captured every 15 min for 6 h using 10X magnifi- cation to analyse at least 100 cells per field of view. Neutrophil elastase activity Neutrophil elastase activity in cell-free BAL fluids was assessed using conventional chromogenic peptide assays [35]. Briefly, BAL aliquots (100 μl) were incu- bated with NE peptide substrate Meo-Suc-Al-Al-Pro- Val-pNA (0.2 mM) for 60 min at 37 °C in a total volume of 200 μl Tris-NaCl buffer (0.1 M Tris and 1 M NaCl, pH 7.4). The reactions were then spun (30 s, 12,000 rpm), and changes in the absorbance of the superna- tants were determined spectrophotometrically at λ 410 nm. Lactate dehydrogenase (LDH) activity assay Changes in LDH activity were used as a marker for cyto- toxicity and were assessed in cell-free BAL fluids by the LDH kit following the manufacturer’s recommendations (Sigma-Aldrich) [37]. LDH catalyses the oxidation of lac- tate to pyruvate. This reaction is coupled with reduction Page 5 of 15 Boxio et al. Respiratory Research (2016) 17:129 but NE was by far the most potent degrading protease (Additional file 1). In the subsequent experiments, we focused on NE for further characterization of E-cad cleavage. of NAD to NADH, which is followed spectrophotomet- rically at 340 nm. The LDH activity is proportional to the rate of absorbance changes. Briefly, 100 μl of BAL samples were added to 900 μl LD-L reagent and changes in absorbance were recorded over 3 min. NE degrades E-cad in an enzyme dose- and incubation time-dependent fashion To determine whether E-cad potentially represents a substrate for NE, we incubated cell protein extracts con- taining E-cad with purified NE. We used protein extracts derived from bronchial cell line 16HBE or alveolar epithelial-like cell line MLE15. Various NE concentra- tions can be encountered in vivo including very high concentrations near sites of granule exocytosis [38]. Accordingly, we examined a wide range of NE concen- trations (1, 5, 10, 25, and 50 nM) against a fixed cell pro- tein extract for varying incubation times. As judged by Western blotting using specific polyclonal antibodies against human or mouse extracellular E-cad domain, identical fragmentation was observed with both human and mouse cell line protein extracts in a manner dependent of NE dose and incubation time (Fig. 1a-c). Progressive cleavage of E-cad started with enzyme con- centration as low as 5 nM. Also, NE catalysed rapid and complete cleavage of intact E-cad (100 % of E-cad was completely degraded by 50 nM of NE within 15 min) (Fig. 1c). Of interest, identical degradation patterns were obtained when incubations were performed in PBS (pH 7.4) or Tris-buffered saline (data not shown). Significantly, E-cad degradation was prevented when NE was preincubated with the specific serine protease inhibitor SLPI indicating that the catalytic activity of the enzyme is required to cleave E-cad (Fig. 1d). Of interest, cleavage of E-cad was also observed in the presence of the other two neutrophil serine-proteases CG and PR3, Statistics Densitometric quantification of western blots was per- formed with the AIDA software (Raytest, Staubenhardt, Germany). Data were analysed by Kruskall-Wallis and Mann–Whitney tests with the StatView software (SAS Institute Inc., Cary, NC, USA). Albumin concentration The ability of NE to degrade E-cad was next examined in cell culture system. After exposure of cells to increas- ing concentrations of NE for a fixed time period, both cell lysates and condition media were analysed by Western blotting. As shown in Fig. 2a-left panel, using an antibody raised against human N-terminal part of E- cad, levels of a distinct cleavage product of about 80 kDa increased progressively in culture supernatants in func- tion of NE concentration coinciding with the disappear- ance of E-cad protein in cell lysates (Additional file 2). Using an antibody raised against the C-terminal part of E-cad, complete degradation of the protein in cell lysates was achieved at 200 nM, four fold higher than that used with protein extracts of Fig. 1 (Fig. 2b, left panel). Ana- lysis of condition media with an antibody against the N-terminal part confirmed loss of intact E-cad, which paralleled the appearance of the distinct 80 kDa degrad- ation fragment (Fig. 2c, left panel). As in Fig. 1, inhib- ition of NE by SLPI, prevented cell-associated E-cad degradation and SLPI alone had no effect (Fig. 2b and c). All these data were further supported by densitomet- ric analyses (Fig. 2a - c; right panels). The albumin level changes were examined using the bromocresol green assay (BCG, Sigma-Aldrich), accord- ing to the manufacturer’s recommendations [37]. Briefly, 100 μl of samples or standards were incubated with 900 μl of BCG substrate for 60 s at room temperature. Changes in absorbance were recorded by spectropho- tometer at 628 nm and were proportional to the albu- min concentrations in the samples. Alteration of epithelial cell monolayer following exposure to purified NE or activated neutrophil-derived NE a-b, incubation with NE (0, 1, 5, 10, 25, 50 nM) for defined time point (30 min) resulted in an enzyme dose-dependent degradation of E-cad derived from 16HBE (a) or MLE15 (b) epithelial cell extracts. c, Incubation of MLE15 epithelial cell extracts with NE (50 nM) for defined time points (0, 10, 15, 20, 25, 30 min) resulted in stepwise degradation of E-cad. d, E-cad cleavage was largely abrogated in both 16HBE and MLE15 protein extracts when NE (50 nM) was pre-incubated with the physiologic serine proteinase inhibitor SLPI (100 nM). Molecular weight (MW) (kDa) standards are between panels. The findings are illustrative of at least three independent experiments of cleaved product (data not shown); findings consist- ent with those in Fig. 2. distributed around cohesive cells (Fig. 4c). In contrast, cells exposed to NE exhibited a dramatic decrease of fluorescence intensity over recording time, concomitant with the appearance of rounded cells and marked inter- cellular space enlargement (Fig. 4d and an additional movie file shows this in more detail [see Additional file 4: Movie S2]). There were less striking morphologic changes and variations in fluorescence intensity in plate wells treated with either NE that was preincubated with SLPI or SLPI alone (Fig. 4e and f and additional movie files show this in more detail [see Additional file 5: Movie S3 and Additional file 6: Movie S4]). Alteration of epithelial cell monolayer following exposure to purified NE or activated neutrophil-derived NE p p Next, to examine the impact of NE-mediated degrad- ation of E-cad on the integrity of epithelial cell mono- layer, purified or activated neutrophil-derived NE (free and/or cell-bound) were added to cultured cells. Immunofluorescence staining of untreated cell mono- layer with N-terminal E-cad antibody found strong im- munoreactive E-cad surrounding cells, consistent with its known membrane localization that confers cells with tight adhesion (Fig. 3a). Incubation with purified NE (200 nM), however, induced loss of E-cadherin immuno- staining resulting in the appearance of patchy gaps within the monolayer (Fig. 3b). We then sought to determine whether secreted NE by activated neutrophils mediates similar effects. The number of neutrophils added per well was calculated to yield concentrations of liberated NE approximately comparable to that shown in Fig. 3b [34]. As for NE, addition of activated neutrophils resulted in loss of epithelial cell layer integrity suggesting the most likely implication of this protease (Fig. 3c). Immunoblotting experiments on protein extracts of cells or culture su- pernatants found loss of intact E-cad and generation Page 6 of 15 Boxio et al. Respiratory Research (2016) 17:129 Fig. 1 Degradation of E-cad is NE concentration and incubation time dependent. Epithelial cell protein extracts containing E-cadherin (10 μg) were incubated alone or in the presence of varying concentrations of NE for various time periods. The reactions were resolved by SDS-PAGE under reducing conditions and visualized by immunoblotting. a-b, incubation with NE (0, 1, 5, 10, 25, 50 nM) for defined time point (30 min) resulted in an enzyme dose-dependent degradation of E-cad derived from 16HBE (a) or MLE15 (b) epithelial cell extracts. c, Incubation of MLE15 epithelial cell extracts with NE (50 nM) for defined time points (0, 10, 15, 20, 25, 30 min) resulted in stepwise degradation of E-cad. d, E-cad cleavage was largely abrogated in both 16HBE and MLE15 protein extracts when NE (50 nM) was pre-incubated with the physiologic serine proteinase inhibitor SLPI (100 nM). Molecular weight (MW) (kDa) standards are between panels. The findings are illustrative of at least three independent experiments Fig. 1 Degradation of E-cad is NE concentration and incubation time dependent. Epithelial cell protein extracts containing E-cadherin (10 μg) were incubated alone or in the presence of varying concentrations of NE for various time periods. The reactions were resolved by SDS-PAGE under reducing conditions and visualized by immunoblotting. Spatio-temporal dynamics of NE-mediated disruption of epithelial cell monolayer To follow changes in epithelial cell monolayer integrity in association with NE-mediated degradation of E-cad, we used time-lapse videomicroscopy approach. Cell behav- iour was analysed first by phase-contrast image record- ings. Initially, cells displayed a flat polygonal morphology (Fig. 4a). Following addition of NE, the morphology of cells changed and became rounded due to detachment of cells from each other, in distinct areas (Fig. 4b and an additional movie file shows this in more detail [see Additional file 3: Movie S1]). At the end of recording time, considerable number of cells was floating in culture medium. Of note, there were areas of the cell monolayer that were not affected by morphologic changes. In vivo detection of E-cad and NE Experiments were repeated three times with similar findings Fig 2 NE degrades cell associated E Cad and generates a distinct extracellular fragment Confluent 16HBE cells were left untreated or treated with Fig. 2 NE degrades cell-associated E-Cad and generates a distinct extracellular fragment. Confluent 16HBE cells were left untreated or treated with varying concentrations of purified NE (0, 2, 20, or 200 nM) for 6 h. Next, equal protein aliquots from culture supernatants and cell lysates (10 μg) were subjected to SDS-PAGE and immunoblotting using antibodies raised against N- and C-terminal parts of E-cad respectively. a, left panel. Anti-E-cad N-terminal antibody revealed a progressive increase of a distinct N-terminal fragment of about 80 kDa that paralleled the increase of NE concentration. a, lower panel, densitometric analysis confirms increased levels of E-cad fragment. Data are mean values ± SD. p < 0.05; Kruskall-Wallis test. b, right panel. Loss of intact E-cad was achieved with NE at 200 nM. Of note, anti-E-cad C-terminal antibody detected varying fragments. NE inhibition with SLPI (400 nM) prevented degradation of cell-associated E-cad. b, lower panel, densitometric analysis found low levels of intact E-cad when cells were exposed to NE alone. However, preincubation of NE with SLPI prevented considerably such degradation. Data are mean values ± SD. p < 0.05; Mann–Whitney test. c, left panel. Immunoblotting analysis of condition media from B with an anti-E-cad N-terminal part confirmed the generation of the distinct 80 kDa degradation product, which paralleled the loss of intact E-cad. c, lower panel, densitometric analysis found increased level of E-cad fragment concomitant with low levels of intact E-cad when cells were exposed to NE alone in B. However, preincubation of NE with SLPI prevented considerably E-cad degradation. MW standards are on wright. a.u., arbitrary unit. Data are mean values ± SD. * corresponds to p < 0.05 for cleaved versus intact E-cad; Mann–Whitney test. Experiments were repeated three times with similar findings (Fig. 5c). Concomitant with E-cad degradation and active NE, analysis of the inflammatory response in BAL fluids found a massive cellular recruitment predominated with neutrophils and increased albumin level and LDH release (Additional file 7, [34, 40]). expression restricted to epithelia, consistent with the known sites of E-cad localization (Fig. 5a) [25]. However, O.N. post-infection with P. aeruginosa, strong E-cad im- munostaining was observed in both epithelial lining and intraalveolar exudates (Fig. 5b, upper panels). In vivo detection of E-cad and NE No stain- ing was seen when the primary antibody was replaced with the preimmune serum (Fig. 5b, lower panels). Next, we analysed lung tissues of control and infected animals to look for evidence of simultaneous presence of active NE and E-cad. NE activity assay in BAL fluids detected significant amounts of NE suggesting the presence of ac- tive neutrophil-derived NE within the alveolar spaces NE contributes to degradation of endogenous E-cad in the setting of lung injury To assess the specific role of NE in neutrophil- mediated degradation of E-cad, WT and NE-deficient (NE−/−) mice were i.n. challenged with P. aeruginosa as described above and sacrificed 4 and 24 h In vivo detection of E-cad and NE In vivo detection of E-cad and NE Next, we determined whether NE mediates E-cad degradation in injured lungs using our previously described mouse P. aeruginosa pneumonia model [34]. To circumvent the confounding effect of P. aeruginosa metalloelatase, known to degrade a variety of host mole- cules, we employed P. aeruginosa strain H103, which lacks this enzyme [39]. Immunostaining of lung sections from unchallenged control wild type mice found E-cad Using immunofluorescence staining combined with time-lapse videomicroscopy, the pattern of E-cad in un- treated cells was similar to that seen in Fig. 3a through- out the recording time. Indeed, E-cad was uniformly Boxio et al. Respiratory Research (2016) 17:129 Page 7 of 15 Fig. 2 NE degrades cell-associated E-Cad and generates a distinct extracellular fragment. Confluent 16HBE cells were left untreated or treated with varying concentrations of purified NE (0, 2, 20, or 200 nM) for 6 h. Next, equal protein aliquots from culture supernatants and cell lysates (10 μg) were subjected to SDS-PAGE and immunoblotting using antibodies raised against N- and C-terminal parts of E-cad respectively. a, left panel. Anti-E-cad N-terminal antibody revealed a progressive increase of a distinct N-terminal fragment of about 80 kDa that paralleled the increase of NE concentration. a, lower panel, densitometric analysis confirms increased levels of E-cad fragment. Data are mean values ± SD. p < 0.05; Kruskall-Wallis test. b, right panel. Loss of intact E-cad was achieved with NE at 200 nM. Of note, anti-E-cad C-terminal antibody detected varying fragments. NE inhibition with SLPI (400 nM) prevented degradation of cell-associated E-cad. b, lower panel, densitometric analysis found low levels of intact E-cad when cells were exposed to NE alone. However, preincubation of NE with SLPI prevented considerably such degradation. Data are mean values ± SD. p < 0.05; Mann–Whitney test. c, left panel. Immunoblotting analysis of condition media from B with an anti-E-cad N-terminal part confirmed the generation of the distinct 80 kDa degradation product, which paralleled the loss of intact E-cad. c, lower panel, densitometric analysis found increased level of E-cad fragment concomitant with low levels of intact E-cad when cells were exposed to NE alone in B. However, preincubation of NE with SLPI prevented considerably E-cad degradation. MW standards are on wright. a.u., arbitrary unit. Data are mean values ± SD. * corresponds to p < 0.05 for cleaved versus intact E-cad; Mann–Whitney test. NE contributes to degradation of endogenous E-cad in the setting of lung injury To assess the specific role of NE in neutrophil- mediated degradation of E-cad, WT and NE-deficient (NE−/−) mice were i.n. challenged with P. aeruginosa as described above and sacrificed 4 and 24 h Boxio et al. Respiratory Research (2016) 17:129 Page 8 of 15 Fig. 3 Purified NE or activated neutrophils alters epithelial monolayer integrity. Confluent cultured MLE15 cells were exposed to purified or activated neutrophils for 6 h. a, immunofluorescence staining of untreated cell monolayer with N-terminal E-cad antibody found strong immunoreactive E-cad surrounding cells. b, exposure of cells to purified NE (200 nM) resulted in considerable loss of E-cadherin immunostaining and disruption of cell-cell adhesion. c, activated neutrophils alter epithelial cell layer integrity with the appearance of patchy gaps. The number of neutrophils added to cells was calculated to yield concentration of liberated NE closely equivalent to that of purified NE. d, fluorescence quantification of cultured cells. Note NE-treated cells or cells co-cultured with neutrophils showed significantly less staining than untreated cells. Data are presented as means ± SEM per experimental condition. ** correspond to p < 0.0001 for conditions of cell treatment with NE or PMNs versus control. a.u., arbitrary unit. Scale bar, 120 μm. Experiments were repeated three times with similar observations Fig. 3 Purified NE or activated neutrophils alters epithelial monolayer integrity. Confluent cultured MLE15 cells were exposed to purified or activated neutrophils for 6 h. a, immunofluorescence staining of untreated cell monolayer with N-terminal E-cad antibody found strong immunoreactive E-cad surrounding cells. b, exposure of cells to purified NE (200 nM) resulted in considerable loss of E-cadherin immunostaining and disruption of cell-cell adhesion. c, activated neutrophils alter epithelial cell layer integrity with the appearance of patchy gaps. The number of neutrophils added to cells was calculated to yield concentration of liberated NE closely equivalent to that of purified NE. d, fluorescence quantification of cultured cells. Note NE-treated cells or cells co-cultured with neutrophils showed significantly less staining than untreated cells. Data are presented as means ± SEM per experimental condition. correspond to p < 0.0001 for conditions of cell treatment with NE or PMNs versus control. a.u., arbitrary unit. Scale bar, 120 μm. Experiments were repeated three times with similar observations or as a consequence of the shear stress of the in and out cycling of PBS during lung lavage. post-challenge. As shown in Fig. NE contributes to degradation of endogenous E-cad in the setting of lung injury 6a and b, immuno- blotting of infected cell-free WT and NE−/−BAL fluids and densitometric analysis revealed gradual generation of endogenous cleavage product in func- tion of time, with similar ~80 kDa size to that generated by purified NE. More importantly, NE−/− cell-free BAL fluids showed less degradation of E-cad as compared to WT cell-free BALs. NE activity assay analysis revealed the presence of active NE in WT, but not NE−/−, cell-free BAL fluids that became abundant at time point 24 h (Fig. 6c). Thus, our find- ings demonstrate that NE contributes to efficient cleavage of E-cad. Of note, low levels of E-cadherin extracellular domain were also seen in PBS-challenged mice. This suggests that either a basal level of E-cadherin is constitutively shed from the epithelium Detection of both cleaved E-cad and NE in BAL of patients with exacerbated COPD To determine the relevance of cell and animal data to human lung diseases, we looked for evidence for the pres- ence of both degraded E-cad and NE in cell-free BAL fluids obtained from exacerbated COPD patients charac- terized by an abnormal influx of neutrophils. As shown in Fig. 7a, there was a marked increase in the levels of ~80 kDa immunoreactive E-cad fragment in all cell-free BAL samples, a finding that corroborated our cell culture and mouse data. Next, after stripping and immunoblotting of the same Western blot membranes for NE, densitomet- ric analyses revealed the presence of increased NE levels Boxio et al. Respiratory Research (2016) 17:129 Page 9 of 15 Fig. 4 Spatio-temporal changes of epithelial cell monolayer following treatment with NE. Confluent monolayers of 16HBE epithelial cells were incubated alone or in the presence of defined NE concentration (200 nM). Briefly, cells were first subjected to immunofluorescence staining of E-cad as described in material and methods. Next, cell monolayers were left untreated or exposed to NE (200 nM) alone or pre-incubated with SLPI (400 nM). Video recording was performed for 6 h. a-b, Representative phase contrast images of untreated (a) or NE-exposed (b) 16HBE cells. Unlike control cells with flat polygonal morphology, NE-treated cells detached from each other and displayed rounded morphology. c-d, Representative immunofluorescence images of untreated or NE-exposed 16HBE cells. Immunofluorescent staining showed E-cad surrounding uniformly cohesive cells (c). In contrast, cells exposed to NE lost their immunostaining for E-cad coinciding with the appearance of intercellular gaps (d). Of note, NE preincubated with SLPI (e) or SLPI alone (f) had less striking effect on E-cad distribution and monolayer integrity. Asterisks depict formed gaps. Scale bar = 40 μm. Experiments were repeated twice with similar observations Fig. 4 Spatio-temporal changes of epithelial cell monolayer following treatment with NE. Confluent monolayers of 16HBE epithelial cells were incubated alone or in the presence of defined NE concentration (200 nM). Briefly, cells were first subjected to immunofluorescence staining of E-cad as described in material and methods. Next, cell monolayers were left untreated or exposed to NE (200 nM) alone or pre-incubated with SLPI (400 nM). Video recording was performed for 6 h. a-b, Representative phase contrast images of untreated (a) or NE-exposed (b) 16HBE cells. Unlike control cells with flat polygonal morphology, NE-treated cells detached from each other and displayed rounded morphology. Detection of both cleaved E-cad and NE in BAL of patients with exacerbated COPD c-d, Representative immunofluorescence images of untreated or NE-exposed 16HBE cells. Immunofluorescent staining showed E-cad surrounding uniformly cohesive cells (c). In contrast, cells exposed to NE lost their immunostaining for E-cad coinciding with the appearance of intercellular gaps (d). Of note, NE preincubated with SLPI (e) or SLPI alone (f) had less striking effect on E-cad distribution and monolayer integrity. Asterisks depict formed gaps. Scale bar = 40 μm. Experiments were repeated twice with similar observations as well confirming the predominance of active neutrophils in the samples (Fig. 7b). Detection of both cleaved E-cad and NE suggests, thus, that both proteins are present in inflamed human lungs and support the hypothesis that NE could target E-cad in vivo contributing potentially to the setting of lung inflammation and injury. several folds. As expected, the inflammatory response of our mouse model of acute Pseudomonas pneumonia was characterized by increased alveolocapillary permeability, neutrophil influx, and LDH. Using genetically engi- neered mice deficient in NE in this experimental model, we provide compelling evidence that NE contributes substantially to E-cad degradation in injured lungs. Videomicroscopy approach showed that the spatial and temporal NE-mediated degradation of E-cad was accom- panied with the abrogation of this latter’s cell-cell adhe- sion function. More importantly, the presence of both Discussion That E-cad is susceptible to NE has been previously re- ported in limited cell culture [27, 41, 42] and experimen- tal model studies [24–26]. The originality of our work is Page 10 of 15 Boxio et al. Respiratory Research (2016) 17:129 Fig. 5 In vivo detection of E-cad and NE. a, Lung tissue sections from saline control mice were immunostained for mouse E-cad. Under physiologic conditions, E-cad is restricted to epithelial lining (arrowheads), consistent with the known localization of E-cad expression. b, Representative mouse lung tissue section following O.N. challenge with P. aeruginosa. b, upper panels, Immunostaining for E-cad detected the antigen within inflamed airspaces and in close spatial contact with the cellular infiltrates (arrows). b, lower panels, absence of E-cad staining in the presence of preimmune serum. c, absence of staining was seen when the primary antibody was replaced with the preimmune serum. c, Enzymatic activity assay detected free active NE in cell-free BAL. Consistent with the neutrophilic response to P. aeruginosa, increasing levels of active NE were detected in cell-free BAL fluids following O.N. infection (infected tissues). No NE activity was detected in mice challenged with sterile saline (PBS). Purified active NE (100 ng) was used as control. Data were consistent in all challenged mice (bars ± SEM; * and ** correspond to p < 0.001 and p < 0.000 respectively for infection versus PBS). O.N. corresponds to 24 h. Scale bar of left images, 150 μm. Scale bar of right images, 45 μm Fig. 5 In vivo detection of E-cad and NE. a, Lung tissue sections from saline control mice were immunostained for mouse E-cad. Under physiologic conditions, E-cad is restricted to epithelial lining (arrowheads), consistent with the known localization of E-cad expression. b, Representative mouse lung tissue section following O.N. challenge with P. aeruginosa. b, upper panels, Immunostaining for E-cad detected the antigen within inflamed airspaces and in close spatial contact with the cellular infiltrates (arrows). b, lower panels, absence of E-cad staining in the presence of preimmune serum. c, absence of staining was seen when the primary antibody was replaced with the preimmune serum. c, Enzymatic activity assay detected free active NE in cell-free BAL. Consistent with the neutrophilic response to P. aeruginosa, increasing levels of active NE were detected in cell-free BAL fluids following O.N. infection (infected tissues). No NE activity was detected in mice challenged with sterile saline (PBS). Discussion Purified active NE (100 ng) was used as control. Data were consistent in all challenged mice (bars ± SEM; * and ** correspond to p < 0.001 and p < 0.000 respectively for infection versus PBS). O.N. corresponds to 24 h. Scale bar of left images, 150 μm. Scale bar of right images, 45 μm degraded E-cad and NE was detected in COPD exacerba- tion, a clinical manifestation characterized by predomin- ant neutrophilic inflammatory response. It must be noted that a limited number of human samples (n = 5) have been examined in this study and to further document the gen- erality of our findings investigation of a large number of human BAL fluids is warranted. Altogether, these data suggest the likelihood contribution of NE-mediated deg- radation of E-cad in the development of inflammation and tissue destruction in the setting of neutrophil-rich lung diseases. Of importance, the concentrations of NE used in both in vitro and cell culture studies are relevant since they can be even exceeded in pulmonary diseases [43, 44]. Also, in the mouse experimental model, E-cad degradation was not completely prevented in the absence of NE sug- gesting contribution of other proteases. In this regard, ac- tivated PMNs release two other members of neutrophil serine protease family, cathepsin G (CG) and proteinase 3 (PR3) known to share the same conserved catalytic cleft as NE [5]. Interestingly, our in vitro data showed that Page 11 of 15 Boxio et al. Respiratory Research (2016) 17:129 Fig. 6 In vivo contribution of NE to degradation of endogenous E-cad. a, Representative image of equal BAL fluid protein aliquots (in 20 μl) from control mice and mice i.n. challenged with P. aeruginosa for 4 h or O.N. that were reduced and processed for immunoblotting using E-cad N-terminal antibody. Both 4 and O.N. time points revealed a gradual degradation of endogenous E-cad with generation of a distinct ~80 kDa cleavage fragment that migrated similar to the cleavage fragment generated by purified NE. Note cell-free NE−/−BAL fluids showed less cleaved E-cad fragment by comparison to cell-free WT BALs. b, Densitometric analysis of immunoblot images corresponding to all cell-free BAL fluids per group, genotype and condition confirmed the protein profile of Fig. a. c, enzymatic activity assay analysis revealed the presence of active NE in cell-free WT, but not NE−/−, BAL fluids that became abundant over time (O.N.). Discussion Data were similar in all challenged mice and mouse experiment was repeated once with reproducible findings (bars ± SEM; corresponds to p < 0.0001for infection versus PBS) Fig. 7 Detection of cleaved endogenous E-cad coincides with NE in human diseased lungs. Equal protein aliquots (in 20 μl) of concentrated cell-free BAL derived from patient with COPD exacerbation (n = 5) were subjected to SDS-PAGE and immunoblotting using antibodies raised against human E-cad extracellular domain and NE respectively. a, densitometric analysis found increased level of immunoreactive E-cad fragment (~80 kDa). b, densitometric analysis revealed the presence of immunoreactive NE in the same samples of (a). Experiments were repeated three times with similar results (bars ± SEM; corresponds to p < 0.0001 for COPD exacerbation versus control) Fig. 6 In vivo contribution of NE to degradation of endogenous E-cad. a, Representative image of equal BAL fluid protein aliquots (in 20 μl) from control mice and mice i.n. challenged with P. aeruginosa for 4 h or O.N. that were reduced and processed for immunoblotting using E-cad N-terminal antibody. Both 4 and O.N. time points revealed a gradual degradation of endogenous E-cad with generation of a distinct ~80 kDa cleavage fragment that migrated similar to the cleavage fragment generated by purified NE. Note cell-free NE−/−BAL fluids showed less cleaved E-cad fragment by comparison to cell-free WT BALs. b, Densitometric analysis of immunoblot images corresponding to all cell-free BAL fluids per group, genotype and condition confirmed the protein profile of Fig. a. c, enzymatic activity assay analysis revealed the presence of active NE in cell-free WT, but not NE−/−, BAL fluids that became abundant over time (O.N.). Data were similar in all challenged mice and mouse experiment was repeated once with reproducible findings (bars ± SEM; corresponds to p < 0.0001for infection versus PBS) Fig. 7 Detection of cleaved endogenous E-cad coincides with NE in human diseased lungs. Equal protein aliquots (in 20 μl) of concentrated cell-free BAL derived from patient with COPD exacerbation (n = 5) were subjected to SDS-PAGE and immunoblotting using antibodies raised against human E-cad extracellular domain and NE respectively. a, densitometric analysis found increased level of immunoreactive E-cad fragment (~80 kDa). b, densitometric analysis revealed the presence of immunoreactive NE in the same samples of (a). Discussion The relative importance of CG and PR3 in cleaving E-cad would be, however, best defined using mice deficient in these proteases. In general, epithelial cell-cell adhesion proteins are grouped in three categories (from apical to basal side): tight junctions (e.g., ZO-1 and occludin), adhe- rens junctions (E-cad) and desmosomes. There is a large body of evidence indicating that E-cad repre- sents a key protein for the establishment and main- tenance of cohesive epithelium. Indeed, the protein mechanically ensures tight adhesion of cells [45]. It is required for the formation of other junctional com- plexes such as tight junctions [46]. E-cad regulates cell proliferation and differentiation [47]. Furthermore, E-cad plays a modulatory role in host immune re- sponses by regulating the expression of growth factors and proinflammatory mediators [48]. Consequently, there are important functional implications of E-cad degradation. Though we have no direct in vivo evi- dence that degradation of E-cad by NE leads to lung epithelial disruption and injury, our findings still sug- gest strongly that this protease contributes to this physiopathologic phenotype. Because the N-terminal region corresponds to E-cad homophilic interaction hence cell-cell adhesion, its cleavage by NE (see below) is likely to disrupt and/or destabilize the epi- thelium integrity. This is consistent with our findings of epithelial monolayer disruption following its expos- ure to NE. It also implies that in the setting of lung injury, NE-mediated E-cad degradation likely partici- pates to epithelial barrier alteration contributing to in- creased alveolocapillary membrane permeability and exudate within airspaces. While our in vivo finding with NE−/−mice show that NE deficiency was associated with considerable decrease of E-cad degradation, lung inflam- mation and injury of these mice was, however, similar to that seen in WT mice including inflammatory cell influx and albumin levels. Among the explanations could be that other proteases (e.g., CG and PR3) cleave E-cad masking therefore the relative contribution of NE. Some matrix metalloproteases (MMP) including MMP-7 cleave also E-cad [49, 50]. Although unlikely, the possibility that NE cleavage of E-cad corresponds to an epiphenomenon is plausible as well. A common denominator of neutrophil-rich lung inflam- matory diseases (e.g., ALI/ARDS, CF, non-CF bronchiec- tasis) is epithelial injury. Thus, cleavage of E-cad by neutrophil-derived NE, whose level and activity are known to be increased, could be anticipated. Such proteolytic event could occur during the migration of PMNs across the epithelium and/or their accumulation in the vicinity of epithelial lining. Discussion Experiments were repeated three times with similar results (bars ± SEM; corresponds to p < 0.0001 for COPD exacerbation versus control) Fig. 7 Detection of cleaved endogenous E-cad coincides with NE in human diseased lungs. Equal protein aliquots (in 20 μl) of concentrated cell-free BAL derived from patient with COPD exacerbation (n = 5) were subjected to SDS-PAGE and immunoblotting using antibodies raised against human E-cad extracellular domain and NE respectively. a, densitometric analysis found increased level of immunoreactive E-cad fragment (~80 kDa). b, densitometric analysis revealed the presence of immunoreactive NE in the same samples of (a). Experiments were repeated three times with similar results (bars ± SEM; ** corresponds to p < 0.0001 for COPD exacerbation versus control) Page 12 of 15 Page 12 of 15 Boxio et al. Respiratory Research (2016) 17:129 estimated size of E-cad fragment detected by Western blotting and the epitope (amino acid sequence Asp157-Val709) that was used to raise anti-E-cad N- terminus antibody, we readily inferred that NE cleaves E-cad within its extracellular region. Furthermore, in- spection of the primary structure of E-cad especially within the N-terminal domain (amino acid sequence ranging from 24 to 713, NCBI accession: Q9R0T4) revealed the presence of peptide bonds that are pre- ferred by NE. Finally, Mayerle J. et al. identified a cleavage site for NE within EC-3 domain of E-cad extracellular portion (starting amino acid at position 394) [24]. Whether this cleaved fragment possesses in vivo biologic functions in inflamed lungs remains to be assessed. Interestingly, analysis of our immuno- stained lung tissue sections of infected mice suggested that E-cad fragments were engulfed by recruited neu- trophils (left panel of Fig. 5b); an observation that confirms previously reported findings by Evans SM et al. [25]. It has been, however, shown in cell culture studies that cleaved E-cad fragment induces expres- sion and activation of proteases such as matrix metal- loproteases [51] suggesting that a hypothetical vicious cycle might exist between proteases and structural proteins (e.g., E-cad in this study), which perpetuates tissue inflammation and injury. Other studies reported that this E-cad fragment exhibits a stimulatory effect on the migratory capability of cells [52]. while the two other members of neutrophil serine prote- ase family, cathepsin G (CG) and proteinase 3 (PR3) de- graded E-cad, NE was the most potent degrading enzyme. Authors’ contributions RB, JW and LM carried out mouse experimental animal model, processed lung tissues and bronchoalveolar lavages and performed tissues staining and immunohistochemistry techniques; RB and RNB performed cell culture experiments and videomicroscopy; BL, LM and TH performed culture studies and biochemical techniques; CT, YP and GD collected and prepared human samples and contributed to the writing of the manuscript, AB conceived the study and wrote the manuscript and together with GD supervised the project. All authors were involved in writing the manuscript and approved the submitted version. Conclusion This work was supported by grants from Inserm Avenir Program, Agence Nationale de la Recherche, Fondation pour la Recherche Médicale, Fonds AGIR pour les Maladies Chroniques. In conclusion, we provide in this study compelling evidence that NE contributes substantially to E-cad deg- radation in inflamed lung situations. However, based on our in vivo studies with mice deficient in NE, it seems that inhibition of this protease alone may not prevent proteolysis-mediated inflammation and tissue degrad- ation seen in neutrophil-rich pathologies. Availability of data and materials Data sharing not applicable to this article as no datasets were generated or analysed during the current study. Acknowledgements We thank Veronique Laplace and Jean-Marie Zahm for their excellent animal care and assistance with videomicroscopy. Discussion time as long as significant amounts of free active NE are available and the microenvironment allows interaction of the enzyme and its target. As mentioned above, other proteases namely MMP have been reported to cleave E-cad [49, 50]. For example, McGuire JK et al. reported that MMP-7 mediates E-cadherin ectodomain shedding in injured lung epithelium [49, 50]. Interestingly, this group proposed that shedding of E-cad is required for epithelial repair. Altogether, previously reported findings along with this work point to E-cad cleavage as “double- edged sword” process and raise a relevant question of whether such cleavage contributes to dysfunctional or normal repair of injured epithelium. Our cell-free and cell culture data show that degradation of E-cad was blocked by NE physiological inhibitor, SLPI. As such, in vivo E-cad cleavage and associated phenotype (i.e., in- jury or repair) are in any case contingent on the equilib- rium of active protease(s) (NE in this study) and their corresponding endogenous inhibitors within lung microenvironment. Additional file 7: Cell influx, albumin levels, and LDH release in BAL fluids of NE−/−and WT mice in response to P. aeruginosa infection 24 h post-challenge. A. Total leukocyte counts in BAL fluids from NE−/−and WT mice (n = 4/genotype) following i.n. challenge with a sub-lethal dose of P. aeruginosa. B. Representative cytospin micrographs of BAL fluids from both WT and NE−/−mice. Left panel, insignificant cell numbers corresponding mostly to resident alveolar macrophages, were detected in BAL fluids from saline control mice. Right panel, predominance of neutrophils. Scale bar, 50 μm. C. Albumin levels in cell-free BAL fluids from mice in A. D. LDH release in cell-free BAL fluids from mice in A. Data are mean values ± SD. p < 0.05; differences between genotypes of mice were tested by two-way analyses of variances with group and time as factors. (TIFF 2703 kb) Ethics approval Use of human data or tissue: “Not applicable”. Use of human data or tissue: “Not applicable”. Use of animal tissue: Animal handling and procedures were approved by the Animal Studies Committee at our institution (Health and Animal Protection Office, Châlons-en-Champagne, France, Authorization number: 51–31) in accordance with the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA) and following the European Directive on the protection of animals used in scientific procedures. Additional file 3: Videomicroscopy 1 (Bright field, addition of NE to cultured cells). (MOV 2864 kb) Additional file 3: Videomicroscopy 1 (Bright field, addition of NE to cultured cells). (MOV 2864 kb) Additional file 4: Videomicroscopy 2, (Immunofluorescence, addition of NE to cultured cells). (MOV 1338 kb) Additional file 4: Videomicroscopy 2, (Immunofluorescence, addition of NE to cultured cells). (MOV 1338 kb) Additional files Additional file 1: Degradation of E-cad by NE, PR3 and CG. MLE15 cell protein extracts containing E-cadherin (10 μg) were incubated for 30 min alone (Ctrl) or in the presence of 25 nM of purified NE, PR3, and CG. The reactions were resolved by SDS-PAGE under reducing conditions and visualized by immunoblotting as in Fig. 1. Note that NE is the most potent protease to cleave E-cad followed by PR3 and CG. Molecular weight (kDa) standards are on the right. The findings are illustrative of at least three independent experiments. (TIFF 2703 kb) Abbreviations ALI: Acute lung injury; ARDS: Acute respiratory distress syndrome; BAL: Bronchoalveolar lavage; CF: Cystic fibrosis; CG: Cathepsin G; COPD: Chronic obstructive pulmonary diseases; E-cad: Epithelial-cadherin; fMLP: formyl-methionyl-leucyl-phenylalanine; i.n.: intranasal; LDH: Lactate dehydrogenase; MMP: Matrix metalloproteases; NE: Neutrophil elastase; NE−/−: NE-deficient mice; PMNs: Polymorphonuclear neutrophils; PR3: proteinase 3; SDS-PAGE: Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis; SLPI: Secretory leukocyte proteinase inhibitor; WT: Wild type Competing interests Competing interests The authors declare that they have no competing interests. Additional file 2: NE degrades cell-associated E-Cad. Confluent 16HBE cells were left untreated or treated with varying concentrations of purified NE (0, 2, 20, or 200 nM) for 6 h. Next, equal protein aliquots from cell lysates (10 μg) were subjected to SDS-PAGE and immunoblotting using antibodies raised against C-terminal parts of E-cad. A, Left panel. Anti-E-cad C-terminal antibody revealed a progressive decrease of E-cad that paralleled the increase of NE concentration. Right panel, densitometric analysis confirms decreased levels of E-cad. Data are mean values ± SD. p < 0.05; Kruskall-Wallis test. Of note, anti-E-cad C-terminal antibody detected varying fragments. Immunoblotting for GAPDH, an internal control, was used as protein loading control of cell lysate proteins. Experiments were repeated three times. (TIFF 2703 kb) Competing interests The authors declare that they have no competing interests. Authors’ information The last name of Bentaher used to be Belaaouaj. Discussion In this regard and as mentioned earlier, NE could degrade first other cell-cell junction proteins be- fore E-cad (unpublished data, [27]. Also, the detected E-cad fragment in our mouse and human studies could in- clude products derived from degraded VE-cad [28]; a hypothesis that warrants investigation. Other scenarios of NE involvement in E-cad degradation and ensued epithe- lium disruption could be envisioned. Recently, we reported that NE activates calpain, which is known to cleave E-cad [53, 54]. As mentioned above, tight junction and adherens junction proteins are spatially and function- ally linked in epithelia. Degradation of E-cad could there- fore lead to dysfunctionning of tight junctions and thus enhanced disruption of the epithelium [55]. NE cleavage of E-cad was marked by the generation of a distinct fragment (about 80 kDa) in both cell culture system and in vivo. In their mouse model challenged with human NE, Evans SM and his colleagues reported the release into BAL fluids of a cleavage product with similar size and suggested NE as the prime protease targeting E-cad [25]. Given the It must be emphasized that in this study, we focused on the time point 24 h post-infection because it corre- sponds to a sharp increase of neutrophil numbers and enhanced NE activity by comparison to the other time points [37]. But, E-cad degradation could be seen at any Page 13 of 15 Boxio et al. Respiratory Research (2016) 17:129 time as long as significant amounts of free active NE are available and the microenvironment allows interaction of the enzyme and its target. As mentioned above, other proteases namely MMP have been reported to cleave E-cad [49, 50]. For example, McGuire JK et al. reported that MMP-7 mediates E-cadherin ectodomain shedding in injured lung epithelium [49, 50]. Interestingly, this group proposed that shedding of E-cad is required for epithelial repair. Altogether, previously reported findings along with this work point to E-cad cleavage as “double- edged sword” process and raise a relevant question of whether such cleavage contributes to dysfunctional or normal repair of injured epithelium. Our cell-free and cell culture data show that degradation of E-cad was blocked by NE physiological inhibitor, SLPI. As such, in vivo E-cad cleavage and associated phenotype (i.e., in- jury or repair) are in any case contingent on the equilib- rium of active protease(s) (NE in this study) and their corresponding endogenous inhibitors within lung microenvironment. References Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. Science. 2000;289(5482):1185–8. 7. Belaaouaj A, Kim KS, Shapiro SD. Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. Science. 2000;289(5482):1185–8. 8. Belaaouaj A, McCarthy R, Baumann M, Gao Z, Ley TJ, Abraham SN, et al. 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(MOV 2084 kb) 1Inflammation and Immunity of the Respiratory Epithelium Group, Faculté de Médecine Lyon Sud, EA 7426, UCBL 1, Inserm U-1111, Pierre Benite - Lyon Sud, France. 2INSERM UMR-S 903, CHU Maison Blanche, Reims, France. 3Department of Pulmonary Medicine, German Clinic for Diagnostics (DKD), Page 14 of 15 Page 14 of 15 Page 14 of 15 Boxio et al. Respiratory Research (2016) 17:129 Boxio et al. Respiratory Research (2016) 17:129 Boxio et al. Respiratory Research (2016) 17:129 Wiesbaden, Germany. 4Centre for Infection and Immunity, Queen’s University Belfast, Belfast, Northern Ireland, UK. 5CHU Croix-Rousse, Lyon, France. 25. Evans SM, Blyth DI, Wong T, Sanjar S, West MR. Decreased distribution of lung epithelial junction proteins after intratracheal antigen or lipopolysaccharide challenge: correlation with neutrophil influx and levels of BALF sE-cadherin. Am J Respir Cell Mol Biol. 2002;27(4):446–54. Received: 28 April 2016 Accepted: 10 October 2016 Received: 28 April 2016 Accepted: 10 October 2016 BALF sE-cadherin. Am J Respir Cell Mol Biol. 2002;27(4):446–54. 26. Zemans RL, Briones N, Campbell M, McClendon J, Young SK, Suzuki T, et al. Neutrophil transmigration triggers repair of the lung epithelium via beta- catenin signaling. Proc Natl Acad Sci U S A. 2011;108(38):15990–5. 27. Tanga A, Saidi A, Jourdan ML, Dallet-Choisy S, Zani ML, Moreau T. Protection of lung epithelial cells from protease-mediated injury by trappin-2 A62L, an engineered inhibitor of neutrophil serine proteases. Biochem Pharmacol. 2012;83(12):1663–73. References 1. Hallstrand TS, Hackett TL, Altemeier WA, Matute-Bello G, Hansbro PM, Knight DA. Airway epithelial regulation of pulmonary immune homeostasis and inflammation. Clin Immunol. 2014;151(1):1–15. 1. Hallstrand TS, Hackett TL, Altemeier WA, Matute-Bello G, Hansbro PM, Knight DA. Airway epithelial regulation of pulmonary immune homeostasis and inflammation. Clin Immunol. 2014;151(1):1–15. 28. Carden D, Xiao F, Moak C, Willis BH, Robinson-Jackson S, Alexander S. Neutrophil elastase promotes lung microvascular injury and proteolysis of endothelial cadherins. Am J Physiol. 1998;275(2 Pt 2):H385–92. 2. Chang EH, Pezzulo AA, Zabner J. Do cell junction protein mutations cause an airway phenotype in mice or humans? Am J Respir Cell Mol Biol. 2011;45(2):202–20. 2. Chang EH, Pezzulo AA, Zabner J. Do cell junction protein mutations cause an airway phenotype in mice or humans? Am J Respir Cell Mol Biol. 2011;45(2):202–20. 29. Decramer ML, Chapman KR, Dahl R, Frith P, Devouassoux G, Fritscher C, et al. Once-daily indacaterol versus tiotropium for patients with severe chronic obstructive pulmonary disease (INVIGORATE): a randomised, blinded, parallel-group study. Lancet Respir Med. 2013;1(7):524–33. 3. 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Extracellular cleavage of E-cadherin by leukocyte elastase during acute experimental pancreatitis in rats. Gastroenterology. 2005;129(4):1251–67. Page 15 of 15 Boxio et al. Respiratory Research (2016) 17:129 Boxio et al. Respiratory Research (2016) 17:129 46. Tunggal JA, Helfrich I, Schmitz A, Schwarz H, Gunzel D, Fromm M, et al. E- cadherin is essential for in vivo epidermal barrier function by regulating tight junctions. EMBO J. 2005;24(6):1146–56. 47. Bajpai S, Correia J, Feng Y, Figueiredo J, Sun SX, Longmore GD, et al. {alpha}- Catenin mediates initial E-cadherin-dependent cell-cell recognition and subsequent bond strengthening. Proc Natl Acad Sci U S A. 2008;105(47):18331–6. 48. Cowell CF, Yan IK, Eiseler T, Leightner AC, Doppler H, Storz P. Loss of cell- cell contacts induces NF-kappaB via RhoA-mediated activation of protein kinase D1. J Cell Biochem. 2009;106(4):714–28. 49. McGuire JK, Li Q, Parks WC. 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https://openalex.org/W4221036811	https://www.nature.com/articles/s41746-022-00569-x.pdf	English	null	Culturally adapting internet- and mobile-based health promotion interventions might not be worth the effort: a systematic review and meta-analysis	npj digital medicine	2,022	cc-by	11,230	REVIEW ARTICLE OPEN Culturally adapting internet- and mobile-based health promotion interventions might not be worth the effort: a systematic review and meta-analysis Sumeyye Balci 1✉, Kerstin Spanhel 2, Lasse Bosse Sander 2 and Harald Baumeister 1 Health promotion interventions offer great potential in advocating a healthy lifestyle and the prevention of diseases. Some barriers to communicating health promotion to people of certain cultural groups might be overcome via the internet- and mobile-based interventions (IMI). This systematic review and meta-analysis aims to explore the effectiveness of culturally adapted IMI for health promotion interventions among culturally diverse populations. We systematically searched on Cochrane Central Register of Controlled Trials (CENTRAL), EbscoHost/MEDLINE, Ovid/Embase, EbscoHost/PsychINFO, and Web of Science databases in October 2020. Out of 9438 records, 13 randomized controlled trials (RCT) investigating culturally adapted health promotion IMI addressing healthy eating, physical activity, alcohol consumption, sexual health behavior, and smoking cessation included. From the included studies 10,747 participants were eligible. Culturally adapted IMI proved to be non-superior over active control conditions in short- (g = 0.10, [95% CI −0.19 to 0.40]) and long-term (g = 0.20, [95% CI −0.11 to 0.51]) in promoting health behavior. However, culturally adapted IMI for physical activity (k = 3, N = 296) compared to active controls yielded a beneﬁcial effect in long-term (g = 0.48, [95% CI 0.25 to 0.71]). Adapting health promotion IMI to the cultural context of different cultural populations seems not yet to be recommendable given the substantial adaption efforts necessary and the mostly non-signiﬁcant ﬁndings. However, these ﬁndings need to be seen as preliminary given the limited number of included trials with varying methodological rigor and the partly substantial between-trial heterogeneity pointing in the direction of potentially useful culturally adapted IMI which now need to be disentangled from the less promising approaches. PROSPERO registration number: 42020152939 npj Digital Medicine (2022) 5:34 ; https://doi.org/10.1038/s41746-022-00569-x Researchers in the ﬁeld of health promotion are encouraged by WHO to approach their practice and research considering diverse human experience and intersectionality of various factors, including culture, gender, immigration status, and ethnicity, which may be related to lower physical and mental health23–25. In order to offer an acceptable and relevant health service to people of a certain cultural background, health promotion interventions could be developed from scratch with cultural sensitivity or a less resource-consuming way can, for example, be tailoring an already existing intervention for speciﬁc cultural groups26. This process is deﬁned as cultural adaptation27. www.nature.com/npjdigitalmed 1Department of Clinical Psychology and Psychotherapy, Institute of Psychology and Education, Ulm University, Lise-Meitner-Str. 16, D-89081 Ulm, Germany. 2Department of Rehabilitation Psychology and Psychotherapy, Institute of Psychology, University of Freiburg, Engelbergerstr. 41, D-79085 Freiburg, Germany. ✉email: sumeyye.balci@uni-ulm.de REVIEW ARTICLE OPEN Culturally adapting internet- and mobile-based health promotion interventions might not be worth the effort: a systematic review and meta-analysis Cultural adaptation could be adopted on surface structure modiﬁcations (pairing materials and messages to apparent features of the target population such as language) or deep structure modiﬁcations (concerning intersect- ing effects of social, cultural, and historical variables on the target behavior)28,29. Culturally adapted face-to-face interventions are shown to be effective in smoking cessation30,31, health education, and healthy eating32. Moreover, IMI developed for ethnic minorities and underserved populations are also shown to be accepted and effective in the promotion of various health behaviors such as physical activity33 and healthy eating16. Effectiveness of culturally adapted IMI of smoking cessation Seven studies were conducted in the USA38,39,43–45,47,48, two in China37,42, two in Brazil40,41, one in Hong Kong46, and one in Australia49. The cultural adaptation was based on a theory or a guideline in three studies38–40. Eight studies based their cultural adaptation on a formative research/pilot study or expert review37,41,42,44,45,47–49. The cultural adaptation was based on a theory or a guideline in three studies38–40. Eight studies based their cultural adaptation on a formative research/pilot study or expert review37,41,42,44,45,47–49. Two studies did not provide information regarding the basis of cultural adaptation43,46. In terms of alterations of the intervention content, four studies incorporated both surface and deep structure changes38,40,43,45, while nine studies used surface structure changes only28. Details of the culturally adapted and original interventions are presented in Table 2. Two studies did not provide information regarding the basis of cultural adaptation43,46. In terms of alterations of the intervention content, four studies incorporated both surface and deep structure changes38,40,43,45, while nine studies used surface structure changes only28. Details of the culturally adapted and original interventions are presented in Table 2. RESULTS All the predeﬁned characteristics of included articles are presented in Table 1. Study selection We identiﬁed a total of 20,012 records. After screening titles and abstracts and full-texts, 13 studies were included in the quantita- tive analyses. The main characteristics of the studies are outlined in Table 1. During the full-text screening, 38 studies were excluded due to lack of cultural adaptation, 29 studies were concerning culturally sensitive interventions, 13 studies did not report relevant outcome data, eight studies were no original articles, four studies were not RCTs, ﬁve were excluded due to other reasons (i.e. novel interventions, study protocols, non-English full-text) and one due to a differing health promotion topic. The meta-analysis of six studies38,43–45,47,48 examining any long- term health promotion interventions revealed that culturally adapted IMI of health promotion were not superior to active control conditions in the long-term. In addition, four stu- dies38,43,44,48 that provided short-term follow-up data were not superior to active control conditions in improving health behavior outcomes. Due to substantial heterogeneity, the results are reported descriptively, see Figs. 3, 4. y g Comparisons with passive control groups could not be pooled given the low number of studies (k = 4), see Fig. 5. One study39 reported only Odds ratios ([OR], 1.13; 95% CI 0.18 to 7.04). Effectiveness of culturally adapted IMI of healthy eating, sexual health behavior, and alcohol consumption Fewer than three studies reported on sexual health behavior44, alcohol consumption48, and healthy eating40,43. Only one generic outcome, health-related quality of life, was reported in one trial42. These studies were not eligible for pooling due to the number of trials in the respective outcome but reported in Figs. 3–5. INTRODUCTION Health promotion interventions are an effective way of disease prevention and improving overall health1,2. Well-established approaches focus amongst others on healthy eating, exercising, avoiding excessive use of alcohol, quitting smoking, and sexual health behaviors, such as condom use3,4. Promoting these health behaviors on a large scale by using health promotion interven- tions might be a promising mean for reducing the burden of disease5–7. However, these intervention offers do not always reach or ﬁt populations equally between and within countries8. p p q y The reach of these interventions could be expanded globally via the internet9 using the Internet- and mobile-based interventions (IMI)10–14. IMI offers time and place ﬂexibility, potential cost- effectiveness, and scalability without necessarily losing effective- ness15. Thereby, IMI could reach a diverse group of people including minorities and people of a cultural background that is not yet well covered by the established health care systems16–19. However, IMI also come with some substantial limitations, most prominently risk of low adherence and uptake20,21. In order to tackle these issues, population-related factors such as the needs and expectations of the users should be taken into account15. Tailoring the intervention content and delivery method to the target group’s culture can thus be a means for increasing engagement and effectiveness22. y y y Previous reviews included IMI for ethnic minority and histori- cally underserved populations in developed countries22,34–36. However, none of them speciﬁcally examined culturally adapted Published in partnership with Seoul National University Bundang Hospital S. Balci et al. 2 bias arising from the randomization process40. This study had an unequal number of clusters, which resulted in signiﬁcant baseline differences in primary outcomes. IMI for health promotion. The number of studies exploring the development and dissemination of culturally adapted health promotion IMI is increasing and thus a systematic review and meta-analysis seems timely. Hence, this review aims to system- atically identify culturally adapted IMI on health promotion and explore their effectiveness among populations that is different from the original intervention’s target group. Risk of bias of in included studies The risk of bias assessment of the included studies is presented in Figs. 1, 2. The interrater reliability suggested substantial agree- ment between the raters, κ = 0.79. Five studies were assessed to have a low risk of bias, ﬁve studies had some risk of bias and three studies were rated to have a high risk of bias. Four studies were assessed to have some risk of bias due to deviations from the intended intervention40,43,46,48, three studies were assessed to have some risk of bias in the measurement of the outcome domain41,44,46. Two studies37,38 were assessed to have some risk of bias due to missing outcome data, one study43 had a high risk of bias in this domain. One study was assessed to have some risk of Effectiveness Nine studies that provided data to calculate standardized mean difference effect sizes were pooled. Individual data points are presented in Supplementary Table 1. Four studies concerning smoking cessation did only provide dichotomous outcomes. We conducted separate meta-analyses for the different areas of health promotion (physical activity, smoking cessation), in the case of at least three studies reported on the same outcome. Since we included only nine studies in the analysis, we refrained from exploring the publication bias via a funnel plot50. Following, we report on pooled effectiveness across health promotion domains. Effectiveness of culturally adapted IMI of physical activity Five studies reported physical activity outcomes, four of which provided data via accelerometers/pedometers38,45,47 and one via a self-reported questionnaire46. Pooling the three studies with active control conditions resulted in a small signiﬁcant long-term effect favoring culturally adapted IMI (N = 296; g = 0.48; 95% CI 0.25 to 0.71; I2 = 41%; ﬁxed effect), see Fig. 6. Four studies dealt with smoking cessation37,39,41,49, two with both healthy eating and physical activity38,42, three with physical activity only45–47, two with healthy eating only40,43, one with sexual health behavior44, and one with alcohol consumption48. Four studies provided follow-up data on short-term effective- ness (one to ﬁve months follow-up)39,41,42,46, three studies on long-term effectiveness (six to 12-months follow-up)40,45,47, six studies provided follow-up data on both assessment points. Effectiveness of culturally adapted IMI of smoking cessation Three RCTs (N = 8,112) reported smoking cessation outcomes measuring short-term abstinence at the end of the intervention versus active controls. The meta-analysis ﬁndings of these studies were not signiﬁcant (Odds Ratio [OR], 1.75; 95% CI 0.51 to 6.05, I2 = 56%), see Fig. 7. The number of included studies was small (k = 3) and the effect size was mainly based on one large-scale study with N = 8000 participants37. Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 34 Study characteristics The 13 RCTs included in this review comprise a total of N = 10,747 randomized participants, mostly adult populations (N = 9710). Trials were conducted between 2012 and 202037–49. The mean age of the participants varied from 14 to 57 (see Table 1). Primary studies focused on smokers (k = 3, N = 2546), individuals diag- nosed with Type 2 diabetes (k = 2, N = 171), and HIV + individuals (k = 1, N = 433). y Due to a small number of studies (k = 9), we did not perform the predeﬁned subgroup and sensitivity analyses. Published in partnership with Seoul National University Bundang Hospital DISCUSSION To our knowledge, this is the ﬁrst systematic review and meta- analysis that assessed the effectiveness of culturally adapted IMI on health promotion among populations which the intervention was originally not developed for. Our results suggested that culturally adapted IMI are not more effective in promoting health behaviors than active conditions in short- (g = 0.10) and long-term (g = 0.20), respectively. When regarding health promotion topics separately, health promotion of physical activity resulted in a small to moderate effect favoring culturally adapted IMI over active npj Digital Medicine (2022) 34 S. Balci et al. Table 1. Characteristics of included articles. 1st author (year) Country Sample Sample size E: experiment group/C: control group Gender Female (%) Mean age (SD) Dropout rate at post- assessment (%) Website vs. Mobile Duration/ No. modules Post randomization follow up in months Comparison Outcome Outcome measures Augustson et al.37 China Adults smokers E: 4000 C: 4000 3.6 – 73 SMS 6 weeks 1,3 & 6 M Active control group The Low-Frequency Text Contact (LFTC) received 1 text message a week, for the 6-week intervention period Smoking cessation Smoking status was based on past-7-day abstinence self- reported via text message Bender et al.38 USA Individuals diagnosed with Type 2 Diabetes E: 22 C: 23 62 57.6 (9.8) 2.5 App/ Social media 26 weeks 3 & 6 M Active control group receives only Fitbit accelerometer and training for daily wear. Physical activity Step count via the Fitbit Zip. (accelerometer data) Bowen et al.39 USA Students (6th to 12th graders) E: 64 C: 49 53 14.6 9 Website 6 weeks 1 M Waitlist control Smoking cessation Smoking status based on “A Smoking Prevention Interactive Experience (ASPIRE)” instrument Brito Beck da Silva et al.40 Brazil Students (7th to 9th grade) E: 428 C: 467 46 14.49 (1.42) 30 Website 16 weeks 12 M Waitlist control Healthy eating BMI Cruvinel et al.41 Brazil Adult smoker post-discharge patients E: 44 C: 22 45 47.7 (11.5) 10 Mobile/ SMS 2 weeks 1&3 M Treatment as usual includes educational materials, brief intervention (BI), and access to NRT (adhesive patch and gum) Smoking cessation Smoking status of smokers (cigarettes a day) and self-reported 7-day point prevalence abstinence post- randomization. DISCUSSION Duan et al.42 China University students E: 270 C: 223 60 19.3 (1.07) 45 Website 8 weeks 2&3 M Waitlist control Physical activity & Quality of life Chinese short version of the International Physical Activity Questionnaire (IPAQ-C) & Hong Kong version of the WHO’s Quality of Life-BREF questionnaire Fortmann et al.43 USA Individuals diagnosed with Type 2 Diabetes E: 63 C: 63 75 48.43 (9.8) 10 SMS 26 weeks 3&6 M Treatment as usual (standard diabetes care provided by primary care providers at the clinic and group Diabetes self- management education- use of these services based on patient and physician’s initiative) Healthy eating BMI Kurth et al.44 USA HIV + individuals E: 226 C: 207 55 47.8 8 Website 52 weeks 3,6 & 9 M Active control group (received computer- based audio-narrated risk assessment, which included questions about sexual risk behaviors, substance use, mental health, social support, partner status and disclosure, ART regimen and adherence in last 7 and 30 days, and side effects.) Sexual health behavior sexual transmission risk behaviors (lack of condom use with either a main or another partner) S. Balci et al. 3 Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 34 Published in partnership with Seoul National University Bundang Hospital Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 34 S. Balci et al. 4 e 1 continued or (year) Country Sample Sample size E: experiment group/C: control group Gender Female (%) Mean age (SD) Dropout rate at post- assessment (%) Website vs. Mobile Duration/ No. Published in partnership with Seoul National University Bundang Hospital DISCUSSION modules Post randomization follow up in months Comparison Outcome Outcome measures n 45 USA Adult male E: 22 C: 24 0 43.04 (10.67) 6 SMS 24 weeks 6 M Active control group (wellness control group received two SMS weekly throughout the study and publicly available print-based materials on health topics different from physical activity) physical activity Minutes/week of moderate to vigorous PA (MVPA) measured by accelerometers t al.a 46 Hong Kong Students aged between 12-16 years old E:13 C: 16 49 13.7 not reported SMS 4 weeks 1 M No treatment physical activity Self-reported physical activity via PAQ-C (Physical activity questionnaire) us 47 USA Inactive adult Latinas E: 104 C: 101 100 39.20 (10.47) not reported Website 26 weeks 6 M Active control group (wellness contact, receive access to a Spanish language website with information on health topics different from physical activity) physical activity Minutes/week via 7-day Physical Activity Recall and accelerometers. ag 48 USA American Indian/ Alaska Native women E: 113 C:134 100 28.6 6 Website 20 min 1,3 & 6 M Treatment as usual (get access to displayed educational brochures about health apart from FASD (fetal alcohol spectrum disorders) related information in the various waiting areas) Alcohol consumption Level of alcohol consumption (number of drinks per week) et al.49 Australia Current Aboriginal smokers (>16 years old) E:25 C: 24 78 42 (14) 6 Mobile App 53 weeks 1&6 M Active control group (encouraged to use any other smoking cessation service or support and were offered Quitline and local ACCHS (Aboriginal Community Controlled Health Services) contact numbers) Smoking cessation Smoking status, self- reported abstinence vided three intervention groups versus a control group comparison, we used the intervention group which had the most exposure to the intervention as a comparator. Published in partnership with Seoul National University Bundang Hospital Published in partnership with Seoul National University Bundang Hospital S. Balci et al. 5 Table 2. Summary of culturally adapted and original IMI. DISCUSSION Balci et al. 6 control conditions in the long-term (g = 0.48). This is in line with a previous umbrella review of health promotion IMI for minority and historically underserved populations, which, however, not exclu- sively included culturally adapted IMI22. Similarly, another meta- analysis of IMI concerning physical activity did highlight the superiority of IMI over a control group or no-treatment condition, without a speciﬁc focus on culturally adapted interventions14. No other signiﬁcant effect was revealed for the other addressed health promotion topics. Table 2 continued 1st author (year) Name Language Target group Ethnicity Health promotion Cultural adaptation theory Cultural adaptation components original IMI original IMI original IMI original IMI original IMI adapted IMI adapted IMI adapted IMI adapted IMI adapted IMI Montag (2015) e-CHUG English General population US American Alcohol consumption Focus groups Content (pictures, logo, color of the layout, example characters, myths) - added video (verbal tradition)- language (not a translation but wording and simplifying) eCHECKUP TO GO English General population (women) American Indian/ Alaska Native (AIAN) Alcohol consumption Peiris et al.49 QuitTxt English General population Australian Smoking cessation Formative research with the expert user group Adaptation of the content and tone of the messages based on the attitudes of the target group towards smoking Can’t Even Quit’ English General population Australian/Aboriginal/ Citizen of Torres Strait Island Smoking cessation Digital Medicine (2022) 34 Subgroup analyses aiming to detangle the substantial between trial heterogeneity were not feasible. Heterogeneity among the included studies was moderate to substantial, I2 ranged from 0.36 to 0.66. Prior research points to differential effects on culturally adapted health promotion interventions in terms of different populations (age and ethnicity51), different intervention features (professional vs. non-professional provi- der)32, intervention duration and follow-up times in culturally adapted face-to-face interventions52, interventions focusing on general population groups53, different methodological decisions (RCT methodology use, different control groups, e.g. tailored website, no-treatment controls53) and cultural adaptation contents (inclusion of social support and/or family members51, integrating cultural beliefs and values54). There might be further possible explanations for between-study heterogeneity, and future research needs to provide a better understanding of the impact such factors have on the effectiveness of culturally adapted IMI for health promotion. The present ﬁndings are inconsistent with previous meta-analyses on IMI in several ways. Among western populations, these meta-analyses yielded positive effects favoring IMI compared to a waitlist and/or active controls (e.g. DISCUSSION 1st author (year) Name Language Target group Ethnicity Health promotion Cultural adaptation theory Cultural adaptation components original IMI original IMI original IMI original IMI original IMI adapted IMI adapted IMI adapted IMI adapted IMI adapted IMI Augustson et al.37 English General population US American Smoking cessation Expert review, focus groups Language, context adaptation Change to Quit China Chinese General population Chinese Smoking cessation Bender et al.38 Diabetes Prevention Program (DPP) English Type 2 Diabetes patients American Healthy eating/ physical activity Bender & Clark (2011)’s theory107 Content (Filipino food photos), delivery (involvement of family members to the ofﬁce visits) language PilAm Go4Health English Type 2 Diabetes patients Filipino Healthy eating/ physical activity Bowen et al.39 SmokingZine English General population (adolescent) Canadian Smoking cessation Based on a guideline from Wisdom2Action Images, context - English General population (adolescents) American Indian Smoking cessation Brito Beck da Silva et al.40 StayingFit English General population US American Healthy eating/ physical activity Based on Barrera et al (2013)108 and Castro et al (2015)109 Language, cultural standards, meanings, and values added StayingFit Brazil Portuguese General population (adolescents) Brazilian Healthy eating/ physical activity Cruvinel et al.41 - English US American Smoking cessation Formative research Language, information from the Brazilian smoking cessation treatment guideline TXT Portuguese Hospitalized smokers Brazilian Smoking cessation Duan et al.42 - - General population US, Germany and Netherlands Healthy eating/ physical activity Formative research Language, content - Chinese General population (students) Chinese Healthy eating/ physical activity Fortmann et al.43 Staged Diabetes Management (SDM) & Dulce Project English General population US American Healthy eating/ diabetes management Based on face-to-face intervention project Dulce110 Language, cultural beliefs that interfere with optimum self- management, shortened content, motivational messages Dulce Digital English and Spanish Type 2 Diabetes patients Hispanic Healthy eating Kurth et al.44 CARE + English HIV + patients US American Sexual health behavior The local expert advisory panel, usability testing Content (Language), expert suggestions CARE + Spanish Spanish HIV + patients Latino Sexual health behavior Larsen et al.45 Seamos Saludables Spanish General population US American physical activity Formative research and pilot (qualitative interviews) Language adaptation, the content of the SMS, and printed materials Activo Spanish General population (men) Latino physical activity Lau et al.46 - English General population US American/Canadian physical activity NA Language, content (colloquial dialogue for adolescents) - English, Dutch, Turkish General population Hong Kong Chinese physical activity Marcus et al.47 - English General population US American physical activity Focus groups Cultural and linguistic adaptation, culturally adapted content and support speciﬁcally for Latinas, ﬂexible scheduling for assessment meetings, reimbursement for travel Pasos Hacia la Salud English General population (women) Latina physical activity ublished in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) Published in partnership with Seoul National University Bundang Hospital S. Published in partnership with Seoul National University Bundang Hospital DISCUSSION Implementing deep structure changes involves core cultural values of a certain population, such as beliefs towards a health issue, or performing a behavior as a member of gender identity. A meta-analysis of face-to-face culturally adapted health interven- tions showed that incorporating family members and religious values, which are considered as deep structure changes, in the intervention was related to improvements31. Future research should consider exploring surface versus deep structure altera- tions on the effectiveness of IMI e.g. in the framework of dismantling and additive clinical trial designs aiming at detangling active components and mechanisms of change of the respective interventions68–71. Another topic of relevance to our ﬁndings is the reach, uptake, and intervention adherence of culturally adapted IMI. Even if such IMI would be effective, they still need to be used in order to exploit their full potential. Although internet technologies are widely used globally78, there are still barriers to utilizing these technologies, which cause inequalities in accessing the internet and mobile technologies and comprehending health informa- tion16,79,80. Moreover, pure mobile-based interventions are seemingly less effective than internet-based or combined interventions81–83, which might also affect for instance minority populations, where they are more likely to use a smartphone to access the internet than non-minority populations where multi- ple device (e.g. tablets, desktops) ownership is common84. To increase adherence, multimodal content and guidance (direct contact with the provider)85 might be useful via diminishing issues of health literacy, motivational and volitional aspects, and the digital divide71,86,87. These aspects might be particularly important in people of certain cultural backgrounds living in a high-income country, people living in low-income countries, and/or vulnerable populations, such as immigrants, given the limited representation of many of these populations in the health research, and high rates of drop-out86,88–91. Moreover, culturally adapting interventions is not free from criticism. It should be taken into account that the majority of the interventions developed in the ﬁelds of psychology and beha- vioral medicine are for a rather homogeneous group (white, educated, middle to high socioeconomic status) but not representative for the majority19,72. In the cultural adaptation process, the same intervention is often altered to cater to the needs of a different group of people that are non-white, occasionally less educated, and/or bilingual. This process could be seen as a form of assimilation for the target group because even the topic of the intervention might also be representative of western, white, educated humans1. DISCUSSION other internet-based or face-to-face interventions): for smoking cessation and abstinence outcome10,55, which is maintained at 12-month follow-up56 and higher effects achieved with the use of tailored messages10,13; healthy eating57–59 and weight loss60; sexual health behavior promotion61 and regarding HIV prevention and condom use62. Another meta-analysis found signiﬁcant positive effects of tailored (based on personal relevance) web-based interventions on health behaviors com- pared to non-tailored web-based interventions53 and a different meta-analysis of SMS-based interventions on various health behavior outcomes suggested that targeted and tailored (based on demographic and psychosocial factors) SMS yielded larger effect sizes, especially for physical activity interventions (g = .51), which yielded a similar effect size to our results (g = 0.48)10. Most surprisingly in this context is our null-ﬁnding regarding the effectiveness of culturally adapted IMI compared to waitlist control conditions, which are known to provide a rather upper benchmark of the beneﬁt of interventions63, usually associated with signiﬁcantly larger between-group effect sizes in IMI for health promotion as well10,13,64. Although some of the meta-analyses mentioned above concern tailored inter- vention contents, none of the above-mentioned meta-analyses were speciﬁcally examining culturally adapted IMI. Therefore, our results cannot be easily compared with prior meta-analyses. However, if culturally adapted IMI for health promotion are not effective at all, even when compared to waitlist controls, we might need to challenge the idea of providing culturally adapted IMI to populations for which the intervention was originally not developed for at large and examine whether IMI developed with cultural sensitivity are effective in the same target groups. Hence, explanations for this surprisingly limited effectiveness seem warranted. One possible explanation of our results might be related to the One possible explanation of our results might be related to the quality of cultural adaptation of the interventions. The cultural adaptation processes were rarely well deﬁned in the included studies. Therefore, it was not clear whether aspects of cultural adaptation were appropriate. In addition, the high dropout among included studies could be an indicator of cultural adaptation not Published in partnership with Seoul National University Bundang Hospital S. Balci et al. 7 Fig. 1 Risk of bias summary. Reviewers’ judgments about each risk of bias item for each included study. g. 1 Risk of bias summary. Reviewers’ judgments about each risk of bias item for each included study. cultural background are not homogenous within themselves, each member’s experience is affected by intersecting factors74. DISCUSSION There- fore, it might be more complicated than often expressed to adapt an intervention for a cultural group75. One possible solution might be to invest in adapting interventions to cultural speciﬁcs of the users, e.g. based on user needs assessments76 or community leaders’ input77. Another solution, especially for migrant/immi- grant populations could be developing interventions based on the target groups’ acculturation levels, i.e. a process on a spectrum of either orientation to the host culture or maintaining the native culture1,76. It seems also worthwhile to pay attention to intersecting factors that might inﬂuence a member of a cultural group, namely gender and literacy. However, we ﬁrst need to establish whether to culturally adapt health promotion IMI at all. The present ﬁndings at least suggest—except for physical activity IMI—a non-favorable cost-beneﬁt ratio, a result that still is in need of stronger evidence. working as intended by the researcher. Moreover, only three studies based their cultural adaptation process on a theory, which might contribute to its quality. A tested theory of cultural adaptation of IMI is missing. However, there are guidelines developed for culturally adapting face-to-face interventions28,65 and researchers could implement these guidelines when adapting an IMI66. A recent taxonomy of cultural adaptation of IMI for mental disorders serves as a basis for future cultural adaptations of IMI67. Adopting a theoretical basis in intervention development is suggested to result in higher effects, as was shown in a meta- analysis14. However, this could not be shown in our results: only one out of the three IMI that utilized a theory of cultural adaptation resulted in an improvement in physical activity outcome38. In the future, culturally adapted intervention studies should consider supporting cultural adaption with an established theory and report the adaptation process in more detail to lead prospective cultural adaptations and replications. In the process of cultural adaptation, some of the included studies sought expert reviews, focus group feedback, and conducted a pilot study, and at least altered one aspect of the intervention. However, the majority of the changes were regarded as taking place only at the surface structure28, which might be one reason for the limited impact of culturally adapted IMI shown in the present review. Surface structure changes aim at improving feasibility while deep structure changes target program’s effect for the participants28. Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 34 Published in partnership with Seoul National University Bundang Hospital while the ﬁndings reported here could guide researchers in what to examine next. In this context, we suggest adding generic outcomes such as health-related quality of life, daily functioning, or self-efﬁcacy to domain/disease-speciﬁc outcomes to allow for Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 34 Published in partnership with Seoul National University Bundang Hospital DISCUSSION Therefore, in order to avoid these issues, the ﬁrst step of cultural adaption might include approaching the problem and deﬁning it with the cultural sensitivity of its target group73. In addition, people of a certain g p This review has some limitations. First, we included interven- tions concerning only ﬁve prominent areas of health promotion thus results might not be generalizable to other health promotion domains. Second, the deﬁnition of cultural adaptation varies, and our operationalization of culturally adapted interventions resulted in the exclusion of studies that investigated IMI that were developed newly in a culturally sensitive way. Comparison of culturally adapted vs. culturally sensitive interventions is an interesting further research topic. Moreover, comparing culturally adapted versus culturally sensitive IMI might present insights into whether it is worthwhile to develop a novel IMI for a group or adapt an already existing one. Third, we were able to pool data from only 13 studies, which further limit the generalizability of our S. Balci et al. ed evidence base, analyses were restricted estions while subgroup analyses were not while the ﬁndings reported here co to examine next. In this context h. Reviewers' judgments about each risk of bias item presented as percentages acr S. Balci et al. mited evidence base, analyses were restricted while the ﬁndings reported here co aph. Reviewers' judgments about each risk of bias item presented as percentages acr 8 Fig. 2 Risk of bias graph. Reviewers' judgments about each risk of bias item presented as percentages across all included studies. results. Due to this limited evidence base, analyses were restricted to the main research questions while subgroup analyses were not feasible yet. Future updates might allow for exploring the between-study heterogeneity highlighted in the present review, while the ﬁndings reported here could guide researchers in what to examine next. In this context, we suggest adding generic outcomes such as health-related quality of life, daily functioning, or self-efﬁcacy to domain/disease-speciﬁc outcomes to allow for results. Due to this limited evidence base, analyses were restricted to the main research questions while subgroup analyses were not feasible yet. Future updates might allow for exploring the between-study heterogeneity highlighted in the present review, Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 34 Fig. 6 Fixed effects meta-analysis of culturally adapted IMI for physical activity vs. active control conditions. Eligibility criteria Studies were included if they (1) were RCTs, (2) had no treatment, treatment as usual (TAU), placebo, waitlist, or active control conditions, (3) were delivered via the internet, (4) were culturally adapted for a population that differed from the original intervention’s target group, (5) examined a health promotion intervention on healthy eating, physical activity, alcohol consumption, sexual health behavior and/or smoking cessation (6) reported one of the respective health promotion- speciﬁc outcomes: body mass index (BMI), time spent exercising, change in condom use, level of smoking, level of alcohol consumption, or one of the following generic outcomes: health- related quality of life and self-efﬁcacy. Fig. 5 Summary of culturally adapted IMI of health promotion vs. passive controls. Due to few numbers of studies (two studies reported data in the long-term, two in the short-term, while one study reported dichotomous outcome) comparing culturally adapted IMI to a passive control group, meta-analytic pooling did not perform. cross-trial cross health promotion domain comparisons in the future. Fourth, although the studies included in this meta-analysis were culturally adapted, the adaptation process was rarely well deﬁned. Fifth, only three out of 13 studies used a theory to adapt the intervention. And last, none of the studies were comparing culturally adapted IMI to non-adapted IMI. This creates a difﬁculty to draw any ﬁrm conclusions about the differential effectiveness of culturally adapted interventions. Despite these limitations, this meta-analysis had some strengths. To our knowledge, this is the DISCUSSION Forest plot present- ing ﬁxed effects meta-analysis of culturally adapted IMI for physical activity vs. active controls. S. Balci et al. 9 S. Balci et al. S. Balci et al. Fig. 3 Summary of culturally adapted IMI of health promotion vs. active controls in the long-term. Due to substantial heterogeneity among the culturally adapted IMI of health promotion vs. active controls in long-term meta-analytical pooling did not perform. Fig. 3 Summary of culturally adapted IMI of health promotion vs active controls in the long-term. Due to substantial heterogeneity among the culturally adapted IMI of health promotion vs. active controls in long-term meta-analytical pooling did not perform. Fig. 6 Fixed effects meta-analysis of culturally adapted IMI for physical activity vs. active control conditions. Forest plot present- ing ﬁxed effects meta-analysis of culturally adapted IMI for physical activity vs. active controls. ﬁrst meta-analysis concerning culturally adapted health promo- tion IMI. Moreover, this a-prior registered systematic review included mostly scientiﬁcally sound RCTs from a broad sample and health behavior topics. A summary of recommendations for future research is represented in Box 1. Fig. 3 Summary of culturally adapted IMI of health promotion vs. active controls in the long-term. Due to substantial heterogeneity among the culturally adapted IMI of health promotion vs. active controls in long-term meta-analytical pooling did not perform. Fig. 4 Summary of culturally adapted IMI of health promotion vs. active controls in the short-term. A summary plot of effect sizes of four studies of culturally adapted IMI of health promotion vs. active controls in short-term are presented. Protocol and registration This systematic review and meta-analysis has been registered at PROSPERO (Registration number: CRD 42020152939) and follows the format of the PRISMA guideline92. Review protocol93 described the aim, methodology, and data analysis plan in advance. Changes to study protocol are listed in the supplementary notes. Fig. 5 Summary of culturally adapted IMI of health promotion vs. passive controls. Due to few numbers of studies (two studies reported data in the long-term, two in the short-term, while one study reported dichotomous outcome) comparing culturally adapted IMI to a passive control group, meta-analytic pooling did not perform. CONCLUSION Based on the present ﬁndings, culturally adapted IMI might not be superior compared to control conditions in the short- and long-term, except for physical activity. Although they might exhibit a more attractive health offer to their target group, their usefulness is questionable or at least need further examination. Thereby, it might be worthwhile to take into consideration intersecting aspects of experiences of people of certain cultural groups regarding health behaviors to assure acceptability and effectiveness when designing interventions and contribute to diminishing health inequalities. Fig. 4 Summary of culturally adapted IMI of health promotion vs. active controls in the short-term. A summary plot of effect sizes of four studies of culturally adapted IMI of health promotion vs. active controls in short-term are presented. Published in partnership with Seoul National University Bundang Hospital Information sources and study selection The initial search was conducted in the following databases on 26. August 2019: Cochrane Central Register of Controlled Trials (CENTRAL), EbscoHost/MEDLINE, Ovid/Embase, EbscoHost/Psy- chINFO, and Web of Science. A combination of keywords (including MeSH terms) indicating culturally adapted IMI for health promotion has been used. The search terms are published in this review’s protocol93 and cover comprehensively both the topic of the present review as well as one of a parallel systematic npj Digital Medicine (2022) 34 Published in partnership with Seoul National University Bundang Hospital Fig. 7 Summary of culturally adapted IMI for smoking cessation vs. active controls in short-term. Three studies reported smoking cessation outcomes measuring short-term abstinence at the end of the intervention vs. active controls are presented on the forest plot. S. Balci et al. 0 Fig. 7 Summary of culturally adapted IMI for smoking cessation vs. active controls in short-term. Three studies reported smoking cessation outcomes measuring short-term abstinence at the end of the intervention vs. active controls are presented on the forest plot. S. Balci et al. 0 S. Balci et al. S. Balci et al. 10 Fig. 7 Summary of culturally adapted IMI for smoking cessation vs. active controls in short-term. Three studies reported smoking cessation outcomes measuring short-term abstinence at the end of the intervention vs. active controls are presented on the forest plot. consumption; smoking cessation assessed via the level of smoking or the abstinence percentage; sexual health behavior assessed with condom use. Generic outcomes were deﬁned as health-related quality of life and self-efﬁcacy, assessed by means of validated self-report questionnaires. Control conditions were categorized into active (placebo, other health promotion interventions & TAU) and passive controls (waitlist & No treatment). When related information could not be extracted, corresponding authors of the articles were contacted to obtain information. The extracted data was tabulated. Risk of bias 4. Particularly compare culturally adapted IMI with the respective non- adapted versions or simple language translations of the IMI. 4. Particularly compare culturally adapted IMI with the respective non- adapted versions or simple language translations of the IMI. Ultimately, the substantial effort necessary for adapting IMI culturally might only be justiﬁed in case of clinically signiﬁcant superiority of the culturally adapted versions compared to active controls. In case this is given (see 3./4.)- at least with regard to some subgroups (see 2.): Two independent reviewers performed quality assessments with Cochrane Collaboration’s Risk of Bias Tool 2.096. The RoB tool 2.0 has ﬁve domains including bias arising from the randomization process, bias due to deviations from intended interventions, bias due to missing outcome data, bias in the measurement of the outcome, bias in the selection of the reported result. A third reviewer solved disagreements following a discussion between the reviewers. The Kappa statistic was used to calculate interrater reliability97. g g g p 5. Examine active components and mechanisms of change of these effective culturally adapted IMI. Particularly provide a better understanding of the impact of surface and deep structure changes on intervention adherence and effectiveness. 6. Examine ways of improving reach, uptake, engagement, and intervention adherence for the effective culturally adapted IMI. 7. Develop evidence-based recommendations and guidelines for adapting IMI culturally delineated from the effective culturally adapted IMI. 7. Develop evidence-based recommendations and guidelines for adapting IMI culturally delineated from the effective culturally adapted IMI. Box 1 Recommendations for future research 1. Improve evidence on whether culturally adapted IMI for health promotion are indeed not effective at all, even when compared to passive control conditions. 1. Improve evidence on whether culturally adapted IMI for health promotion are indeed not effective at all, even when compared to passive control conditions. 2. Provide a better understanding of the impact of population and intervention characteristics on the differential effects of culturally adapted IMI for health promotion. 2. Provide a better understanding of the impact of population and intervention characteristics on the differential effects of culturally adapted IMI for health promotion. If culturally adapted IMI are effective at least compared to passive control conditions (see 1.) and/or at least with regard to some subgroups (see 2.): p If culturally adapted IMI are effective at least compared to passive control conditions (see 1.) and/or at least with regard to some subgroups (see 2.): 3. Improve evidence on whether culturally adapted IMI are effective compared to active control conditions. Meta-analysis For each study, a standardized mean difference (SMD) and 95% conﬁdence intervals (CI) were calculated with mean scores of intervention and control groups. In order to decrease the bias of small samples, Hedges’ g was calculated98. Effect sizes were recoded when higher scores of an outcome assessment indicated worsening results (e.g. BMI and level of alcohol consumption). For continuous outcomes, Hedges’ g and 95% CIs were reported; for dichotomous outcomes, odds ratios and CIs were reported. Random effects model was chosen for analyses due to an expected diversity among IMI of health promotion, sample size, and duration of intervention among studies99. Data were pooled to calculate a standardized mean effect size for each outcome and a forest plot with 95% CIs, in the case of at least three studies reporting the respective outcome. Otherwise, results were presented descriptively. Sensitivity analysis was planned to assess the impact of studies with a high risk of bias. Analyses were performed in R package meta and metafor, and Review Manager 5100–103. Continuous effect sizes were categorized along with Cohen’s rule of thumb with 0.20 considered a small effect, 0.50 medium effect, and 0.80 large effects104. In order to assess publication bias, we planned to conduct funnel plots. review on culturally adapted IMI for mental health conditions94,67. There were no restrictions on the publication date. A search update was conducted on 15 October 2020. All search results were merged into Covidence95 and duplicates were automatically removed. Two reviewers screened titles and abstracts of the identiﬁed articles against the inclusion criteria and selected potentially relevant articles for the full-text screening. Full-text screening has been performed by two reviewers independently, disagreements have been solved by consensus or a third reviewer where needed. The study selection is illustrated in the PRISMA ﬂow diagram (see Fig. 8). Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 34 Published in partnership with Seoul National University Bundang Hospital DATA AVAILABILITY Data supporting the ﬁndings of this study are available within the respective articles cited in this review and from the corresponding author on reasonable request. 5. Di Cesare, M. et al. Inequalities in non-communicable diseases and effective responses. Lancet 381, 585–597 (2013). 6. World Health Organization. Global Status Report On Noncommunicable Diseases 2014 (World Health Organization, 2014). Reporting summary 2. Cerf, M. E. Healthy lifestyles and noncommunicable diseases: nutrition, the life‐ course, and health promotion. Lifestyle Med. 2, 1–12 (2021). 2. Cerf, M. E. Healthy lifestyles and noncommunicable diseases: nutrition, the life‐ course, and health promotion. Lifestyle Med. 2, 1–12 (2021). Further information on research design is available in the Nature Research Reporting Summary linked to this article. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 3. Preston, S. H., Stokes, A., Mehta, N. K. & Cao, B. Projecting the effect of changes in smoking and obesity on future life expectancy in the United States. Demo- graphy 51, 27–49 (2014). 3. Preston, S. H., Stokes, A., Mehta, N. K. & Cao, B. Projecting the effect of changes in smoking and obesity on future life expectancy in the United States. Demo- graphy 51, 27–49 (2014). 4. World Health Organization. Sexual Health and its Linkages to Reproductive Health: an Operational Approach 1–12 (World Health Organization, 2017). 4. World Health Organization. Sexual Health and its Linkages to Reproductive Health: an Operational Approach 1–12 (World Health Organization, 2017). Data extraction Data extraction was conducted by two independent reviewers and then extracted data was then checked by a third reviewer. The following data were extracted from the included studies: publication details, study participants (demographics and cultural background, baseline characteristics), study design, study setting, characteristics of the original and culturally adapted intervention, health behavior-speciﬁc and generic outcome measures, information regarding cultural adaptation (content, utilization of theoretical or evidence-based compo- nents). Behavioral outcomes are deﬁned as: physical activity measured via physical activity minutes per week with accel- erometers or self-report questionnaires; healthy eating measured via BMI; alcohol consumption assessed via the level of alcohol Statistical heterogeneity among studies was analyzed with the I2 statistics97,105. Statistical heterogeneity refers to the variability among effect sizes in a meta-analysis106. However, the veracity of measures of heterogeneity is arguable; therefore, their inter- pretation should be made with caution97. Statistical hetero- geneity was calculated with the I2 test for each outcome domain. Heterogeneity I2 ≥60% was regarded as substantial heterogene- ity, in which case no pooled effect sizes are reported. Subgroup analyses were planned to explore possible sources of npj Digital Medicine (2022) 34 S. Balci et al. Fig. 8 Prisma Flow chart92. Study identiﬁcation, selection, and inclusion represented on the diagram. 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Telehealth methods to deliver dietary interventions in adults with chronic disease: a systematic review and meta-analysis1,2. Am. J. Clin. Nutr. 104, 1693–1702 (2016). y g p to deliver dietary interventions in adults with chronic disease: a systematic review and meta-analysis1,2. Am. J. Clin. Nutr. 104, 1693–1702 (2016). 35. Montague, E. & Perchonok, J. Health and wellness technology use by his- torically underserved health consumers: Systematic review. J. Med. Internet Res. 14, e78 (2012). 60. Hutchesson, M. J. et al. eHealth interventions for the prevention and treatment of overweight and obesity in adults: A systematic review with meta-analysis. Obes. Rev. 16, 376–392 (2015). npj Digital Medicine (2022) 34 Published in partnership with Seoul National University Bundang Hospital FUNDING Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Open Access funding enabled and organized by Projekt DEAL. S. Balci et al. 14 Published in partnership with Seoul National University Bundang Hospital COMPETING INTERESTS Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative 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/. 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. AUTHOR CONTRIBUTIONS S.B., K.S., L.S., and H.B. conceived the study design. S.B., K.S., and L.S. developed the search strategy, study selection, and extraction. S.B. wrote the draft of the manuscript. All authors read, provided feedback, and approved the ﬁnal version. S.B. is the guarantor of the review. 88. James, D. C., Harville, C., Sears, C., Efunbumi, O. & Bondoc, I. Participation of African Americans in e-Health and m-Health studies: a systematic review. Tele- med. e-Health 23, 351–364 (2017). Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2022) 34 Published in partnership with Seoul National University Bundang Hospital ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41746-022-00569-x. Correspondence and requests for materials should be addressed to Sumeyye Balci. Reprints and permission information is available at http://www.nature.com/ reprints © The Author(s) 2022 npj Digital Medicine (2022) 34 npj Digital Medicine (2022) 34
https://openalex.org/W4298009026	https://repozitorij.uni-lj.si/Dokument.php?id=165761&dn=	English	null	Reflectance calibration of multimode optical fiber probes by probe-to-target distance reflectance profile modeling	Measurement	2,022	cc-by	13,187	1. Introduction lipid concentrations [6] and characterization of optical phantoms [7]. Extraction of turbid medium optical properties using a multimode optical fiber probe requires an accurate light propagation model that relates the optical and geometrical properties of the turbid phantoms and multimode optical fiber probes to the acquired reflectance. While various models have been proposed, a general consensus is that the gold standard for light propagation modeling is the stochastic Monte Carlo (MC) method. Optical properties can be subsequently extracted from the acquired reflectance in an inverse manner by using lookup tables [8], predictive models [9] or neural networks [10] determined or trained by the MC method. Multimode optical fiber probes represent an indispensable tool for light delivery and collection of re-emitted light from turbid media. In comparison to other configurations such as hyperspectral imaging sys tems and integrating spheres, multimode optical fiber probes present a cheap, portable, durable, and flexible option, which can be utilized in various environments including endoscopic and contact applications, where disinfection and cleaning are required. Since light that has propagated and interacted with the turbid me dium carries an abundance of information, one of the main uses of multimode optical fiber probes is extraction of optical properties such as the absorption and scattering coefficients, and scattering phase function of the investigated turbid medium from the acquired reflectance. The absorption coefficient can be related to the chemical composition of the turbid medium (i.e., concentration of various chromophores), while the scattering coefficient and scattering phase function can be related to the morphological microstructure responsible for fluctuations of the refractive index. The extracted optical properties can be used for various applications including spatial and temporal characterization of sea ice [1], generating photodynamic therapy treatment plans [2,3], detection of changes in optical and hemodynamic properties [4], property and quality assessment of horticultural products [5], estimation of water and A crucial step in the extraction of optical properties from the ac quired reflectance is the so-called calibration of the reflectance pre dicted by the light propagation model. On the one hand, the modeled reflectance is provided relative to the energy of the light supplied by the source fiber. On the other hand, the measured reflectance is normalized to a signal acquired from a reflective standard such as Spectralon®. Reflectance calibration of multimode optical fiber probes by probe-to-target distance reflectance profile modeling Peter Nagliˇc , Franjo Pernuˇs , Miran Bürmen Laboratory of Imaging Technologies, Faculty of Electrical Engineering, University of Ljubljana, Trˇzaˇska cesta 25, SI-1000 Ljubljana, Slovenia Laboratory of Imaging Technologies, Faculty of Electrical Engineering, University of Ljubljana, Trˇzaˇska cesta 25, SI-1000 Ljubljana, Slovenia A R T I C L E I N F O Keywords: Optical fiber probes Reflectance calibration Reflectance modeling Monte Carlo simulations Optical properties Reflectance acquired with a multimode optical fiber probe can be related to optical properties of an investigated turbid medium by utilizing a light propagation model. During this step, a calibration of the light propagation model is required, as the modeled reflectance is normalized to the light energy of the source fiber, while the experimentally acquired reflectance is normalized to a reflective standard. Since currently established calibration methods based on liquid and solid turbid phantoms suffer from drawbacks such as low stability and dependence on other characterization methods, we propose a new method for reflectance calibration that is based on modeling and acquisition of probe-to-target distance reflectance profiles from first surface mirrors. We show that the spectrally resolved calibration factors can be estimated with a repeatability of 2% and agree within 10% with the reference values obtained by using turbid phantoms based on aqueous suspensions of polystyrene microspheres. lipid concentrations [6] and characterization of optical phantoms [7]. Corresponding author. E-mail address: peter.naglic@fe.uni-lj.si (P. Nagliˇc). Measurement 203 (2022) 112002 Measurement 203 (2022) 112002 Available online 29 September 2022 0263-2241/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.measurement.2022.112002 Received 30 June 2022; Received in revised form 5 September 2022; Accepted 25 September 2022 p 0263-2241/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author. E-mail address: peter.na Available online 29 September 2022 0263-2241/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Co espo d g aut o . E-mail address: peter.naglic@fe.uni-lj.si (P. Nagliˇc). https://doi.org/10.1016/j.measurement.2022.112002 Received 30 June 2022; Received in revised form 5 September 2022; Accepted 25 September 2022 2.1.1. Irradiance at the optical probe face The fundamental equation of the radiative transfer in a non- absorbing and non-scattering medium assumes the following form [17] dΦ = Lmed dA1cosθ1dA2cosθ2 ρ2 , dΦ = Lmed dA1cosθ1dA2cosθ2 ρ2 , (1) (1) where Lmed is the radiance in the medium between the infinitesimal areas dA1 and dA2 at a distance ρ. The parameters θ1 and θ2 represent the angles between the normals of the infinitesimal areas and the connecting line. While Eq. (1) is sufficient for modeling light propagation between optical fiber probe face and first surface mirror, it is not applicable for absorbing and scattering media such as aqueous suspensions of poly styrene microspheres. In this case, we have modeled light propagation using a modified version of the MC method based on the Boltzmann transport equation described in detail by Wang and Jacques [18]. Our modifications are summarized in Sect. 3.3. ili i The light originates from the source fiber, reflects from the first surface mirror, and enters the detector fiber (see Fig. 1(a)). According to Eq. (1), the irradiance E (W/m2) within the detector fiber area at probe- to-target distance h and wavelength λ can be expressed as Zhang et al. [12] has tested various calibration methods for single fiber reflectance spectroscopy and found that Fresnel reflection from the tip of the fiber is the most repeatable and reliable. However, such an approach can only be utilized for single fiber reflectance spectroscopy where the same fiber acts as the source and detector. Such an approach is not possible for optical fiber probes used to acquire spatially resolved reflectance. The same study also investigated first surface mirrors for calibration, as their reflectivity can be measured very precisely and accurately. However, a lower repeatability of the calibration factor was observed since the acquired signal depended significantly on the established contact between the probe and the mirror. While mirrors might not be appropriate candidates for calibration of single fiber probes in contact, they seem a promising candidate for calibration of optical fiber probes by contactless acquisition of distance reflectance profiles. 1. Introduction Reflective standard is usually mounted in a special holder that maintains a fixed and repeatable distance between the multimode optical fiber probe and reflective standard [11]. Since the reflective standard only backscatters a fraction of the light supplied by the source fiber, the measured reflectance is not on the same scale as the modeled reflec tance. In this case a spectrally resolved calibration factor must be Measurement 203 (2022) 112002 P. Nagliˇc et al. significantly influence the relative radiant power received by the de tector fiber for small probe-to-target distances. determined that represents a ratio between the measured and modeled reflectance. l The calibration is usually performed by using turbid phantoms with known optical properties for which reflectance can be measured and accurately modeled using the MC method. For this purpose, liquid turbid phantoms based on Intralipid-20 % fat emulsions [2,5,12] and aqueous suspensions of polystyrene microspheres [1,8,13] are frequently uti lized. Intralipid-20 % is especially common since it is relatively cheap, easy to prepare, and optical properties are provided in the literature [14]. However, since optical properties are measured using an alterna tive optical modality, any uncertainty regarding the original measure ment setup are propagated to the optical properties and subsequently into the calibration curve. On the other hand, aqueous suspensions of polystyrene microspheres are quite advantageous in terms of optical properties, since the absorption and scattering coefficients and scat tering phase function can be accurately calculated using the Mie theory [15]. However, the polystyrene microspheres are expensive, and the Mie theory depends on the input parameters such as the diameter, refractive index, and number density of the microspheres, which can affect the optical properties and calibration factors. Moreover, liquid phantoms are known to exhibit a short shelf time due to evaporation and settling or possible agglomeration of the dispersed phase. In this aspect, solid phantoms exhibiting longer shelf lives have been proposed [7,16]. However, similarly to Intralipid-20 % the optical properties must be measured and can be potentially inaccurate, which could influence the spectrally resolved calibration factors. 2.1.1. Irradiance at the optical probe face l E(r2,h,λ) = dΦ dA2 (r2,h,λ) = ∫∫ source fiber area Rmir(λ)Tsrc ( θfib,λ ) Tdet(θ,λ)Lmed(λ) cos2θ ρ2 dA1, (2) (2) where θ1 = θ2 = θ = arccos(2h/ρ), r2 denotes the distance of the infinitesimal area dA2 from the center of the source fiber area, Rmir(λ) corresponds to the first surface mirror reflectivity, and dA1 and dA2 correspond to an infinitesimal area at the source and detector fibers. Since light is radiated from within the source fiber and received within the detector fiber, we introduced two Fresnel transmission coefficients Tsrc ( θfib, λ ) and Tdet(θ, λ) that depend on the refractive index of the fiber core nfib(λ) and medium nmed(λ). The angle θfib represents the incident angle of light within the source fiber exiting into the medium and can be expressed using Snell’s law and cosθ (Fig. 1(a)): ili In this study, we propose a new method for reflectance calibration using first surface mirrors, which is based on modeling and acquisition of probe-to-target distance reflectance profiles (DRPs). In the first part of the study, we lay out and derive the required models to predict the DRPs, present the measurement setup for acquisition of the DRPs and define the calibration method. Subsequently, we thoroughly validate and test the derived models and use them to fit the measured DRPs to obtain spectrally resolved calibration factors. Finally, we compare the obtained spectrally resolved calibration factors to the reference ones that are determined by turbid phantoms based on aqueous suspensions of poly styrene microspheres. θfib = arcsin (nmed(λ) nfib(λ) ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ 1 −cos2θ √ ) = arcsin ⎛ ⎝nmed(λ) nfib(λ) ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ 1 − (2h ρ )2 √ ⎞ ⎠. θfib = arcsin (nmed(λ) nfib(λ) ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ 1 −cos2θ √ ) = arcsin ⎛ ⎝nmed(λ) nfib(λ) ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ 1 − (2h ρ )2 √ ⎞ ⎠. (3) (3) To calculate the irradiance E at a specific point within the detector fiber area, the integration in Eq. (2) is performed over the source fiber area utilizing polar coordinates (r, ϕ), i.e., dA1 = r dr dϕ. Distance ρ can be geometrically related to coordinates (r, ϕ) by ρ2 = r2 + r2 2 + 4h2 −2 r r2cosϕ.i r ∈ ( max{0, r2 −2htanθNA}, rfib ) , ϕ ∈(0, arccos(min{max{cosϕx, −1}, 1 } ) ), (4) 2. Theory The integration limits over the area of the source fiber depend on the emittance and acceptance characteristics of the source and detector fi bers. When using a simple Lambertian emittance and angularly uniform acceptance characteristics for source and detector fibers with a cutoff angle θNA defined by the numerical aperture (NA), at short probe-to- target distances h, only a part of the source fiber area contributes to the irradiance integral in Eq. (2) within the area of the detector fiber. The integration limits for (r, ϕ) therefore depend on the probe-to-target distance h and distance r2 In this section, we present theoretical foundations for modeling DRPs based on light propagation between the optical fiber probe and the first surface mirror. DRPs represent relative radiant power received by the detector fiber as a function of distance between the optical fiber probe face and the first surface mirror (probe-to-target distance). Firstly, we describe deterministic and stochastic physical models based on a single reflection from the first surface mirror. The deterministic model is based on the radiative transfer equation, while the stochastic model is based on a random propagation of light energy packets also known as the MC method. Subsequently, we enhance the stochastic model to include multiple reflections between the optical fiber probe face and first surface mirror, since we experimentally found that multiple reflections r ∈ ( max{0, r2 −2htanθNA}, rfib ) , (4) ϕ ∈(0, arccos(min{max{cosϕx, −1}, 1 } ) ), (4) 2 P. Nagliˇc et al. Measurement 203 (2022) 112002 Fig. 1. Schematic of the source (yellow) and detector (blue) fiber areas and corresponding notation for (a) the deterministic and (b) stochastic model. Fig. 1. Schematic of the source (yellow) and detector (blue) fiber areas and corresponding notation for (a) the deterministic and (b) stochastic model. Fig. 2. (a) Experimental setup for light delivery and acquisition. LS: light source, SPEC: multichannel spectrometer, OF: multimode optical fiber. (b) Setup for DRP measurements of first surface mirrors. RM: rotation mount, MIR: first surface mirror, LIS: linear stage, OP: optical fiber probe, h: probe-to-target distance. (c) Setup for reflectance measurements of turbid samples. dsds: source-detector separation. (d) An example of spectrally resolved reflectance acquired from a turbid sample using five source-detector separations. (e) Flowchart of the proposed calibration method. Fig. 2. (a) Experimental setup for light delivery and acquisition. LS: light source, SPEC: multichannel spectrometer, OF: multimode optical fiber. The integration of Eq. (14) yields ϕ ∈(0, π) . (8) (8) ϕ ∈(0, π) . P0(λ) = K(λ) 2π2r2 fib ∫1 0 s ( cosθfib, λ ) cosθfib dcosθfib. (15) Note that the integration limits now include the full range of r and ϕ since the measured characteristics are not limited by the NA , albeit they reduce to 0 near the NA (see Sect. 3.5). (15) It is now clear that the constant K(λ) could be in principle deduced from Eq. (15) by measuring the initial radiant power P0 and the distri bution of the emittance characteristic of the source fiber s ( cosθfib, λ ) . The relative irradiance can now be expressed as 2.1.2. Relative irradiance at the optical probe face i 2.1.2. Relative irradiance at the optical probe face Radiant power received by the detector fiber is usually expressed E(r2, h, λ) P0(λ) = 2 h2 Rmir(λ) π2r2 fib ∫1 0 s ( cosθfib, λ ) cosθfib dcosθfib ∫∫ source fiber area s ( cosθfib, λ ) T ( θfib, λ ) T(θ, λ)a ( cosθfib, λ ) 1 ρ4 r drdϕ. (16) relative to the initial radiant power of light P0 originating from within the source fiber, which is more practical in an experimental setting since the units can be arbitrary and related to photon counts of the sensor. i relative to the initial radiant power of light P0 originating from within the source fiber, which is more practical in an experimental setting since the units can be arbitrary and related to photon counts of the sensor. i 2.1.3. Received relative radiant power (reflectance) 2.1.3. Received relative radiant power (reflectance) 2.1.3. Received relative radiant power (reflectance) The initial radiant power P0 originating from within the source fiber is given by l The relative radiant power received by the detector fiber, termed as reflectance RdM fib , can be obtained by integrating relative irradiance given in Eqs. (13) and (16) over the area of the detector fiber as shown in Fig. 1 (a) with a radius rfib and center-to-center source-detector separation dsds P0(λ) = ∫∫ Lfib(λ)dA1cosθfibdΩ, (9) (9) where the integral is evaluated over the source fiber area dA1 and over the solid angle dΩ defined by the NA or measured emittance characteristic of the source fiber. RdM fib (dsds, h, λ) = ∫∫ source fiber area E(r2, h, λ) P0(λ) dA2, (17) (17) Using the invariance relation provided in Eq. The integration of Eq. (14) yields (6) and the Lambertian emittance characteristic, the initial radiant power P0 can be obtained by where the integration can be done in polar coordinates using dA2 = r2dr2dϕ2. We explicitly note that the reflectance RdM fib is a function of source-detector separation dsds, probe-to-target distance h and the wavelength of light λ. Considering that for non-overlapping source and detector fiber openings rfib2 = r2 2 + d2 sds −2r2dsdscosϕ2, the integration can be carried out over ϕ2, which yields where the integration can be done in polar coordinates using dA2 = r2dr2dϕ2. We explicitly note that the reflectance RdM fib is a function of source-detector separation dsds, probe-to-target distance h and the wavelength of light λ. Considering that for non-overlapping source and detector fiber openings rfib2 = r2 2 + d2 sds −2r2dsdscosϕ2, the integration can be carried out over ϕ2, which yields P0(λ) = Lmed(λ) n2 med(λ)n2 fib(λ) ∫rfib 0 r dr ∫2π 0 dϕ′ ∫1 cosθNA,fib cosθfib dcosθfib ∫2π 0 dϕ, (10) (10) (10) where cosθNA,fib = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ 1 − ( NA nfib(λ) )2 √ , (11) i cosθNA,fib = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ 1 − ( NA nfib(λ) )2 √ , (11) RdM fib (dsds, h, λ) = 2 ∫dsds+rfib dsds−rfib arccos (r2 2 + d2 sds −rfib2 2r2dsds ) E(r2, h, λ) P0 r2dr2. (18) l (18) finally resulting in finally resulting in The reflectance RdM fib (dsds, h, λ) as estimated by the deterministic model can be directly compared to the reflectance obtained by the MC method, which is discussed next. P0(λ) = Lmed(λ)π2r2 fib ( NA n2 med(λ) )2 . (12) (12) It follows from Eqs. (2) and (12), that the relative irradiance is It follows from Eqs. (2) and (12), that the relative irradiance is E(r2, h, λ) P0(λ) = 4 h2 Rmir(λ) π2r2 fib ( NA nmed(λ) )2 ∫∫ source fiber area T ( θfib, λ ) T(θ, λ) 1 ρ4 r dr dϕ. (13) 2.2. Received relative radiant power estimated by the Monte Carlo (MC) method 2. Theory Note that the integration since the measured character reduce to 0 near the NA (see 2.1.2. Relative irradiance at Radiant power received relative to the initial radian the source fiber, which is mo the units can be arbitrary an The initial radiant power is given by P0(λ) = ∫∫ Lfib(λ)dA1cosθfibdΩ where the integral is eva over the solid angle dΩ de characteristic of the source f Using the invariance rela emittance characteristic, the P0(λ) = Lmed(λ) n2 med(λ)n2 fib(λ) ∫rfib 0 r d where cosθNA,fib = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ 1 − ( NA nfib(λ) )2 √ , finally resulting in P0(λ) = Lmed(λ)π2r2 fib ( NA n2 med(λ) It follows from Eqs. (2) a E(r2, h, λ) = 4 h2 Rmir(λ) K(λ) ( E(r2, h, λ) P0(λ) = 2 h2 R π2r2 fib ∫1 0 s ( cosθfib P. Nagliˇc et al. P. Nagliˇc et al. Measurement 203 (2022) 112002 E(r2, h, λ) = 4 h2 Rmir(λ) K(λ) (nmed nfib )2 ∫∫ source fiber area s ( cosθfib, λ ) Tsrc ( θfib, λ ) Tdet(θ, λ)a ( cosθfib, λ ) 1 ρ4 dA1, (7) E(r2, h, λ) = 4 h2 Rmir(λ) K(λ) (nmed nfib )2 ∫∫ source fiber area s ( cosθfib, λ ) Tsrc ( θfib, λ ) Tdet(θ, λ)a ( cosθfib, λ ) 1 ρ4 dA1, (7) P0(λ) = K(λ) ∫rfib 0 r dr ∫2π 0 dϕ′ ∫1 0 s ( cosθfib, λ ) cosθfib dcosθfib ∫2π 0 dϕ. (14) where the integration limits in polar coordinates are P0(λ) = K(λ) ∫rfib r r ∈ ( 0, rfib ) , (14) 2. Theory (b) Setup for DRP measurements of first surface mirrors. RM: rotation mount, MIR: first surface mirror, LIS: linear stage, OP: optical fiber probe, h: probe-to-target distance. (c) Setup for reflectance measurements of turbid samples. dsds: source-detector separation. (d) An example of spectrally resolved reflectance acquired from a turbid sample using five source-detector separations. (e) Flowchart of the proposed calibration method. received by the detector fiber. We assume that the acceptance and emittance characteristics only depend on the polar angle θfib and are uniformly distributed across the source fiber opening. Let s ( cosθfib, λ ) and a ( cosθfib, λ ) denote the wavelength-dependent emittance and acceptance characteristic defined within the source and detector fibers, respectively. The radiance within the source fiber can then be written as Lfib(θ, λ) = K(λ)s ( cosθfib, λ ) , where K(λ) is a wavelength-dependent normalization constant related to the total radiant power originating from the source fiber. Using the invariance relation between the radi ance in the fiber Lfib and in the medium Lmed that accounts for the change in solid angle due to refraction [17] where where cosϕx = r2 + r2 2 −4 h2tan2θNA 2 r r2 . (5) cosϕx = r2 + r2 2 −4 h2tan2θNA 2 r r2 . (5) The integration limits over the angle ϕ consider the symmetry over the line connecting the source and detector fiber centers, and thus the integral in Eq. (2) must be counted twice. Note that the integration limits in Eq. (4) are valid for h > rfib/(2tanθNA), while for smaller h the inte gration must be performed over a smaller circular area situated within the source fiber area. However, for standard multimode fibers as used in this study with NA = 0.22 and rfib = 100 μm, the integration limits in Eq. (4) are valid for h > 222 μm. Probe-to-target distances smaller than 222 μm are not utilized in this study. Lfib(λ) n2 fib(λ) = Lmed(λ) n2 med(λ), (6) Lfib(λ) n2 fib(λ) = Lmed(λ) n2 med(λ), (6) As will be shown later, the emittance and acceptance characteristics must be measured to obtain a realistic simulation of the radiant power the irradiance E in Eq. (2) takes the form the irradiance E in Eq. (2) takes the form where the integration limits r ∈ ( 0, rfib ) , ϕ ∈(0, π) . P. Nagliˇc et al. The angles ϕ and θfib can be sampled from the given probability density functions by equating their cumula tive distribution function to uniformly distributed and independent random numbers ξ1 and ξ2 from the interval [0, 1] The reflection from the mirror surface is performed in the frame of the mirror. The direction pr of the reflected energy packet is given by pr = MTRMp, (27) ϕξ1 = 2πξ1 , pr = MTRMp, (27) ϕξ1 = 2πξ1 , sinθξ2 = ̅̅̅̅̅ ξ2 √ NA nfib . (21) sinθξ2 = ̅̅̅̅̅ ξ2 √ NA nfib . (21) where M is a rotation matrix sinθξ2 = ̅̅̅̅̅ ξ2 √ NA nfib . (21) M = ⎡ ⎣ cosϕ ′cosθ ′ sinϕ ′cosθ ′ −sinθ ′ −sinϕ ′ cosϕ ′ 0 cosϕ ′sinθ ′ sinϕ ′sinθ ′ cosθ ′ ⎤ ⎦, (28) (28) For a measured emittance characteristic, the probability density p ( cosθfib ) is proportional to the radiance Lfib(θ, λ) = K(λ)s ( cosθfib, λ ) and cosθfib with angles ϕ ′ and θ ′ defining the mirror surface normal in the frame of the optical fiber probe (see Fig. 1(b)). Matrix R performs a reflection by changing the sign of the z-direction in the frame of the mirror p ( cosθfib ) ∝K(λ)s ( cosθfib, λ ) cosθfib. (22) (22) The polar angle θξ2 can then be sampled via the following cumulative distribution function R = ⎡ ⎣ 1 0 0 0 1 0 0 0 −1 ⎤ ⎦. (29) l (29) ξ2 = CDF ( cosθξ2 ) = ∫cosθξ2 0 s ( cosθfib ) cosθfib dcosθfib ∫1 0 s ( cosθfib ) cosθfibdcosθfib . (23) (23) Finally, the intersection rd between the reflected energy packet and the optical fiber plane with normal (0, 0, 1) is obtained using (Fig. 1) Finally, the intersection rd between the reflected energy packet and the optical fiber plane with normal (0, 0, 1) is obtained using (Fig. 1) The cumulative function CDF ( cosθξ2 ) can be inverted, re- interpolated and provided as a linear lookup table that yields cosθξ2 upon a drawn uniformly distributed random number ξ2. To retain a high level of accuracy when sampling from a linear lookup table, its size was kept in the order of 104. i rd = rc −rc,z pr,z pr . (30) rd = rc −rc,z pr,z pr . P. Nagliˇc et al. P. Nagliˇc et al. Measurement 203 (2022) 112002 characteristics as described in the previous section. The Lambertian launching scheme is based on a cumulative distri bution function derived from a probability density function of an energy packet launched into a solid angle dΩ = dcosθfibdϕ proportional to the cosine of the polar angle θfib within the source fiber z = 0 , (25) (25) z = 0 , z = 0 , where ξ3 and ξ4 are also uniformly distributed random numbers drawn from the interval [0, 1]. p ( ϕ, cosθfib ) = dP dΩ∝Lfibcosθfib, (19) (19) Upon launching the energy packet, its weight is dropped due to transmission through the fiber-medium boundary and the direction is updated according to the Snell’s law. Subsequently, the intersection rc of the energy packet with the first surface mirror is calculated by where Lfib is the radiance which is constant for a Lambertian emittance. The proportionality constant is obtained through normalization of the probability density function. i rc = r0 + (r0 −h) • ̂n ̂p • ̂n ̂p, (26) Under the assumption that the multimode fiber used in this study illuminates in a rotationally symmetric fashion and that the polar angle θfib and azimuth angle ϕ are completely independent, the probability density function can be written as p ( ϕ, cosθfib ) = p(ϕ)p ( cosθfib ) , where (26) where ro denotes the initial position and ̂p the initial direction of the photon packet, h = (0, 0, h) the vertical distance of the mirror from the centre of the source fiber, and ̂n = (cosϕ ′sinθ ′, sinϕ ′sinθ ′, cosθ ′) the mirror surface normal (see Fig. 1(b)). While we strive to keep the mirror surface in the experiments in parallel to the optical fiber probe face with a normal ̂n = (0, 0, −1), the MC method allows a straightforward way of simulating reflections from a tilted first surface mirror, which allows us to numerically evaluate the sensitivity of the method to the tilt of the mirror. l p(ϕ) = 1 2π , p ( cosθfib ) = 2cosθfib 1 −cos2θNA,fib , (20) p(ϕ) = 1 2π , p ( cosθfib ) = 2cosθfib 1 −cos2θNA,fib , (20) (20) and θNA,fib given by Eq. (11). P. Nagliˇc et al. (30) At the mirror and detector fiber plane, the energy packet weight is multiplied by the reflectivity of the first surface mirror and Fresnel transmission coefficient for unpolarized light, respectively. For an angularly uniform acceptance characteristic, the energy packets are only accepted, if they arrive within the detector fiber area and the acceptance cone defined by Finally, we assume that the multimode source fiber core is uniformly illuminated and thus the energy packets are launched uniformly from within the surface of the fiber core. This assumption is based on the premise that longer multimode fibers act as a mixing rod, sufficiently mixing any initial radial distribution of light into a uniform flat-top profile. −pr,z > ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ 1 − ( NA nmed )2 √ . (31) (31) i To summarize the launching scheme, the initial direction (px, py, pz) of the energy packet is given by In the realistic case, the energy packet weight is multiplied by the acceptance characteristic a ( cosθfib, λ ) after the Fresnel transmission and refraction into the detector fiber. px = cos 2πξ1sinθξ2 , py = sin 2πξ1sinθξ2 , pz = cosθξ2 , px = cos 2πξ1sinθξ2 , py = sin 2πξ1sinθξ2 , pz = cosθξ2 , (24) px = cos 2πξ1sinθξ2 , py = sin 2πξ1sinθξ2 , pz = cosθξ2 , (24) px = cos 2πξ1sinθξ2 , py = sin 2πξ1sinθξ2 , i Finally, the energy packet weights are accumulated and divided by the total number of launched energy packets to yield the received relative radiant power or reflectance RsMC fib (dsds, h, λ) at the selected de tector fiber. (24) pz = cosθξ2 , pz = cosθξ2 , where θξ2 can be obtained from either a Lambertian or measured emit tance characteristic. The initial position (x, y, z) is given by where θξ2 can be obtained from either a Lambertian or measured emit tance characteristic. The initial position (x, y, z) is given by 2.2.1. Multiple reflections between the optical probe face and the first surface mirror l x = ̅̅̅̅̅ ξ3 √ rfibcos2πξ4 , 2.2. Received relative radiant power estimated by the Monte Carlo (MC) method (13) (13) The MC method is based on random propagation of light energy packets. A light energy packet is launched with a weight of 1 from within the source fiber according to a launching scheme which assigns the packet its initial direction and position. We have implemented two launching schemes based on the Lambertian and measured emittance The initial radiant power P0 for a measured emittance characteristic of the source fiber s ( cosθfib, λ ) with radiance Lfib(θ, λ) = K(λ)s ( cosθfib, λ ) can be calculated using Eq. (9) 4 2.2.1. Multiple reflections between the optical probe face and the first surface mirror l To account for multiple reflections, the proposed MC method was 5 P. Nagliˇc et al. Measurement 203 (2022) 112002 Fig. 3. A summary of the models and input parameters used in this study. Fig. 3. A summary of the models and input parameters used in this study. Apeldoorn, The Netherlands) and the detector fibers to a custom 10- channel spectrometer which is based on a multi-fiber-to-slit coupler, spectrograph (spectral range 400–1000 nm, Imspector V10E, Specim, Spectral Imaging ltd., Oulu, Finland) and a camera (Basler ace acA1920- 155um, Basler AG, Ahrensburg, Germany). l Apeldoorn, The Netherlands) and the detector fibers to a custom 10- channel spectrometer which is based on a multi-fiber-to-slit coupler, spectrograph (spectral range 400–1000 nm, Imspector V10E, Specim, Spectral Imaging ltd., Oulu, Finland) and a camera (Basler ace acA1920- 155um, Basler AG, Ahrensburg, Germany). l modified to allow iterative propagation of energy packets between the optical fiber probe face and the first surface mirror. ii When at each iteration an energy packet is incident on the optical fiber probe face, a reflection coefficient is calculated and compared to a random number drawn uniformly from an interval [0,1]. If the random number is less or equal to the reflection coefficient, the energy packet is reflected. Otherwise, the energy packet is terminated, except if located within the detector fiber opening in which case it is refracted, attenuated by the acceptance characteristic a ( cosθfib, λ ) and accumulated. Finally, if lateral position of the energy packet exceeds the probe, the energy packet is terminated by default. li In general, the measured reflectance at a source-detector separation dsds, probe-to-target distance h, and wavelength of light λ is defined as Rmeas fib (dsds, h, λ) = Im(dsds, h, λ) −D(dsds, λ) Is(dsds, λ) −D(dsds, λ) , (32) (32) where Im represents signal of light reflected from the first surface mirror, Is white signal acquired from a reflective standard Spectralon®, and D dark current. The repeatability of a reflective standard was sustained by mounting the Spectralon® in a custom 3D printed holder that kept the probe at a fixed distance from the standard. Note that normalization via the reflective standard signal Is is necessary since the intensity of the light source and sensitivity of the sensor in the multichannel spec trometer might change due to changes in the ambient temperature. 2.2.1. Multiple reflections between the optical probe face and the first surface mirror l i The reflection coefficient depends on the lateral position of the en ergy packet at the optical probe face (see probe parameters in Fig. 3). The reflection coefficient is calculated according to the refractive index mismatch between the medium nmed(λ) and silica nfib(λ) if the lateral position is within the fiber opening, or medium and epoxy resin nepoxy(λ) if the lateral position is within the epoxy filler. If the lateral position of the energy packet is within the optical probe face but not within the cutout for the fibers, the reflection coefficient is equal to the reflectivity Rs of the polished stainless-steel housing. l Fig. 2(b) shows the optical fiber probe mounted in a two-axis tilting system for acquisition of DRPs, while Fig. 2(c) shows a standard reflectance measurement setup for liquid turbid samples. While for DRPs the acquired signal comprises a fraction of light that is reflected from the first surface mirror, contact reflectance signal comprises a fraction of light that is scattered in the medium. Exemplary reflectance of a turbid sample for five source-detector separations is presented in Fig. 2(d). l We denote the received relative radiant power or reflectance esti mated by this advanced model as RmMC fib (dsds, h, λ). A summary of the models is given in Fig. 3. 3. Materials and methods i Fig. 2(e) shows the flowchart of the proposed calibration method. The measured DRPs as a function of probe-to-target distance h (acquired by the setup in Fig. 2(a) and 2(b)) are fitted by modeled DRPs using the multi-reflection MC model for each source-detector separation dsds and wavelength λ (details described in Sect. 3.4). The fitting procedure yields the so-called calibration factor as a function of dsds and λ, which can then be utilized for calibrating the reflectance modeled by an MC model for turbid media. The calibrated modeled reflectance can subse quently be fitted to the measured reflectance experimentally acquired from an unknown turbid sample for extraction of optical properties such 3.2. Probe-to-target distance reflectance profile (DRPs) measurements of the first surface mirrors In this way, the detector fiber could simultaneously illumi nate the first surface mirror and acquire the reflected light which was then transmitted to the camera. The optical fiber probe was perpen dicularly aligned to the first surface mirror by finding the maximum intensity of the reflected light signal measured by the camera. Our OpenCL™ implementation allows arbitrary scattering phase functions by using a lookup table approach that is especially useful for Mie scattering phase functions [23]. Furthermore, we implemented a completely realistic description of the optical fiber probe that included reflectivity of the stainless steel housing Rs, refractive index of the black epoxy fixing agent nepoxy and refractive index of the fiber nfib. The description of the optical fiber probe is therefore identical to the description of the probe in the deterministic and stochastic MC model used for simulations of DRPs (see Fig. 3). l First surface mirrors were mounted on a linear stage (LES 4, Isel Germany AG, Eichenzell, Germany) with positioning repeatability of ± 20 µm. It should be noted that only the relative distance from the first surface mirror was required since the shape of the DRP uniquely defines the absolute distances from the mirror. The DRPs were recorded with an integrating time of 0.9 ms. The closest probe-to-target distance was selected by observing the signal at the shortest source-detector separa tion dsds which was required to decrease by approximately 50 % while the optical fiber probe was approaching the first surface mirror. This way, colliding with the mirror was avoided, while the most critical and characteristic part of the DRP which includes the peak was recorded even for the shortest dsds (see Fig. 3 for an exemplary DRP). The total distance of the recorded DRPs was 10 mm. The source and the detector optical fibers were not removed from their respective input or output channels throughout the entire study to ensure that the signals were not disturbed due to changes in coupling. i Finally, the aqueous suspension of polystyrene microspheres was modeled as a semi-infinite homogeneous layer with the refractive index nmed of water and with optical properties calculated according to the Mie theory as described above. To speed up the simulations, a termination radius of 15 mm was selected at which the packets that negligibly contributed to the reflectance signal were terminated. 3.4. Calibration factor determination The calibration factor is defined as a ratio between the measured Rmeas and modeled reflectance Rmodel at a specific source-detector sep aration dsds and wavelength point λ We recorded the DRP profiles of two silver (PF10-03-P01, Thorlabs Inc.) and one aluminium mirrors (PF10-03-G01, Thorlabs Inc.) over three consecutive days to test the measurement repeatability and in fluence of different mirror reflectivities. The reflectivity of the silver and aluminium mirrors as a function of wavelength was obtained from the supplier for unpolarized light and 12◦angle of incidence. In total, we recorded nine DRP profiles for each source-detector separation dsds. Furthermore, perpendicular alignment was performed each time the first surface mirror was changed. C(dsds, λ) = Rmeas(dsds, λ) Rmodel(dsds, λ) . (33) C(dsds, λ) = Rmeas(dsds, λ) Rmodel(dsds, λ) . (33) In our proposed approach, the calibration factor was obtained by firstly shifting the position of the measured DRP Rmeas fib until the peak aligned with its modeled counterpart RmMC fib (Fig. 4). It should be noted that the absolute position of the Rmeas fib is not known a priori (note the h* in Fig. 4). The alignment of the two DRPs was performed by comparing Rmeas fib to RmMC fib within an interval in which the values of Rmeas fib exceed 65 % of the peak value (denoted by [h0, h0 + Δh] in Fig. 4). This was done to ensure that the highest emphasis of the alignment procedure was given to the area around the peak and that the interval was well-defined. The parameter h0 influences the interval offset within which the RmMC fib is compared to the selected measurement points Rmeas fib , effectively shifting the Rmeas fib relative to the RmMC fib . The optimization of the parameter h0 was based on the following metric 3.1. Optical fiber probe measurement principle and calibration workflow 3.1. Optical fiber probe measurement principle and calibration workflow 3.1. Optical fiber probe measurement principle and calibration workflow Optical probe used in this study is based on an all-silica linear multimode fiber array (NA = 0.22, 220 µm fiber–fiber distance, 200 µm fiber core diameter, Fibertech Optica Inc., Kitchener, Canada). The first fiber is used as the source fiber and the remaining five as detector fibers enabling source-detector separations dsds of 220, 440, 660, 880 and 1100 μm. As depicted in Fig. 2(a), the source fiber was at all times coupled to a broadband halogen light source (AvaLight-Hal LS, Avantes, 6 P. Nagliˇc et al. Measurement 203 (2022) 112002 as the absorption and reduced scattering coefficients. as the absorption and reduced scattering coefficients. Germany) and solid content of 0.05 g/ml. To calculate the scattering coefficient and the scattering phase function, the refractive index of the water was obtained from Schiebener and Straub [20], while the refractive index of the polystyrene was adopted from Nikolov et al. [21]. Similarly to the DRPs, the reflectance from turbid phantoms was recorded over three consecutive days. The initial suspension was diluted three times to obtain reduced scattering coefficients of 30, 20 and 10 cm−1 at 600 nm. In total, 9 reflectance spectra were recorded for each source-detector separation dsds. l Germany) and solid content of 0.05 g/ml. To calculate the scattering coefficient and the scattering phase function, the refractive index of the water was obtained from Schiebener and Straub [20], while the refractive index of the polystyrene was adopted from Nikolov et al. [21]. Similarly to the DRPs, the reflectance from turbid phantoms was recorded over three consecutive days. The initial suspension was diluted three times to obtain reduced scattering coefficients of 30, 20 and 10 cm−1 at 600 nm. In total, 9 reflectance spectra were recorded for each source-detector separation dsds. l 3.2. Probe-to-target distance reflectance profile (DRPs) measurements of the first surface mirrors Reflected light signal Im in Eq. (32) was measured by the optical fiber probe fixed to a two-axis tilting system (shown in Fig. 2(b)) that enabled precise angular positioning of the probe relative to the mounted metallic mirror surface (to approx. 0.1◦degree). As will be shown in the Results and discussion, perpendicular orientation of the probe to the metallic mirror is crucial for accurate fitting of the modeled and measured DRPs. For this task (not shown in Fig. 2), one of the detector fibers was de- coupled from the multichannel spectrometer and coupled to a beam splitter cube with three mounted collimating lenses, allowing it to be used simultaneously as a source and detector. One arm of the beam splitter cube was coupled to a separate broadband light source (SLS201L/M, Thorlabs Inc., New Jersey, USA), while the other arm was coupled to a camera (Basler ace acA1920-40um, Basler AG, Ahrensburg, Germany). In this way, the detector fiber could simultaneously illumi nate the first surface mirror and acquire the reflected light which was then transmitted to the camera. The optical fiber probe was perpen dicularly aligned to the first surface mirror by finding the maximum intensity of the reflected light signal measured by the camera. The reflectance RscatMC ph (dsds, λ) was modeled with an in-laboratory developed and extensively validated OpenCL™ implementation of the MC method for turbid media [22]. It should be noted that our imple mentation allows the use of a custom emittance and acceptance fiber characteristic, which is required to establish correspondence between the deterministic and stochastic models defined in this study. The energy packets are launched uniformly over the source fiber opening following the emittance characteristic and detected only, if they arrive within the detector opening located at a predefined source-detector separation by reducing their weight according to the acceptance characteristic. i For this task (not shown in Fig. 2), one of the detector fibers was de- coupled from the multichannel spectrometer and coupled to a beam splitter cube with three mounted collimating lenses, allowing it to be used simultaneously as a source and detector. One arm of the beam splitter cube was coupled to a separate broadband light source (SLS201L/M, Thorlabs Inc., New Jersey, USA), while the other arm was coupled to a camera (Basler ace acA1920-40um, Basler AG, Ahrensburg, Germany). 3.3. Reflectance measurement and modeling of turbid phantoms For an objective validation of the spectrally resolved calibration factors obtained through DRPs, we acquired reflectance Rmeas ph (dsds, λ) from turbid phantoms. For a particular source-detector separation dsds, Rmeas ph (dsds, λ) can be defined by Eq. (32) with h set to 0 mm, which effectively means that the probe is in contact with the medium and only the scattered light signal is collected. The turbid phantoms were based on an aqueous suspension of polystyrene microsphere with known sphere diameter and number density. This allowed us to calculate the optical properties of turbid phantoms such as the scattering coefficient and the scattering phase function using Mie theory [15]. The calculated values were then used for modeling light propagation and reflectance RscatMC ph (dsds, λ) by multiple-scattering stochastic MC method. L (h0) = ∑ n−1 i=0 ∑ n−1 j=0 ⎛ ⎜ ⎜ ⎝ Rmeas fib ( dsds, i Δh n−1, λ ) RmMC fib ( dsds, h0 + i Δh n−1, λ ) − Rmeas fib ( dsds, j Δh n−1, λ ) RmMC fib ( dsds, h0 + j Δh n−1, λ ) ⎞ ⎟ ⎟ ⎠ 2 , (34) (34) where n denotes the number of the measurement points of Rmeas fib in the selected interval. The L (h0) was defined in a way that circumvents the In this study, we utilized aqueous suspensions of polystyrene mi crospheres with a diameter of 0.721 µm (Microparticles GmbH, Berlin, 7 P. Nagliˇc et al. Measurement 203 (2022) 112002 Measurement 203 (2022) 112002 Fig. 4. Detailed schematic of the calibration method using the acquired DRPs or reflectance from turbid phantoms based on an aqueous suspension of polystyrene microspheres. P. Naglic et al. schematic of the calibration method using the acquired DRPs or reflectance from turbid phantoms based on an aqueous susp bration method using the acquired DRPs or reflectance from turbid phantoms based on an aqueous suspension of polystyrene Fig. 4. Detailed schematic of the calibration method using the acquired DRPs or reflectance from turbid phantoms b microspheres. Fig. 4. Detailed schematic of the calibration method using the acquired DRPs or reflectance from turbid phantoms based on an aqueous suspension of polystyrene microspheres. Fig. 5. Experimental setup for (a) acceptance and (b) emittance characteristic measurement of the source and detector fibers used in the optical fiber probe. 3.5. Emittance and acceptance characteristic measurements of optical fibers y gl Fig. 6(a) shows the DRPs modeled by the deterministic model (Eq. (18)) based on a simplified Lambertian emittance and angularly uniform acceptance characteristics (dashed line), and realistic emittance and acceptance characteristics measured at 600 nm (solid line) for five source-detector separations. The modeled DRPs approach zero at short and long probe-to-mirror distances. On the one hand, short probe-to- mirror distances restrict the light to propagate from the source to the detector fibers when assuming a single reflection from the first surface mirror. On the other hand, long probe-to-mirror distances decrease the apparent area of the detector fibers that collect the reflected light and thus decrease the acquired reflectance. The peaks of the DRPs also decrease and shift to longer probe-to-mirror distances with increasing source-detector separation. The simplified and realistic emittance and acceptance characteristics result in significant differences in magnitude and positions of the peaks in modeled DRPs (marked with vertical lines in Fig. 6 for the realistic case) emphasizing the necessity to consider realistic characteristics for optimal fitting of the measured DRPs. The acceptance characteristic of the detector fibers and the emit tance characteristic of the source fiber were measured sequentially by a goniometric setup shown in Fig. 5. The goniometer comprised an all- silica multimode optical fiber (NA = 0.22, 200 µm core diameter, 2 m length, Avantes, Apeldoorn, The Netherlands) which was terminated with a collimating lens (COL-UV/VIS, 8.7 mm focal length, Avantes) attached to the goniometer arm at an approximate distance of 25 cm from the rotation axis. The optical probe face was positioned and fixed in the center of the rotation axis. The acceptance characteristic measure ments were carried out by coupling the fiber of the goniometer arm to a broadband light source (SLS201L/M, Thorlabs Inc., New Jersey, USA) producing a collimated beam of approximately 5 mm in diameter and divergence of 0.66◦in the center of the goniometer. During these mea surements, the light coupled into the optical fiber probe was blocked. On the other hand, the emittance characteristic measurements were carried out by coupling the fiber from the goniometer arm to a free channel of the custom 10-channel spectrometer and acquiring the light emission from the source fiber of the optical fiber probe. The goniometric mea surements were performed in a dark room. 4. Results and discussion It is essential that the Rmeas ph is obtained using the same reflective standard as Rmeas fib and that the optical fiber probe remains attached to the source and spectrometer. Only in such case are the calibration factors C(dsds, λ) comparable. 4.1. Comparison of the deterministic and stochastic models of probe-to- target distance reflectance profiles (DRPs) The stochastic single-reflection MC model was validated against the deterministic model by comparing the modeled DRPs. The validation of the single-reflection MC model is an important step since the latter is incrementally upgraded to multiple-reflection MC model. 3.3. Reflectance measurement and modeling of turbid phantoms C(dsds, λ) = Rmeas ph (dsds, λ) RscatMC ph (dsds, λ). (36) C(dsds, λ) = Rmeas ph (dsds, λ) RscatMC ph (dsds, λ). (36) 3.3. Reflectance measurement and modeling of turbid phantoms LS: broadband light source, OF: multimode optical fiber, CL: collimating lens, RS: rotation stage, OP: optical fiber probe, SPEC: multichannel spectrometer. Fig. 5. Experimental setup for (a) acceptance and (b) emittance characteristic measurement of the source and detector fibe broadband light source, OF: multimode optical fiber, CL: collimating lens, RS: rotation stage, OP: optical fiber probe, SPEC: ceptance and (b) emittance characteristic measurement of the source and detector fibers used in the optical fiber probe. LS de optical fiber, CL: collimating lens, RS: rotation stage, OP: optical fiber probe, SPEC: multichannel spectrometer. Fig. 5. Experimental setup for (a) acceptance and (b) emittance characteristic measurement of the source and detector fibers used in the optical fiber probe. LS: broadband light source, OF: multimode optical fiber, CL: collimating lens, RS: rotation stage, OP: optical fiber probe, SPEC: multichannel spectrometer. mismatch in magnitude between the Rmeas fib and RmMC fib without requiring an additional optimization parameter. Once h0 is obtained, the Rmeas fib and RmMC fib are aligned and the spectrally resolved calibration factor C(dsds, λ) can be estimated by a ratio between the Rmeas fib and RmMC fib averaged over the n measurement points of Rmeas fib in the selected interval mismatch in magnitude between the Rmeas fib and RmMC fib without requiring an additional optimization parameter. Once h0 is obtained, the Rmeas fib and RmMC fib are aligned and the spectrally resolved calibration factor C(dsds, λ) can be estimated by a ratio between the Rmeas fib and RmMC fib averaged over the n measurement points of Rmeas fib in the selected interval mismatch in magnitude between the Rmeas fib and RmMC fib without requiring an additional optimization parameter. Once h0 is obtained, the Rmeas fib and RmMC fib are aligned and the spectrally resolved calibration factor C(dsds, λ) can be estimated by a ratio between the Rmeas fib and RmMC fib averaged over the n measurement points of Rmeas fib in the selected interval C(dsds, λ) = 1 n ∑ h∈[h0,h0+Δh] Rmeas fib (dsds, h, λ) RmMC fib (dsds, h, λ). (35) (35) For an objective comparison, we derived the same calibration factor C(dsds, λ) as a ratio of the measured reflectance Rmeas ph and modeled reflectance RscatMC ph (shown in Fig. 4) obtained from turbid phantoms based on an aqueous suspension of polystyrene microspheres 8 P. Nagliˇc et al. Measurement 203 (2022) 112002 Fig. 5 for λ = 600 nm. 3.5. Emittance and acceptance characteristic measurements of optical fibers Any remaining stray light was eliminated by recording a dark signal at sufficiently high angles θ ≈ 30 ◦to avoid light signal coupling into the fibers. i Fig. 6(b) shows relative deviations between the DRPs predicted by the deterministic and single-reflection MC models based on a simplified and realistic emittance and acceptance characteristics corresponding to Fig. 6(a). Slightly higher relative deviation (less than 0.25 %) at longer probe-to-mirror distances for the realistic case stems from the differ ences in sampling of the realistic emittance profiles between the deter ministic and single-reflection models. In contrast, relative deviation for the simplified case never exceeds 0.1 % for h greater than 2 mm. For short probe-to-mirror distances, relative deviation rapidly increases since the reflectance approaches zero. Such short probe-to-mirror dis tances are not considered in our analysis of the measured DRPs. ll i The recorded raw emittance and acceptance characteristic signals were centered, symmetrically merged across the center, and corrected for the transmittance losses at the air-fiber boundary. The latter step is necessary because the characteristics are defined within the source and detector fibers in our theoretical models. The refractive index of the fi bers nfib was based on fused silica [19], while the refractive index of air nmed was equal to unity over the utilized spectrum. Furthermore, the acceptance characteristic was divided by cosθ, where θ is the incident angle of the collimated light beam onto the optical fiber probe face, to account for the detector fiber area projection. Finally, the acceptance profile was normalized to the maximum value at θ = 0 ◦. In such case, an ideal acceptance profile would equal to unity with a sharp cut-off at the maximum acceptance angle θ given by the NA. Fig. 7 shows the influence of multiple reflections between the optical fiber probe tip and first surface mirror on the DRPs. The DRPs predicted by a multiple-reflection MC model are significantly higher than DRPs predicted by a single-reflection MC model, especially for probe-to-target distances shorter than the positions of the DRP peaks. The latter can be easily observed in Fig. 7(b) showing relative deviations between the discussed DRPs. 3.5. Emittance and acceptance characteristic measurements of optical fibers Since our analysis of the measured DRPs focuses on the region around the peak of the DRPs for each source-detector separation, Examples of the measured acceptance a ( cosθfib, λ ) and emittance s ( cosθfib, λ ) characteristics used in our theoretical models are given in Fig. 6. (a) DRPs for five source-detector separations (see the legend) predicted by the deterministic model using simplified (dashed) and realistic (solid) emittance and acceptance characteristics with peak positions denoted by vertical lines. (b) Relative deviation between the DRPs predicted by the deterministic and single- reflection MC models for example (a). Fig. 6. (a) DRPs for five source-detector separations (see the legend) predicted by the deterministic model using simplified (dashed) and realistic (solid) emittance and acceptance characteristics with peak positions denoted by vertical lines. (b) Relative deviation between the DRPs predicted by the deterministic and single- reflection MC models for example (a). P. Nagliˇc et al. Measurement 203 (2022) 112002 Fig. 7. (a) DRPs modeled by a single-reflection (solid) and multiple-reflection (dashed) MC models for five source-detector separations (see the legend). (b) Relative deviation between the DRPs modeled by a single-reflection and multiple-reflection MC models. Fig. 7. (a) DRPs modeled by a single-reflection (solid) and multiple-reflection (dashed) MC models for five source-detector separations (see the legend). (b) Relative deviation between the DRPs modeled by a single-reflection and multiple-reflection MC models. multiple reflection must be accounted for to obtain as accurate cali bration factors as possible. i detector separations for the deterministic, single-reflection MC and multiple-reflection MC models based on realistic emittance and accep Table 1 Reflectance values (R) and relative deviations (r) for deterministic model (RdM fib ), single-reflection MC model (RsMC fib ) and multiple-reflection MC model (RmMC fib ) evaluated for each dsds at h corresponding to the peak of the RsMC fib . Relative deviations (rCdm and rCmMC) between the calibration factors based on RdM fib and RmMC fib , and the reference calibration factor based on aqueous suspensions of polystyrene microspheres are also provided. dsds[μm] RdM fib RsMC fib RmMC fib rdM−sMC[%] rdM−mMC[%] rCdm [%] rCmMC [%] 220 33.35 33.35 33.92 −0.008 −1.74 4.86 2.67 440 7.79 7.79 7.92 −0.008 −1.62 6.49 4.82 660 3.89 3.89 3.95 −0.008 −1.54 8.48 6.63 880 2.46 2.46 2.50 0.000 −1.35 8.08 6.63 1100 1.35 1.35 1.37 −0.008 −1.62 10.72 8.81 Fig. 8. 3.5. Emittance and acceptance characteristic measurements of optical fibers Relative deviation of the calibration factors at five source-detector separations (see the legend) obtained by inclining the mirror for ± 1◦around axis (a) along and (b) perpendicular to the line connecting the centers of the fibers (details in text). Examples of modeled DRPs (solid line) with parameters (c) ϕ ′ = 0 ◦and mirror inclination 1◦, and (d) ϕ ′ = 90 ◦and mirror inclination 0.2◦with corresponding fitted DRPs (dashed line). 4.2. Sensitivity of the calibration factors to mirror inclination Through experiments we observed a high sensitivity of the acquired DRPs to the inclination of the first surface mirror relative to the optical fiber probe. To study the sensitivity, we modeled DRPs from inclined mirrors by a multi-reflection MC model and calculated the calibration factors utilizing the procedure described in Sect. 3.4. Since, ideally, the calibration factors between two modeled DRPs should be 1, we calcu lated relative deviation of the fitted calibration factors from 1 for various inclinations of the first surface mirror. i i Fig. 8(a) shows the relative deviation of the calibration factors at five source-detector separations obtained by inclining the mirror for ϕ ′ = 0 ◦ and θ ′ between 179◦and 181◦(see Fig. 1(b) for the definition of the parameters) corresponding to inclination angles of ± 1◦. Fig. 8(b) shows relative deviation of the calibration factor obtained by the same incli nation angle but for ϕ ′ = 90 ◦. For ϕ ′ = 0 ◦the mirror is symmetrically inclined around an axis connecting the centers of the source and detector fibers, which results in symmetric relative deviation of the calibration factors for positive and negative inclinations. In contrast, for ϕ ′ = 90 ◦ the mirror is inclined around an axis perpendicular to the direction of the source and detector fibers. Thus, the pathlength that light propagates from the source to the detector fiber can decrease or increase. Change in the light pathlength can dramatically influence the acquired reflectance and thus the ± 1◦inclination for ϕ ′ = 90 ◦causes a much greater relative deviation of the calibration factors (up to ± 20 %) than for ϕ ′ = 0 ◦(does not exceed 2.5 %). The sensitivity analysis suggests that the mirror inclination must not exceed 0.1◦to retain the accuracy of calibration factors within 2 %. i Spectrally resolved calibration factors for five source-detector sepa rations obtained through the procedure defined in Sect. 3.4 and Eq. (35) are presented in Fig. 10(a). The spectrally resolved calibration factors include a total of 9 measurements of DRPs from three different first surface mirrors performed on three consecutive days. Calibration factors exhibit a clear positive spectral dependence since the acquired DRPs are normalized against the reflective standard Spectralon® as described in Sect. 3.2 and Eq. (32). 4.3. Validation of the calibration factors reflection MC model. Single-reflection model clearly underestimates the DRPs at the peak, which results in significantly higher relative deviation of the calibration factors obtained by the single-reflection model than the multiple-reflection model in comparison to the reference calibration factors based on the aqueous suspensions of polystyrene microspheres (denoted by rCdm and rCmMC). This further showcases the importance for considering multiple-reflection model. Validation of the calibration factors based on the first surface mirrors was performed using the calibration factors based on aqueous suspen sions of polystyrene microspheres with precisely known optical properties. i Two selected examples of fitted DRPs utilizing the multiple- reflection MC model are shown in Fig. 9 for five source-detector sepa rations at a wavelength of 600 nm. The DRPs in Fig. 9(a) and 9(b) correspond to the first surface silver and aluminium mirror targets, respectively. Note that the fitted DRPs overlap nicely with the measured counterparts. However, the relative deviation between the fitted and measured DRPs reveals a progressively higher discrepancies at longer probe-to-mirror distances. This is because DRPs were fitted in the region around the peak. Nevertheless, the relative deviations exceed 2.5 % only for longer probe-to-target distances. It should be noted that as the probe- to-target distance increases the apparent detection angle of the detector fiber decreases and the detector fiber samples only a fraction of the source fiber emittance characteristic. In this case, any assumptions made about the source fiber emittance characteristic and resulting errors can be amplified at longer probe-to-target distances and negatively affect the modeled DRPs. Especially critical is the assumption of the same emit tance characteristic at any exit point of the source fiber area. For shorter probe-to-target distances a higher relative deviation between the fitted and measured DRPs might be explained by the fact that while the stainless-steel tip of the optical fiber probe is assumed as smooth and mirror-like, continuous usage can cause scratches which can scatter the light and at very small probe-to-target distance significantly contribute to the signal at the detector fiber. i Table 1 Table 1 Reflectance values (R) and relative deviations (r) for deterministic model (RdM fib ), single-reflection MC model (RsMC fib ) and multiple-reflection MC model (RmMC fib ) evaluated for each dsds at h corresponding to the peak of the RsMC fib . Relative deviations (rCdm and rCmMC) between the calibration factors based on RdM fib and RmMC fib , and the reference calibration factor based on aqueous suspensions of polystyrene microspheres are also provided. Reflectance values (R) and relative deviations (r) for deterministic model (RdM fib ), single-reflection MC model (RsMC fib ) and multiple-reflection MC model (RmMC fib ) evaluated for each dsds at h corresponding to the peak of the RsMC fib . Relative deviations (rCdm and rCmMC) between the calibration factors based on RdM fib and RmMC fib , and the reference calibration factor based on aqueous suspensions of polystyrene microspheres are also provided. Fig. 8. Relative deviation of the calibration factors at five source-detector separations (see the legend) obtained by inclining the mirror for ± 1◦around axis (a) along and (b) perpendicular to the line connecting the centers of the fibers (details in text). Examples of modeled DRPs (solid line) with parameters (c) ϕ ′ = 0 ◦and mirror inclination 1◦, and (d) ϕ ′ = 90 ◦and mirror inclination 0.2◦with corresponding fitted DRPs (dashed line). multiple reflection must be accounted for to obtain as accurate cali bration factors as possible. i detector separations for the deterministic, single-reflection MC and multiple-reflection MC models based on realistic emittance and accep tance characteristic evaluated at the peak position of the single- p Table 1 shows the DRP values and relative deviations for five source- 10 P. Nagliˇc et al. Measurement 203 (2022) 112002 4.3. Validation of the calibration factors 4.2. Sensitivity of the calibration factors to mirror inclination (a) Mean (dashed) and range (colored region) of spectrally resolved calibration factors based on DRPs of the first surface mirrors obtained for five source- detector separations (see the legend) acquired across 9 independent measurements (details in the text). (b) Relative repeatability of the spectrally resolved calibration factors calculated as a ratio between the standard deviation and mean of the measurements. Fig. 11. (a) Mean (dashed) and range (colored region) of spectrally resolved calibration factors based on aqueous suspensions of polystyrene microspheres obtained for five source-detector separations (see the legend) acquired across 9 independent measurements (details in the text). (b) Relative repeatability of the spectrally resolved calibration factors calculated as a ratio between the standard deviation and mean of the measurements. Fig. 11. (a) Mean (dashed) and range (colored region) of spectrally resolved calibration factors based on aqueous suspensions of polystyrene microspheres obtained for five source-detector separations (see the legend) acquired across 9 independent measurements (details in the text). (b) Relative repeatability of the spectrally resolved calibration factors calculated as a ratio between the standard deviation and mean of the measurements. separation obtained by Eq. (36) through acquisition of reflectance from turbid phantoms based on aqueous suspensions of polystyrene micro spheres are presented in Fig. 11(a). Note that the mean, minimum and maximum regions shown in Fig. 11 are calculated across 9 calibration factors from turbid phantoms with three different number densities of microspheres prepared independently each day over three consecutive days. The relative repeatability, presented in Fig. 11(b), is notably better than in our proposed method. Excellent repeatability stems from the fact that aqueous turbid phantoms can be made highly homogeneous, and optical probe is usually in good contact with the surrounding medium. Fig. 12. Relative deviation of the calibration factors for five source-detector separations (see the legend) obtained through the DRPs of the first surface mirrors and reflectance from turbid phantoms based on aqueous suspension of polystyrene microspheres. Finally, relative comparison of the calibration factors based on the DRPs from the first surface mirrors and reflectance from turbid phan toms is presented in Fig. 12. Remarkably, the calibration factors differ by no more than 10 %. 4.2. Sensitivity of the calibration factors to mirror inclination Spectralon® can introduce a spectral dependence due to the contribution of subsurface scattering which is governed by the scattering coefficient of the reflective standard. Such spectral depen dence of the calibration factor cannot be accounted for by a simple constant multiplicative factor that can be determined simultaneously to extraction of optical properties as suggested by Jacques [11]. Further more, simultaneous extraction of the calibration factor and optical properties might lead to unwanted crosstalk. Fig. 8(c) and 8(d) show two selected examples of modeled DRPs (solid line) with parameters ϕ ′ = 0 ◦and mirror inclination 1◦, and ϕ ′ = 90 ◦and mirror inclination 0.2◦, and the fitted DRPs (dashed line) used to calculate the calibration factors. While the fitted DRPs seem to match, corresponding relative deviations shown in the same graph suggest that even an inclination of 0.2◦significantly distorts the DRPs so that the fitted DRPs at longer probe-to-mirror distances deviate by approxi mately 5 %. This result further underlines the necessity to carefully align and position the probe during the acquisition of the DRPs. The relative repeatability given as a ratio between the standard de viation and the mean of the measured calibration factor at each wave length and source-detector separation is provided in Fig. 10(b). The relative repeatability of the calibration factors is equal or less than 2 % Fig. 9. Examples of measured (solid) and fitted (dashed) DRPs by utilizing the multiple-reflection MC model for five source-detector separations (see the legend) at a wavelength of 600 nm. (a) DRPs corresponding to the first surface silver mirror target and (b) aluminium mirror target. Fig. 9. Examples of measured (solid) and fitted (dashed) DRPs by utilizing the multiple-reflection MC model for five source-detector separations (see the legend) at a wavelength of 600 nm. (a) DRPs corresponding to the first surface silver mirror target and (b) aluminium mirror target. 11 P. Nagliˇc et al. Measurement 203 (2022) 112002 Measurement 203 (2022) 112002 Fig. 10. (a) Mean (dashed) and range (colored region) of spectrally resolved calibration factors based on DRPs of the first surface mirrors obtained for five source- detector separations (see the legend) acquired across 9 independent measurements (details in the text). (b) Relative repeatability of the spectrally resolved calibration factors calculated as a ratio between the standard deviation and mean of the measurements. P. Nagliˇc et al. aglic et al. Fig. 10. 4.2. Sensitivity of the calibration factors to mirror inclination From this perspective, at larger source-detector separations the detector fiber samples a smaller fraction of the source fiber emittance charac teristic at the peak of the DRP. To investigate this point, further research is required where the full emittance characteristic of the multimode source fiber would have to be acquired including the angular depen dence at each exit point of the source fiber area. Finally, an important and often overlooked assumption when using the Mie theory for aqueous suspensions of polystyrene microspheres is that the calculation of the scattering coefficient and the scattering phase function is based on the scattering of a plane electromagnetic wave on a single microsphere. In case the distance between the microspheres is small, the light scattering by a group of microspheres cannot be generalized from scattering on a single microsphere. While testing this hypothesis falls out of the scope of this study, the reflectance from turbid phantoms would have to be calculated using solutions to the scattered unpolarized light from an ensemble of spheres such as T-matrix theory [24]. As a counterargu ment, it should also be noted that the spectral calibration factor for all source-detector separations nicely overlapped across all the number densities of the spheres. The issue with the applicability of the Mie theory would likely appear as a divergence of the spectral calibration factors that depends on the number density of the polystyrene micro spheres. The latter was not observed. which has not yet been measured accurately. Other proposed turbid phantoms are based on measurement of optical properties with alter native optical modalities which can often introduce significant inac curacies. Our proposed calibration method is therefore especially promising for wavelengths in the near and short wavelength infrared region, where turbid phantoms with accurately known optical proper ties are missing. Future work on the proposed calibration procedure should include investigation of whether the inclination angles can be included in the fitting procedure thus also enabling estimation of the optical fiber probe tilt. For this purpose, an array of detector fibers would likely have to be utilized and the measured DRPs would have to be fitted simultaneously to estimate the inclination angles. To make the measurements and alignment of the optical fiber probe relative to the first surface mirror more straightforward, a special holder could be produced. 4.2. Sensitivity of the calibration factors to mirror inclination It should be noted that the calibration factors have been obtained by two completely different approaches, one with light propagation modeling through air between the optical fiber probe and first surface mirrors, while the other utilized the MC method for light propagation in turbid media which is governed by scattering. A 10 % difference is a promising indication of equivalence between the two approaches described in this study. Nevertheless, a trend can be observed where for short source-detector separations the relative devi ation is less than 2 %, while for the longest source-detector separation the relative deviation settles around 9 %. Fig. 12. Relative deviation of the calibration factors for five source-detector separations (see the legend) obtained through the DRPs of the first surface mirrors and reflectance from turbid phantoms based on aqueous suspension of polystyrene microspheres. for all but the 660 μm source-detector separation. The latter is an outlier since it was utilized to precisely align the optical fiber probe perpen dicularly to the first surface mirror and had to be coupled and decoupled from the spectrometer frequently. To alleviate this shortcoming and use our proposed calibration method without requiring an additional alignment optical fiber, a mechanical alignment of the probe to the mirror surface should be established. i for all but the 660 μm source-detector separation. The latter is an outlier since it was utilized to precisely align the optical fiber probe perpen dicularly to the first surface mirror and had to be coupled and decoupled from the spectrometer frequently. To alleviate this shortcoming and use our proposed calibration method without requiring an additional alignment optical fiber, a mechanical alignment of the probe to the mirror surface should be established. i The observed discrepancy can be attributed to the following reasons. Firstly, the perpendicular alignment of the probe relative to the mirror might not be within the targeted 0.1◦. However, perpendicular Spectrally resolved calibration factors for five source-detector 12 P. Nagliˇc et al. Measurement 203 (2022) 112002 alignment of the probe would also affect the shortest source-detector separation, which is not observed in Fig. 12. Secondly, assumptions about the emittance characteristic can differently affect detectors at different source-detector separations. For example, the larger the source-detector separation, the higher the probe-to-target distance at which the light is allowed to propagate from the source to detector fiber. CRediT authorship contribution statement Peter Nagliˇc: Conceptualization, Methodology, Software, Valida tion, Formal analysis, Investigation, Writing – original draft, Visualiza tion. Franjo Pernuˇs: Supervision, Project administration, Funding acquisition. Miran Bürmen: Conceptualization, Methodology, Soft ware, Writing – review & editing. 4.2. Sensitivity of the calibration factors to mirror inclination The holder would have to allow translation of the probe, since capturing the region around the peak of the DRPs alleviates the requirement to know the absolute probe-to-target distance. Finally, our procedure could poten tially be useful for measuring the probe-to-target distances and incli nation angles, thus providing a foundation for a precise optical fiber sensor. Data availability Data will be made available on request. Data will be made available on request. Declaration of Competing Interest We proposed a new calibration method based on fitting measured DRP profiles from first surface mirror using a multi-reflection MC model. The multi-reflection MC model was indirectly validated by comparing the deterministic and single-reflection MC models resulting in an excellent agreement. We found that the calibration factors were highly sensitive to the inclination of the optical fiber probe relative to the first surface mirror and thus the perpendicular alignment must be well within 0.1◦for accurate determination of the calibration factors. The proposed method yields calibration factors with 2 % repeatability, which is slightly less than what can be achieved with the standard method involving aqueous turbid phantoms based on polystyrene microspheres. Furthermore, comparison of spectrally resolved calibration factors based on the two methods reveals less than a 10 % discrepancy. We believe such an agreement is remarkable, since the two methods are based on completely different light propagation models. The proposed method is based on a ray-tracing MC model of light propagation through a non-scattering medium, while the standard method involves light propagation modeling by an MC model for scattering media. The values of the calibration factors obtained by the proposed and standard method agreed particularly well for the shortest source-detector separation with relative deviations below 2 % for the most part of the spectrum. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors acknowledge the financial support from the Slovenian Research Agency for research core funding No. P2-0232 and projects No. BI-LV/20-22-007, J2-1732, J2-2502, J2-2500 and L2-4455. Furthermore, comparison of spectrally resolved calibration factors based on the two methods reveals less than a 10 % discrepancy. We believe such an agreement is remarkable, since the two methods are based on completely different light propagation models. The proposed method is based on a ray-tracing MC model of light propagation through a non-scattering medium, while the standard method involves light propagation modeling by an MC model for scattering media. The values of the calibration factors obtained by the proposed and standard method agreed particularly well for the shortest source-detector separation with relative deviations below 2 % for the most part of the spectrum. References [1] C. Perron, C. Katlein, S. Lambert-Girard, E. Leymarie, L.-P. Guinard, P. Marquet, M. 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https://openalex.org/W2145198235	https://europepmc.org/articles/pmc3966718?pdf=render	English	null	Accommodating Measurements Below a Limit of Detection: A Novel Application of Cox Regression	American journal of epidemiology	2,014	public-domain	7,058	Accommodating Measurements Below a Limit of Detection: A Novel Application of Cox Regression Gregg E. Dinse, Todd A. Jusko, Lindsey A. Ho, Kaushik Annam, Barry I. Graubard, Irva Hertz-Picciotto, Frederick W. Miller, Brenda W. Gillespie, and Clarice R. Weinberg* * Correspondence to Dr. Clarice R. Weinberg, Mail Drop A3-03, P.O. Box 12233, Research Triangle Park, NC 27709 (e-mail: weinber2@niehs.nih.gov). Initially submitted April 28, 2013; accepted for publication January 15, 2014. Gregg E. Dinse, Todd A. Jusko, Lindsey A. Ho, Kaushik Annam, Barry I. Graubard, Irva Hertz-Picciotto, Frederick W. Miller, Brenda W. Gillespie, and Clarice R. Weinberg* * Correspondence to Dr. Clarice R. Weinberg, Mail Drop A3-03, P.O. Box 12233, Research Triangle Park, NC 27709 (e-mail: weinber2@niehs.nih.gov). Initially submitted April 28, 2013; accepted for publication January 15, 2014. regg E. Dinse, Todd A. Jusko, Lindsey A. Ho, Kaushik Annam, Barry I. Graubard, va Hertz-Picciotto, Frederick W. Miller, Brenda W. Gillespie, and Clarice R. Weinberg Initially submitted April 28, 2013; accepted for publication January 15, 2014. In environmental epidemiology, measurements of exposure biomarkers often fall below the assay’s limit of detection. Existing methods for handling this problem, including deletion, substitution, parametric regression, and multiple imputation, can perform poorly if the proportion of “nondetects” is high or parametric models are mis- specified. We propose an approach that treats the measured analyte as the modeled outcome, implying a role reversal when the analyte is a putative cause of a health outcome. Following a scale reversal as well, our approach uses Cox regression to model the analyte, with confounder adjustment. The method makes full use of quantifiable analyte measures, while appropriately treating nondetects as censored. Under the proportional hazards assump- tion, the hazard ratio for a binary health outcome is interpretable as an adjusted odds ratio: the odds for the outcome at any particular analyte concentration divided by the odds given a lower concentration. Our approach is broadly applicable to cohort studies, case-control studies (frequency matched or not), and cross-sectional studies con- ducted to identify determinants of exposure. We illustrate the method with cross-sectional survey data to assess sex as a determinant of 2,3,7,8-tetrachlorodibenzo-p-dioxin concentration and with prospective cohort data to as- sess the association between 2,4,40-trichlorobiphenyl exposure and psychomotor development. 2,3,7,8-tetrachlorodibenzo-p-dioxin; 2,4,40-trichlorobiphenyl; hazard identification; limit of detection; National Health and Nutrition Examination Survey; nondetects; proportional hazards Abbreviations: LOD, limit of detection; NHANES, National Health and Nutrition Examination Survey; PCB, polychlorinated biphenyl; PCB-28, 2,4,40-trichlorobiphenyl; PDI, psychomotor development index; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Vol. 179, No. 8 DOI: 10.1093/aje/kwu017 Advance Access publication: March 4, 2014 Vol. 179, No. 8 DOI: 10.1093/aje/kwu017 Advance Access publication: March 4, 2014 American Journal of Epidemiology American Journal of Epidemiology Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US. Practice of Epidemiology Vol. 179, No. 8 DOI: 10.1093/aje/kwu017 Advance Access publication: March 4, 2014 Practice of Epidemiology Accommodating Measurements Below a Limit of Detection: A Novel Application of Cox Regression Accommodating Measurements Below a Limit of Detection: A Novel Application of Cox Regression The increasing availability of informative exposure bio- markers presents both opportunities and challenges for envi- ronmental epidemiologists as we try to identify determinants of exposure and assess the health effects of environmental pollutants. Many exposure biomarkers have low concentra- tions in serum, urine, or other biological matrices, with mea- surements often falling below the assay’s limit of detection (LOD), the lowest level at which a substance’s presence is distinguishable from its absence (1). Here we propose a new approach for managing these “nondetects,” verify its ap- propriate conﬁdence interval coverage under the null through simulations, and illustrate its use through application to 2 data examples. method is inefﬁcient (2) and prone to bias; in our exam- ple involving 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the National Health and Nutrition Examination Survey (NHANES), the deletion approach would discard 82% of the participants. Replacing nondetects with a speciﬁc value such as LOD / 2 or LOD / ﬃﬃﬃ 2 p can produce bias and overly narrow conﬁdence intervals, as can model misspeciﬁcation in parametric substitution schemes based on conditional ex- pectations; distribution-free substitution of the average de- tectable value can perform poorly as well (2). Multiple imputation methods can also produce bias and overly narrow conﬁdence intervals when the assumed parametric model is misspeciﬁed, especially in applications with many measure- ments below the LOD (3). In view of these difﬁculties, we sought a confounder-adjusted approach that would avoid Existing methods for handling nondetects include discarding them, replacing them, or multiply imputing them. The deletion 1018 Am J Epidemiol. 2014;179(8):1018–1024 Accommodating Detection Limits via Cox Regression 1019 restrictive parametric assumptions while accommodating a high proportion of nondetects. with some remarks about the strengths and limitations of the proposed method. g p p Our method treats the biomarker as the outcome, which sometimes implies switching the roles of exposure and out- come. We use Cox regression (4) to model analyte concentra- tion as a function of the health measure, while adjusting for confounders. Without assuming a parametric concentration distribution, Cox regression easily accommodates nondetects by appropriately treating them as censored. We reverse the analyte’s scale to transform the provided left-censored obser- vations into more easily handled right-censored observations. Although Cox’s proportional hazards model was developed for censored event-time data, it can be applied to any variable that is subject to (potentially heavy) censoring. Background Survival methods are standard statistical tools typically used for analyzing censored positive-valued data such as event times. Most survival methods were originally devel- oped to handle noninformatively right-censored outcomes, known only to exceed a given limit. For example, the death time for a patient in a prospective cohort study is right cen- sored if the patient withdraws from the study or is still alive when the study concludes; in either situation, we simply know that the time to death exceeds the observed time on study. Role reversal of an exposure and its effect raises questions about the use of directed acyclic graphs (5) to identify possi- ble confounders for inclusion as covariates. However, the roles of the analyte and the health measure are typically sym- metrical (i.e., confounder selection is invariant to ﬂipping the direction of that causal arrow), so that the same confounders will usually be appropriate to include as covariates. (The ap- proach we propose would not be useful, however, for infer- ence involving nonconfounder intermediates.) Accommodating Measurements Below a Limit of Detection: A Novel Application of Cox Regression In addition to adjusting for confounders, the Cox model does not require event times (or analyte concentrations in this setting) to fol- low any particular parametric distribution. Reversing the outcome scale to analyze left-censored data Outcomes may also be left censored, known only to be less than some lower limit. Such data can be analyzed with meth- ods designed for right-censored data by reversing the out- come scale (9). Speciﬁcally, and without loss of generality, one can choose a constant, say M, that equals (or exceeds) the largest observation, subtract all uncensored and left- censored outcomes from M, and treat those differences as un- censored and right-censored outcomes, respectively. Other than requiring that it equal or exceed all measured (or cen- sored) values, the choice of M is arbitrary but will have no practical consequence on nonparametric or semiparametric inferences. This maneuver converts values that are left cen- sored by the assay LOD to values that are right censored at M −LOD. Note that LODs can vary across participants, as occurs in our examples. This work was originally motivated by a study of xenobi- otic exposures measured in NHANES from 1999 to 2004 (6). One chemical measured was TCDD, and 82% of its concen- trations were below the LOD. Using the same NHANES data to assess associations between polychlorinated biphenyls (PCBs) and antinuclear antibodies, Gallagher et al. (7) re- stricted their logistic analyses to the last 2 years (2003– 2004),excludingtheﬁrst4years(1999–2002)becausetheless sensitive assay used earlier had generated a high proportion of nondetects. Our second example involves data on infant development in relation to in utero 2,4,40-trichlorobiphenyl (PCB-28) exposure, where a previous analysis omitted sev- eral measured chemicals because too many values were below the LOD (8). We wanted a method that could use all of the available data. Furthermore, we wanted a method that could be applied in a wide range of settings, including case-control studies (with or without frequency matching), randomized controlled trials, and cross-sectional studies, and for which the health outcome could be dichotomous, multilevel, or continuous. Following scale reversal, existing nonparametric methods can estimate the outcome distribution (or at least its tail) for a single group (9) or formally compare distributions for 2 or more groups (10). Here, we extend these ideas to use Cox regression (4) to adjust for confounders when assessing the association between a health measure and a quantitative ex- posure that is subject to substantial censoring. Cox regression analysis of left-censored data For a given individual, let T be the true concentration of an analyte, let Y be a health measure, and let Z be a vector of co- variates. Deﬁne a censoring indicator, Δ = I(T > LOD), and a concentration variable, C = max(T, LOD), where Δ is 1 if C equals the true concentration (i.e., T is large enough to be measured), and Δ is 0 if C equals the LOD (i.e., T is non- detectable). Suppose we have a sample of N observations, denoted {(ci, δi, yi, zi): i = 1, . . . , N}, where (ci, δi, yi, zi) are the observed values of (C, Δ, Y, Z) for the ith individual (i = 1, . . . , N). Choose M ≥max(c1, . . . , cN) to be a ﬁxed constant that equals or exceeds the largest true concentration (and the largest LOD), and deﬁne xi = M −ci (i = 1, . . . , N) to be the observed value of some hypothetical “reverse-scale” concentration represented by X = M −C. We begin by describing the method and showing how the Cox-based hazard ratio can be interpreted as an odds ratio. We present results of simulations to assess conﬁdence inter- val coverage under the correct model and a null hypothesis of no association, comparing coverage percentages for our method with those for logistic regression and linear regres- sion, based on both single impute substitution and multiple imputation. We then illustrate the reverse-scale Cox method by applying it to cross-sectional NHANES survey data to as- sess sex as a determinant of TCDD concentration in the US population. We also apply it to prospective cohort data exam- ining the association between in utero PCB-28 exposure and psychomotor development, where predictor and outcome roles are reversed to accommodate nondetects. We conclude Am J Epidemiol. 2014;179(8):1018–1024 1020 Dinse et al. quantitative confounder Z, and a concentration T subject to a high proportion of nondetects. quantitative confounder Z, and a concentration T subject to a high proportion of nondetects. Because most event-time software is designed for right- censored data, one can reverse the scale of left-censored data and apply standard software to the transformed data. Thus, one can use existing Cox regression software to analyze the data {(xi, δi, yi, zi): i = 1, . . . Design of simulations In addition to our earlier notation, let R denote a reverse-scale version of T. There are n1 cases (Y = 1), n0 con- trols (Y = 0), and we set N = n0 + n1. We generated ln(Z) as normally distributed with mean μ1 for cases (Y = 1), mean μ0 for controls (Y = 0), and variance σ2 for both. Simulating concentrations that have a proportional hazards relationship with the confounder and case/control status on the reverse concentration scale is tricky (see the Appendix for details). In general, the concentration distribution involves a hazard ratio and a baseline (i.e., covariate-free) distribution, where the former depends on Z and Y but not R, and the latter de- pends on R but not Z or Y. In our simulated data, the log haz- ard ratio for R was a linear function of Y and Z. We used a standard Weibull model, with scale parameter α and shape parameter γ, for the baseline distribution of R. Cox regression assumes that hazards are proportional to a baseline hazard; its parameters are log hazard ratios. A posi- tive coefﬁcient for Y implies that M −T tends to be smaller for larger Y, and thus T tends to be larger. Let F(t) = Pr(T ≤t) be the cumulative distribution function for true concentration T, where t is a particular value of T, and let f(t) be its density function. In contrast to the hazard function for T, given by f(t) / [1 −F(t)], the hazard function for reverse-scale concentration, M −T, when rewritten in terms of T, is f(t) / F(t). We wanted to assess performance under a null model of no association between exposure T (or equivalently R) and case/ control status Y, so we set the coefﬁcient of Y to 0. Without loss of generality, we set μ0 = 0 and regulated the dependence of Y on Z through μ1, and we set α = ln(2) to scale the baseline concentration distribution. We set β = ln(2) to obtain a hazard ratio of 2 for the effect of a 1-unit change in Z on R. We report results for lightly (μ1 = 1, σ = 0.25) and heavily (μ1 = 0.5, σ = 1) skewed confounder distributions, the former being nearly bell shaped, and for lightly (γ = 5) and heavily (γ = 1) skewed baseline distributions. Cox regression analysis of left-censored data , N}, treating each xi as an outcome subject to right censoring, δi as a censor- ing indicator, and yi and zi as covariates (i = 1, . . . , N). Be- cause 1 of our examples involves NHANES data obtained from a multistage stratiﬁed cluster sample, we used the SURVEYPHREG procedure in SAS, version 9.3, software (SAS Institute, Inc., Cary, North Carolina), which incorpo- rates information on sampling strata, clusters, and weights to provide appropriate standard errors when analyzing com- plex sample survey data. For our other example, we used the PHREG procedure in SAS, which is appropriate for nonsur- vey Cox analyses. f1ðtÞ=F1ðtÞ f0ðtÞ=F0ðtÞ g g p p We simulated 10,000 data sets for each combination of confounder distribution, baseline concentration distribution, and case/control sample sizes, and we calculated the percent- age of those simulated studies for which the 95% conﬁdence interval covered the true null value. For comparison, we also calculated coverages for 2 substitution methods: 1 based on logistic regression, with Y as the outcome and T and Z as covariates, and 1 based on linear regression, with T as the out- come and Y and Z as covariates. In both analyses, each non- detectable value of T was replaced by LOD / ﬃﬃﬃ 2 p (11). We also calculated coverages for 2 multiple imputation methods based on the same logistic and linear models. Using the meth- ods of Lubin et al. (12), we bootstrapped to estimate param- eters under a lognormal model for T, and we generated 10 “ﬁll-in” data sets (with imputed values for nondetects) for each of the 10,000 simulated data sets. ¼ limε!0 Prðt T < t þ ε j Y ¼ 1Þ=PrðT t j Y ¼ 1Þ limε!0Prðt T < t þ ε j Y ¼ 0Þ=PrðT t j Y ¼ 0Þ ¼ PrðY ¼ 1 j T ¼ tÞ=PrðY ¼ 0 j T ¼ tÞ PrðY ¼ 1 j T tÞ=PrðY ¼ 0 j T tÞ ; ð1Þ ð1Þ which is the odds of the health outcome at concentration t divided by the odds of the health outcome for the aggregate of concentrations at or below t. Thus, the usual proportional hazards assumption made in Cox regression corresponds to an assumption that this odds ratio is the same across all values of t (i.e., all concentrations). If Y is continuous, an analogous argument applies, leading to an odds ratio for a 1-unit change in Y. Design of simulations We studied the following 5 pairs of sam- ple sizes: (n0, n1) = (100, 100), (100, 400), (400, 100), (400, 400), and (700, 700); and we imposed 3 ﬁxed LODs to achieve average censoring proportions of 50%, 70%, and 90%. If Y is binary, the hazard ratio parameter is interpretable as an odds ratio. When comparing people who are positive for the binary health measure (Y = 1) with those who are negative (Y = 0), the hazard ratio is [f1(t) / F1(t)] / [f0(t) / F0(t)], where fy(t) and Fy(t) are the respective density and distribution func- tions for persons with health measure Y = y (for y = 0,1). Re- versing the conditional probabilities, this hazard ratio can be rewritten as follows: f1ðtÞ=F1ðtÞ f0ðtÞ=F0ðtÞ ¼ limε!0 Prðt T < t þ ε j Y ¼ 1Þ=PrðT t j Y ¼ 1Þ limε!0Prðt T < t þ ε j Y ¼ 0Þ=PrðT t j Y ¼ 0Þ ¼ PrðY ¼ 1 j T ¼ tÞ=PrðY ¼ 0 j T ¼ tÞ PrðY ¼ 1 j T tÞ=PrðY ¼ 0 j T tÞ ; ð1Þ SIMULATIONS Table 1 reports conﬁdence interval coverages for these 5 methods in each of the 20 null scenarios with a censoring proportion of 70%. The Cox method maintained the nominal 95% coverage in all scenarios. Linear regression often per- formed poorly, with coverage as low as 44% when using sub- stitution for a lightly skewed (i.e., nearly symmetrical) covariate distribution and 56% when using multiple imputation To assess the validity of our approach, we performed sim- ulations to verify coverage for nominal 95% conﬁdence inter- vals under a correctly speciﬁed null model. In addition to the Cox method, we evaluated several other methods across a range of scenarios. We simulated data with observations on a binary health outcome Y (e.g., case/control status), a Am J Epidemiol. 2014;179(8):1018–1024 Accommodating Detection Limits via Cox Regression 1021 1021 Table 1. Null Coverage Percentages for 95% Confidence Intervals by Shape of Covariate Distribution, Shape of Baseline Concentration Distribution, and Sample Sizes (With 70% Censoring) Level of Skewness Sample Size Coverage Percentage for Regression Methodsa Covariate Distribution Concentration Distribution n0 n1 Reverse Scale Cox Linear with Substitution Logistic with Substitution Linear with MI Logistic with MI Light Light 100 100 95 86 97 95 100 100 400 95 70 96 95 100 400 100 95 89 96 96 99 400 400 95 64 95 95 99 700 700 95 44 95 94 99 Light Heavy 100 100 95 90 97 95 100 100 400 95 80 96 94 100 400 100 95 91 96 96 99 400 400 95 75 95 95 99 700 700 95 61 95 95 99 Heavy Light 100 100 95 92 94 92 95 100 400 95 91 94 88 93 400 100 95 91 91 92 92 400 400 95 84 93 79 90 700 700 95 75 93 67 85 Heavy Heavy 100 100 95 92 94 91 94 100 400 95 87 94 84 92 400 100 95 90 90 90 90 400 400 95 77 91 73 86 700 700 95 63 88 56 78 Abbreviations: LOD, limit of detection; MI, multiple imputation. a Coverage percentages are based on 10,000 replicate data sets. SIMULATIONS The 5 methods are as follows: reverse-scale Cox regression, linear regression with substitution of LOD / ﬃﬃﬃ 2 p for nondetects, logistic regression with substitution of LOD / ﬃﬃﬃ 2 p for nondetects, linear regression with MI for nondetects, and logistic regression with MI for nondetects. See the Simulations section and the Appendix for details. ble 1. Null Coverage Percentages for 95% Confidence Intervals by Shape of Covariate Distribution, Shape of seline Concentration Distribution, and Sample Sizes (With 70% Censoring) Coverage Percentage for Regression Methodsa Abbreviations: LOD, limit of detection; MI, multiple imputation. a Coverage percentages are based on 10,000 replicate data sets. The 5 methods are as follows: reverse-scale Cox regression, linear regression with substitution of LOD / ﬃﬃﬃ 2 p for nondetects, logistic regression with substitution of LOD / ﬃﬃﬃ 2 p for nondetects, linear regression with MI for nondetects, and logistic regression with MI for nondetects. See the Simulations section and the Appendix for details. used TCDD concentrations expressed on a per-lipid basis (pg/g lipid), as supplied through NHANES. These data are anonymous and publicly available, and the National Insti- tutes of Health Human Research Protection Program deemed this study exempt from further ethical or institutional review board review. for a heavily skewed covariate distribution. Logistic regres- sion fared better than linear regression but not as well as Cox regression, with coverages ranging from 88% to 97% when using substitution and from 78% to 100% when using multiple imputation. In some scenarios, for both linear regression and logistic regression, multiple imputation was worse than single impute substitution. Only the Cox method maintained the nominal 95% coverage in all 20 scenarios with 50% censoring (Web Table 1 available at http://aje. oxfordjournals.org/), and its coverages ranged from 94% to 96% when the censoring proportion rose to 90% (Web Table 2). The original data set involved 7,433 participants who were probability sampled with oversampling of certain age, race, ethnicity, and income categories (15). Of these, the Centers for Disease Control and Prevention assessed TCDD for 5,002. We excluded pregnant women and any participant without information on sex or age, further reducing our sam- ple size to 4,756. Within this subsample, 82% of the TCDD concentrations were below the LOD. The medians were 3.5 (range, 0.8–42.7) for the measured TCDD concentrations and 3.1 (range, 0.4–11.9) for the LODs. Abbreviations: LOD, limit of detection; MI, multiple imputation. a Coverage percentages are based on 10,000 replicate data sets. The 5 methods are as follows: reverse-scale Cox regression, linear regression with substitution of LOD / ﬃﬃﬃ 2 p for nondetects, logistic regression with substitution of LOD / ﬃﬃﬃ 2 p for nondetects, linear regression with MI for nondetects, and logistic regression with MI for nondetects. See the Simulations section and the Appendix for details. Example 1: sex and TCDD exposure (8) for details about this study, which was approved by the institutional review boards at the Slovak Medial Uni- versity (Bratislava, Slovakia) and the University of California at Davis (Davis, California). Of the 15 PCB congeners evaluated in maternal sera, Park et al. (8) included 6 in their linear regressions because those 6 “had most values above their LODs” and excluded the other 9 because of substantial censoring. Using 1 of the originally omitted congeners, PCB-28, we illustrate our Cox approach by assessing the association between maternal PCB-28 con- centration and infant PDI score, adjusting for confounding. Data on PDI scores and the 4 potential confounders were available for 666 of the original 1,134 mother-infant pairs in the birth cohort, and among these, 59% of the maternal PCB-28 concentrations were below the LOD. Figure 1. Differences in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) concentration by sex, National Health and Nutrition Examination Sur- vey, 1999–2004. Separate reverse Kaplan-Meier curves (9, 19) esti- mate the TCDD concentration distribution for men and women. At each (x,y) point along a curve, the y-value is the estimated sex-specific proportion of TCDD measurements below the TCDD concentration (in pg/g lipid) represented by the x-value. TCDD concentrations below the limit of detection were treated as left censored when constructing these curves, which are not adjusted for age or the survey design. For comparison, we also performed 2 linear regressions, where nondetects either were replaced by LOD / ﬃﬃﬃ 2 p or were multiply imputed with bootstrapping and 10 “ﬁll-in” data sets, as described by Lubin et al. (12). Both linear re- gression analyses used PDI score as the outcome, with natural-log-transformed PCB-28 concentration as the expo- sure of interest and with the same 4 potential confounders. The Cox analysis strongly suggests that lower maternal PCB-28 concentrations are associated with higher infant PDI scores (P = 0.01), whereas the substitution (P = 0.13) and multiple imputation (P = 0.12) analyses provide only weak evidence for such an association. Infant PDI scores ranged from 51 to 148, and the estimated Cox hazard ratio was 0.88 (95% conﬁdence interval: 0.79, 0.97) per 10-unit change. Thus, for 2 infants whose PDI scores differed by 10 points, the odds ratio was 0.88 for the mother of the higher-scoring child having a given PCB-28 concentration versus all lower concentrations. Example 1: sex and TCDD exposure g We applied the proposed Cox method to these data, treating lipid-adjusted TCDD concentration as the (heavily censored) outcome and incorporating sex and age as covariates. For com- parison, we also performed linear regression, with nondetects replaced by LOD / ﬃﬃﬃ 2 p . Both analyses included a binary indi- cator for sex (1 = male, 0 = female); a simple quantitative term for age; and stratum, cluster, and weight variables to adjust for the clustered probability sampling (using SAS procedures SURVEYPHREG and SURVEYREG). We also performed analyses that instead used a categorical variable or a spline for age, and these analyses gave results very similar to our orig- inal analysis. We did not perform multiple imputation because of the lack of software able to impute “ﬁll-in” data sets within the context of a complex survey sample. Identifying determinants of environmental exposures is an important public health problem. Understanding what pre- dicts exposure can lead to strategies to reduce exposure, and for causal models, provide causal background for select- ing potential confounding variables. As 1 illustration of our method, we investigated sex as a possible determinant of TCDD exposure, using cross-sectional NHANES survey datafrom 1999 to 2004. Serum TCDD concentrations were de- termined by the Centers for Disease Control and Prevention, using high-resolution gas chromatography/isotope-dilution high-resolution mass spectrometry (13). Serum lipid concen- trations were also determined, and total lipid concentration was estimated by the Akins summation method (14). We Am J Epidemiol. 2014;179(8):1018–1024 1022 Dinse et al. Figure 1. Differences in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) concentration by sex, National Health and Nutrition Examination Sur- vey, 1999–2004. Separate reverse Kaplan-Meier curves (9, 19) esti- mate the TCDD concentration distribution for men and women. At each (x,y) point along a curve, the y-value is the estimated sex-specific proportion of TCDD measurements below the TCDD concentration (in pg/g lipid) represented by the x-value. TCDD concentrations below the limit of detection were treated as left censored when constructing these curves, which are not adjusted for age or the survey design. maternal PCB concentrations and infant PDI scores, we included in our regression models district of residence (Michalovce or Svidnik), infant sex, maternal Raven score (a quantitative measure of nonverbal intelligence), and HOME score (a quantitative measure of quality and quantity of stimulation given to the infant at home). See the article by Park et al. Example 1: sex and TCDD exposure As for the linear regression analyses, the estimated changes in PDI score when doubling the PCB-28 concentration were −0.53 (95% conﬁdence in- terval: −1.22, 0.15) with substitution and −0.47 (95% conﬁ- dence interval: −1.06, 0.13) with multiple imputation. Note that the linear regression parameters have a different interpre- tation than the Cox regression parameter. The Cox regression analysis showed both sex and age to be important determinants of TCDD exposure (P < 0.0001), with men having lower serum TCDD concentrations than women (Figure 1) and with TCDD concentrations increasing with age. On the reverse concentration scale, the estimated Cox regression coefﬁcient for men was −0.45, and the esti- mated hazard ratio was exp(−0.45) = 0.64 (95% conﬁdence interval: 0.53, 0.76). In contrast, when linear regression was used with substitution of LOD / ﬃﬃﬃ 2 p for each nondetect, age remained a signiﬁcant predictor (P < 0.0001), but sex did not (P = 0.30). Example 2: PCB exposure and neurodevelopment Though our approach is most useful with a high proportion of nondetects, we applied it to the 6 congeners investigated by Park et al. (8) to conﬁrm that it also performs well with minimal censoring. The Cox method found signiﬁcant asso- ciations between PDI score and the same 2 congeners that had been identiﬁed by Park et al. (8) using linear regression with substitution, and the signs of the regression coefﬁcients in the Cox and linear models agreed. To examine associations between in utero PCB exposures and neurodevelopment, Park et al. (8) used cohort data from mother-infant pairs residing in 2 districts of eastern Slovakia, with births between 2002 and 2004. In utero PCB exposure was estimated from concentrations measured in serum col- lected from the mother at the time of her child’s birth. Fifteen PCB congeners were measured on a wet weight basis (as ng/ mL) and then adjusted for serum lipids (as ng/g lipid) by the Akins method (14). Infant neurodevelopment was assessed using the Bayley Scales of Infant Development-II (16) at 16 months of age; our example focuses on results from the Psychomotor Development Index (PDI), 1 of 2 indices that comprise the Bayley Scales. To adjust for potential con- founders when evaluating the association between individual Am J Epidemiol. 2014;179(8):1018–1024 DISCUSSION When used to relate exposure biomarker analytes to a health outcome of interest, our method begins by reversing the concentration scale and then applies Cox regression with adjustment for potential confounders, that is, for factors Am J Epidemiol. 2014;179(8):1018–1024 Accommodating Detection Limits via Cox Regression 1023 a prospective formulation, as a consequence of results by Prentice and Pyke (18). On the other hand, if pair matching has been performed on the basis of nonquantiﬁed factors (e.g., with each case matched to a friend control or a sibling control), then an analyte with a small number of known con- centrations cannot be readily studied using our method. How- ever, such a design presents an intractable and possibly insurmountable challenge for other approaches, too. For ex- ample, suppose 80% are nondetects. Even if analyte concen- trations are independent within pairs, 64% of pairs would be completely noninformative, and only 4% of pairs would have 2 measured concentrations to compare. Consequently, we caution against such a design if the number of known analyte concentrations is small. that may be causal “ancestors” of both the analyte concentra- tion and the health outcome. Directed acyclic graph methods (5) can be used to identify those ancestors for inclusion in the model. Our simulations show that the approach is valid even with both confounding and extreme LOD censoring (i.e., a high proportion of nondetects), provided the model is cor- rectly speciﬁed, whereas alternative approaches can perform poorly. In our NHANES example, we were in fact testing whether sex inﬂuences TCDD concentrations, so sex was the cause and TCDD the outcome. In other applications, reversal of the roles of exposure and outcome may be needed, and in such settings the reverse-scale Cox method can be regarded as simply assessing association. Our method has some important limitations. As with any complex model involving confounding covariates, misspeci- ﬁcation of the model can invalidate inferences. The Cox anal- ysis does not assume any parametric distributions, but it does require that the hazard functions for different covariate values be proportional across concentrations of the analyte under study. This assumption would be important to check, partic- ularly because we have not here assessed the method’s sensi- tivity to violations of that assumption. Other strategies for optimizing ﬁt should be used, such as considering transfor- mations for continuous confounders, though goodness of ﬁt can be challenging to evaluate under the Cox model (17). ACKNOWLEDGMENTS A second potential limitation is that characterizing a dose-response relationship may be difﬁcult under the pro- posed approach, especially in situations in which the roles of the exposure and the outcome are reversed. Although es- timation of a dose-response curve may be nearly impossible when LOD censoring is extreme, the proposed method will be useful for detecting risks from chemicals or metabolites of biomarkers under investigation. For substances identiﬁed, further testing could be done with a more sensitive assay to study dose-response relationships. Author afﬁliations: Biostatistics Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (Gregg E. Dinse, Clarice R. Weinberg); Department of Public Health Sciences, Division of Epidemiology, University of Rochester School of Medicine and Dentistry, Rochester, New York (Todd A. Jusko); Health Sciences Research Group, Survey Research Associates International, Durham, North Carolina (Lindsey A. Ho); Department of Biology, The Wharton School, University of Pennsylvania, Philadelphia, Pennsyl- vania (Kaushik Annam); Department of Statistics, The Whar- ton School, University of Pennsylvania, Philadelphia, Pennsylvania (Kaushik Annam); Biostatistics Branch, Divi- sion of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (Barry I. Graubard); Department of Public Health Sciences, School of Medicine, University of California Davis, Davis, California (Irva Hertz-Picciotto); Environmental Autoimmu- nity Group, Program of Clinical Research, National Institute of Environmental Health Sciences, National Institutes of Health Clinical Research Center, Bethesda, Maryland (Frederick W. Miller); and Department of Biostatistics, School of Public Health, University of Michigan, Ann Arbor, Michigan (Brenda W. Gillespie). Other limitations relate to censoring. Our method may not be useful in a cohort study when the outcome of primary in- terest is the time to an event (e.g., death), and the predictor of interest is an exposure biomarker subject to a high proportion of nondetects. The potential problem with such a setting is that both the outcome and its predictor are subject to cen- soring. Also, Cox analysis typically assumes censoring is noninformative. In our application, censoring is inherently noninformative because knowing that the analyte concentra- tion falls below a particular LOD should not tell us more than that about its actual value. The proposed method has somenotable strengths. Unlike ap- proaches that discard nondetects or analyze detect/nondetect dichotomies, our method allows full use of the available data for a quantitative exposure biomarker. DISCUSSION In summary, a reverse-scale Cox model can be used to assess a confounder-adjusted association between a health outcome (or an exposure determinant) and an exposure bio- marker whose concentrations often fall below its assay’s LOD. The method fully uses quantiﬁable analyte measures, appropriately treats nondetects as censored, avoids speciﬁc parametric assumptions about the concentration distribution, and enables adjustment for confounders. Am J Epidemiol. 2014;179(8):1018–1024 REFERENCES Deﬁne a binary outcome Y, a quantitative confounder Z, and a quantitative reverse-scale concentration R. We simu- lated various null data under a proportional hazards model for R, as assumed by the Cox method. True concentration T, observed concentration C = max(T, LOD), and censoring indicator Δ = I(T > LOD) can be obtained from R and the LOD. 1. Browne RW, Whitcomb BW. Procedures for determination of detection limits: application to high-performance liquid chromatography analysis of fat-soluble vitamins in human serum. Epidemiology. 2010;21(suppl 4):S4–S9. 2. Nie L, Chu H, Liu C, et al. Linear regression with an independent variable subject to a detection limit. Epidemiology. 2010;21(suppl 4):S17–S24. ( ) The usual formulation of the proportional hazards model (4, 17) implies 3. Arunajadai SG, Rauh VA. Handling covariates subject to limits of detection in regression. Environ Ecol Stat. 2012;19(3):369–391. ln½Sðr j y; zÞ ¼ expðϕy þ βzÞ ln½Sðr j 0; 0Þ; ðA1Þ ðA1Þ 4. Cox DR. Regression models and life-tables. J Roy Statist Soc Ser B. 1972;34(2):187–220. where S(r \| y,z) = Pr(R > r \| Y = y, Z = z) is the “survival” (or upper-tail distribution) function for R given Y and Z, and S(r \| 0,0) is a baseline survival function that depends only on R. Regression coefﬁcients φ and β are log hazard ratios that gauge the effect on R of Y and Z, respectively. 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REFERENCES We assigned yi = 0 for n0 persons and yi = 1 for n1 persons, where N = n0 + n1. Given yi, we sampled ln(zi) from a normal distribution with mean μ0 if yi = 0, mean μ1 if yi = 1, and variance σ2 for either value of yi and then exponentiated to get zi. We set μ0 = 0 and con- trolled the dependence of Z on Y through μ1; we chose pairs (μ1 = 1, σ = 0.25) and (μ1 = 0.5, σ = 1) to produce lightly and heavily skewed distributions for Z, respectively. Given yi and zi, we generated ri using formulas A1 and A2. We set φ = 0 to enforce the null hypothesis of no association between Y and R (or equivalently T) and β = ln(2) to obtain a hazard ratio of 2 for the effect of a 1-unit change in Z on R. With respect to the baseline survival function, we set α = ln(2) to yield a median of 1, and we chose shapes γ = 5 and γ = 1 to produce light and heavy skewness, respectively. We cre- ated a reverse-scale limit of detection U by determining the value that yields an average S(U \| yi,zi), in large samples, equal to the desired censoring rate. Then we deﬁned V = max(r1, . . . , rN) and LOD = V −U. Finally, we set (δi = 1, ci = V −ri) if ri < U, and we set (δi = 0, ci = LOD) otherwise. This procedure produced “observed” data of the form {(yi, zi, ci, δi): i = 1, . . . , N} for the Cox analysis. The linear and lo- gistic regression analyses, based on substitution or multiple imputation, required data on T. If δi = 1, then T was uncen- sored, and we set ti = ci in either case. If δi = 0, the substitution approach set ti = LOD / ﬃﬃﬃ 2 p , and the imputation approach used missing data methods to impute a value of ti from the estimated distributions of the observed data (12). 9. Gillespie BW, Chen Q, Reichert H, et al. Estimating population distributions when some data are below a limit of detection by using a reverse Kaplan-Meier estimator. Epidemiology. 2010; 21(suppl 4):S64–S70. 10. Zhang D, Fan C, Zhang J, et al. ACKNOWLEDGMENTS Unlike approaches that substitute speciﬁc values (e.g., LOD / 2 or LOD / ﬃﬃﬃ 2 p ) for non- detects, our method does not assume that unknown values are known and thus should not experience the same biases (both in estimation and testing) and underestimations of variability. Application to matched case-control studies is also possi- ble with the proposed method. If frequency matching has been applied, the usual adjustments for the matching factors must be included in the model. The adjusted odds ratio from the Cox model can then be viewed as equivalent to one from The proposed method has somenotable strengths. Unlike ap- proaches that discard nondetects or analyze detect/nondetect dichotomies, our method allows full use of the available data for a quantitative exposure biomarker. Unlike approaches that substitute speciﬁc values (e.g., LOD / 2 or LOD / ﬃﬃﬃ 2 p ) for non- detects, our method does not assume that unknown values are known and thus should not experience the same biases (both in estimation and testing) and underestimations of variability. This research was supported in part by the intramural re- search program of the National Institutes of Health, National Institute of Environmental Health Sciences (projects ZIA ES040006 and ZIA ES101074) and by the National Institutes of Health (grants K12 ES019852, P30 ES001247, and R01 CA096525). Application to matched case-control studies is also possi- ble with the proposed method. If frequency matching has been applied, the usual adjustments for the matching factors must be included in the model. The adjusted odds ratio from the Cox model can then be viewed as equivalent to one from We are grateful to Drs. G. Kissling, M. Longnecker, and R. Morris for helpful discussions, to C. Co and R. Jaramillo for programming assistance, and to members of the National Am J Epidemiol. 2014;179(8):1018–1024 1024 Dinse et al. 1024 Health and Nutrition Examination Study Autoimmunity Study Group (Drs. M. Satoh, E. Chan, K. Rose, C. Parks, R. Cohn, N. Walker, D. Germolec, I. Whitt, L. Birnbaum, and D. Zeldin) for initiating the studies that motivated much of this research. 18. Prentice RL, Pyke R. Logistic disease incidence models and case-control studies. Biometrika. 1979;66(3): 403–411. 19. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc. 1958;53(282):457–481. Conﬂict of interest: none declared. REFERENCES Nonparametric methods for measurements below detection limit. Stat Med. 2009;28(4): 700–715. 11. Hornung RW, Reed LD. Estimation of average concentration in the presence of nondetectable values. Appl Occup Environ Hyg. 1990;5(1):46–51. 12. Lubin JH, Colt JS, Camann D, et al. Epidemiologic evaluation of measurement data in the presence of detection limits. Environ Health Perspect. 2004;112(17):1691–1696. 13. Patterson DG, Hampton L, Lapeza CR, et al. High-resolution gas chromatographic/high-resolution mass spectrometric analysis of human serum on a whole-weight and lipid basis for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Anal Chem. 1987;59(15): 2000–2005. 14. Akins JR, Waldrep K, Bernert JT. The estimation of total serum lipids by a completely enzymatic ‘summation’ method. Clin Chim Acta. 1989;184(3):219–226. 15. Mohadjer L, Curtin LR. Balancing sample design goals for the National Health and Nutrition Examination Survey. Surv Methodol. 2008;34(1):119–126. 16. Bayley N. Bayley Scales of Infant Development. San Antonio, TX: The Psychological Corporation; 1993. 17. Hosmer DW, Lemeshow S, May S. Applied Survival Analysis: Regression Modeling of Time-to-Event Data. 2nd ed. Hoboken, NJ: John Wiley & Sons; 2008. Am J Epidemiol. 2014;179(8):1018–1024
https://openalex.org/W2738421076	http://editorarevistas.mackenzie.br/index.php/ptp/article/download/8318/11928	English	null	Recriando a vida: o luto das mães e a experiência materna	Psicologia	2,017	cc-by	6,255	Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43. Sistema de avaliação: às cegas por pares (double blind review). Universidade Presbiteriana Mackenzie. Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43. Sistema de avaliação: às cegas por pares (double blind review). Universidade Presbiteriana Mackenzie RECRIANDO A VIDA: O LUTO DAS MÃES E A EXPERIÊNCIA MATERNA Resumo: A perda de um filho rompe com o equilíbrio familiar. Para retomar a vida após a perda, as mães precisam expressar seus sentimentos e agir de forma espontânea. Neste estudo, foi investigada a experiência materna de mulheres que perderam seus filhos ainda crianças. Foram utilizados uma entrevista semiestruturada e o Procedimen- to de Desenhos de Famílias com Estórias (DF-E). A análise dos dados foi feita de acordo com o método da livre inspeção do material clínico-qualitativo. As mães demonstraram dificuldades na expressão de seus sentimentos, o que agravou a vivência da perda e a elaboração do luto, tornando difícil o exercício da maternagem com os filhos sobrevi- ventes. O apoio do ambiente mostrou-se essencial para que as mães possam elaborar os sentimentos derivados do luto e assim, acreditar na sua capacidade de agir de forma espontânea para auxiliar e cuidar dos seus filhos, ressignificando a vida após a perda. Palavras-chave: luto; família; psicanálise; maternidade; crianças. Recreating life: the grief of the mothers and the maternal experience Marcela Lança de Andrade1 Universidade de São Paulo, Ribeirão Preto, Brasil Fernanda Kimie Tavares Mishima-Gomes Universidade de São Paulo, Ribeirão Preto, Brasil Valéria Barbieri Universidade de São Paulo, Ribeirão Preto, Brasil Translation: Sofie Tortelboom Translation: Sofie Tortelboom Abstract: The loss of children disturbs the family balance. In order to resume life after the loss, mothers need to express their feelings and act spontaneously. This study inves- tigates the maternal experience of women who lost their young children. This study used a semi-structured interview and the Draw a Family – Tell a Story Procedure. The data was analyzed by means of the free inspection method of clinical-qualitative mate- rial. The mothers demonstrated difficulty in expressing their feelings, which worsened the experience of the loss and the elaboration of the grief, making it difficult for these women to be mothers for the surviving children. The support from the environment showed to be essential for the mothers to elaborate the feelings deriving from the grief and thus believe in their capacity to act spontaneously to help and take care of their children, giving a new meaning to life after the loss. Keywords: grief; family; psychoanalysis; motherhood; children. 1 Mailing address: Marcela Lança de Andrade, Rua Batatais, 654, Casa 10, Parque do Bandeirante. CEP: 14090-425. Ribeirão Preto – SP. E-mail: marcela.andrade@usp.br; marcela.ldandrade@gmail.com. Introduction Experiencing the loss of a loved one can mean coping with many difficulties. The set of reactions to a loss and the attempt to rebuild and organize life are part of the grief experience (Mazorra, 2009). Accepting the reality of death, experiencing pain completely, seeking to adjust to the new life, integrating aspects of the deceased lo- ved one into one’s own identity and finding meaning in the loss to initiate new rela- tionships are tasks that can help the mourner reconcile with life after the loss (Cohen, Mannarino, & Knudsen, 2004). Grief being the result of individual reactions, it will always occur differently from person to person. The pain may reappear as memories of the departed person arise, depending on the relationship between the mourner and the deceased (Webb, 2011). When losing a child, it is necessary for parents to build a new reality without him/ her, deconstructing all expectations regarding the child’s development and growth. Parental grief is complex, non-linear and continuous, as the loss breaks with family balance and compromises the quality of the environment (Carter & McGoldrick, 2001). Many negative consequences can be generated in the parents’ lives, in their work, marital and social relationship. They need to feel supported and secure not to unders- tand the loss as a failure in their function (Bittencourt et al., 2011). The family has an important influence in the experience of the grief process and its understanding. Their support is essential for parents, as well as the existence of pro- grams that offer professional assistance so that they can feel supported and unders- tood. Mourners should be encouraged to communicate their feelings (Parkes et al., 2011). Trust in the environment allows them to be able to act creative and sponta- neously, managing to experience grief and proceeding with their emotional develop- ment (Winnicott, 1958; 2012). For Winnicott (1958; 2012), emotional development occurs based on the conti- nuous and creative interaction of one individual with the other. Every individual has an innate tendency towards maturing which, together with the presence of a suffi- ciently good environment, provides for the achievement of autonomy and indepen- dence. Winnicott (1958; 2012) explored the child’s relationships with his family, espe- cially with his mother (the baby’s first environment). RECREANDO LA VIDA: LA PÉRDIDA DE UN NIÑO Y LA EXPERIENCIA MATERNA Resumen: La pérdida de un hijo rompe el equilibrio familiar. Para reanudar la vida des- pués de la pérdida, las madres necesitan expresar sus sentimientos y ser espontáneas. Este estudio investigó la experiencia materna de mujeres que perdieran sus hijos cuando pequeños. Para esta investigación, se ha utilizado una entrevista semiestructurada y el 1 Mailing address: Marcela Lança de Andrade, Rua Batatais, 654, Casa 10, Parque do Bandeirante. CEP: 14090-425. Ribeirão Preto – SP. E-mail: marcela.andrade@usp.br; marcela.ldandrade@gmail.com. 33 Psico 19-1_miolo.indd 33 7/31/17 3:33 PM Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri Procedimiento de Dibujos de Familias con Historias. El análisis de datos se ha realizado acuerdo con el método de libre inspección del material clínico cualitativo. Las madres han demostrado dificultades en la expresión de sus sentimientos, lo que perjudicó la experiencia de la pérdida y de la elaboración del duelo, lo que dificulta el ejercicio de la maternidad con los niños sobrevivientes. El apoyo del ambiente fue esencial para que las madres pudieran elaborar los sentimientos derivados del duelo, y agir espontánea- mente para auxiliar y cuidar de sus hijos, dando un nuevo significado a la vida después de la pérdida. Palabras clave: duelo; familia; psicoanálisis; maternidad; niños. g , ( ), , , j ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. Introduction The mother may be able to meet the needs of the child, offer her physical and emotional support (holding) and present the world gradually, so that the baby can bear it. Through this care (initially offered by the mother, then extended to other people), children are able to mature, expres- sing their creative potential. 34 Revist ISSN Psico 19-1_miolo.indd 34 34 7/31/17 3:33 PM Psico 19-1_miolo.indd 34 Grief and the maternal experience Grief and the maternal experience Creativity is essential in the elaboration of the experiences of loss because, through it, reality can coexist and be supported, without negatively affecting the emotional development. Thus, in addition to family and social support, to prepare for grief, it is necessary for the bereaved to use their own creative capacity to repair the loss suffe- red. The use of creativity enables individuals to re-signify the loss and elaborate their feelings, so that the pain does not compromise their ability to give continuity to life. In this sense, the presence of a sufficiently good environment allows the bereaved to continue to become, as they can express themselves in a creative and spontaneous way. The grief experience happens when the individual manages to be creative again, even in the pain of loss, and feel that life is worthwhile (Barone, 2004). In the light of the literature, this study aimed to investigate the maternal experien- ce of women who lost their young children. In this way, we intend to understand as- pects related to the grief of mothers, the possibility of elaborating the losses and the continuity of the mothering. Instruments Semi-structured interview and the Draw a Family - Tell a Story Procedure (DF- Method This paper is part of the Master’s Thesis entitled: “After the storm: a psychodyna- mic study about the bereaved child and his parents”, whose objective was to unders- tand the experience of the child who lost a sibling, considering the grief experienced by the parents. In the present study, the results of the meetings with the mothers will be presented. Participants Three mothers aged 38 (Helena), 39 (Rosário) and 41 (Regina), who had lost a child due to some chronic illness, and who had another child alive. All participants found out about the research after seeking psychological care for one of their surviving children. Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Case 1 – Regina Regina has three living children: the oldest, Jane, 22, from her first marriage, who currently lives with her maternal grandmother and the other two from her current marriage (Julia, 14, and Rodrigo, 12). Her husband was hospitalized for substance abuse treatment. She sought psychological assistance for her son Rodrigo, at the suggestion of the school, who claimed learning difficulties. In her second marriage, Regina lost two children when her eldest daughter was two years old: a girl, who was born prematurely and died after contracting a hospital infection and a boy who died at six months of age, a victim of meningitis. Regina did not mention the names of the deceased children and referred to them as “the little girl” and “the little boy”. In reporting her life story, she did so very briefly, with short and direct lines, saying: “My life is normal... typical of the Brazilian family”. She repor- ted that she had always wanted to be a mother, that she would like to have more children, but that she was afraid of realizing that desire after the babies’ death. In her drawings, she demonstrated the need to keep the family together, without differentiation (family members are drawn as part of a single block). Difficulties are felt as possible disintegrators of the family nucleus, so it is necessary to invest in this union (in her history, she claims that the family makes an effort to be together despi- te the difficulties). This task demands a lot from her though, revealing the need to be cared for and supported, especially by the mother and daughter figures (these are highlighted in her drawings). When drawing her own family, she fills the entire sheet of paper and shows herself lost as to the number of people to draw. Finally, she draws herself, her husband, her mother and all (living) children, including two more people of whom “she does not know who they are.” Results Case 1 – Regina Procedures A psychological interview was conducted with the guiding question “Tell me about your family” and “Tell me a typical family day”, in which the participants reported what they wanted freely. Then, the DF-E took place, in which the participant made four drawings with the themes: “Draw a family,” “Draw a family that you would like to have”, “Draw a family where someone is not well” and “Draw your family”, con- cluding them with a story about the production and giving a title to it. All meetings were individual. Data analysis was done according to the free inspection method of the clinical-qua- litative material (Trinca, 1984), considering the psychoanalytic theoretical framework. 35 Psico 19-1_miolo.indd 35 7/31/17 3:33 PM Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri In the interview with the mothers, special attention was paid to issues related to grief, feelings, anxieties and desires generated in the experience of loss and the attempted family reconstruction, especially regarding the relationship of the mother to the living child. As for the drawings, interpretative analyses of each production were made. In the interview with the mothers, special attention was paid to issues related to grief, feelings, anxieties and desires generated in the experience of loss and the attempted family reconstruction, especially regarding the relationship of the mother to the living child. As for the drawings, interpretative analyses of each production were made. g ( ) j ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Case 2 – Rosário Rosário has four living children and three who died. In her first marriage, the el- dest son, Lucas, died when she was eight months pregnant, shortly after birth, due to cardiac arrest. She then had three more children in this union: Sílvio (19 years), Leandro (17 years) and Ana; she died at the age of 14, after becoming ill as a result of cancer. Later, in her second marriage, Rosário had two more pregnancies: Edgar (14 years) and Leonardo (11 years); the pregnancy of the latter would be of twins, but one of them passed away before being born, having discovered his death at 36 36 Revist ISSN Psico 19-1_miolo.indd 36 7/31/17 3:33 PM 7/31/17 3:33 PM Psico 19-1_miolo.indd 36 Grief and the maternal experience childbirth. Currently, Rosario is in her third relationship and does not have children with this man. Rosário made a long report about Ana’s death. As the girl was ill for a few months, the family suffered many changes in daily life, “a very heavy burden we had to get through”. As a result, she reported how much the children missed her, because they had to settle in the maternal grandmother’s house while her mother took care of Ana. After Ana’s death, the two older children continued to live at their grandmother’s house and distanced themselves a lot from the family, showing difficulty to stay at their mother’s house, as Rosário said: “You realize that they... locked themselves in.” The younger children also had difficulties after their sister’s death. Edgar was con- sidered a possible bone marrow donor for his sister, but Ana died before surgery, which made him feel guilty and depressed, claiming to see his sister in his bedroom and at school. Leonardo was diagnosed as hyperactive, refused to go to school, had learning difficulties and episodes of frequent fainting without a diagnosed cause. As a result of the refusal to attend school, he initiated psychological care by order of the tutelary council. Rosário pointed out that these two children wanted to make sure to remember their sister, and insisted that the mother kept objects belonging to her and visited the cemetery regularly to take flowers and to take care of Ana’s tomb. Case 2 – Rosário She al- leged that this was the way the children “worshipped their sister” and that she could not deprive them of these desires, for they would suffer due to the interruption of the rituals. During the production of the drawings, Rosario included people from the family of origin (like her parents and brother). There are signs of difficulty in the relationship with the mother figure (the mother is the only one drawn with a sad mouth). Her stories are confusing and difficult to understand, especially when she wants to explain who each child is (they are drawn without any mutual distinction). In the drawing of the “family that is not well”, she is confused about how many children to draw, says that one of them is missing, the eldest, who is locked up, so the family must get orga- nized to “take things to him” (sic). She reported the feeling of discontinuity in the family when someone is distant (by death or imprisonment). Mobilized by this reflec- tion, in her last drawing, Rosario draws her living children and ends with a drawing of Ana. Realizing that the absence of her daughter can be an important factor of disin- tegration of the family union causes her to be paralyzed in the face of a situation that is “very difficult”, without being able to elaborate a history. Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Case 3 – Helena Helena had two children from her first marriage: Paula (20 years old) who lives alone in her mother’s hometown and Davi, who suddenly died at the age of 14 after showing common flu symptoms. One year after his death, Helena had Isabela (8 years) from her second marriage. At the time of her son’s death, Helena had gone out for a walk with her current husband, and when she returned, she had to take her son to the 37 Psico 19-1_miolo.indd 37 Psico 19-1_miolo.indd 37 7/31/17 3:33 PM Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri hospital quickly: “That’s when he died... For me, it was all over, you know? I did not feel like doing anything. “ Davi died without being able to verify the reason for his death. Helena felt very depressed about the death of her son, but she received help from other people, which made her feel better. So, after a month of intense suffering, she resumed her activities, relying on her work, her friends, and a puppy that she had just gained from her husband. Through her relationship with this dog, Helena felt suppor- ted in her loss. When she needed to move, she left this animal in the care of her daughter Paula, but a few months later he died, which made her feel guilty, believing that the death occurred because the dog felt she had abandoned him. After the death of Davi, the daughter Paula began to harm herself, claiming to hear voices. These symptoms improved over time, but others, such as feelings of revolt and rebellious behavior, especially opposed to the mother, intensified with the birth of Isabela. Helena did not seek professional help for this daughter and, in her account, these opposing behaviors decreased over time. For Isabela, she sought psychological care, with complaints that she felt tachycardia, nausea, belly cramps, fear, difficulty sleeping and recurrent thoughts about death, accompanied by “voices” in her head. During the application of the drawings, Helena included the characters she had lost (her son Davi and the dog), putting her daughter Paula more distant from the rest of the family. The figure of Isabela is the only one drawn with her feet close to the ground (possibly representing the fact that, through her symptoms, she demonstrates that something is not right). Case 3 – Helena Moreover, the figures of Davi and Isabela are the only ones who seem to have their eyes open, as if they represented contact with the outsi- de world. In the second drawing (“family that she would like to have”), Helena inser- ted the figure of her own father (representing the function of care and protection) and of Davi. This design signals the loss of male figures and, consequently the loss of limits, protection and care. Finally, in drawing “her family”, Helena did not draw her son Davi (as if she had retrieved the understanding that her son’s absence is irrepara- ble) and added: “I think it’s the real family.” g , ( ), , , j ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516 3687 (impresso) ISSN 1980 6906 (on line) http://dx doi org/10 5935/1980 6906/psicologia v19n1p33 43 Discussion The process of grief is understood as a subjective experience, and it is difficult to establish a period of completion, as some feelings and aspects of loss will always ac- company the mourner (Hangman, 2001). It is considered that the mourning has taken place when grief is no longer frequent and intense, resulting in a kind of reconcilia- tion with life after the loss (Franco, 2002). Being able to re-signify life, however, does not mean forgetting the loved one, or necessarily, ending mourning (Rangel, 2008). According to Stedeford (1986), when there are difficulties in the process of grief, the mourner can settle for the loss without, however, elaborating his feelings. Defen- ses such as denial, affective detachment and trivialization of events would be a way of ensuring that life can continue despite suffering. 38 Revist ISSN Psico 19-1_miolo.indd 38 7/31/17 3:33 PM 7/31/17 3:33 PM Psico 19-1_miolo.indd 38 Grief and the maternal experience The mothers in this study demonstrated affective involvement, especially in con- flicts that were not directly related to the loss of their children. It is as if these situa- tions occupy them and cause them to distance themselves somewhat from the pain of loss. An attempt was made to trivialize the problems, making them feel less painful and cause anxiety, as in Regina’s account, who claimed that her family was “typical of the Brazilian family” (sic). The position of these mothers was of passivity and of little affective involvement, defending themselves against a possible disintegration that would make it impossible to take care of the family. Among other feelings involved in the loss, there was deep sadness, the feeling of paralysis and guilt. Regina, Rosário and Helena showed signs that they felt responsible for the death of their children, either because they felt that they transmitted something harmful to them or because they believed that they were unable to offer enough holding, taking care of their ill- ness or being present when they needed it. Mothers used rationalization as a form of defense, explaining the events that preoccupied them, giving concrete reasons for the feelings they expressed (mainly related to their routine, work or family life), avoiding to relate them to the losses of the children and the insecurities deriving from this experience. In the difficulty to ex- press oneself and the lack of confidence in the environment, the mind assumes con- trol. Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Discussion In this sense, Rosário demonstrated strictness in Ana’s recollections, in dis- cussing the need for the entire family to always remember her, through the care for her objects and rituals. For her, these actions represent her love for her daughter and the certainty that Ana will never be forgotten. Recollecting deceased children through memories is a common behavior of be reaved parents (Rangel, 2008). For mourning to take place, however, mothers need to be able to internalize the lost object so that the child’s memories take place without prejudice to the changes necessary to continue life. This situation does not seem to be possible for Rosário, as the daughter has to exist concretely (through objects and rit uals) to also exist in her mental world. Giving up going to the cemetery means letting her daughter be forgotten. To face the reality generated by the loss of a child makes these mothers feel weak ened by the pain, as well as the fear of dealing with everything that may represent an end. In this sense, mothers may find it difficult to offer holding and caring to surviving children (Alam et al., 2012). For the mothers in this study, the relationship with the children was made difficult after the experience of the loss, due to maternal doubts about the ability to perform their function. Faced with this intense suffering, support and care of the family is essential for these mothers to be able to recover and exercise their motherhood (Heath & Cole, 2011). Mourning may be related to how mothers’ families lived their grievances and how they assisted them in the first losses, from the beginning of their lives (Pincus, 1989). Thus, the participants reported the history of their families of origin, demon strating that the relationship between them and their own parents influenced and resemble their relationship with their own children. When asked about their families, they reported on their childhood and memories of how they were raised (they said they lived with grandparents, sibling rivalry and other issues that made it difficult for the parents to offer their holding), highlighting the importance of the family envi- ronment in emotional development (Winnicott, 1958; 2012). g , ( ), , , j ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Discussion Thus, in a situation of disorganization, the individual seeks the help of the mental functioning to restrain himself (Winnicott, 1958; 2012). In order to avoid the feelings of loss and the resulting memories, mothers also made use of projection and displacement, especially of the feelings associated with hatred and sadness. Helena seems to have displaced the feelings and memories of Davi’s mourning to the loss of her dog. Rosário projected in her children her difficulties with mourning and the children took responsibility for representing the maternal feelings. All these defenses became necessary because, in losing their children, parents feel confident in their own creative capacity and face difficulty to address their own fee- lings (Rangel, 2008). Schoen et al. (2004) described communication as one of the ways to help the ela- boration process of mourning, to recall the deceased and to express oneself. Helena and Regina had difficulty in talking about the deceased children, due to the absence of a space and a place they occupied, as if it were necessary not to remember death so as not to feel it. Regina did not refer to her children by their names and showed a desire to distance herself from the feelings of their loss. Helena, then, briefly discussed the story of her son’s death, turning more to her current life, to the loss of the dog and memories of her daughter Paula’s reactions after her brother’s death. Rosario was the only one who spoke at length about her deceased daughter, however, presenting dif- ficulties in reporting on the death of her other children. For Rangel (2008), the re- membrance of the deceased child continues for the parents without this representing a disease, but the strictness of these memories may represent a pathological freeze. Miller (2002) argues that this freezing can undermine the elaboration of feelings of 39 Psico 19-1_miolo.indd 39 Psico 19-1_miolo.indd 39 7/31/17 3:33 PM Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri mourning. In this sense, Rosário demonstrated strictness in Ana’s recollections, in dis- cussing the need for the entire family to always remember her, through the care for her objects and rituals. For her, these actions represent her love for her daughter and the certainty that Ana will never be forgotten. mourning. Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-4 Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. Discussion In the case of the loss of a loved one, this importance is even more evident, as the elaboration of the feelings of mourning can only be successful if the mourner is supported for as long as he needs it. The death of a child shakes the family, which needs shelter, usually sought in the family of origin (Parkes et al., 2011). The participants demonstrated that they did not have this support, evidencing the need to be cared for and protected, so that they could survive the difficult conditions of the reality of the loss. The perception that they still need the care of the family places these mothers in a situation of fragility, hindering the exercise of motherhood. Thus, they demonstrated the need to keep their families together, perceiving this union as a protection against difficulties. To remain united, however, negative feelings, which could cause destabi- lization of the family structure, need to be bypassed. Thus, in order to assist in the development of the children, the mothers concluded that they needed to be strong, avoiding and denying their own feelings. This effort 40 Revist ISSN Psico 19-1_miolo.indd 40 40 7/31/17 3:33 PM Psico 19-1_miolo.indd 40 Grief and the maternal experience makes them feel overwhelmed and tired though, with their own limited resources to focus on themselves. In this context, the relationship with the child, despite being very close, becomes conflicting because, in order to avoid feeling helpless in the face of situations that can prove the (in)effectiveness of their function in the child’s life, the mothers end up distancing themselves from them affectively (Alam et al., 2012). The participants expressed their concern to reaffirm their commitment to their maternal role. They sought to feel successful as mothers, to believe in their competen- cy as caregivers, which seems to have been questioned due to the death of their chil- dren, unconsciously or not, feeling that they were not able to exercise their function of protecting them. Rosário, for example, reported the difficulties she experienced during the time her daughter was hospitalized, emphasizing her effort to be with her, even if it meant distancing herself from her other children. Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Discussion In addition, she demons- trated her attitudes as responsible for maintaining the health and well-being of the family when talking about her living children: “I have to be strong or the world will collapse for them”, thus demonstrating that mourning cannot be fully lived, because you have to be complete and willing to offer what your children need. Confronting the feelings derived from the loss of their children and creating satis- factory solutions for the pain and all the other feelings that need elaboration is a diffi- cult process. Some parents experience loss constantly, even over time and with the most intense feelings mitigated, because the connection with the child will never be undone (Rangel, 2008). Thus, it was observed in mothers that the experience of loss is constant, even if it is not assumed. The elaboration of mourning, therefore, is only possible based on the understanding of the reality and the experience of the feelings awakened in the loss, as a possibility of a spontaneous action in the world. Living the feelings of loss, respecting one’s own rhythm, makes it possible to recreate reality through the unders- tanding that the deceased child will always be connected in some way to his parents. For this to be possible, however, it becomes essential that the bereaved be able to make use of their ability to create (Barone, 2004). The participating mothers showed impairment in relation to this capacity, with difficulty to make use of their own creati- vity, making the process of mourning more lasting and complicated and making it difficult to resume life after the loss. The affections need to be contained and the inte- raction between the inner and outer world occurs in an ineffective way. The concrete object is still very important, there is no symbolization of the loss. Thus, the experience of the mother’s role becomes difficult for a mother who, by not being able to express herself spontaneously, makes it difficult to prepare the mourning for herself and the children, because she does not accept their spontaneity (Musachio & Daudt, 2003). Without the possibility of recreating reality through creative and spontaneous im- pulse, it is not possible to experience mourning that permits a healthy relationship with reality. Once again, the experience of mothers’ mothering is impaired by the difficulty to elaborate the mourning of their children, resulting from the difficulty to elaborate their own mourning. Discussion 41 Psico 19-1_miolo.indd 41 Psico 19-1_miolo.indd 41 7/31/17 3:33 PM Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri Marcela Lança de Andrade, Fernanda Kimie Tavares Mishima-Gomes, Valéria Barbieri Conclusion The death of a child is a difficult situation that interferes in many aspects of the mothers’ life. In this study, the mothers sought to deny the issues related to mourning, as there was fear of facing death, with their own fragility and with new possibilities of loss. The total knowledge of reality can make them not restore themselves. If affec- tive appropriation of situations means depressing, one needs to take distance from difficult feelings in order to continue to live, even in a rather spontaneous way. The possibility of mourning is thus impaired. Survival becomes necessary and care for the other children occurs with great effort. The experience of mothering is hampered by the impossibility to live their feelings fully and act spontaneously. For these mothers, the help and support of their families, friends and health professionals is essential if they are to find each other, feeling con- fident that they are able to provide their children with the care they need, facing the difficulties and transforming the most painful of losses. It is important to emphasize that, as mourning is a subjective experience, bereaved families need to be welcomed and cared for with respect and attention to their parti- cularities. In relation to the maternal experience, we add the need for children to be heard so that the reflection of this relationship, which after all consists of at least two people, can also be considered. Mourning in mothers is a complex subject that needs to be discussed in order to improve the support offered to this population, which is fragile in the face of one of the most difficult losses. ev sta s co og a: eo a e át ca, ( ), 33 3. São au o, S , ja . ab . 0 7. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. ISSN 1516-3687 (impresso), ISSN 1980-6906 (on-line). http://dx.doi.org/10.5935/1980-6906/psicologia.v19n1p33-43 Revista Psicologia: Teoria e Prática, 19(1), 33-43. São Paulo, SP, jan.-abr. 2017. References Alam, R., Barrera, M., D’Agostino, N., Nicholas, D. B., & Schneiderman, G. (2012). 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https://openalex.org/W3201841192	https://bmcmedinformdecismak.biomedcentral.com/track/pdf/10.1186/s12911-021-01634-3	English	null	A framework for validating AI in precision medicine: considerations from the European ITFoC consortium	BMC medical informatics and decision making	2,021	cc-by	10,134	A framework for validating AI in precision medicine: considerations from the European ITFoC consortium Rosy Tsopra1,2,3,10* , Xose Fernandez4, Claudio Luchinat5, Lilia Alberghina6, Hans Lehrach7,8, Marco Vanoni6, Felix Dreher8, O.Ugur Sezerman9, Marc Cuggia10, Marie de Tayrac11, Edvins Miklasevics12, Lucian Mihai Itu13, Marius Geanta14, Lesley Ogilvie7,8, Florence Godey15,16, Cristian Nicolae Boldisor13, Boris Campillo‑Gimenez17, Cosmina Cioroboiu14, Costin Florian Ciusdel13, Simona Coman13, Oliver Hijano Cubelos4, Alina Itu13, Bodo Lange8, Matthieu Le Gallo15,16, Alexandra Lespagnol18, Giancarlo Mauri19, H.Okan Soykam20, B ti R 1 2 3 P l T 5 L d T i5 Al i Vi li5 Ch i t h Wi li 8 N B h bil 21 d Bastien Rance1,2,3, Paola Turano5, Leonardo Tenori5, Alessia Vignoli5, Christoph Wierling8, Nora Benhabiles21 and Anita Burgun1,2,3,22 © 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. Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 https://doi.org/10.1186/s12911-021-01634-3 Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 https://doi.org/10.1186/s12911-021-01634-3 Abstract Background: Artificial intelligence (AI) has the potential to transform our healthcare systems significantly. New AI technologies based on machine learning approaches should play a key role in clinical decision-making in the future. However, their implementation in health care settings remains limited, mostly due to a lack of robust validation pro‑ cedures. There is a need to develop reliable assessment frameworks for the clinical validation of AI. We present here an approach for assessing AI for predicting treatment response in triple-negative breast cancer (TNBC), using real-world data and molecular -omics data from clinical data warehouses and biobanks. Methods: The European “ITFoC (Information Technology for the Future Of Cancer)” consortium designed a frame‑ work for the clinical validation of AI technologies for predicting treatment response in oncology. Results: This framework is based on seven key steps specifying: (1) the intended use of AI, (2) the target population, (3) the timing of AI evaluation, (4) the datasets used for evaluation, (5) the procedures used for ensuring data safety (including data quality, privacy and security), (6) the metrics used for measuring performance, and (7) the procedures used to ensure that the AI is explainable. This framework forms the basis of a validation platform that we are building for the “ITFoC Challenge”. This community-wide competition will make it possible to assess and compare AI algorithms for predicting the response to TNBC treatments with external real-world datasets. Conclusions: The predictive performance and safety of AI technologies must be assessed in a robust, unbiased and transparent manner before their implementation in healthcare settings. We believe that the consideration of the ITFoC consortium will contribute to the safe transfer and implementation of AI in clinical settings, in the context of precision oncology and personalized care. Correspondence: rosy.tsopra@nhs.net 1 Centre de Recherche Des Cordeliers, Inserm, Université de Paris, Sorbonne Université, 75006 Paris, France Full list of author information is available at the end of the article Correspondence: rosy.tsopra@nhs.net 1 Centre de Recherche Des Cordeliers, Inserm, Université de Paris, Sorbonne Université, 75006 Paris, France Full list of author information is available at the end of the article © 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. Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Page 2 of 14 Keywords: Artificial intelligence, Precision medicine, Personalized medicine, Computerized decision support systems, Cancer, Oncology Backgroundi valuable source of reliable external datasets for validating AI before its implementation in healthcare settings. Artificial intelligence (AI) has the potential to transform our healthcare systems considerably and will play a key role in clinical decision-making in the future [1]. AI has been in the spotlight since the 1980’s, when the first “expert systems” simulating the clinical reasoning for clinical decisions emerged [2]. With the huge increase in medical data over the last few decades, new approaches have been developed (principally machine learning (ML), including neural networks). ML techniques trained on clinical datasets [2] have already proved useful for diag- nostic applications [3–5] and risk prediction [6]. Guidelines on the regulation of AI technologies include high-level directions, but not specific guidance on the practical steps in AI evaluation [19]. Here, we propose a framework for assessing the clinical performance and safety of AI in the context of precision oncology. More precisely, the objective is to use real-world data collected from clinical data warehouses and biobanks to assess AI technologies for predicting the response to anti-cancer drugs. We developed this framework as part of the Euro- pean Flag-Era project ‘ITFoC (Information Technology for the Future of Cancer)’ [20], to validate AI algorithms with -omics and clinical data for the prediction of treat- ment response in triple-negative breast cancer (TNBC). This framework could help AI developers and institutions to design clinically trustworthy decision support systems, and to assess them with a robust methodology. Despite the enthusiasm surrounding AI, their use in healthcare settings remains limited. AI technologies require rigorous assessment before they can be used in clinical practice [7]. For example, the first AI-based device to receive market authorization from the FDA was assessed with a large prospective comparative clini- cal trial including 900 patients from multiple sites [4]. AI technologies must satisfy stringent regulations for approval as medical devices, because (1) the decision support provided is optimized and personalized con- tinuously in real time, according to the phenotype of the patient [7]; (2) the performance of AI depends strongly on the training datasets used [8], resulting in a large risk of AI performing less well in real practice [9–11] or on another group of patients or institutions [9]. It is, there- fore, essential to assess the performance and safety of AI before its introduction into routine clinical use. Step 1: Specify the intended use of AIhi Step 1: Specify the intended use of AIhi approach involves evaluating the performance and safety of these AI models through robust clinical evaluation with reliable and external real-world datasets, before their implementation in healthcare settings. The ITFoC consortium has designed a framework to meet this goal. This framework is based on seven key steps specifying (Fig. 1): (1) the intended use of AI, (2) the target popula- tion, (3) the timing of AI evaluation, (4) the datasets used for evaluation, (5) the procedures used for ensuring data safety (including data quality, privacy and security), (6) the metrics used for measuring performance, and (7) the procedures used to ensure that the AI is explainable. The first step in AI assessment is accurately defining its intended use (for medical purposes) [7], together with its input (i.e. the data required to run the AI), and out- put (i.e. the results provided by AI) parameters. Once the intended use of AI is clearly stated, it is important to be sure that: • AI is used only to address questions that are relevant and meaningful for the medical community. Indeed, AI may be irrelevant if it is used in a correct, but not useful manner in healthcare settings [27]. It is, there- fore, important to define clearly the benefits of AI for a particular clinical scenario. Methods Breast cancer is the most common cancer in women worldwide [21, 22]. The most aggressive type is triple- negative breast cancer (TNBC), characterized by a lack of estrogen receptor, progesterone receptor and human epidermal growth factor expression, together with a high histologic grade and a high rate of mitosis [23]. TNBC accounts for 10–20% of all breast cancers, and has a very poor prognosis, with chemotherapy the main therapeutic option [23, 24]. New targeted and personalized therapies are, therefore, urgently required [23]. Robust evaluations are required for AI to be trans- ferred to clinical settings, but, in practice, only a few such systems have been validated with external datasets [12, 13]. A recent literature review reported that most studies assessing AI did not include the recommended design features for the robust validation of AI [9]. There is, therefore, a need to develop frameworks for the robust validation of the performance and safety of AI with reli- able external datasets [14, 15]. In recent decades, cancer treatments has followed a “one-size-fits-all” approach based on a limited set of clinical criteria. Recent advances, rendering sequenc- ing techniques more widely available, are providing new opportunities for precision oncology, the personaliza- tion of treatment based on a combination of clinical and molecular data, and improvements in drug efficacy, with fewer side effects. Finding, accessing and re-using reliable datasets is a real challenge in medicine (contrasting with other FAIR data collections [16]). However, with the development of clinical data warehouses within hospitals, it should become easier to obtain access to “real datasets”. The benefit of using real-world data for research purposes [17], and, particularly, for generating complementary evi- dence during AI life cycles, has been highlighted by the European Medicines Agency [18]. Real-world data from clinical data warehouses may, therefore, constitute a f In this context, many AI models have been developed, based on the detailed molecular characterization of indi- vidual tumors and patients. They model the effects and adverse effects of drugs in the context of TNBC treat- ment [25, 26]. However, these AI models often lack clini- cal validation, and require further external evaluation. The ITFoC (Information Technology for the Future of Cancer) consortium [20], a multidisciplinary group from six European countries, has proposed a new approach to the unbiased validation of these AI models. This Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Page 3 of 14 Resultsh The framework designed by the “ITFoC consortium” follows seven principles that we consider essential for the assessment of AI technologies. This framework was developed to support a community-based programming contest to be held during “Pink October”. This “ITFoC challenge”, will open a platform enabling various teams (academic, research, and MedTech organizations) to test their AI-based approaches with TNBC datasets provided by our partners for the purpose of this competition. • AI complies with ethical, legal and social standards [27, 28]. As stated by the High-Level Expert Group on AI established by the European Commission [29], AI should (1) comply with all applicable laws and regulations, (2) adhere to ethical principles and val- ues, (3) not disadvantage people from particular soci- odemographic backgrounds or suffering from certain conditions, (4) not increase discrimination based on ethnicity or sex. We describe here the framework and the paral- lel actions planned for the setting up of the “ITFoC challenge”. Fig. 1 The seven key steps needed for the clinical validation of AI technologies Fig. 1 The seven key steps needed for the clinical validation of AI technologies Fig. 1 The seven key steps needed for the clinical validation of AI technologies Page 4 of 14 Page 4 of 14 Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Step 4: Specify the datasets used for AI evaluation The fourth step in AI assessment is the selection of reli- able and representative datasets: In the “ITFoC challenge”, the target population is “women who have been diagnosed with TNBC”. We need to assess AI performance in terms of treatment response. We must therefore select patients who have already received first-line treatment (making it possible to compare the predicted and observed responses in a retrospective mul- ticentre cohort of TNBC patients). • Publicly accessible datasets [1] are available through public repositories (e.g. ArrayExpress [30], GEO [31]) or are released by research and/or medical institu- tions (e.g. TCGA, or ICGC collections). However, most are more suitable for bioinformatics than for clinical informatics [1]. Planned actions phase” in drug development, when drugs are tested in a laboratory setting. Here, AI is evaluated inter- nally in three steps: training, internal validation, and testing. The training step involves training the algo- rithm on a subset of so-called “training” data. The internal validation involves fine-tuning the algorithm or selecting the most optimized parameters. The test step corresponds to the final internal assessment of the performance of the algorithm. In the “ITFoC challenge”, we aim to assess AI with the fol- lowing intended use: predicting the response of TNBC patients to treatment, regardless of their origin or ethnic background. More precisely, AI should be able to predict, at the time of diagnosis, whether particular patients are likely to respond to standard treatment, so that prob- able non-responders can be offered alternative treatment options. The expected clinical impact is an improvement in survival rates for TNBC patients, particularly those not responding to standard treatment. The “clinical validation” phase follows the internal validation and testing of AI. It is equivalent to phases I and II of clinical trials, in which drug efficacy and safety are assessed in a limited number of patients. Here, the performance and safety of AI are assessed with external data. The goal is to check that AI will not result in lost opportunities for patients through the generation of false-positive or false-negative pre- dictions (i.e. for patients predicted to respond to a treatment who do not in reality, and vice-versa). The “clinical validation” phase follows the internal validation and testing of AI. It is equivalent to phases I and II of clinical trials, in which drug efficacy and safety are assessed in a limited number of patients. Here, the performance and safety of AI are assessed with external data. The goal is to check that AI will not result in lost opportunities for patients through the generation of false-positive or false-negative pre- dictions (i.e. for patients predicted to respond to a treatment who do not in reality, and vice-versa). Planned actions In the “ITFoC challenge”, we will focus on the “clini- cal validation” phase. Akin to early-phase drug trials, the goal will be to determine whether the AI developed is sufficiently accurate and safe for transfer into clinical practice for further assessment in RCTs. Step 2: Clearly specify the target populationh The second step in AI assessment is accurately defining the target population. AI must be evaluated on inde- pendent datasets similar to the target population of the AI technology. The population is defined during the development phase, by specifying patient and disease characteristics, in a similar manner to the definition of eligibility criteria in conventional clinical trials. The sets of patients selected for the assessment should be representative of the target population, and consecu- tive inclusion or random selection should be used for patient recruitment, at multiple sites, to limit the risk of spectrum bias (i.e. the risk of the patients selected not reflecting the target population) [15], and to ensure that the results can be generalized. • Finally, patient outcomes are assessed after clini- cal validation with external datasets. This phase is equivalent to the phase III of clinical trials, in which new drugs are compared to standard treatment in randomized controlled trials (RCT). Here, AI is implemented in healthcare settings, and its effect on patient outcomes and the efficiency of the healthcare system is assessed with real patients, via a RCT. • Finally, patient outcomes are assessed after clini- cal validation with external datasets. This phase is equivalent to the phase III of clinical trials, in which new drugs are compared to standard treatment in randomized controlled trials (RCT). Here, AI is implemented in healthcare settings, and its effect on patient outcomes and the efficiency of the healthcare system is assessed with real patients, via a RCT. Contrary to the AI validation and training stages, which require large datasets, AI evaluation does not necessarily require ‘big data’ [15]. As in randomized clinical trials, the study sample should be determined according to the study hypothesis, expected effect (e.g. superiority, non-inferiority) and degree of importance (differences important or unimportant) [15]. Step 3: Specify the timing of AI evaluationh The third step in AI assessment is clearly defining the timing of the evaluation. As in drug development, vari- ous phases can be distinguished for AI evaluation (Fig. 2): • Patient databases store retrospective or prospective datasets generated by clinical trials or routine care (real-world data). • The “fine-tuning” phase is an essential part of AI development. It is equivalent to the “preclinical • ‘Clinical trial’ datasets are collected in the con- trolled environment of a specific clinical trial Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Page 5 of 14 Fig. 2 Evaluation of AI-timing Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Page 6 of 14 (Table 1), from a restricted population that may not be representative of the general population. The data collection process is time-consuming and costly, but the resulting data should be homo- geneous, highly reliable and should have a well- structured format. However, such datasets are not generally made publicly available, for the follow- ing reasons [32]: the potential loss of competitive advantage for the organization funding the study; the possibility of invalidating the results published through secondary analyses; the costs associated with data sharing and, finally, due to ethical and scientific considerations. Moreover, data collec- tion is usually limited to predefined sets of vari- ables, and it may, therefore, be difficult to re-use secondarily these data to address questions not included in the initial protocol [32]. spectrum bias (i.e. the internal dataset is not represent- ative of the population on which the AI will be used). Validation on completely independent external data- sets is required to overcome these limitations and for validation of the generalizability of AI [15]. Geographic sampling (i.e. using datasets collected by independent investigators from different sites) could considerably limit both biases, and improve the estimation of AI generalizability in healthcare settings [15]. Data quality modeling and exchange standards) have been developed for microarray experiments [41]. The most widely used format for variant identification is VCF (variant clinical format), which includes a number of fields for genomic coordinates, reference nucleotide, and variant nucleotide, for example, but also metadata adding meaningful infor- mation relating to variants: e.g. gene symbol, location, type, HGVS (human genome variation society) nomen- clature, predicted protein sequence alterations and additional resources, such as cross-references to cancer- specific and general genomic databases and prior in silico algorithm-based predictions. Standardization is strongly recommended, to guarantee the quality, sharing, portability and reusability of data for AI evaluation [38]. Standardization is defined as the representation of heterogeneous data with consensual specifications [38]. It includes specifications for both data fields (i.e. variables) and their value sets (i.e. codes) [38]. Standardization is highly dependent on the type of data- sets involved. Clinical data Clinical data are highly complex, for sev- eral reasons: (1) they come from different sources (e.g. electronic health records, reimbursement claims data), (2) they have various formats (e.g. free text, numbers, images), and representations (e.g. structured, semi-struc- tured, unstructured); (3) the level of granularity is highly variable, ranging from general to fine-grained concepts; (4) datasets are not complete (e.g. missing data); (5) data- set content varies within and between institutions. Standardization of clinical and ‑omics data Standardi- zation makes it possible to combine data from multiple institutions. It also ensures the consistency of datasets, and improves the quality and reliability of clinical and -omics data. These aspects are crucial, to maximize the chances of predicting the real impact of AI on the health- care process. Indeed, the ultimate performance of AI depends strongly on the quality of data used for evalua- tion [12, 13]. Various common data models can be used to standard- ize clinical datasets. These models include the CDISC (Clinical Data Interchange Standards Consortium) model for “clinical trial datasets”, which can be used to ensure information system interoperability between healthcare and clinical research, and the OMOP (Observational Medical Outcomes Partnership) common data model for real-world datasets. Data quality The data values must also be harmo- nized by the use of terminologies ensuring interoperabil- ity between AI systems, such as the ICD 10 (International Classification of Diseases) for the standardization of medical diagnoses, LOINC (Logical Observation Iden- tifiers Names and Codes) for biological tests, Med- DRA (Medical Dictionary for Regulatory Activities) for adverse events, and so on. Most standard terminologies are integrated into the UMLS (Unified Medical Language System) metathesaurus, which can be used as a global thesaurus in the biomedical domain. Planned actions In the “ITFoC” challenge, we will apply a range of internationally accepted standards for breast cancer data, to overcome issues of data heterogeneity and variability associated with the use of data of different provenances [34, 35] and to ensure access to high-quality real-world datasets [38] Clinical datasets will be standardized with the OMOP common data model [42] for data structure and the OSI- RIS model [43] for data content. The OMOP CDM is sup- ported by the OHDSI consortium (Observational Health Data Sciences and Informatics), and OSIRIS is supported by the French National Institute of Cancer. Both stand- ards include a list of concepts and source values, con- sidered the minimal dataset necessary for the sharing of clinical and biological data in oncology. Items and values are structured and standardized according to interna- tional medical terminologies, such as ICD 10, LOINC, SNOMED CT. A standardized TNBC data model based on these models will be used: items will be added, removed and/or transformed, and values will be adapted to TNBC data (e.g. the values of the “biomarker” item are limited to RO, RP and HER2 receptors, Ki67). The instan- tiated model contains the dataset specifications provided to participants in this challenge. The database will be populated locally through dedicated extract-transform- load pipelines. ‑Omics data -Omics data are complex: (1) they are generated by different techniques, with different bioin- formatic tools; (2) they may be based on different types of NGS (next-generation sequencing) data, such as WGS (whole-genome sequencing), WES (whole-exome sequencing), and RNA-sequencing, or on data from prot- eomics and metabolomics platforms; (3) their integration and interpretation remain challenging, due to their size and complexity, and the possibility of experimental and technical errors during sample preparation, sequencing and data analysis [39]. -Omics data can be standardized at any stage from data generation to data interpretation. Planned actions BMC Med Inform Decis Mak (2021) 21:274 Page 7 of 14 Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Planned actions In the “ITFoC challenge”, we are working with retrospec- tive real-world datasets collected from the clinical data warehouses and biobanks of multiple hospitals, ensuring that the TNBC population is broadly represented. The inclusion criteria for datasets are: • A follow-up period of at least three years, to ensure the standardized evaluation of treatment response • Real-world datasets are usually stored in clinical data warehouses (Table 1). These datasets are col- lected throughout patient care and have various clinical sources (structured and unstructured clin- ical records, laboratory, pharmacy, and radiology results, etc.) [17, 33]. The collection of these data is less time-consuming and costly than that for clinical trial datasets. However, their exploitation requires careful data quality management, because they are highly variable and were initially collected for clinical purposes rather than for research [34– 37]. • High-quality data extracted from a clinical data ware- house or from a dedicated cancer database • Biological samples must be available in biobanks for additional -omics analyses, if required. • Patients must have signed a consent form for the reuse of their data and the reuse of their samples for research purposes The objective is not to acquire thousands of patient datasets of variable quality, but to collect a representative set of high-quality patient data. Split-sample validation involves randomly splitting datasets into separate parts, which are then used for both the development and internal evaluation of AI [12, 15]. This method is relevant only during the develop- ment phase, and cannot be used to validate the gen- eralizability of AI. Indeed, there is a risk of overfitting bias (i.e. the AI fits too exactly to the training data), and Step 5: Specify the procedures used to ensure data safety The fifth step in AI assessment is ensuring data safety, including data quality, privacy and security, during the evaluation phase. Table 1 Clinical trial versus Real-world datasets for AI evaluation Table 1 Clinical trial versus Real-world datasets for AI evaluation Clinical trial datasets Real-world datasets Setting Experimental Real world Population Representativeness Selective sample Large sample Type Homogeneous Heterogeneous Size +/− ++++ Time period for recruitment and follow- up Limited Long Data Type Clinical +/− -omics Clinical +/− -omics Collected by Dedicated specialist professionals Various healthcare professionals Quality +++ +/− Need for data management +/− +++ Need for anonymization + + Tsopra et al. Data privacy Th ’ The patients’ right to privacy must be respected. Patients must be informed about the storage and use of their data, and must have signed a consent form authorizing the collection and use of their data for research [44, 45]. Within Europe, data privacy is regulated by the General Data Protection Regulation (GDPR) [45]), which protects patients against the inappropriate use of their data. Such regulations ensure that (1) patients can choose whether or not to consent to the collection of their data, (2) patients are informed about the storage and use of their data (principle of transparency), (3) data are stored in an appropriate manner (principle of integrity), (4) data are used only for certain well-defined purposes, and (5) patients have the right to change their minds and to with- draw consent at any time. Data quality For example, MIAME (minimum information about a microarray experi- ment) [40] and MAGE (microarray gene expression data It may not be possible to extract -omics data directly from clinical data warehouses, because these data are Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Page 8 of 14 not widely collected in routine care. If not already pre- sent in the electronic health record of the patient, -omics data will be generated from patient samples stored in biobanks. For the challenge, WES data, RNA-sequencing data, microRNA expression levels and metabolomic data will be obtained from primary tumor samples, and from blood samples as a control. Data quality will be ensured by using only freshly frozen tumors with a celll content of more than 30% (as determined by a pathologist). Multi- level -omics data contain a wealth of potentially relevant information, including molecular variants (directly or indirectly) affecting clinically significant pathways. Their incorporation into the challenge dataset should greatly increase the predictive power of the AI technologies evaluated. compromised deliberately or accidentally [44]. Any plat- form used for AI evaluation should implement the strict- est control over access, to ensure that data are available only to authorized parties [44], only for the duration of the evaluation [44], and that any personal data (including both data directly linked to a patient, such as surname, and indirectly linked to the patient, such as diagnosis date) are removed [47]. Planned actions In the “ITFoC challenge”, data security will be ensured by using a dedicated ITFoC data space. Workflows will be created between local clinical data warehouses and the local ITFoC data space, for standardi- zation of the datasets with respect to the standard TNBC model. Each standardized dataset will be transferred to a secure platform, on which it will be stored (Fig. 3). Participants will assess their AI technologies with the same datasets hosted on a secure platform, but they will not be allowed to access datasets directly. Clinical and -omics data will be inaccessible throughout the duration of the challenge, and participants will be provided only with the specifications of the datasets. Step 6: Specify the metrics used for measuring AI performancehi The sixth step in AI assessment is defining the metrics used to evaluate the performance of the AI algorithm. The intrinsic performance of the AI itself is assessed during the “fine-tuning” and the “clinical validation” phases. Discrimination performance is measured in terms of sensitivity and specificity for binary outputs [15]. By plotting the effects of different levels of sensitivity and specificity for different thresholds, a ROC (receiver oper- ating characteristics) curve can be generated [48]. This ROC curve represents the discrimination performance of a particular predictive algorithm [15]. The most common metric used is the AUC (area under the ROC Curve), the values of which lie between 0 and 1. Algorithms with high levels of performance have a high sensitivity and specific- ity, resulting in an AUC close to 1 [15, 48]. Planned actions In the “ITFoC challenge”, data privacy will be respected: • Only datasets from patients who have signed a con- sent form authorizing the reuse of their data and samples for research will be included in the chal- lenge. Calibration performance is measured for quantitative outputs, such as probabilities [15]. It is used to determine whether predicted probabilities agree with the real prob- abilities [15]. The predicted probabilities are plotted on the x-axis, and the observed real probabilities are plotted on the y-axis, to generate a calibration plot [15]. This plot can be used to estimate the goodness of fit between the predicted and real probabilities [49]. Bland–Altman plots can also be used to analyze the agreement between the predicted and the observed probabilities [50]. • The clinical data will be pseudo-anonymized by state-of-the-art methods (and in accordance with the GDPR), without altering the scientific content. Any clinical information that could be used, directly or indirectly, to identify the individual will be removed (e.g. dates will be transformed into durations (com- puted as a number of days)). Data security A more detailed discussion of the statistical methods used to measure AI performance is beyond the scope of this article but can be found elsewhere [49]. AI evaluation should be hosted and managed on a secure platform [46], that can ensure that confidentiality, integ- rity and/or the availability of patient information are not Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Page 9 of 14 Fig. 3 Data workflow for the ITFoC challenge Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Page 10 of 14 Page 10 of 14 Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 The clinical performance of AI in real clinical settings is assessed during the “patient outcome assessment” phase. AI metrics, such as AUC, are not always understood by clinicians [51], and do not necessarily reflect clinical effi- cacy [52]. There is a need to determine the effect of AI on patient outcomes in real-life conditions. Ideally, the effects of AI should be compared to a gold standard [53] or baseline (i.e. standard procedure) in an RCT using standard statistical approaches [15]. Planned actions In the “ITFoC challenge”, we aim at explain why some AI successfully predict treatment response, whereas oth- ers fail. Each AI developer participating in the challenge should provide the data specifications used by the AI. We will encourage the AI developers to share their codes openly. Alternatively, they could opt for restricted code sharing with the scientific committee (the scientific com- mittee will sign a confidentiality agreement). Use of real‑world datasets for validating AI technologies Th d l d f h l Use of real‑world datasets for validating AI technologies The systematic and rigorous validation of AI technologies is essential before their integration into clinical practice. Such evaluation is the only way to prevent unintentional harm, such as misdiagnosis, inappropriate treatment or adverse effects, potentially decreasing patient survival. To date, only a few AI-based solutions have actually been clinically validated [9], mostly exclusively on internal datasets, with no external validation. RCTs in which AI technologies are compared to the gold standard (i.e. rou- tine care delivered by medical experts) are the strongest and most reliable approach for assessing AI performance and safety [56]. Such trials provide a more detailed evalu- ation, including a range of relevant parameters, such as patient benefits in terms of quality of life, acceptance by physicians, integration into the clinical workflow, and economic impact. However, RCTs are costly, both finan- cially and in terms of time required, and should be pre- ceded by early-phase studies [4]. Discussion We describe here the framework designed by the ITFoC consortium for the assessment of AI technologies for predicting treatment response in oncology. This frame- work will be used to construct a validation platform for the “ITFoC Challenge”, a community-wide competition for assessing and comparing AI algorithms predicting the response to treatments in TNBC patients from real- world datasets. In the “ITFoC challenge”, we will assess the performance of AI itself with the binary criterion “predicted response to treatment” during the clinical validation phase. For each AI algorithm, various metrics will be reported, including AUC, confusion matrix, sensitivity, specificity, positive and negative predictive values. The evaluation will be carried out by a scientific com- mittee, independent of the ITFoC organizational com- mittee. This scientific committee will include members from various disciplines (e.g. bioinformaticians, medical doctors, data scientists, statistical and machine-learning experts) and from various international institutions (aca- demic, research and hospital institutions). Step 7: Specify the procedures to ensure AI explainability Th h h f h Step 7: Specify the procedures to ensure AI explainability The seventh step in the assessment of AI is examining the underlying algorithm [54, 55]. This step has two expected benefits. First, it may prevent an inappropriate represen- tation of the dataset used for training/validation. Sec- ond, it may reveal the learning of unanticipated artifacts instead of relevant inputs [54].hih The input data must be analyzed first [54]. The type (structured or unstructured), format (e.g. text, numbers, images), and specifications (e.g. variables used) of the data must be assessed. A better comprehension of the input data should ensure that the data used by the AI are comprehensive and relevant to clinical practice.h The underlying algorithm should also be analyzed [54]. The code, documented scripts, and the computer envi- ronment should be evaluated by independent research- ers. Ideally, independent researchers should even run the pipeline, check the underlying AI methods and evaluate the explainability of the outputs [54]. However, AI devel- opers may be reluctant to share their codes openly, for scientific or economic reasons. In such cases, alternatives can be found, such as a trusted neutral third party signing a confidentiality form, or a virtual computing machine running the code with new datasets [54], or the provision of documentations about the AI. Here, we support the idea that when AI technologies reach a state of sufficient “maturity”, they should undergo clinical validation with external real-world datasets. This would make it possible to measure the performance and safety of AI quickly and reliably in conditions close to those encountered in real-life. This validation pro- cess would save both money and time, due to the use of real-world datasets from clinical data warehouses. At the end of this early validation step, if the performance of a specific AI technology falls short of expectations (e.g. if it fails to predict response to treatment, or is considered unsafe), then it can be rejected (as in early-phase trials for drugs), and no further evaluation in RCTs is required. If an AI is validated clinically with these real-world Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Page 11 of 14 Page 11 of 14 to other questions in oncology, with minor adaptations. For instance, for diagnosis, other datasets could be con- sidered (e.g. images, signals). Step 7: Specify the procedures to ensure AI explainability Th h h f h Likewise, we propose here the use of real world dataset from various healthcare centres, to guarantee the volume and representativeness of the dataset. Similarly, when dealing with rare can- cers, the datasets may come from various centers, and may even be extended to other sources, such as clinical research data. Dataset from other sources have already been successfully used for the assessment of AI in breast and prostate cancers [46, 58]. Furthermore, the metrics used to assess AI performance may also differ, depend- ing on the type of cancer and the intended use of AI (e.g. for diagnosis, the primary outcome could be compared to the diagnosis made by an oncologist). datasets, it can be considered a good candidate and allowed to progress to the next stage in evaluation (i.e. an RCT). The validation process outlined here (“validation step with retrospective real-world datasets”) should thus be an integral part of the entire AI evaluation process, constituting the decisive step concerning whether or not to perform a RCT. Supporting precision medicine We derived a framework from these competition- based approaches. Our approach is based on the same principles as these existing challenges, but focusing on the combination of real-world data collected from clini- cal data warehouses (rather than data collected through RCTs), and -omics data generated by next-generation sequencing techniques. The results of the “ITFoC chal- lenge” will provide essential proof-of-principle evidence for the use of real-world datasets for validating AI tech- nologies in a competition setting, as an essential precur- sor to RCTs. Clinical care decision are traditionally driven by patient symptoms and disease characteristics. In precision oncology, the scope is extended to the patient pheno- type, preclinical symptoms, tumor characteristics and the complex molecular mechanisms underlying disease [61]. Recent advances in genetics and sequencing tech- nologies are now enabling clinicians to include molec- ular aspects of the disease in their clinical decision processes, and advances in metabolomics have facili- tated considerations of the functional activity of can- cer cells [62, 63]. The use of -omics data in routine care (e.g. genomic, metabolomic or proteomic data [64]), is strongly supported by the European Medicines Agency [18], and could lead to significant improvements in patient care. Use of a community‑wide competition to assess AI technologies We propose here to organize the “validation step” in the form of a community-wide competition. Competition- based approaches are increasingly being seen as relevant in the medical informatics domain, with participating teams usually tackling a challenge over a limited time period, with access to an anonymized dataset for the test- ing of methods. For example, the i2b2 (Informatics for Integrating Biology and the Bedside) project includes a “Natural Language Processing” challenge for assess- ing methods for understanding clinical narratives [57]. Competition-based approaches have also been developed in oncology (e.g. the Sage Bionetworks—DREAM Breast Cancer Prognosis Challenge, designed for developing computational models that can predict breast cancer survival [58, 59]; and the Prostate DREAM Challenge, for identifying prognostic models capable of predicting survival in patients with metastatic castration-resistant prostate cancer [46]). The utility of these crowdsourced challenges for the community has clearly been demon- strated. They have multiple advantages: (1) they allow the development of models that outperform those developed with traditional research approaches [58, 60], (2) they encourage collaboration between teams for the improve- ment of models [60], and (3) they provide more trans- parent results, because both favorable and unfavorable results are published [58, 60]. We believe that a platform, as described here, could help to accelerate AI transfer to healthcare settings in oncology. AI systems are currently considered to be medical devices that can only be implemented in health centers after the demonstration of their safety and effi- cacy through a large prospective RCT [4]. However, this is time-consuming and expensive, and there is a risk of patient outcome studies becoming obsolete by the time the results become available [15]. The use of a valida- tion platform has several advantages: (1) several AI tech- nologies can be assessed in parallel for the same price (whereas a RCT is usually designed to assess a single AI technology); (2) the platform can be re-used for fur- ther AI evaluations; (3) new datasets can easily be added to the platform; (4) transparency is guaranteed, as the results are communicated even if unfavorable. For all these reasons, validation platforms constitute a credible route towards establishing a rigorous, unbiased, trans- parent and durable approach to the assessment of AI technologies. Availability of data and materials Data sharing is not applicable to this article as no datasets were generated or analyzed during this study. Funding This work was supported by the ITFoC project (Information Technology for the Future of Cancer) – FLAG-ERA support. Abbreviations AI A tifi i l i t ll AI: Artificial intelligence; CDISC: Clinical data interchange standards con‑ sortium; GDPR: General data protection regulation; HGVS: Human genome variation society; ICD: International classification of diseases; LOINC: Logical observation identifiers names and codes; MedDRA: Medical dictionary for regulatory activities; OMOP: Observational medical outcomes partner‑ ship; MIAME: Minimum information about a microarray experiment; MAGE: MicroArray gene expression; ML: Machine learning; NGS: Next-generation sequencing; OHDSI: Observational health data sciences and informatics; RCT : Randomized controlled trials; ROC: Receiver operating characteristics; TNBC: Triple-negative breast cancer; UMLS: Unified medical language system; VCF: Variant clinical format; WGS: Whole-genome sequencing; WES: Whole-exome sequencing. Received: 18 June 2020 Accepted: 22 September 2021 Received: 18 June 2020 Accepted: 22 September 2021 Accelerating AI transfer to healthcare settings We propose a framework for the clinical validation of AI technologies before their transfer to clinical settings and clear actions in the domain of TNBC treatment. Both the framework and the planned actions can be generalized Page 12 of 14 Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Here, we provide support for the idea that -omics anal- ysis should be part of the clinical decision process. The “ITFoC Challenge” aims to demonstrate the benefits of integrating clinical data warehouses and biobanks into the clinical care process, in accordance with the findings of previous studies [65, 66]. By combining clinical and -omics data, AI tools may facilitate the delivery of treat- ments that are personalized according to the characteris- tics of the patients and their tumors, thereby increasing of the chances of survival and decreasing side effects. By designing the “ITFoC Challenge”, we aim to encourage the development of AI based on clinical and -omics data for the prediction of treatment response in cancer, and the personalization of cancer treatment. References 1. Paton C, Kobayashi S. An open science approach to artificial intelligence in healthcare. Yearb Med Inform. 2019;28:47–51. Competing interests p g Hans Lehrach is a member of the board of Alacris Theranostics GmbH. Felix Dreher is an employee of Alacris Theranostics GmbH. Lesley Ogilvie is an employee of Alacris Theranostics GmbH. Bodo Lange is the CEO of Alacris Theranostics GmbH. Christoph Wierling is an employee of Alacris Theranostics GmbH. The other authors have no conflicts of interest to declare. Conclusions h b We hereby propose a framework for assessing AI tech- nologies based on real-world data, before their use in healthcare settings. This framework includes seven key steps specifying: (1) the intended use of AI, (2) the target population, (3) the timing for AI evaluation, (4) the datasets selected for evaluation, (5) the proce- dures used to ensure data safety, (6) the metrics used to measure performance, and (7) the procedures used to ensure that the AI is explainable. The proposed frame- work has the potential to accelerate the transfer of AI into clinical settings, and to boost the development of AI solutions using clinical and -omics data to predict treatment responses and to personalize treatment in oncology. Here, we applied this framework to the estab- lishment of a community-wide competition in the con- text of predicting treatment responses in TNBC. Consent for publication Not applicable. Consent for publication Not applicable. Author details 1 Centre de Recherche Des Cordeliers, Inserm, Université de Paris, Sorbonne Université, 75006 Paris, France. 2 Inria, HeKA, Inria Paris, France. 3 Department of Medical Informatics, Hôpital Européen Georges-Pompidou, AP-HP, Paris, France. 4 Institut Curie, 25 Rue d’Ulm, 75005 Paris, France. 5 Centro Risonanze Magnetiche ‑ CERM/CIRMMP and Department of Chemistry, University of Flor‑ ence, 50019 Sesto Fiorentino (Florence), Italy. 6 Department of Biotechnology and Biosciences, University of Milano Bicocca and ISBE-Italy/SYSBIO - Candi‑ date National Node of Italy for ISBE, Research Infrastructure for Systems Biol‑ ogy Europe, Milan, Italy. 7 Max Planck Institute for Molecular Genetics, Berlin, Germany. 8 Alacris Theranostics GmbH, Berlin, Germany. 9 School of Medicine Biostatistics and Medical Informatics Dept., Acibadem University, Istanbul, Turkey. 10 Univ Rennes, CHU Rennes, Inserm, LTSI - UMR 1099, 35000 Rennes, France. 11 Univ Rennes, Department of Molecular Genetics and Genomics, CHU Rennes, IGDR-UMR6290, CNRS, 35000 Rennes, France. 12 RSU Institute of Oncol‑ ogy, Dzirciema str. 16, Riga 1010, Latvia. 13 Transilvania University of Brasov, Brasov, Romania. 14 Centre for Innovation in Medicine, Bucharest, Romania. 15 INSERM U1242 « Chemistry, Oncogenesis Stress Signaling », Université de Rennes, 35042 CEDEX, Rennes, France. 16 Centre de Lutte Contre Le Cancer Eugène Marquis, CRB Santé (BRIF Number: BB-0033-00056), 35042 CEDEX, Rennes, France. 17 Univ Rennes, CLCC Eugène Marquis, INSERM, LTSI - UMR 1099, 35000 Rennes, France. 18 Department of Molecular Genetics and Genom‑ ics, CHU Rennes, 35000 Rennes, France. 19 Department of Informatics, Systems and Communication, University of Milano Bicocca and ISBE-Italy/SYSBIO - Candidate National Node of Italy for ISBE, Research Infrastructure for Systems Biology Europe, Milan, Italy. 20 EPIGENETICS Inc. 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Magrabi F, Ammenwerth E, McNair JB, De Keizer NF, Hyppönen H, Nykänen P, et al. Artificial intelligence in clinical decision support: chal‑ lenges for evaluating AI and practical implications. Yearb Med Inform. 2019;28:128–34. 49. Steyerberg E. Clinical prediction models: a practical approach to develop‑ ment, validation, and updating. New York: Springer; 2009. https://doi.org/ 10.1007/978-0-387-77244-8. 50. Altman DG, Bland JM. Measurement in medicine: the analysis of method comparison studies. J R Stat Soc Ser Stat. Authors’ contributions Design: RT, XF, CL, LA, HL, MV, FD, OUS, MC, MDT, EM, LMI, MG, LO, FG, CNB, BCG, CC, CFC, SC, OHC, AI, BL, MLG; AL, GM, HOS, BR, PT, LT, AV, CW, NB, AB. Writing original manuscript: RT, AB. Agreement with all aspects of the work: RT, XF, CL, LA, HL, MV, FD, OUS, MC, MDT, EM, LMI, MG, LO, FG, CNB, BCG, CC, CFC, SC, OHC, AI, BL, MLG; AL, GM, HOS, BR, PT, LT, AV, CW, NB, AB. All authors read and approved the final manuscript. 2. 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Systems biology of metabolism: a driver for developing person‑ alized and precision medicine. Cell Metab. 2017;25:572–9. 54. Norgeot B, Quer G, Beaulieu-Jones BK, Torkamani A, Dias R, Gianfrancesco M, et al. Minimum information about clinical artificial intelligence mod‑ eling: the MI-CLAIM checklist. Nat Med. 2020;26:1320–4. 63. Sun L, Suo C, Li S-T, Zhang H, Gao P. Metabolic reprogramming for cancer cells and their microenvironment: beyond the Warburg effect. Biochim Biophys Acta Rev Cancer. 2018;1870:51–66. 55. Haibe-Kains B, Adam GA, Hosny A, Khodakarami F, Board MS, Waldron L, et al. The importance of transparency and reproducibility in artificial intel‑ ligence research. 2020.https://doi.org/10.1038/s41586-020-2766-y y 64. Wang F, Preininger A. AI in health: state of the art, challenges, and future directions. Yearb Med Inform. 2019;28:16–26. 56. Moja L, Polo Friz H, Capobussi M, Kwag K, Banzi R, Ruggiero F, et al. Effec‑ tiveness of a hospital-based computerized decision support system on clinician recommendations and patient outcomes: a randomized clinical trial. JAMA Netw Open. 2019;2:e1917094. 65. Rance B, Canuel V, Countouris H, Laurent-Puig P, Burgun A. Integrating heterogeneous biomedical data for cancer research: the CARPEM infra‑ structure. Appl Clin Inform. 2016;7:260–74. 66. Danciu I, Cowan JD, Basford M, Wang X, Saip A, Osgood S, et al. Tsopra et al. BMC Med Inform Decis Mak (2021) 21:274 Second‑ ary use of clinical data: the Vanderbilt approach. J Biomed Inform. 2014;52:28–35. 57. Uzuner Ö, Goldstein I, Luo Y, Kohane I. Identifying patient smoking status from medical discharge records. J Am Med Inform Assoc JAMIA. 2008;15:14–24. 58. Margolin AA, Bilal E, Huang E, Norman TC, Ottestad L, Mecham BH, et al. Systematic analysis of challenge-driven improvements in molecular prognostic models for breast cancer. Sci Transl Med. 2013;5:181re1. Publisher’s Note S N Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 59. Cheng W-Y, Ou Yang T-H, Anastassiou D. Development of a prognostic model for breast cancer survival in an open challenge environment. Sci Transl Med. 2013;5:181ra50. 59. Cheng W-Y, Ou Yang T-H, Anastassiou D. Development of a prognostic model for breast cancer survival in an open challenge environment. Sci Transl Med. 2013;5:181ra50. • 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 ? 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https://openalex.org/W4307195823	https://www.scielo.br/j/ref/a/q4pXS5HfCvXHJrwz7TGdg6s/?lang=pt&format=pdf	Portuguese	null	Uma “perspectiva parcial” sobre ser mulher, cientista e nordestina no Brasil	DOAJ (DOAJ: Directory of Open Access Journals)	2,016	cc-by	11,250	http://dx.doi.org/10.1590/1806-9584-2016v24n3p801 http://dx.doi.org/10.1590/1806-9584-2016v24n3p801 Vívian Matias dos Santos Universidade Federal de Pernambuco, Recife, PE, Brasil Uma “perspectiva parcial” sobre Uma “perspectiva parcial” sobre Uma “perspectiva parcial” sobre Uma “perspectiva parcial” sobre Uma “perspectiva parcial” sobre ser mulher ser mulher ser mulher ser mulher ser mulher, cientista e nordestina , cientista e nordestina , cientista e nordestina , cientista e nordestina , cientista e nordestina no Brasil no Brasil no Brasil no Brasil no Brasil no Brasil no Brasil no Brasil no Brasil no Brasil Resumo: Resumo: Resumo: Resumo: Resumo: Este artigo propõe compreender como mulheres cientistas estão inseridas na produção de conhecimento científico e tecnológico em universidades públicas federais específicas da Região Nordeste do Brasil. A realização de entrevistas e observações diretas nos seus cotidianos de trabalho tornaram possível a construção de reflexões alicerçadas nas experiências sociais de mulheres cientistas pertencentes a dois grandes ramos de saberes: humanidades e as ciências supostamente “exatas”. Por meio desta abordagem, situada e parcial, sobre a inserção e permanência de mulheres nas ciências contemporâneas, pudemos observar a conservação de antigas questões que ainda se colocam como prementes na compreensão feminista e de gênero das ciências. Palavras-chave: Palavras-chave: Palavras-chave: Palavras-chave: Palavras-chave: Mulheres cientistas; gênero; ciências. 1 Introdução: pontos de partida para um 1 Introdução: pontos de partida para um 1 Introdução: pontos de partida para um 1 Introdução: pontos de partida para um 1 Introdução: pontos de partida para um olhar feminista situado nas universidades olhar feminista situado nas universidades olhar feminista situado nas universidades olhar feminista situado nas universidades olhar feminista situado nas universidades nordestinas nordestinas nordestinas nordestinas nordestinas Esta obra está sob licença Creative Commons. Como as mulheres se inserem nas ciências dos dias atuais? Como as especificidades carregadas pela produção de conhecimento científico e tecnológico nas diversas regiões do Brasil relacionam-se com a reiteração de mecanismos discriminatórios de gênero? Pensar em ciências no contexto nacional, hoje, diz respeito a pensar, também, sobre o conhecimento construído nos cotidianos institucionais das universidades. Neste artigo, propomos construir reflexões sobre como mulheres cientistas estão inseridas na produção de conhecimento científico e tecnológico em universidades específicas da Região Nordeste. Partimos da defesa de que, no campo dos estudos sobre gênero e ciências, deve-se destacar a relevância das 801 Estudos Feministas, Florianópolis, 24(3): 398, setembro/dezembro/2016 VÍVIAN MATIAS DOS SANTOS VÍVIAN MATIAS DOS SANTOS epistemologias feministas (Maria Cecília Bacellar SARDENBERG, 2002) por sua preocupação que ultrapassa a dimensão da denúncia ao sexismo nas ciências, questionando profunda- mente o fazer científico em suas dimensões conceituais, em seus métodos, práticas, e mais: constituindo-se como bases analítico-conceituais diversas que ampliam as possibilidades criativas de dispositivos metodológicos por meio dos quais se torna possível uma apreensão/compreensão das múltiplas discriminações no campo científico e tecnológico. Dentre as contribuições feministas, neste estudo, destacam-se como alicerces teórico-metodológicos os olhares de Sandra Harding (1993; 1996) e Donna Haraway (1995; 2001). Com Harding, afirmamos haver um recorrente desafio na abordagem feminista e de gênero das ciências: ultrapassar o projeto “mulheres notáveis” (1996) – que se refere ao esforço de construir um levantamento de nomes de mulheres que se destacam/destacaram na construção de conhecimentos científicos – e tomar como preocupação fundamental as múltiplas dimensões dos mecanismos discriminatórios. Neste aspecto, o presente estudo preocupa-se com os discursos e práticas que permeiam a atuação das mulheres nas ciências levando em consideração duas questões fundamentais: primeiro, as relações construídas a partir da organização social do trabalho científico; segundo, as porosidades existentes entre o cotidiano de trabalho nas instituições e as outras esferas de sociabilidade nas quais estão inseridas as cientistas, tal como a vida doméstica/familiar. Por sua vez, Donna Haraway (1995; 2001) proporciona uma contribuição que nos permite um alicerce consistente para as proposições de Harding (1993; 1996) ao afirmar que: toda e qualquer ciência, todo e qualquer conhecimento científico, por ser uma construção social, deve ser “situado” em seu tempo, espaço e sujeitos. 1 Introdução: pontos de partida para um 1 Introdução: pontos de partida para um 1 Introdução: pontos de partida para um 1 Introdução: pontos de partida para um 1 Introdução: pontos de partida para um olhar feminista situado nas universidades olhar feminista situado nas universidades olhar feminista situado nas universidades olhar feminista situado nas universidades olhar feminista situado nas universidades nordestinas nordestinas nordestinas nordestinas nordestinas Reconhecemos com Haraway que as ciências são formadas por uma “multiplicidade de visões”, já que são constituídas por uma pluralidade de sujeitos, em distintos contextos sociais, econômicos e culturais. Deste modo, nas palavras de Haraway, “(...) apenas uma perspectiva parcial promete uma visão objetiva” (1995, p. 21). A abordagem parcial reconhece a diversidade e, portanto, complexidade das visões que compõem as ciências. Assim, é partindo desta perspectiva parcial que pensamos, aqui, a problemática participação de mulheres nas ciências. UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL federais nordestinas pertencentes aos estados do Ceará e Pernambuco: UFC – Universidade Federal do Ceará e UFPE – Universidade Federal de Pernambuco. Para mais bem localizar o cenário que nos rendeu as reflexões aqui esboçadas, é indispensável explicitar que estas universidades, referências empíricas de nossas análises, situam-se numa região periférica no contexto científico e tecnológico nacional: no Nordeste. A Política Nacional de Ciência, Tecnologia e Inovação (CT&I) foi construída articulando- se às desigualdades socioeconômicas e culturais entre as regiões do país. Historicamente, o eixo Norte-Nordeste e Centro-Oeste representa as regiões que movimentam menores recursos financeiros em CT&I no Brasil.1 1 De acordo com o Mapa de Investimentos do CNPq, em 2014, o Sudeste e Sul são as regiões que movimentaram maiores recursos, 45% e 22%, em ordem. Por outro lado, as regiões Nordeste, Norte e Centro-Oeste movimentaram 19%, 5% e 9% destes recursos, respectivamente. Neste contexto de periferia científica, não é objetivo desvendar as ciências e seu processo de construção em si mesmo. O mais relevante, e objeto desta investigação, é a elaboração de uma abordagem feminista que compreenda o processo de inserção e permanência de mulheres nestes espaços acadêmicos onde são produzidos conhecimentos científicos, particularmente, em dois grandes ramos de saberes2 que consideramos estratégicos: nas humanidades, incluindo-se as ciências sociais e suas áreas aplicadas; e nas ciências supostamente “exatas”,3 as quais, para esta análise, incluem áreas como engenharias e agrárias. 2 A ideia dos dois grandes ramos de saberes, ao invés das nove gran- des áreas do conhecimento clas- sificadas e reconhecidas oficial- mente pelo CNPq, nos permite manobras reflexivas para tentar situar e perceber “distinções” ou diferenciações no fazer científico de cada uma das mulheres entrevistadas, tendo em vista a possibilidade de haver discursos e práticas específicas em cada ramo de saber. Por um lado, a escolha pelo campo de conhecimentos considerados “exatos” se deu de forma a tentar compreender a atuação de mulheres cientistas em espaços onde sua presença, até hoje, ainda é pouco expressiva. Além disso, a intenção é pensar os discursos e práticas que envolvem a participação de mulheres neste ramo de saberes que, historicamente, se fez hegemônico nos marcos da modernidade das ciências ocidentais. 1.1 Situando o cenário 1.1 Situando o cenário 1.1 Situando o cenário 1.1 Situando o cenário 1.1 Situando o cenário Partindo deste diálogo feminista, as reflexões contidas neste artigo alicerçam-se em uma abordagem parcial e situada na realidade específica de universidades públicas 802 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL 3 Embora as várias áreas carreguem em si especificidades no que se referem aos seus campos de refle- xão e aos seus modos de “fazer ciência”, defendemos que todos os conhecimentos científicos são construtos históricos e sociais e, assim, todas as ciências são sociais, inclusive as “exatas”. Os saberes “exatos” alicerçam-se, sobretudo, numa racionalidade orquestrada pelas “ideias matemáticas” que deram fundamentação paradigmática ao surgimento e consolidação de uma proposta monolítica e monocolor de “Ciência Moderna Ocidental”, a qual produziu como ausentes os sujeitos (e seus saberes) que não se enquadravam nos seus parâmetros sexistas, racistas, lesbo-homo-transfóbicos, eurocêntricos e ocidentalizantes (Boaventura de Sousa SANTOS, 2006; 2010). Inicialmente, a dita “Ciência Moderna” tinha como pretensão primeira a neutralidade, cuja condição fundamental seria a construção de conhecimento resultado de um tipo específico de racionalidade: aquela utilizada nas chamadas Hard Sciences. Neste contexto, a entrada de mulheres em muitos departamentos, tais como os de matemática, física, engenharias – nas universidades ocidentais – somente se deu já no século XX (Londa SCHIENBINGER, 2001). Os saberes 803 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 VÍVIAN MATIAS DOS SANTOS VÍVIAN MATIAS DOS SANTOS ditos “exatos” representam, para os estudos de gênero e ciências, um lugar relevante para tentar compreender quais as especificidades das discriminações que permeiam o processo de construção e consolidação das carreiras científicas de mulheres em campos consolidados como espaços de predominância masculina. Por outro lado, a seleção das humanidades ocorreu por ser neste ramo onde encontramos como objeto a vida em sociedade de uma forma geral e, particularmente, por ter sido neste espaço onde a crítica feminista e os estudos de gênero surgiram. As ciências humanas representam uma das áreas que primeiro permitiram uma entrada mais considerável de mulheres. Em termos de matrículas: “As ciências humanas no Brasil é a única área do conhecimento [predominantemente] feminina” (Neide Mayumi OSADA & Maria Conceição COSTA, 2006, p. 289). Todavia, neste ramo, se observado de perto, ainda se percebem alguns mecanismos sutis – ou não – de discriminação de gênero, como pudemos constatar por meio desta abordagem parcial. 804 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 1.2 Mulheres cientistas como sujeitos 1.2 Mulheres cientistas como sujeitos 1.2 Mulheres cientistas como sujeitos 1.2 Mulheres cientistas como sujeitos 1.2 Mulheres cientistas como sujeitos “Não se pode imaginar saber mais do ofício do[a] cientista do que aquele[a] que pratica a ciência” (Gabriel PUGLIESE, 2007, p. 351). Então, esta proposta é elaborada a partir das experiências sociais de mulheres cientistas, obtidas por meio de entrevistas e observação direta nos departamentos4 onde constroem suas carreiras. Aqui, pensar sobre mulheres cientistas significa olhar para a lógica organizacional e ocupacional das universidades públicas federais, pensar sobre docentes e pesquisadoras doutoras – sujeitos que produzem conhecimentos científicos e tecnológicos. 4 Na UFC, foram entrevistadas e biografadas 3 (três) mulheres vinculadas aos Departamentos de Ciências Sociais, Física e Engenharia de Pesca. Na UFPE, foram coletados relatos orais autobiográficos de 2 (duas) mulheres pertencentes ao Departamento de Antropologia e Museologia, 1 (uma) vinculada ao Departamento de Ciência Política, e 2 (duas) atuantes no Departamento de Serviço Social. Estas mulheres foram escolhidas devido às suas performances acadêmicas reconhecidas em seus campos de estudos. Se o processo de construção da carreira das cientistas tem sido, muitas vezes, marcado pela invisibilidade – embora as mulheres sempre estivessem presentes na produção de conhecimento ao longo da história ocidental, estas foram produzidas discursivamente como ausentes, inexistentes (SANTOS, 2006) – vale explicitar que as cientistas, sujeitos desta abordagem parcial, possuem destaque e reconhecimento em seus departamentos, universidades e ramos de saberes, inseridas em redes nacionais e/ou internacionais de pesquisa. Se o processo de construção da carreira das cientistas tem sido, muitas vezes, marcado pela invisibilidade – embora as mulheres sempre estivessem presentes na produção de conhecimento ao longo da história ocidental, estas foram produzidas discursivamente como ausentes, inexistentes (SANTOS, 2006) – vale explicitar que as cientistas, sujeitos desta abordagem parcial, possuem destaque e reconhecimento em seus departamentos, universidades e ramos de saberes, inseridas em redes nacionais e/ou internacionais de pesquisa. Por meio deste olhar, situado e parcial, não pretendemos edificar generalizações, pois as reflexões aqui construídas tomam como referência trajetórias de mulheres específicas, que não compõem uma amostragem estatisticamente representativa do universo de cientistas nordestinas. Para esta abordagem de base socioantropológica, o relevante não seria ‘quantas’, mas ‘quem’ daria vida a este estudo. Por meio deste olhar, situado e parcial, não pretendemos edificar generalizações, pois as reflexões aqui construídas tomam como referência trajetórias de mulheres específicas, que não compõem uma amostragem estatisticamente representativa do universo de cientistas nordestinas. Para esta abordagem de base socioantropológica, o relevante não seria ‘quantas’, mas ‘quem’ daria vida a este estudo. UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL Isso não nos garante ponderações aplicáveis às realidades de todas e quaisquer cientistas, mas garante uma abordagem em profundidade e parcial que reconhece a pluralidade das experiências de mulheres nas ciências. Isso não nos garante ponderações aplicáveis às realidades de todas e quaisquer cientistas, mas garante uma abordagem em profundidade e parcial que reconhece a pluralidade das experiências de mulheres nas ciências. A parcialidade deste estudo nos permitiu observar a conservação de antigas questões que ainda se colocam prementes na compreensão feminista e de gênero das ciências: as mulheres ainda encontram na conciliação/articulação entre os tempos dedicados à família e à carreira um dos obstáculos mais frequentes e dificilmente transponíveis; na composição de suas carreiras científicas, ainda se percebem discursos e práticas que reiteram a “segregação sexual territorial e hierárquica” (SCHIENBINGER, 2001); e, agravando as discriminações de gênero, emerge a condição de pertencer e atuar em universidades situadas no Nordeste, região historicamente periférica na política científica e tecnológica do país. 5 Cientista “E” do Departamento de Serviço Social da UFPE entrevistada durante os meses do segundo semestre de 2013. 2.1 O conflito permanente: família 2.1 O conflito permanente: família 2.1 O conflito permanente: família 2.1 O conflito permanente: família 2.1 O conflito permanente: família versus versus versus versus versus trabalho científico trabalho científico trabalho científico trabalho científico trabalho científico O peso da responsabilidade conferida pelas atividades relativas ao âmbito doméstico, familiar, ainda aparece como um dos aspectos que mais dificulta a inserção, permanência e o reconhecimento das mulheres na carreira científica. As trajetórias das mulheres entrevistadas não expressam somente dados da vida de indivíduos, mas carregam em si normas, padrões que se estabeleceram discursivamente nos contextos onde vivenciaram suas experiências. Sem exceção, as cientistas entrevistadas percebem que, de fato, as mulheres tendem a ter menos tempo para as ciências. Elas continuam tendo que dar conta dos papéis de esposa, mãe e responsável pelo lar. Estes papéis discursivamente construídos como femininos, acarretando um acúmulo de atividades, fazem com que as cientistas tenham que construir táticas cotidianas para conseguirem ter a mesma produtividade de seus colegas do sexo masculino. Melhor dizendo, para conseguirem firmar suas carreiras científicas, as mulheres necessitam de um esforço maior do que aquele realizado por homens. Neste âmbito, vale saber que o matrimônio no Ocidente, inserido na lógica binária da diferença sexual (masculino/ feminino), atribui assimetricamente responsabilidades e deveres para mulheres e homens. Para muitas feministas e estudiosas de gênero, o casamento significa um “contrato sexual” (Carole PATEMAN, 1993) em que aos cônjuges são 805 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 VÍVIAN MATIAS DOS SANTOS VÍVIAN MATIAS DOS SANTOS determinados comportamentos, responsabilidades diferentes e desiguais. Esta conjugalidade tem suas raízes numa sociabilidade heteronormativa (Judith BUTLER, 2010), em que o parentesco, tido como heterossexual (BUTLER, 2003), assenta os seus alicerces nas imagens simbólicas de “mãe” e “pai”. As práticas discursivas que envolvem a percepção heterossexual do parentesco estão intimamente relacionadas com o ethos que preside uma sociedade ocidental firmada sobre uma noção de “direitos e deveres” calcados em relações de dominação e subordinação. Neste sentido, pode- se perceber que a família tem sido “rainha e prisioneira” do social, como sugere Jacques Donzelot (1986). O discurso “familista” tem firmado normas ao mesmo tempo em que é aprisionado por estas mesmas normas. É bem verdade que as famílias e as relações familiares têm sofrido transformações ao longo da história ocidental (Elisabeth ROUDINESCO, 2003; DONZELOT, 1986). Entretanto, ainda hoje mulheres casadas com homens têm maior dificuldade em progredir em suas carreiras, e esta realidade não é diferente para mulheres cientistas (Vivian Matias dos S. ALBUQUERQUE & Maria Helena de P. FROTA, 2006; Vívian MATIAS DOS SANTOS, 2006). 806 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 6 Cientista “B”, antropóloga do Departamento de Antropologia e Museologia da UFPE, entrevistada durante os meses do primeiro semestre de 2014. 2.1.1 Mulheres infiéis: o tempo para a ciência 2.1.1 Mulheres infiéis: o tempo para a ciência 2.1.1 Mulheres infiéis: o tempo para a ciência 2.1.1 Mulheres infiéis: o tempo para a ciência 2.1.1 Mulheres infiéis: o tempo para a ciência como o tempo roubado da família como o tempo roubado da família como o tempo roubado da família como o tempo roubado da família como o tempo roubado da família O tempo para a ciência representa, muitas vezes, nas narrativas das cientistas, a negação do tempo para a família. Indo além, o tempo dedicado ao trabalho científico é percebido como o tempo que legitimamente pertence à família e, que, todavia, lhe foi roubado. Eu gostaria de ter tido mais tempo para conviver com os meus filhos. E eu não tinha tempo porque trabalhava muito. Sempre trabalhei muito e isso significa: o trabalho formal dentro da instituição, e fora, porque para você produzir, escrever, sistematizar, planejar, você precisa de outro tempo. Então era no final de semana, noites, madrugadas. Por isso me habituei muito a trabalhar pelas madrugadas. Então isso, na verdade, te rouba tempo da tua relação com família.5 A ciência furtaria da família a presença da mãe e da esposa. Para a família conjugal heterossexual estabelecer- se na carreira científica significaria um comportamento “infiel” na medida em que subverteria a prioridade que uma mulher deveria dar aos seus papéis de mãe e esposa. E, ao contrário da infidelidade do marido, no âmbito do casamento, a infidelidade da mulher é “literalmente impensável” (ROUDINESCO, 2003, p. 22). 806 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL Enquanto, para mulheres cientistas, o casamento pode significar um obstáculo a mais, para homens, o casamento pode significar a construção da estrutura que dará o suporte necessário à intenção de consolidar sua carreira por meio de uma performance que lhe garanta o reconhecimento diante de seus pares concorrentes. Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 807 7 Cientista “A”, antropóloga do Departamento de Antropologia e Museologia da UFPE, entrevistada durante os meses do segundo semestre de 2013 e do primeiro semestre de 2014. 2.1.2 O peso da maternidade 2.1.2 O peso da maternidade 2.1.2 O peso da maternidade 2.1.2 O peso da maternidade 2.1.2 O peso da maternidade A lógica em que o tempo dedicado à ciência é sentido e compreendido como o tempo roubado da família é mais profunda quando as cientistas são mães. Nas trajetórias das entrevistadas, assim como é comum na trajetória de grande parte das mulheres, a maternidade é peça fundamental para compreender as engrenagens discursivas que permeiam a dinâmica cotidiana de conciliação entre trabalho científico e família. Em muitos estudos, a conciliação entre profissão e maternidade tem sido um dilema persistente na vida de mulheres cientistas (Attico CHASSOT, 2003; Betania MACIEL, 2004; SCHIENBINGER, 2001). Muitas mulheres optam por não terem filhos, pois percebem na maternidade um obstáculo, algumas vezes, intransponível. “Como conciliar profissão e maternidade tem sido um dos dilemas apresentados nas biografias de cientistas, artistas e literatas do século XX” (Miriam Pillar GROSSI, 2006, p. 252) e nas carreiras das cientistas sujeitos deste estudo, a maternidade não é percebida de forma diferente. As mulheres que tem filhos de zero a quinze anos, se não tiverem um suporte de apoio, não podem fazer isso [ciência]. Não adianta! Pela forma que é dividida a educação dos filhos. Então, se o marido dela não se colocar como corresponsável, ela não poderá fazer isso. Eu, depois que me separei, não passei por isso porque minha mãe morava comigo e se disponibilizava. Eu tenho amigas que não puderam fazer doutorado fora porque optaram pela família. E, se tiver filhos, é muito pior.6 É frequente, nas narrativas das cientistas, a necessidade de um suporte familiar para dar conta dos cuidados indispen- sáveis ao desenvolvimento de filhos e filhas. Neste aspecto, percebemos que tal suporte é mais frequentemente proporcionado por outras mulheres da família, ou, mesmo, empregadas domésticas. Com pouca frequência as entrevistadas consideram que as várias atividades que envolvem a criação de filhos e filhas são equitativamente divididas com seus maridos e/ou companheiros. Dentre as entrevistadas, duas optaram por não terem filhos. Coincidentemente ou não, as duas cientistas que 807 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 VÍVIAN MATIAS DOS SANTOS VÍVIAN MATIAS DOS SANTOS perceberam a impossibilidade de – ou que não desejaram – maternar atuam em espaços tradicionalmente ocupados por homens: o Departamento de Física da UFC; e o Departamento de Ciência Política da UFPE. 808 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 8 Cientista “F”, socióloga do Departamento de Ciências Sociais da UFC, entrevistada durante os meses do primeiro semestre e do segundo semestre de 2011. 2.1.2 O peso da maternidade 2.1.2 O peso da maternidade 2.1.2 O peso da maternidade 2.1.2 O peso da maternidade 2.1.2 O peso da maternidade Entretanto, o fato de não serem mães não significa que estejam imunes às críticas e constrangimentos: “a falta de filhos compromete, em muitos casos, a imagem de mulher destas cientistas, colocando-as sob suspeição de anormalidade e desvio” (GROSSI, 2006, p. 252). Escolher não ter filhos, mesmo estando inseridas no contexto da heteroconjugalidade, vai de encontro à lógica de maternidade que emerge na sociedade ocidental pós-século XVIII como o destino feminino “normal”, “natural” (Elisabeth BADINTER, 1985). A glorificação da maternidade e o exagero de responsabilidades atribuídas às mães são relativamente recentes, tendo sido intensamente reforçadas apenas nos séculos XVIII e XIX. Antes disso, a criação dos filhos estava integrada a outros afazeres das mulheres, e não era considerada uma de suas principais tarefas. No contexto ocidental, a necessidade de sobrevivência na economia pré-industrial requeria, não apenas dos homens, mas, também, das mulheres, que o trabalho de produção fosse priorizado sobre as preocupações reprodutivas (Maria Lúcia ROCHA-COUTINHO, 2005, p. 122). Nos dias atuais, nos lugares e sujeitos tomados como nossas referências, há um complexo paradoxo: as mulheres são alvos de comentários depreciativos por parte de seus pares por serem mães e por não o serem. E, mais grave, o processo de gravidez e maternidade, na experiência de uma cientista específica, chega a ser elemento desencadeador de escárnio ou/e assédio moral. Na primeira reunião, com todo o corpo docente do curso de Antropologia e Museologia – eu tinha feito o concurso e engravidado depois e, até eu ser convocada, já estava com cinco meses de gestação [...]. Aí um colega disse: ‘Estamos aqui reunidos, infelizmente aconteceu isso!’. Então, ele apontou para minha barriga. Tipo: ‘Infelizmente!’. Falou como se minha gravidez fosse algo que prejudicaria o grupo. Então, tem-se uma visão bem instantânea da coisa: ‘Essa professora vai passar seis meses fora?’ [...] Não se leva em consideração que poderei passar trinta anos na UFPE.7 Na experiência narrada acima, nota-se que, mesmo nas humanidades, os mecanismos discriminatórios de gênero não necessariamente se dão de forma sutil. Observa- se, por meio desta experiência particular, que “as instituições científicas – universidades, academias e indústrias – foram estruturadas sobre a suposição de que os cientistas seriam homens com esposas em casa para cuidar deles e de suas 808 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL famílias” (SCHIENBINGER, 2001, p. 69). Isso implica pensar como as instituições científicas não são espaços apropriados para as mulheres. As demandas específicas do contexto científico contemporâneo nos países capitalistas, no Brasil e no Nordeste, recaem de forma especificamente desgastante e desumana quando as cientistas são mulheres. Como ser esposa, mãe, docente e pesquisadora produtiva? 2.1.3 Ser mulher e produtiva 2.1.3 Ser mulher e produtiva 2.1.3 Ser mulher e produtiva 2.1.3 Ser mulher e produtiva 2.1.3 Ser mulher e produtiva Assim como pode ser percebido em outros campos profissionais, permanecem, na carreira científica de mulheres, os conflitos provenientes da conciliação entre trabalho e família. Entretanto, há, no trabalho científico, uma especificidade: o tempo de permanência na universidade – salas de aula, laboratórios e demais espaços onde exercem suas funções de docentes e pesquisadoras – não é suficiente para dar conta de todas as atividades necessárias às suas produções. ç Na vida das entrevistadas emerge como necessária a extensão do trabalho científico para o espaço doméstico. É trabalhando em suas casas, além do tempo de permanência nos departamentos, que estas mulheres elaboram estratégias para produzir, conforme as exigências hegemônicas no campo político-científico. Então, assim [trabalhando em casa] eu aprendi. Às vezes meu marido diz: ‘Ah! Você é muito concentrada!’. Isso porque eu aprendi a escrever ouvindo choro de menino, ouvindo telefone. Às vezes, a casa cai e eu estou escrevendo. [...] Para compensar a dispersão, eu tenho várias estratégias na minha vida. Tipo: de manhã, bem cedinho, eu amanheço com mais lucidez, aí é quando eu escrevo. [...] Quando eu estou muito cansada e atarefada [...], eu pego, por exemplo, cinco horas da manhã. Se durante uma semana você acordar todo dia às cinco, você vai ter vários escritos. Ou, quando eu não posso escrever um texto porque estou cheia de trabalho, eu escrevo assim: três linhas num dia, para não deixar morrer. É como se você aguasse uma planta. Aqui, aqui, aqui... Todo dia... Com uma semana ele está um pouquinho grande. Aí já não é folha em branco. Folha em branco é angustiante!8 Para manter performances produtivas em seus ramos de saberes, é comum as cientistas atribuírem importância às táticas e estratégias por elas elaboradas cotidianamente. Para estas mulheres, sem tais negociações estratégicas de seus tempos e das atividades executadas por seus corpos, ser produtiva e obter reconhecimento em suas carreiras significaria um feito impossível. 809 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 VÍVIAN MATIAS DOS SANTOS Destaca-se, nas narrativas das cientistas, que ser produtiva significa não somente pesquisar, mas divulgar os resultados de suas pesquisas publicando em eventos científicos, livros e, sobretudo, em periódicos reconhecidos em suas áreas. Neste sentido, a escrita aparece como um dos aspectos mais presentes em suas trajetórias científicas. A ideia de ser produtiva está vinculada visceralmente à ideia de escrever, publicar. 9 Cientista “E” do Departamento de Serviço Social da UFPE. 10 Cientista “G” do Departamento de Engenharia de Pesca da UFC, entrevistada durante os meses do primeiro e segundo semestres de 2011. 2.1.3 Ser mulher e produtiva 2.1.3 Ser mulher e produtiva 2.1.3 Ser mulher e produtiva 2.1.3 Ser mulher e produtiva 2.1.3 Ser mulher e produtiva Publicar, por sua vez, somente é possível pela prática da pesquisa e, além disso, as pesquisas somente encontram repercussão e reconhecimento mediante a sua publicização. Ao publicarem, tornam-se conhecidas, poderão ser referências para gerações seguintes de textos publicados em seus campos de estudo (Bruno LATOUR, 2000). “Ser citada” significa um ponto articulador indispensável para suas carreiras. Neste sentido, publicar não é um fim em si mesmo, mas um meio pelo qual pesquisas são divulgadas, tornam-se conhecidas, podendo tornar-se reconhecidas. Para as entrevistadas, este é o caminho para a consolidação de suas carreiras científicas. Estas cientistas têm performances de alta produtivi- dade, superando as médias brasileiras de publicação de artigos científicos em suas respectivas grandes áreas do conhecimento. Observando os seus cotidianos na universida- de, alicerçando tal produtividade, percebemos haver uma intensa carga de trabalho, considerada por estas mulheres como “pesada” e até “desumana”, porém, “inevitável”: além das atividades de ensino no nível de graduação e/ou pós- graduação, orientando monografias, dissertações e teses, todas as cientistas entrevistadas coordenam laboratórios, grupos e/ou núcleos de pesquisa; em algum momento de suas trajetórias dedicaram-se a cargos de gestão universitária; e, com frequência, viajam para participarem de eventos científicos e firmarem parcerias com redes de pesquisa no Brasil e no exterior. Na busca por reconhecimento e por consolidar suas carreiras, estas mulheres, em algumas de suas narrativas, deixam claro haver na lógica acadêmica das universidades públicas federais uma hierarquização entre as atividades de ensino, extensão e pesquisa. A pesquisa aparece como prioridade na política das universidades. Na verdade, você faz um concurso para ser professor, mas o que vai te identificar, não é se você é um excelente professor, é se você é um pesquisador, se tem os títulos que universalmente, no âmbito nacional, são consagrados a um bom pesquisador. O teu referencial não é ser um docente, mas passa a ser: o pesquisador. Nessa hora, eu acho que há uma dissociação entre o ensino, a extensão e a pesquisa.9 810 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 810 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL F t El b ã i d í l d i ti t t i t d di í i Pl t f Produtividade das cientistas – 2000-2014 Produtividade das cientistas – 2000-2014 q * Classificação referente aos ramos de saberes considerados estratégicos para esse estudo. ** Referente à média de produtividade de pesquisadores e pesquisadoras, no Brasil, nas respectivas grandes áreas do conhecimento das cientistas entrevistadas. Elaboração por meio de dados fornecidos pelo Diretório de Grupos de Pesquisa no Brasil – Lattes/CNPq (2000 a 2014). Embora as cientistas entrevistadas sejam produtivas, constroem críticas veementes ao produtivismo, cuja palavra de ordem é “publicar, publicar e publicar!”10 em detrimento das atividades de ensino e extensão. Por meio dos olhares das cientistas, percebemos que a lógica produtivista é compreendida como algo contem- porâneo, novo, e que há, intrínseco ao disciplinamento necessário aos padrões de produtividade, um processo de proletarização do trabalho científico. Hoje em dia o tempo se inseriu no nosso trabalho. Antes a gente associava o tempo ao trabalho do operário. O operário era aquele que tinha o tempo como um elemento, quase de escravização do seu trabalho. Marx relata isso lindamente na sua obra. [...] E hoje nós temos o tempo de trabalho que talvez não 811 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 VÍVIAN MATIAS DOS SANTOS VÍVIAN MATIAS DOS SANTOS chegue a ser uma “mais-valia”, mas é uma “mais qualquer coisa”. Alguém vai ter que discutir o que é esse trabalho que se carrega no corpo. Que você tem que ir para casa e ficar espremido por esse tempo e por essa necessidade absoluta de permanente grau de crescimento que nós temos.11 Esta sensação de controle do tempo, dos corpos e do que se produz não se trata de uma questão presente somente nas realidades dos departamentos aos quais as cientistas entrevistadas são vinculadas, conforme afirma Santos: Por um lado, a comunidade científica estratificou-se, as relações de poder entre cientistas tornaram-se mais autoritárias e desiguais e a esmagadora maioria dos cientistas foi submetida a um processo de proletarização no interior dos laboratórios e dos centros de investigação. Por outro lado, a investigação capital- intensiva (assente em instrumentos caros e raros) tornou impossível o livre acesso ao equipamento, o que contribuiu para o aprofundamento do fosso, em termos de desenvolvimento científico e tecnológico, entre países centrais e países periféricos (2005, p. 57). 12 Elaboração nossa por meio do entrecruzamento dos dados fornecidos pelo Diretório dos Grupos de Pesquisa no Brasil e Fomento do CNPq (2000-2014) e das informações provenientes dos currículos lattes das docentes pesquisadoras. 11 Cientista “F”, socióloga do Departamento de Ciências Sociais da UFC. UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL Trata-se de um processo amplo que acompanha o desenvolvimento das sociedades capitalistas, embora em cada lugar isso se dê de formas particulares. E, no caso das universidades do Nordeste brasileiro, a proletarização é percebida como intensificada, conforme será discutido mais adiante. 812 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 13 Cientista “H” do Departamento de Física da UFC, entrevistada durante os meses do segundo semestre de 2010 e primeiro semestre de 2011. UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL tendem a movimentar menores recursos com suas pesquisas (MATIAS DOS SANTOS, 2012). Sabendo que “a luta mais importante no seio da comunidade científica é a luta pela utilização dos investimentos públicos e privados” (SANTOS, 1978, p. 41), por que as mulheres movimentam menores recursos? Que mecanismos se estabelecem para que seus projetos sejam mais ‘baratos’ que os de seus colegas homens? Neste aspecto, a fala de uma cientista suscitou-nos reflexões esclarecedoras: Suspeito que um pesquisador solicita recursos para executar projetos de acordo com a sua disponibilidade de tempo. E, como as mulheres têm que dar conta de várias outras atividades em seu cotidiano, tal como sua vida familiar, elas pedem menores recursos para projetos menores, visto que seu tempo é reduzido para a ciência.13 Aparentemente, a constatação preocupante de que as mulheres acessam menores recursos da política de fomento à pesquisa está intrinsecamente vinculada ao fato de as mulheres terem menos tempo para as ciências. Isso implica pensar que não poderemos compreender as desigualdades de gênero no campo científico sem olhar, também, para o cotidiano dessas mulheres fora das universidades, em seus lares, com as suas famílias. 2.1.4 Acesso desigual aos recursos em CT&I 2.1.4 Acesso desigual aos recursos em CT&I 2.1.4 Acesso desigual aos recursos em CT&I 2.1.4 Acesso desigual aos recursos em CT&I 2.1.4 Acesso desigual aos recursos em CT&I Devido à sobrecarga de trabalho – doméstico e científico –, e não por serem menos qualificadas, de forma geral, as mulheres tendem a publicar menos (MATIAS DOS SANTOS, 2012). Todavia, as cientistas entrevistadas chegam a ter uma média de publicação anual entre 8,15 e 0,07 pontos acima da média nacional de publicação12 de artigos em suas respectivas grandes áreas. Mesmo sendo mais produtivas que a média de pesquisadores/as no Brasil, suas produtividades não são constantes: no período em que os filhos são pequenos, suas produtividades tendem a declinar. Cuidando das crianças, as mulheres publicam menos e participam de eventos científicos com menor frequência. Na conflituosa conciliação entre o tempo para a família e para o trabalho, estas cientistas acabam tendo menos tempo para as ciências se comparadas aos seus pares do sexo masculino. Isso repercute no acesso desigual aos recursos destinados à produção de conhecimentos: as mulheres 2.2.1 Lugares para homens ou para mulheres? 2.2.1 Lugares para homens ou para mulheres? 2.2.1 Lugares para homens ou para mulheres? 2.2.1 Lugares para homens ou para mulheres? 2.2.1 Lugares para homens ou para mulheres? As consequências da transgressão das fronteiras As consequências da transgressão das fronteiras As consequências da transgressão das fronteiras As consequências da transgressão das fronteiras As consequências da transgressão das fronteiras Há uma intensificação dos mecanismos discrimina- tórios de gênero quando a luta cotidiana por consolidação da carreira das cientistas se dá em áreas tradicionalmente masculinas. A lógica sexista parece permanecer na organização social do trabalho científico (HARDING, 1996) nas diversas áreas do conhecimento, no seio dos distintos ramos de saberes. Olhar para o ramo de saberes dito “exato” nos remete à reflexão sobre espaços científicos ocupados tradicional- mente por homens.16 Em contrapartida, pensar nas humani- dades, frequentemente, faz emergir a (pré)noção de que este ramo de saberes é o mais equitativo do ponto de vista das relações de gênero. Entretanto, as narrativas das cientistas nos mostram que, mesmo em departamentos em que a presença de homens e mulheres se dá numericamente equilibrada, isso não implica a ausência de práticas discriminatórias. 16 Neste sentido, um trecho do diário de campo construído por ocasião do primeiro contato com o Departamento de Física da UFPE faz-nos perceber a “naturalização” da ideia que afirma as ciências ditas “exatas”, no caso, a Física, como um lugar para homens. Segue: “Eu não sabia onde se localizava este Centro de Ciências Exatas no interior do campus de Recife e por isso resolvi pedir informação a um rapaz que passou pelo caminho. Por sorte, ele afirmou que estava indo para o mesmo local, então fomos juntos, cami- nhando. Nesta oportunidade ele questionou sobre o que eu desejava no Centro e sobre o que eu pesquisava, então expliquei que eu não era pertencente àquele Centro e que estava desenvolvendo uma pesquisa sobre a inserção de mulheres na ciência, que buscava entender questões como: por que o número de mulheres no curso de Física é tão reduzido? Prontamente aquele estudante de doutorado em Química me respondeu: ‘Mas isso é obvio, mulheres não estudam física por que física é difícil!’”. E, ainda, no seio das humanidades, podemos encon- trar campos de estudos e disciplinas permeadas pela lógica da divisão sexual do conhecimento, como, por exemplo, o caso da Ciência Política que emerge nas ciências sociais construída como uma disciplina tradicionalmente masculina. Especificamente, o Departamento de Ciência Política estudado é um espaço de predominância masculina: dos 17 docentes efetivos do Departamento, apenas 3 (três) são mulheres. Neste sentido, pudemos perceber, mais uma vez, que também no seio das ciências sociais opera a lógica da segregação sexual territorial e hierárquica. Como isso acontece? 2.2 Segregação sexual territorial e 2.2 Segregação sexual territorial e 2.2 Segregação sexual territorial e 2.2 Segregação sexual territorial e 2.2 Segregação sexual territorial e hierárquica hierárquica hierárquica hierárquica hierárquica No Brasil, não somente nas universidades, mas no contexto da Política de CT&I Nacional, ainda há uma explícita segregação sexual territorial (divisão sexual das áreas de conhecimento) agravada pela segregação sexual hierárquica (SCHIENBINGER, 2001): em todas as áreas, as mulheres ainda encontram maiores dificuldades para ocupar cargos de prestígio nas universidades, bem como continuam pouco expressivas nos cargos decisórios da política científica e tecnológica nacional.14 14 Historicamente, as principais instituições deliberativas e de fomento no Brasil representam lugares “feitos por homens e para homens”. Como exemplo, temos o caso do CNPq, principal agência de fomento à pesquisa no país, que se constituiu como espaço ocupado – principalmente em cargos decisórios – por homens (MATIAS DOS SANTOS, 2012). Referindo-se às que serviram como referência para este estudo, temos universidades em que, durante toda a sua existência, jamais uma mulher ocupou a reitoria. Segundo as narrativas das cientistas, as mulheres têm maiores dificuldades em se inserirem nos cargos mais elevados da gestão institucional, em acumular “capital científico-político” (Pierre BOURDIEU, 2004). Isso pode ser constatado na seguinte fala:15 “Quando eu fui pró-reitora eu me destaquei, então fizeram uma manobra política para que eu não lançasse candidatura para a reitoria. Todas as chapas que saíram eram lideradas por homens”. 15 Cientista “D” do Departamento de Serviço Social da UFPE, entrevis- tada no mês de outubro de 2014. Na realidade dos departamentos destas cientistas, a segregação sexual territorial e hierárquica parece ser um 813 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 VÍVIAN MATIAS DOS SANTOS VÍVIAN MATIAS DOS SANTOS dos mecanismos mais explícitos e presentes, capazes de continuar reiterando discursos e práticas discriminatórias. dos mecanismos mais explícitos e presentes, capazes de continuar reiterando discursos e práticas discriminatórias. Dentro das ciências eu acho que a ciência política é a mais masculina, na verdade. Comparando não só com 17 Cientista “C” do Departamento de Ciência Política da UFPE, entrevistada no mês de março de 2014. 18 Cientista “C” do Departamento de Ciência Política da UFPE. 2.2.1 Lugares para homens ou para mulheres? 2.2.1 Lugares para homens ou para mulheres? 2.2.1 Lugares para homens ou para mulheres? 2.2.1 Lugares para homens ou para mulheres? 2.2.1 Lugares para homens ou para mulheres? As consequências da transgressão das fronteiras As consequências da transgressão das fronteiras As consequências da transgressão das fronteiras As consequências da transgressão das fronteiras As consequências da transgressão das fronteiras Sabendo-se que a inserção das mulheres no campo político, no Brasil, se dá de forma desigual, haveria um reflexo desta questão no que se refere ao interesse de mulheres pesquisadoras por problemáticas relativas à política? p q p p p De acordo com Miriam Pillar Grossi e Sônia Malheiros Miguel (2001), ainda parece haver, no Brasil, um processo que constrói os cargos políticos como lugares a serem ocupados por homens. Em seu texto, as autoras citam uma entrevista realizada com uma Deputada Estadual de São Paulo, que afirma: “Espera-se que as mulheres façam o dobro dos homens, na metade do tempo e sem mérito algum [...]. Acho que isso marca muito o nosso trabalho” (GROSSI & MIGUEL, 2001, p. 181). Dentro das ciências eu acho que a ciência política é a mais masculina, na verdade. Comparando não só com 814 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL o departamento da UFPE, mas com o departamento de outras universidades, isso é bastante comum: a gente ter um departamento composto quase 100% por homens. Isso acaba se reproduzindo. Tanto é que aqui na UFPE acho que a primeira professora mulher foi aparecer com três anos atrás [...]. É um pouco complicado para você se inserir dentro do departamento. E também, em relação aos alunos, você sempre tem essa limitação: pelo fato de você ser mulher já deve ter certo cuidado. E no meu caso, como sendo uma mulher mais nova, se torna mais complicado um pouquinho. Você não é vista como pesquisadora, como professora. Você na verdade é sempre vista como a monitora, né? Então isso é bastante comum aqui.17 Com esta narrativa, parece que a ciência política, seguindo uma tradição, se estabelece como uma área não somente de predominância numérica masculina, mas como uma área em que, para conseguirem reconhecimento, as mulheres enfrentam maiores obstáculos do que aqueles enfrentados por mulheres atuantes em áreas que não têm predominância de homens. Entretanto, esta possível discriminação aparenta não existir. Seria uma discriminação “velada”. Para a cientista “C”, fica evidente que o fato de ser uma mulher, em um departamento de maioria masculina, acarreta alguns tensionamentos no trabalho: Eu acho que a discriminação que existe é muito mais velada. É muito mais inconsciente, certo? Em alguns sentidos você tem uma preocupação: ‘Não, não vamos falar isso porque ela está aqui, ela não pode ouvir!’, ou, ‘Não vamos pedir para ela fazer tal coisa porque isso não é atividade de mulher’, ou, ‘Não vamos deixar ela sozinha com os alunos porque pode pegar mal!’. Então, tem assim, alguma coisa mais inconsciente. Na verdade, os próprios professores nem notam que estão tendo esse tipo de comportamento.18 Esta narrativa nos fez perceber que as situações em que se sentiu discriminada revelaram-se alicerçadas por duas dimensões principais: a suposta incapacidade feminina para certos assuntos; e a percepção das mulheres como objetos do desejo sexual dos homens. A primeira dimensão destas práticas discriminatórias diz respeito à crença numa suposta incapacidade feminina para a execução de determinadas atividades. Ao ouvir de seus pares do sexo masculino frases do tipo “Não vamos pedir para ela fazer tal coisa porque isso não é atividade de mulher”, a cientista sente-se tolhida da possibilidade de participar efetivamente das diversas atividades e dos distintos espaços do Departamento. UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL A segunda dimensão é a percepção das mulheres como objetos dos desejos sexuais dos homens do Departamento: 815 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 VÍVIAN MATIAS DOS SANTOS VÍVIAN MATIAS DOS SANTOS alunos e docentes. A ideia recorrente expressa na frase “Não vamos deixar ela sozinha com os alunos porque pode pegar mal!” pode significar, na execução das atividades cotidianas, uma forte vigilância sobre o corpo e a sexualidade feminina. O discurso ambíguo onde os docentes, aparentemente, estão tentando proteger a reputação da colega, traduz-se como um dos mais potentes entraves para a conquista de espaço, de reconhecimento de seu trabalho. ç Por outro lado, as dificuldades que as mulheres enfrentam em áreas de predomínio masculino não parecem ser vivenciadas por homens atuantes em carreiras tradicionalmente femininas: Durante minha carreira de 30 anos no Departamento de Serviço Social, acho que nunca vi um homem ter mais dificuldades que as mulheres. Ao contrário, percebo que a competição é estabelecida entre as mulheres, como se os homens ficassem fora da disputa. Os homens – que são minoria – são bem recebidos, acolhidos. Eu diria, até, que são cuidados e protegidos pelas colegas. Facilmente os homens atuantes no Serviço Social conseguem um lugar de destaque.19 Sendo o Serviço Social um espaço tradicionalmente ocupado por mulheres, na UFPE, poucos foram os homens que atuaram como docentes nesta área. Entretanto, segundo as narrativas da cientista acima citada, mesmo sendo minoria numérica neste espaço, isso não significou uma condição de subalternidade no Departamento. “Eles são minoria, mas terminam desenvolvendo um relacionamento muito afetuoso com as mulheres. [...] A concorrência se dá entre as próprias mulheres. Os homens são enaltecidos”.20 Esta fala explicita uma questão complexa no âmbito da discriminação de gênero nas ciências: por meio desta e de outras pesquisas anteriores, sabemos que, em uma mão, há uma difícil inserção e consolidação da carreira de mulheres cientistas em áreas tradicionalmente masculinas, como na Física, por exemplo; em outra, isso parece não ocorrer com homens atuantes em carreiras construídas historicamente como femininas. Esta problemática nos fez perceber que, para compreender a complexidade pela qual os mecanismos discriminatórios incidem sobre as carreiras das mulheres cientistas, emergem como necessários estudos que abordem, também, a presença de homens em espaços de predomi- nância feminina. 20 Cientista “D” do Departamento de Serviço Social da UFPE. 19 Cientista “D” do Departamento de Serviço Social da UFPE. 2.3 “Humanidades” e “exatas”: fazeres 2.3 “Humanidades” e “exatas”: fazeres 2.3 “Humanidades” e “exatas”: fazeres 2.3 “Humanidades” e “exatas”: fazeres 2.3 “Humanidades” e “exatas”: fazeres distintos? distintos? distintos? distintos? distintos? Observando os fazeres das cientistas em seus ramos de saberes, pudemos reafirmar que todos os conhecimentos, 816 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 21 Analisar a distribuição de Bolsas de Produtividade significa analisar como se estabelecem as hierar- quias entre pesquisadores/as das diversas regiões na política científica nacional. Neste âmbito, percebe-se a liderança do eixo Sudeste-Sul, detentor de 81% das Bolsas de Produtividade, enquanto que o Nordeste, Norte e Centro- Oeste, juntos, possuem apenas 19% destas bolsas (Mapa de Investimentos do CNPq, 2014). UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL inclusive os ditos “exatos”, são socialmente construídos: é indispensável o diálogo com estudos produzidos por outros/ as pesquisadores/as anteriormente e em outros lugares, inserindo as cientistas em “linhagens” (Mariza PEIRANO, 1995) acadêmicas; ao mesmo tempo, tais conhecimentos são socializados por meio do processo de orientação de estudantes, garantindo-se descendentes. Assim é assegurada a perenidade das ideias. Neste aspecto, sobressaiu-se outro ponto comum importante: tanto nas humanas quanto nas “exatas”, posicionar-se teoricamente demarca a posição das cientistas em seus departamentos: as disputas teóricas traduzem-se como disputas políticas no seio da comunidade científica. Por outro lado, mesmo identificando alicerces comuns, por meio dos fazeres das cientistas, percebemos nos ramos de saberes elementos de diferenciação, tanto no que se referem às performances científicas, quanto aos aspectos objetivos/ estruturais que envolvem a produção de conhecimentos. Nas performances científicas das entrevistadas percebemos: primeiro: nas “exatas”, as cientistas dedicam maior quantidade de horas aos laboratórios, executando e coordenando experimentos, enquanto nas humanas as incursões em campo são percebidas como aquilo que demanda um tempo significativo; segundo: nas “exatas”, as características da organização do trabalho promovem a prática mais intensa de publicação em coautorias, já que diversos pesquisadores/as e estudantes participam das várias etapas procedimentais dos experimentos. Copartícipes são potenciais coautore/as. Vale dizer que as cientistas, nas humanas e sociais aplicadas, reconhecem como necessário um deslocamento para “estar fora em contato com o outro” e, paradoxalmente, na construção de saberes e nas suas escritas explicitam como relevante certo isolamento, sendo frequente a ideia da produção científica como um processo solitário. Esta dimensão torna-se reafirmada ao analisarmos as “redes de colaboração” nos currículos lattes das cientistas: enquanto, nas humanas, observamos uma presença média de 3 (três) coautorias, nas “exatas”, identificamos uma média de 52,5 coautorias. Referindo-se aos aspectos objetivos/estruturais que perpassam tais performances científicas, percebemos que os projetos de pesquisa das cientistas atuantes nas “exatas” movimentam maiores recursos, bem como os laboratórios por elas coordenados necessitam de um investimento financeiro consideravelmente maior em equipamentos. A relevância do acesso aos recursos para aquisição de equipamentos é ressaltada pelas cientistas na física e na engenharia. UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL O fato de possuir ou não determinados equipamentos, máquinas e 817 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 VÍVIAN MATIAS DOS SANTOS VÍVIAN MATIAS DOS SANTOS materiais em geral repercute diretamente nas possibilidades de produção de conhecimento de um grupo, bem como nas condições de reconhecimento no seio da comunidade científica (Bruno LATOUR & Steve WOOLGAR, 1997). Nas universidades analisadas, em 2014, os ramos de saberes “exatos” – que nesta abordagem agregam as Exatas e da Terra, Agrárias, Engenharias e Computação – foram contemplados com 64% e 62% dos recursos destinados ao apoio à pesquisa pelo CNPq (2014) na UFPE e UFC, respectivamente. Já as humanidades – somando as Humanas e Sociais Aplicadas – não ultrapassaram a captação de 5% dos recursos nas duas universidades. Como um reflexo da política científica nacional, nas universidades onde as cientistas, sujeitos deste estudo, tecem suas carreiras, observa-se a hegemonia dos saberes “exatos” em termos capital-intensivos. E, considerando que é neste ramo onde se encontra maior parte das áreas de predominância masculina, como as mulheres poderiam ter acesso igualitário aos recursos destinados à pesquisa? UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL Difícil!... Bastante difícil!... Na verdade, a gente tem uma série de limitações para desenvolver ciência no Brasil. E, especificamente em Pernambuco, e especificamente como mulher, que se torna um pouco mais difícil. [...] Principalmente na área de ciência política, porque a gente está falando de uma área tradicionalmente masculina, tradicionalmente Sudeste. Então, às vezes, a gente tem pouca inserção, pouco acesso a certas discussões também por uma limitação já inicial. Mesmo que a gente tenha qualificação para fazer algumas atividades, a gente tem várias dificuldades para fazer, principalmente aqui. [...] Eu acho que a principal limitação é de infraestrutura e de recursos.22 Difícil!... Bastante difícil!... Na verdade, a gente tem uma série de limitações para desenvolver ciência no Brasil. E, especificamente em Pernambuco, e especificamente como mulher, que se torna um pouco mais difícil. [...] Principalmente na área de ciência política, porque a gente está falando de uma área tradicionalmente masculina, tradicionalmente Sudeste. Então, às vezes, a gente tem pouca inserção, pouco acesso a certas discussões também por uma limitação já inicial. Mesmo que a gente tenha qualificação para fazer algumas atividades, a gente tem várias dificuldades para fazer, principalmente aqui. [...] Eu acho que a principal limitação é de infraestrutura e de recursos.22 Segundo a cientista “B”, do Departamento de Antropologia e Museologia da UFPE, há um “espírito de colonizado” que assombra os/as docentes, que, apesar de criticarem os parâmetros estipulados pelo eixo sudeste-sul, batalham para se adequar a estes, abrindo mão de sua autonomia e reforçando/legitimando as desigualdades regionais na política científica. Indo além, tivemos como sujeitos deste estudo mulheres pertencentes a distintas gerações de cientistas – desde mulheres com menos de 10 anos de carreira até mulheres com 30 anos de atuação acadêmica. Conhecer as trajetórias destas mulheres pertencentes a distintas gerações possibilitou-nos notar que, se agregando à dupla discriminação – de gênero e regional – destacam-se, nos departamentos analisados, ainda, desigualdades geracionais. As desigualdades geracionais ocorrem de forma contraditória: se, por um lado, os critérios de produtividade – intensificados a partir dos anos 1980/1990 no Brasil – são mais bem assimilados nos ritmos de trabalho de jovens pesquisadoras, por outro, observa-se que pesquisadoras em início de carreira inserem-se de forma mais precária nos contextos dos departamentos analisados. 3.1 Gênero, geração e pertencimento regional 3.1 Gênero, geração e pertencimento regional 3.1 Gênero, geração e pertencimento regional 3.1 Gênero, geração e pertencimento regional 3.1 Gênero, geração e pertencimento regional Historicamente, o desenvolvimento capitalista no Brasil se deu de forma desigual entre as distintas regiões. Sudeste e Sul protagonizaram o processo de industrialização e urba- nização do país e, acompanhando este desenvolvimento desigual, também se construiu a política científica nacional.21 Olhar para a realidade específica destas universidades nordestinas fez-nos perceber uma questão relevante: embora estas universidades não tenham acesso aos mesmos recursos e não disponham de infraestruturas semelhantes às das universidades do eixo Sudeste-Sul, há uma cobrança por produtividade baseada nos parâmetros daquelas instituições – que lideram a produção científica e tecnológica no país. A cobrança por alta produtividade em um contexto de condições de trabalho precárias atinge todas/os as/os docentes e pesquisadores/as, independente de seu sexo e identidade de gênero. Todavia, atinge homens e mulheres de formas distintas. Historicamente, o desenvolvimento capitalista no Brasil se deu de forma desigual entre as distintas regiões. Sudeste e Sul protagonizaram o processo de industrialização e urba- nização do país e, acompanhando este desenvolvimento desigual, também se construiu a política científica nacional.21 Olhar para a realidade específica destas universidades nordestinas fez-nos perceber uma questão relevante: embora estas universidades não tenham acesso aos mesmos recursos e não disponham de infraestruturas semelhantes às das universidades do eixo Sudeste-Sul, há uma cobrança por produtividade baseada nos parâmetros daquelas instituições – que lideram a produção científica e tecnológica no país. A cobrança por alta produtividade em um contexto de condições de trabalho precárias atinge todas/os as/os docentes e pesquisadores/as, independente de seu sexo e identidade de gênero. Todavia, atinge homens e mulheres de formas distintas. Por meio das experiências das cientistas, percebemos que, na composição de suas carreiras, permeia uma dupla discriminação: ser mulher e nordestina no campo científico brasileiro. As desigualdades regionais que marcam historicamente a Política de CT&I no Brasil são percebidas pelas mulheres entrevistadas como definidoras e limitantes em suas carreiras. 818 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 818 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 22 Cientista “C” do Departamento de Ciência Política da UFPE. Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 819 UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL As entrevistadas, ao narrarem os cotidianos de trabalho em seus Departamentos, proporcionaram-nos a percepção das seguintes questões sobre mulheres em início de carreira científica: 1) quando não são doutoras – não podem concorrer aos editais de apoio a projetos de pesquisa, nem podem pleitear bolsas de iniciação científica –, as pesquisas são executadas de forma precária, as quais, sem o auxílio de bolsistas, tornam-se sobrecarga de trabalho. Na mesma medida, acabam sendo executadas de forma limitada, com recursos próprios, uma vez que não houve financiamento institucional para o seu desenvolvimento; 2) quando são contratadas temporariamente, a exemplo do cargo de professor/a substituto/a, mesmo participando de grupos de pesquisa consolidados, devem dedicar-se exclusivamente 819 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 VÍVIAN MATIAS DOS SANTOS VÍVIAN MATIAS DOS SANTOS ao ensino de graduação, sendo-lhes subtraídas a pesquisa e a extensão. Por outro lado, paradoxalmente, algumas mulheres reconhecem que as exigências produtivistas no cenário científico, refletidas nas universidades como supervalorização das atividades de pesquisa e desvalorização do ensino e extensão, se dão permeadas, também, por discriminações geracionais, tendo em vista que o ritmo de produção daqueles e daquelas que já estão nas universidades há mais de duas décadas não é o mesmo dos/as jovens recém- ingressos/as. Essa política é burra, é idiota! Um Departamento não se faz só de publicações, tem várias outras questões que precisam ser feitas. Como é que você não pontua o ensino, a produção técnica? Ela é uma política estúpida e extremamente neoliberal! Ela é massacrante, desumana! E ela desconsidera as pessoas mais velhas, que não foram formadas nesse perfil.23 23 Cientista “B” do Departamento de Antropologia e Museologia da UFPE. 24 Compreendemos por cientistas que possuem destaque aquelas que lideram laboratórios, núcleos e/ou grupos de pesquisa inseridos em redes de pesquisa nacional e/ou internacional, que possuem uma média de produtividade (publicações) acima da média de seus pares em suas respectivas áreas, podendo ser bolsistas de produtividade do CNPq. 820 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 820 Estudos Feministas, Florianópolis, 24(3): 801-824, setembro-dezembro/2016 3.2 Quem são as cientistas que se destacam? 3.2 Quem são as cientistas que se destacam? 3.2 Quem são as cientistas que se destacam? 3.2 Quem são as cientistas que se destacam? 3.2 Quem são as cientistas que se destacam? Mulheres cis, brancas, heterossexuais, Mulheres cis, brancas, heterossexuais, Mulheres cis, brancas, heterossexuais, Mulheres cis, brancas, heterossexuais, Mulheres cis, brancas, heterossexuais, pertencentes à classe média pertencentes à classe média pertencentes à classe média pertencentes à classe média pertencentes à classe média As cientistas entrevistadas são mulheres pertencentes aos dois ramos de saberes – humanidades e “exatas” –, atuantes nas universidades nordestinas selecionadas, que possuem destaque em seus departamentos, em seus campos de estudos – este foi o critério inicial de inclusão destes sujeitos nesta pesquisa.24 Neste âmbito, emerge uma das lacunas deste estudo: não tomamos como condição para inclusão de sujeitos os marcadores de raça/etnia, trans* identidades de gênero, orientação sexual e classe social. g , ç Contudo, mesmo não partindo destas questões, no decorrer da pesquisa, notamos um perfil comum às mulheres entrevistadas. Ao buscarmos cientistas reconhecidas em seus ramos de saberes, quem encontramos? Nos meandros de suas identidades cisgênero, encontramos mulheres brancas, heterossexuais, provenientes de classe média. Deste modo, após a conclusão deste estudo, emergiram muitos questiona- mentos, dentre eles: E se as mulheres nordestinas, sujeitos deste estudo, fossem negras, indígenas, bissexuais, lésbicas ou transgêneros? Como se daria as suas inserções na carreira científica? Que outros discursos e práticas discriminatórias permeariam suas trajetórias nas ciências, nas universidades? Estes questionamentos explicitam que as reflexões aqui contidas tratam-se de uma “visão parcial” sobre mulheres cis, brancas, heterossexuais e de classe média em universidades nordestinas públicas federais específicas. E, reconhecendo a sua parcialidade, acreditamos que esta é Contudo, mesmo não partindo destas questões, no decorrer da pesquisa, notamos um perfil comum às mulheres entrevistadas. Ao buscarmos cientistas reconhecidas em seus ramos de saberes, quem encontramos? Nos meandros de suas identidades cisgênero, encontramos mulheres brancas, heterossexuais, provenientes de classe média. Deste modo, após a conclusão deste estudo, emergiram muitos questiona- mentos, dentre eles: E se as mulheres nordestinas, sujeitos deste estudo, fossem negras, indígenas, bissexuais, lésbicas ou transgêneros? Como se daria as suas inserções na carreira científica? Que outros discursos e práticas discriminatórias permeariam suas trajetórias nas ciências, nas universidades? Estes questionamentos explicitam que as reflexões aqui contidas tratam-se de uma “visão parcial” sobre mulheres cis, brancas, heterossexuais e de classe média em universidades nordestinas públicas federais específicas. E, reconhecendo a sua parcialidade, acreditamos que esta é UMA “PERSPECTIVA PARCIAL” SOBRE SER MULHER, CIENTISTA E NORDESTINA NO BRASIL uma discussão inacabada, não havendo, aqui, a pretensão de edificarmos generalizações. Destaca-se, então, a relevância da realização de pesquisas nas diversas regiões e em cada estado brasileiro que contemplem as desigualdades em suas “interseccionali- dades” (Adriana PISCITELLI, 2008) como modo de consolidar- mos as diversas abordagens feministas. É inquestionável haver uma articulação entre as diversas discriminações. Ao gênero, articulam-se as discriminações de raça (Kimberlé CRENSHAW, 2002) e etnicidade, geracionais, lesbo-homo- transfóbicas, classistas (HARDING, 1996; Danièle KERGOAT, 2010) e relativas ao pertencimento territorial. Há uma neces- sidade de construção de outras várias abordagens parciais e interseccionais que nos proporcionem conhecer a parti- cipação de mulheres nas ciências no país em sua plura- lidade e complexidade. Portanto, apostamos que, para entendermos com maior profundidade a problemática participação de mulheres nas ciências no Brasil, tem-se que apostar na vantagem epistemológica das abordagens parciais, como defende Haraway (2001), no esforço de situar as mulheres cientistas e os conhecimentos por elas produzidos nas especificidades de seus tempos, espaços, condições objetivas e subjetivas. No Brasil, observamos avanços nos estudos sobre a participação de mulheres no âmbito da pesquisa financiada pelas agências nacionais, principalmente no que diz respeito à participação feminina na concessão de recursos pelo CNPq (Hildete Pereira MELO & Helena Maria M. LASTRES, 2006). Todavia, ainda está por ser conhecida a atuação de mulheres na pesquisa financiada pelas diversas fundações estaduais. Também nos cabe o esforço em desvendar as particularidades das práticas e discursos que permeiam a inserção e perma- nência de mulheres cientistas nos diversos institutos de pes- quisa, nas universidades privadas e públicas, federais e estaduais. No campo dos estudos de gênero e feministas das ciências, muito trabalho ainda teremos pela frente! ALBUQUERQUE, Vivian Matias; FROTA, Maria Helena de Paula. “Na penumbra da ciência”. O Público e o Privado, v. 1, n. 8, p. 87-107, jun./dez. 2006. BADINTER, Elisabeth. Um amor conquistado: O mito do amor materno. Rio de Janeiro: Nova Fronteira, 1985. BOURDIEU, Pierre. Os usos sociais da ciência: por uma sociologia clínica do campo científico. São Paulo: UNESP, 2004. BUTLER, Judith. “Corpos que pesam: sobre os limites discursivos do sexo”. In: LOURO, Guacira Lopes (Org.). O corpo educado Referências Referências Referências Referências Referências ALBUQUERQUE, Vivian Matias; FROTA, Maria Helena de Paula. “Na penumbra da ciência”. 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Mulheres e homens na política de ciência e tecnologia. Fortaleza: UECE/Edmeta, 2012. MELO, Hildete Pereira; LASTRES, Helena Maria M. “Ciência e Tecnologia numa perspectiva de gênero: o caso CNPq”. Centro Brasileiro de Pesquisas Físicas, 2006. p. 1-27. OSADA, Neide Mayumi; COSTA, Maria Conceição. “A construção social de gênero na Biologia: preconceitos e obstáculos na biologia molecular”. Cadernos Pagu, Campinas, n. 27, p. 279-299, jul./dez. 2006. PATEMAN, Carole. O Contrato Sexual. São Paulo/Rio de Janeiro: Paz e Terra, 1993. PEIRANO, Mariza G. S. “Os antropólogos e suas linhagens”. In: PEIRANO, Mariza G. S. A favor da etnografia. Rio de Janeiro: Relume Dumará, 1995. p. 13-30. PISCITELLI, Adriana. “Interseccionalidades, categorias de articulação e experiências de migrantes brasileiras”. Sociedade e Cultura, v. 11, n. 2, p. 263-274, jul/dez. 2008. PUGLIESE, Gabriel. “Um sobrevôo no “Caso Marie Curie”: um experimento de antropologia, gênero e ciência”. 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[Recebido em 06/05/2015, reapresentado em 05/01/2016 e aceito para publicação em 13/04/2016] [Recebido em 06/05/2015, reapresentado em 05/01/2016 e aceito para publicação em 13/04/2016] A “Partial Perspective” about Being a Woman, Scientist and Northeastern in Brazil A “Partial Perspective” about Being a Woman, Scientist and Northeastern in Brazil A “Partial Perspective” about Being a Woman, Scientist and Northeastern in Brazil A “Partial Perspective” about Being a Woman, Scientist and Northeastern in Brazil A “Partial Perspective” about Being a Woman, Scientist and Northeastern in Brazil Abstract Abstract Abstract Abstract Abstract: This paper proposes to understand how women scientists are embedded in the production of scientific and technological knowledge in specific federal public universities in the Northeast of Brazil. The interviews and direct observations in daily life scientific work made possible the construction of reflections grounded on the social experiences of women scientists belonging to two knowledge’s branches: humanities and supposed “hard sciences”. Through this partial and situated approach, about insertion and permanence of women in contemporary science, we could observe the conservation of old issues that remain as important for feminist and gender studies of science. Key words: Key words: Key words: Key words: Key words: Women Scientists; Gender; Science
https://openalex.org/W2625675002	http://liu.diva-portal.org/smash/get/diva2:1120972/FULLTEXT01	English	null	Altered somatosensory profile according to quantitative sensory testing in patients with degenerative lumbar spine disorders scheduled for surgery	BMC musculoskeletal disorders	2,017	cc-by	9,147	Lindbäck et al. BMC Musculoskeletal Disorders (2017) 18:264 DOI 10.1186/s12891-017-1581-6 Lindbäck et al. BMC Musculoskeletal Disorders (2017) 18:264 DOI 10.1186/s12891-017-1581-6 Open Access * Correspondence: yvonne.lindback@liu.se 1Department of Medical and Health Sciences, Division of Physiotherapy, Faculty of Medicine and Health Sciences, Linköping University, SE-581 83 Linköping, Sweden 6http://www.imh.liu.se/fysioterapi Full list of author information is available at the end of the article Altered somatosensory profile according to quantitative sensory testing in patients with degenerative lumbar spine disorders scheduled for surgery Yvonne Lindbäck1,6, Hans Tropp2,3, Paul Enthoven1,6, Björn Gerdle4, Allan Abbott1,5,6 and dbäck1,6, Hans Tropp2,3, Paul Enthoven1,6, Björn Gerdle4, Allan Abbott1,5,6 and Birgitta Öberg1,6 Abstract Background: Somatosensory profiling in affected and non-affected body regions can strengthen our insight regarding the underlying pain mechanisms, which can be valuable in treatment decision making and to improve outcomes, in patients with degenerative lumbar spine disorders pre-surgery. The aim was to describe somatosensory profiles in patients with degenerative lumbar spine disorders, to identify the proportion with altered somatosensory profile, and to analyze demographic characteristics, self-reported function, pain, and health pre- and 3 months post-surgery. Methods: In this prospective cohort study in a Spine Clinic, 105 patients scheduled for surgery for spinal stenosis, disc herniation, degenerative disc disease, or spondylolisthesis were consecutively recruited. Exclusion criteria were; indication for acute surgery or previous surgery at the same spinal level or severe grade of pathology. Quantitative sensory testing (QST) and self-reported function, pain, and health was measured pre- and 3 months post-surgery. The somatosensory profile included cold detection threshold, warmth detection threshold, cold pain threshold, heat pain threshold and pressure pain threshold in affected and non-affected body regions. Results: On a group level, the patients’ somatosensory profiles were within the 95% confidence interval (CI) from normative reference data means. On an individual level, an altered somatosensory profile was defined as having two or more body regions (including a non-affected region) with QST values outside of normal ranges for reference data. The 23 patients (22%) with altered somatosensory profiles, with mostly loss of function, were older (P = 0.031), more often female (P = 0.005), had higher back and leg pain (P = 0.016, 0.020), lower mental health component summary score (SF-36 MCS) (P = 0.004) and larger pain distribution (P = 0.047), compared to others in the cohort. Post-surgery there was a tendency to worse pain, function and health in the group with altered somatosensory profile pre-surgery. (Continued on next page) * Correspondence: yvonne.lindback@liu.se 1Department of Medical and Health Sciences, Division of Physiotherapy, Faculty of Medicine and Health Sciences, Linköping University, SE-581 83 Linköping, Sweden 6http://www.imh.liu.se/fysioterapi Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Background disc herniation scheduled for surgery, one study with 21 participants has reported both hypoesthesia and hypoalgesia in affected dermatome [11]. There is a lack of data about somatosensory function in patients with degenerative lumbar spine disorders, as for instance spinal stenosis or disc herniation, which represent the two largest groups that undergo spinal surgery [12]. Somatosensory profiling in both affected and non- affected body regions together with analysis of other biopsychosocial factors can strengthen our insight re- garding the underlying pain mechanisms, which may help to guide the pre-surgical clinical decision-making process and need of additional management that may improve surgical outcomes [5]. The aim was to describe somatosensory profiles in patients with degenerative lumbar spine disorders, to identify the proportion with altered somatosensory profile, and to analyze demo- graphic characteristics, self-reported function, pain, and health pre- and 3 months post-surgery. Among patients scheduled for spinal surgery due to de- generative lumbar spine disorders (e.g., disc herniation, spinal stenosis, spondylolisthesis, or degenerative disc disease), a majority have experienced pain for over three months. Persistent pain results from a complex interplay of many factors. Maladaptive neuroplastic changes in the central nervous system can be influenced by orthopedic structural pathology, as well as biochemical and psycho- social factors [1]. Such changes can result in increased pain sensitivity, and alterations of somatosensory or sen- sory function leading to amplified responses to nocicep- tion from localized spinal pathology, reduced pain thresholds, or widening distribution of pain [1]. In patients with low back pain (LBP), bedside neuro- logical testing is recommended [2]. However, quantitative sensory testing (QST) can provide more specific and quantifiable somatosensory or sensory profiling [3]. QST assesses the function of; small myelinated A-delta fibers that conduct cold sensations and deep pain sensitivity, small unmyelinated C-fibers that conduct warmth, heat- pain sensations and deep pain sensitivity [4], and large A- beta fibers for light touch [3]. Perception in response to mechanical, thermal, or electrical stimulation at a con- trolled intensity [4, 5] can therefore be used to test sensory detection, pain thresholds, and pain summation [3]. (Continued from previous page) (Continued from previous page) Conclusions: On a group level, patients with degenerative lumbar spine disorders scheduled for surgery were within normal range for the QST measurements compared to reference values. On an individual level, an altered somatosensory profile outside of normal range in both affected and non-affected body regions occurred in 22% of patients, which may indicate disturbed somatosensory function. Those patients had mostly loss of sensory function and had worse self-reported outcome pre-surgery, compared to the rest of the cohort. Future prospective studies are needed to further examine whether these dimensions can be useful in predicting post-surgery outcome and guide need of additional treatments. Keywords: Disc herniation, Spinal stenosis, Spondylolisthesis, Degenerative disc disease, Spine surgery, Quantitative sensory testing, Outcome Methods This prospective cohort study with cross sectional and prospective analysis investigated pre-surgery sensory profiles and biopsychosocial factors. The study conforms to the STROBE statement checklist for cohort studies. The patients received oral and written information about the study 1week before the measurements. All partici- pants provided signed consent at the time of QST mea- surements. The study was approved by the Regional Ethics Committee (Dnr 2013/410-31). QST can detect gain of sensory function in the form of lowered sensory thresholds (e.g., hyperesthesia and hyper- algesia) and loss of sensory function (e.g., hypoesthesia and hypoalgesia) [3, 4]. Through QST profiling in both affected and non-affected body regions, LBP symptoms can be more thoroughly assessed to determine the extent of localized hypoesthesia and hyperalgesia, due to in- creased nociceptive input in affected body region [6] or generalized hyperalgesia when there is alteration even in other body regions (arm and leg) not affected by LBP [7]. In patients with disc herniation, generalized deep tissue hyperalgesia has been reported using QST on the infraspi- natus and anterior tibialis muscles [8]. Hypoesthesia to thermal detection in affected dermatome was reported among patients with sciatica [9, 10] and in patients with Abstract The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 10 Lindbäck et al. BMC Musculoskeletal Disorders (2017) 18:264 Participants and settings A total of 105 patients were consecutively recruited at a Spine Clinic at a University Hospital, in Sweden, be- tween September 2013 and December 2014. Inclusion criteria were; patients scheduled for surgery due to: disc herniation, spinal stenosis, spondylolisthesis (Grade 4) or degenerative disc disease, age between 25 and 80 years and fluent in Swedish. Exclusion criteria were; indication for acute surgery, previous surgery at the same spinal level or severe grade of pathology. Seventeen eligible Page 3 of 10 Lindbäck et al. BMC Musculoskeletal Disorders (2017) 18:264 Page 3 of 10 patients chose not to participate due to the requirement of additional appointments at the hospital for QST. patients were comfortably seated or lying down in a quiet room with an air temperature of 22 °C. At the start of test- ing, patients were asked to use a visual analogue scale (VAS) to rate the average pain intensity in their back and legs during the last 2 weeks, as well as their current pain intensity at rest [16]. The patient’s most symptomatic side in the back or leg was also registered. The patients re- ported whether they had been able to refrain from using any stronger analgesics during the 24 h prior to QST, as had been recommended. In cases of analgesic use, the dosage was documented. Each test was initially performed on the non-dominant hand, with the purpose of familiar- izing the patient with the QST protocol. These tests were not included in the analysis. Subsequently, the thermal sensory measurements were performed and finally the PPT measurements. Procedure for QST measurements Sensory profile investigation included the following QST measurements: cold detection threshold (CDT), warmth detection threshold (WDT), cold pain threshold (CPT), heat pain threshold (HPT), and pressure pain threshold (PPT). A standardized QST protocol [13, 14] was ap- plied for all patients. CDT and WDT, and subsequently CPT and HPT, were measured using a thermic stimula- tor (Somedic, Hörby, Sweden). A thermode containing a Peltier element with a stimulating area of 25 × 50 mm was used. CDT, WDT, CPT, and HPT reportedly show a high degree of repeatability in healthy subjects [15], and acceptable repeatability in patients with sciatica [10]. For thermal tests, the baseline temperature was 32 °C, and the temperature was decreased or increased at a rate of 1 °C/s within a range of 10–50 °C. During the thermal measurements, the thermode was held on the test site. When measuring CDT or WDT, the patients were instructed to push a stop button when they first per- ceived a decreasing temperature or an increasing temperature, respectively. For CPT or HPT measure- ments, respectively, patients were instructed to push the stop button when the cold or heat sensation was first perceived as painful [13, 14]. Thermal measurements were performed on the following seven body regions: lower back (2 cm lateral of the spinal column on the most painful side, i.e., the symptomatic side), thighs (lower part of quadriceps muscle, 7–10 cm above the patella upper border, bilaterally), and lower legs (upper part of tibialis anterior muscle, 7–10 cm below the pa- tella lower border, bilaterally) and two non-affected body-regions according to the degenerative lumbar spine disorders; hand (thenar eminence muscle on the domin- ant hand) and upper back (on the lower thoracic spine, contralateral to the lower back region). The two non- affected body regions were added to detect if there were patients with generalized alteration in sensory profile. Each thermal measurement was repeated five times, and the mean value was calculated for each patient. Evaluation of function, pain, and health To collect demographics and data regarding function, pain, and health pre-surgery the patients completed the questionnaire from the Swedish National Spine Register for spinal surgery patients (SweSpine) [12] and comple- mentary questionnaires, including pain drawing [17], the Hospital Anxiety and Depression Scale (HADS) [18], the Self-Efficacy Scale (SES) [19], the fear avoidance beliefs questionnaire (FABQ) [20], and questions about lifestyle habits and expectations. Data regarding function, pain and health (EQ-5D, HADS and SES) was also collected 3 months post-surgery. Function was measured using Oswestry Disability Index (ODI) [21], which includes ten items related to different functions and back pain, with six answer options (0–5) for each item, generating a sum score of between 0–100% dis- ability [21]. ODI is the most commonly used instrument for this purpose [22, 23]. The patients rated their pain intensity the last week in the back and legs using a VAS with a horizontal line of 0–100 mm containing endpoints named “no pain” and “worse imaginable pain” [16]. Patients also reported their pain duration in the back and legs, with responses including “I don’t have pain”, “less than 3 months”, “3 to 12 months,” “1 to 2 years,” and “more than 2 years”. A pain drawing was used to identify whether patients had unilateral, bilateral, or no leg pain, as well as pain distribution in other body regions [17]. ODI and VAS pain are recommended instruments for measuring function and pain, respectively, in chronic LBP [22] and after spine surgery [24], with respect to validity and responsiveness [22, 24]. PPT was measured using a handheld electrical pressure algometer (Somedic, Hörby, Sweden) with a 1-cm diam- eter probe. The patient was instructed to state when the pressure started to become painful, at which point the applied pressure was released [13, 14]. Pressure was applied at a rate of 30 kPa/s up to a maximal pressure of 700 kPa. PPT was measured once at each of five body regions, which included the same body regions used for thermal measurements, excluding the two spinal regions. Health-related quality of life was measured using the European Quality of Life instrument (EQ-5D) [25, 26], which includes two scales: EQ-index and EQ-VAS. EQ- index includes five dimensions; mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Statistical methods Statistical analysis was performed using IBM SPSS statistics version 23. The level of significance was set to P = 0.05. Statistical analysis was performed using IBM SPSS statistics version 23. The level of significance was set to P = 0.05. Sample size calculation: Using CPT, HPT and PPT as outcomes at least 23 patients in each group were required based on calculations from a previous study that com- pared healthy subjects and subjects with chronic WAD [14]. For comparison between two groups of patients at least 17 patients in each group was required, based on sample size calculation in two subgroups of patients with WAD [13]. Sample size calculations were done using the computer program Power and Sample Size Calculations, version. 3.0.43 Vanderbilt University, US [36]. p p Health was also measured with the Short Form Health Survey (SF-36) [28], which includes eight multi-items scales: bodily pain; vitality; general mental health; general health perceptions; limitations in physical func- tioning; limitations in usual role activities due to physical health; limitations in usual role activities due to personal and emotional problems; and limitations in social func- tioning due to physical or mental health problems. The subscales are summarized as physical and mental health component summary scores (PCS and MCS, respect- ively), each ranging from 0–100 with a higher score indi- cating better health [28]. Studies in a general population in Sweden show that the SF-36 has satisfactory reliabil- ity, construct-based validity, [29, 30] and criterion-based validity [31]. Another study reported that the EQ-5D, SF-36 PCS, and SF-36 MCS show a medium responsive- ness after lumbar surgery, while the SF-36 total score shows low responsiveness [24]. Patient demographics were analyzed using descriptive statistics, and are presented as mean and SD for con- tinuous variables, and as frequencies and percentages for categorical variables. For between-group comparisons of demographic data, function, pain, and health, the un- paired Student’s t-test or Mann–Whitney U test for continuous variables, and the Chi-Square test or Fisher Exact probability test for categorical variables were used. In the t-test, if Levine’s test was significant, the P value for “equal variance not assumed” was reported. Symptoms of depression and anxiety was measured using the Hospital Anxiety and Depression Scale (HADS) [18], which includes seven items for anxiety and seven items for depression. The HADS total score ranges from 0–21, with a higher score indicating more signs of anxiety or depression. Evaluation of function, pain, and health Each dimension receives a score of 1–3, based on three QST measurements were performed 1 to 2 weeks prior to surgery by a single investigator, a physiotherapist work- ing at the Spine clinic. During QST measurements, the QST measurements were performed 1 to 2 weeks prior to surgery by a single investigator, a physiotherapist work- ing at the Spine clinic. During QST measurements, the Lindbäck et al. BMC Musculoskeletal Disorders (2017) 18:264 Page 4 of 10 Page 4 of 10 Each item is answered on a 7-grade scale, where a higher number indicates a higher level of fear avoidance beliefs. The English version of FABQ shows good test-retest reliability and internal consistency in patients with chronic LBP [20], and the Swedish version shows good internal consistency among patients with whiplash- associated disorder [34]. possible answer options: “no problems”, “some prob- lems”, “extreme problems”. The final EQ-index ranges from −0.594 to 1, with a higher score indicating better health status. The EQ-VAS is a 20 cm vertical scale ran- ging from a score of 0 indicating the “worst imaginable health state” (score 0) to a score of 100 indicating the “best imaginable health state”. EQ-5D is one of the five most commonly used questionnaires to measure health- related quality of life [22]. Mean EQ-5D index has been reported as 0.86 in a population in the UK, 0.84 in a Swedish population, and 0.66 among responders in the Swedish population with LBP [27]. Statistical methods The Swedish version of HADS is reportedly a robust instrument with regard to reliability, discriminant validity, concurrent validity, and ability to be a case finder for anxiety and depression [32]. q p The data in the sensory profiles data were compared with reference data, obtained of healthy subjects from studies of the German Research Network on Neuro- pathic Pain (DFNS) [37, 38]. QST data for the hand, thigh, and lower leg were compared with reference data for hand and feet from Magerl et al. [37]. QST data re- garding the upper and lower back were compared with reference data reported by Pfau et al. [38]. CDT, WDT, and PPT were log-transformed. To compare the patient’s sensory profile with reference data, all QST data were standardized with Z-transformation, meaning that each QST value was matched for age and sex in the reference values [3]. The following expression was used for Z-transformation: The Self-Efficacy Scale (SES) is a 20-item scale for assessing a patient’s confidence regarding activities of daily living [19]. The scores range from 0 indicating “not at all confident” to 10 indicating “very confident”, with a higher score indicating better self-efficacy. The English version was developed for use in patients with LBP, and shows good internal consistency [19]. The Swedish version has been modified to be suitable for use in pa- tients with all kinds of pain [33], shown good internal consistency [34] and test-retest reliability among patients with whiplash-associated disorder (WAD) [35]. Proportion of patients on an individual level with altered somatosensory profile A total of 23 patients had altered somatosensory profiles for at least one QST measurement in the hand or upper back and at least one additional body region (Table 2). Compared to the other 82 patients, the patients with altered somatosensory profiles had a significantly higher mean age. Additionally, more women than men had an altered somatosensory profile. Demographics Table 1 presents demographic data and self-reported function, pain, and health for the 105 patients. The mean age was 59.8 ± 12.9 years, and the cohort in- cluded 55 women (52%). The represented degenerative lumbar spine disorders included spinal stenosis (n = 61; 58.1%), disc herniation (n = 30; 28.6%), degenerative disc disease (DDD; n = 8; 7.6%), and spondylolysis/ spondylolisthesis (n = 6; 5.7%). Patients with spinal sten- osis were significantly older (67.6 ± 7.6 years) than patients with disc herniation (47.7 ± 11.4 years; P < 0.001). A pain duration of > 2 years was reported by 55 patients (52.5%), and was significantly more common among patients with spinal stenosis compared to patients with disc herniation (P = 0.003). Z–score ¼ Xsingle patient– Meanreferences =SDreferences: Z–score ¼ Xsingle patient– Meanreferences =SDreferences: A Z-score of < −2 or >2 is outside of the 95% confi- dence interval (CI) of a normal standard distribution for healthy subjects [39]. Moreover, Z-score values of <0 indicate a loss of sensory function, while Z-score values of >0 indicate r a gain of sensory function [39]. For the comparison between symptomatic and non-symptomatic Fear avoidance was measured using FABQ [20], which is a 16-item questionnaire focused on a patient’s beliefs. FABQ questions comprise a 4-item subscale describing how physical activity affects the patient’s pain (FABQ-PA). Lindbäck et al. BMC Musculoskeletal Disorders (2017) 18:264 Page 5 of 10 Page 5 of 10 Somatosensory profiles of patients on a group level compared to reference data body regions in the legs, as identified by the pain drawing, only the patients with unilateral leg pain were included. An altered somatosensory profile was defined as hav- ing two or more body regions including a normally non-affected body region, i.e., the hand and/or upper back—with a Z-score of < −2 or >2 compared to refer- ence data for CDT, WDT, CPT, HPT, or PPT. Somatosensory profiles of patients on a group level compared to reference data body regions in the legs, as identified by the pain drawing, only the patients with unilateral leg pain were included. An altered somatosensory profile was defined as hav- ing two or more body regions including a normally non-affected body region, i.e., the hand and/or upper back—with a Z-score of < −2 or >2 compared to refer- ence data for CDT, WDT, CPT, HPT, or PPT. On a group level the somatosensory profiles of the patients showed that all QST measurements had mean Z-score values between −2 and 2, meaning that the group values were within the 95% CI of the reference data (Fig. 1). The Z-scores for CDT and HPT were below “0” in all body regions tested. The Z-scores for WDT were below “0” in all body regions except for symptomatic and non-symptomatic thigh. Furthermore, the Z-scores for CPT were above “0” in all body regions tested. Forty-eight patients had unilateral pain with no significant differences between symptomatic and non-symptomatic side in the thigh or lower leg (Fig. 1). SD standard deviation, ODI Oswestry Disability Index (0–100) (higher score indicate higher disability), VAS visual analogue scale (0–100) (higher score indicate higher pain intensity); EQ-5D (−0.594 - 1) and EQ-VAS EuroQol (higher score indicate better health), HADS Hospital anxiety and depression scale (0–21) (higher score indicate more signs of symptoms), SES Self-Efficacy Scale (0–200) (higher score indicate better self-efficacy), SF-36 PCS physical component summery and MCS mental component summery (0–100) (higher score indicate better health), FABQ-PA fear avoidance beliefs questionnaire – physical activity (0–24) (higher score indicates higher level of fear avoidance beliefs) aMaximum missing data in each column: n = 11 bn =9 cn = 4 Proportion of patients on an individual level with altered somatosensory profile The two groups showed no significant differences in the proportions of patients with Table 1 Pre-surgery measures for all patients, comparison between those with spinal stenosis and disc herni Table 1 Pre-surgery measures for all patients, comparison between those with spinal stenosis and disc herniation All patients n = 105a Spinal stenosis n = 61b Disc herniation n = 30c Spinal stenosis/ Disc herniation Age, mean (SD) 59.8 (12.9) 67.6 (7.6) 47.7 (11.4) <0.001 Women, n (%) 55 (52.4) 32 (52.5) 16 (53.3) 0.937 Pain duration back/leg > 2 years, n (%) 55 (52.5) 38 (63.3) 8 (30.7) 0.003 ODI, mean (SD) 38.4 (15.6) 37.9 (15.3) 40.2 (16.0) 0.539 VAS back pain, mean (SD) 53.8 (26.4) 54.1 (25.0) 49.9 (28.8) 0.493 VAS leg pain, mean (SD) 61.2 (25.2) 61.9 (22.0) 67.4 (24.3) 0.298 EQ-5D index, mean (SD) 0.42 (0.31) 0.41 (0.32) 0.41 (0.29) 0.924 EQ-VAS, mean (SD) 50.5 (21.8) 51.4 (21.6) 50.7 (22.0) 0.896 HADS anxiety, mean (SD) 6.0 (3.8) 5.6 (3.5) 5.9 (4.1) 0.744 HADS depression, mean (SD) 4.7 (3.3) 4.3 (3.1) 4.5 (3.5) 0.773 SES, mean (SD) 129.8 (40.8) 132.5 (40.3) 125.1 (44.0) 0.451 SF-36 PCS, mean (SD) 29.4 (9.1) 28.5 (9.4) 30.3 (8.9) 0.417 SF-36 MCS, mean (SD) 46.4 (12.5) 47.9 (11.4) 45.5 (15.0) 0.419 FABQ-PA, mean (SD) 14.8 (6.0) 14.0 (6.3) 15.5 (5.6) 0.321 Pain drawing n (%): Back and/or unilateral leg pain 41 (39.8) 14 (23.35) 19 (65.5) <0.001 Bilateral leg pain 43 (41.7) 34 (56.7) 6 (20.7) Back- leg pain and other pain locations 19 (18.4) 12 (20.0) 4 (13.8) SD standard deviation, ODI Oswestry Disability Index (0–100) (higher score indicate higher disability), VAS visual analogue scale (0–100) (higher score indicate higher pain intensity); EQ-5D (−0.594 - 1) and EQ-VAS EuroQol (higher score indicate better health), HADS Hospital anxiety and depression scale (0–21) (higher score indicate more signs of symptoms), SES Self-Efficacy Scale (0–200) (higher score indicate better self-efficacy), SF-36 PCS physical component summery and MCS mental component summery (0–100) (higher score indicate better health), FABQ-PA fear avoidance beliefs questionnaire – physical activity (0–24) (higher score indicates higher level of fear avoidance beliefs) aMaximum missing data in each column: n = 11, bn =9, cn = 4 r all patients, comparison between those with spinal stenosis and disc herniation Lindbäck et al. BMC Musculoskeletal Disorders (2017) 18:264 Page 6 of 10 a b c d Fig. Discussion In line with the study’s aim to investigate the propor- tion of individuals showing an altered somatosensory profile, 22% of patients were detected. These patients had alterations outside of normal range in two or more body regions, including a non-affected region. This can be considered a sign of generalized alteration of somatosensory function. Most of these patients had loss of sensory function. The group with altered som- atosensory profile showed worse back and leg pain VAS and SF-36 MCS before surgery, and also had larger distribution of pain as reported in pain drawings pre-surgery. The 3 months post-surgery results with patient reported outcome measures could not confirm the pre-surgery differences between the group with- respectively without altered somatosensory function, but it showed a tendency with worse function, pain and health in the group with altered somatosensory profile before surgery. The study does not have the power to detect small differences since the group with altered somatosensory profile turned out to be small. Considering that 20–35% of the patients with spinal stenosis and disc herniation are doubtful or dissatis- fied with the results at 1-year follow up post-surgery [12], it still needs to be proven if the use of a somato- sensory profile could be helpful to understand effects of treatment in larger studies. Of the 23 patients with altered somatosensory profiles, 19 (83%) had Z-scores of < −2 in one of the QST mea- surements in at least two body regions compared to the reference data, indicating that they had loss of sensory function. The remaining 4 patients with altered somato- sensory profiles showed Z-scores of >2 compared to references data, indicating that they had a gain of sensory function and were more sensitive for one test in at least two body regions. Z-scores of < −2 or >2 were observed in all QST measurements (CDT, WDT, CPT, HPT and PPT), except for Z-scores of < −2 in CPT. Proportion of patients on an individual level with altered somatosensory profile 1 Somatosensory profile of patients compared to reference data. Somatosensory profiles from the hand (a) (n = 105), upper and lower back (b) (n = 105), thigh (c) (n = 48), and lower leg (d) (n = 48). Z-scores were calculated to standardize the study population values according to the mean and SD from the reference data [37, 38]. A Z-score of >0 indicates a gain of function where the patient is more sensitive, while a Z-score of <0 indicates a loss of function where the patient is less sensitive to test stimuli compared to controls. A Z-score of between −2 and 2 indicates that data is within the 95% confidence interval of the normal standard distribution [39]. CDT, cold detection threshold; WDT, warm detection threshold; CPT, cold pain threshold; HPT, heat pain threshold, PPT, pressure pain threshold a c b d b c d d c Fig. 1 Somatosensory profile of patients compared to reference data. Somatosensory profiles from the hand (a) (n = 105), upper and lower back (b) (n = 105), thigh (c) (n = 48), and lower leg (d) (n = 48). Z-scores were calculated to standardize the study population values according to the mean and SD from the reference data [37, 38]. A Z-score of >0 indicates a gain of function where the patient is more sensitive, while a Z-score of <0 indicates a loss of function where the patient is less sensitive to test stimuli compared to controls. A Z-score of between −2 and 2 indicates that data is within the 95% confidence interval of the normal standard distribution [39]. CDT, cold detection threshold; WDT, warm detection threshold; CPT, cold pain threshold; HPT, heat pain threshold, PPT, pressure pain threshold spinal stenosis and disc herniation, or the proportions of patients with pain duration of greater than 2 years. Function, pain, and health in patients with or without altered somatosensory profile before and after surgery Compared to patients without an altered somatosensory profile, those with an altered somatosensory profile re- ported significantly higher pre-surgery back and leg pain VAS and lower score on SF-36 MCS (Table 2). The post- surgery results showed no significant differences between the groups but there was a tendency towards worse func- tion, pain and health in the group with altered somatosen- sory profile pre-surgery. Pain drawings pre-surgery showed that patients with altered somatosensory profile had larger distribution of pain than the rest of the cohort (P = 0.047). Lindbäck et al. Function, pain, and health in patients with or without altered somatosensory profile before and after surgery BMC Musculoskeletal Disorders (2017) 18:264 Page 7 of 10 Table 2 Pre- and post-surgery measurements, comparison between those with and without altered sensory profile in QST Pre-surgery measurements Post-surgery measurements Without altered sensory profile n = 82a Altered sensory profile n = 23b P Without altered sensory profile n = 79c Altered sensory profile n = 21d P Age, mean (SD) 58.6 (12.5) 64.0 (13.8) 0.031 Women, n (%) 37 (45) 18 (78) 0.005 Spinal stenosis, n (%) 45 (55) 16 (70) 0.207 Disc herniation, n (%) 24 (29) 6 (26) 0.765 Pain duration >2 years, n (%) 44 (56) 11 (50) 0.859 ODI, mean (SD) 37.1 (15.9) 42.9 (15.7) 0.112 28.4 (17.2) 30.8 (18.0) 0.566 VAS back pain, mean (SD) 50.9 (27.5) 63.6 (19.3) 0.016 28.8 (26.2) 38.3 (26.6) 0.147 ODI > 40% n (%) 37 (48.1) 15 65.2) 0.148 14 (17.9) 6 (28.6) 0.641 VAS leg pain, mean (SD) 58.8 (27.0) 69.3 (15.1) 0.020 23.7 (28.5) 34.0 (27.5) 0.146 EQ-5D index, mean (SD) 0.43 (0.30) 0.37 (0.32) 0.430 0.62 (0.28) 0.68 (0.16) 0.356 EQ-VAS, mean (SD) 51.9 (22.2) 45.9 (21.1) 0.250 66.3 (21.3) 73.4 (19.7) 0.173 HADS anxiety, mean (SD) 5.8 (3.6) 6.6 (4.49) 0.345 4.05 (3.1) 5.4 (4.0) 0.110 HADS depression, mean (SD) 4.6 (3.2) 4.8 (3.9) 0.832 3.27 (2.93) 3.84 (3.30) 0.455 SES, mean (SD) 132.9 (41.5) 116.3 (35.4) 0.120 155.0 (32.7) 146.2 (41.9) 0.348 SF-36 PCS, mean (SD) 30.1 (9.4) 26.9 (7.3) 0.117 SF-36 MCS, mean (SD) 48.3 (11.69) 39.8 (13.06) 0.004 FABQ-PA, mean (SD) 14.8 (6.1) 15.0 (6.0) 0.870 Pain drawing n (%): Back or unilateral leg pain 37 (46.3) 4 (17.4) 0.029 Bilateral leg pain 31 (38.8) 12 (52.1) Back- leg pain and other pain locations 12 (15) 7 (30.4) QST Quantitative sensory testing, SD standard deviation, ODI Oswestry Disability Index (0–100) (higher score indicate higher disability), VAS visual analogue scale (0–100) (higher score indicate higher pain intensity); EQ-5D (−0.594 - 1) and EQ-VAS EuroQol (higher score indicate better health), HADS Hospital anxiety and depression scale (0–21) (higher score indicate more signs of symptoms), SES Self-Efficacy Scale (0–200) (higher score indicate better self-efficacy), SF-36 PCS, physical component summery and MCS, mental component summery (0 – 100) (higher score indicate better health), FABQ-PA fear avoidance beliefs questionnaire – physical activity (0–24) (higher score indicates higher level of fear avoidance beliefs) aMaximum missing data in each column: n = 7, bn =4, cn = 6, dn = 4 QST Quantitative sensory testing, SD standard deviation, ODI Oswestry Disability Index (0–100) (higher score indicate higher disability), VAS visual analogue scale (0–100) (higher score indicate higher pain intensity); EQ-5D (−0.594 - 1) and EQ-VAS EuroQol (higher score indicate better health), HADS Hospital anxiety and depression scale (0–21) (higher score indicate more signs of symptoms), SES Self-Efficacy Scale (0–200) (higher score indicate better self-efficacy), SF-36 PCS, physical component summery and MCS, mental component summery (0 – 100) (higher score indicate better health), FABQ-PA fear avoidance beliefs questionnaire – physical activity (0–24) (higher score indicates higher level of fear avoidance beliefs) aMaximum missing data in each column: n = 7, bn =4, cn = 6, dn = 4 QST Quantitative sensory testing, SD standard deviation, ODI Oswestry Disability Index (0–100) (higher score indicate higher disability), VAS visual analogue scale (0–100) (higher score indicate higher pain intensity); EQ-5D (−0.594 - 1) and EQ-VAS EuroQol (higher score indicate better health), HADS Hospital anxiety and depression scale (0–21) (higher score indicate more signs of symptoms), SES Self-Efficacy Scale (0–200) (higher score indicate better self-efficacy), SF-36 PCS, physical component summery and MCS, mental component summery (0 – 100) (higher score indicate better health), FABQ-PA fear avoidance beliefs questionnaire – physical activity (0–24) (higher score indicates higher level of fear avoidance beliefs) aMaximum missing data in each column: n = 7, bn =4, cn = 6, dn = 4 When considering the other study aim, to describe somatosensory profiles of patients on a group level, Z- scores for QST data compared to reference data showed that our cohort was within the 95% CI of the reference values [37, 38]. Function, pain, and health in patients with or without altered somatosensory profile before and after surgery Studies point out that QST [48] or cold sensitivity [43] is one part of an exam- ination, and should be seen together with other findings to further study the effects of different treatments. One limitation in the study was, in the protocol used in this study tests for cold stopped at +10°, while DFNS used 0°, indicating a risk for underestimation rather than overestimation of the results. When CPT in reference data had the broad normal range 32–0 °C in most age groups [37, 38], that difference in degrees at end-points had minor influence on the result. Another limitation was that as there were no reference data available for thigh and lower leg, reference data for the foot were used instead [37]. Although this was not optimal, CPT and HPT measurements are generally quite uniform among different body regions [3]. Moreover, PPT mea- sured on muscle tissue shows less variability between body regions compared to measurements on bone or nailbed tissue [51]. PPT measurements on thigh and lower leg were on muscle tissue, suggesting minimal influence of variability regarding the use of foot reference data. y Patients with altered somatosensory profile had mostly loss of sensory function in current study. Similar result with loss of sensory function pre-surgery has previous been presented in patients with disc herniation [11], that study also reported that complete recovery after surgery was associated with normalization of QST. Another study reported a trend towards higher loss of sensory function in QST in patients with higher degree of nerve root compression in a cohort of patients with MRI veri- fied disc herniation [49]. The patients with altered som- atosensory profile in the current study were older and included more women, compared to the rest of the co- hort, even though each individual QST measurement was compared to age and gender adjusted reference data in the Z-score. Therefore, we suggest that the different profile in this particular group is related to other factors, which can be potential mediators of outcomes after treatment. Associations between sensory profiles and psychological variables have also been reported in stud- ies of non-specific LBP [50], WAD [14], and fibromyal- gia [40]. Function, pain, and health in patients with or without altered somatosensory profile before and after surgery In many of the QST measurements, the patient group had a tendency to loss of sensory function (Z-score below “0”), while CPT showed a tendency to gain of sensory function (Z-score above “0”) in all body regions tested, which means that the patient group had a tendency to be more sensitive to cold pain than the reference data. function (greater sensitivity) in all body regions com- pared to reference data. Greater sensitivity to cold could indicate disturbed sensory function [3]. Roussel et al. [42] reported conflicting results concerning responsive- ness to various stimuli including different aspects of sensory testing in patients with chronic LBP. QST meas- urement in chronic nonspecific LBP divided into mech- anical pain and non-mechanical pain showed higher odds for cold hyperalgesia in the non-mechanical than in the mechanical LBP group [43]. Also patients with acute non-specific LBP have shown increased sensitivity to cold pain as well as to mechanical stimuli in compari- son to pain-free controls [44]. In patients with fibro- myalgia higher sensitivity in CPT was associated with higher pain intensity, more tender points, and poorer sleep [40]. Beside CPT, PPT has been suggested as the most promising QST measurement to discriminating pain in osteoarthritis [45] and PPT and electrical pain detection thresholds in chronic LBP [46]. In patients The choice of QST measurements is of importance and in subjects with persistent pain, thermal pain thresholds are of greater importance than detection thresholds [40]. People are more sensitive to cold than warmth, partly because the receptors for cold are more superficially located and are present in larger amounts compared to receptors for warmth [41]. With regard to CPT, this patient cohort showed a tendency to gain of Lindbäck et al. BMC Musculoskeletal Disorders (2017) 18:264 Page 8 of 10 Page 8 of 10 this reference data has a broad range of variation in normal values — especially for CPT [37, 38], which may limit the possibility of identifying patients with altered sensory profiles. scheduled for hip- or knee replacement, higher sensitiv- ity to PPT in a non-affected body region was associated with higher pain intensity [47]. In the current study on group level, Z-scores for PPT for all body regions were close to “0”, indicating that there were no differences in PPT compared to reference values. Acknowledgements Ö Acknowledgements We thank Maria Öberg, RPT, for performing the QST measurements of all participants. Acknowledgements We thank Maria Öberg, RPT, for performing the QST measurements of all participants. It has been reported that QST may have predictive value for identifying allodynia and hyperalgesia [48], but com- parison with reference data is necessary for decision- making. In the current study, the best existing reference data was used, the DFNS reference data [37, 38]. However, Conclusions O On a group level, patients with degenerative lumbar spine disorders scheduled for surgery were within the normal range for the QST measurements compared to age and gender adjusted reference values. This might be interpreted as a well-selected patient group for surgery according to QST. However, on an individual level, 22% of the patients showed altered somatosensory profiles, when defined as alterations in two or more body regions, including a non-affected region, which may indicate dis- turbed somatosensory function. These patients had mostly loss of sensory function and reported worse pain and mental health components as well as larger pain distribution pre-surgery compared to the rest of the cohort. Differences 3 months post-surgery could not be confirmed partly due to limited study power. It remains to be determined whether baseline QST measurements have predictive value, and whether change over time is important for the outcome following interventions. Generalizability is one important issue. The question- naires used are internationally recommended self-reported questionnaires of function, pain, and health providing a thorough biopsychosocial description of the patient cohort. In the study, all patients are screened by an orthopedic surgeon and assessed as suitable candidates for surgery, in a shared decision with the patient. The population is repre- sentative of the population scheduled for surgery in Sweden when compared to the population included in the national registry SweSpine with regard to the proportions of different diagnoses, ages, genders, functions, and pain [12]. Additionally, in the present study, all tests were per- formed by one independent physiotherapist, potentially strengthening reliability of the testing procedure. Function, pain, and health in patients with or without altered somatosensory profile before and after surgery To our knowledge, our present study is the first investigation of patients with degenerative lumbar spine disorders prior to surgery to report differences in pain intensity, mental health components and pain distribu- tion in pain drawings between patients with or without altered sensory profiles. Abbreviations QST Q i i Abbreviations QST: Quantitative sensory testing; LBP: Low back pain; CDT: Cold detection threshold; WDT: Warmth detection threshold; CPT: Cold pain threshold; HPT: Heat pain threshold; PPT: Pressure pain threshold Abbreviations QST: Quantitative sensory testing; LBP: Low back pain; CDT: Cold detection threshold; WDT: Warmth detection threshold; CPT: Cold pain threshold; HPT: Heat pain threshold; PPT: Pressure pain threshold Ethics and consent to participate The study was approved by the Regional Ethics Committee in Linköping, Sweden 2013-11-13 (Dnr 2013/410-31). All participants provided signed consent at the time of QST measurements. 18. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand. 1983;67(6):361–70. 18. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand. 1983;67(6):361–70. 19. Altmaier E. Role of self-efficacy in rehabilitation outcome among chronic low back pain patients. J Couns Psychol. 1993;40(3):335–9. 19. Altmaier E. Role of self-efficacy in rehabilitation outcome among chronic low back pain patients. J Couns Psychol. 1993;40(3):335–9. Funding g The study received financial support from the Swedish Research Council (No 521-2019-3578), the Faculty of Medicine and Health, Linköping University, and the County Council of Östergötland, Linköping, Sweden. Page 9 of 10 Page 9 of 10 Lindbäck et al. BMC Musculoskeletal Disorders (2017) 18:264 Consent for publication Not applicable. Consent for publication Not applicable. 16. Scott J, Huskisson EC. Graphic representation of pain. Pain. 1976;2(2):175–84. 16. Scott J, Huskisson EC. Graphic representation of pain. Pain. 1976;2(2):175–84. 17. Southerst D, Cote P, Stupar M, Stern P, Mior S. The reliability of body pain diagrams in the quantitative measurement of pain distribution and location in patients with musculoskeletal pain: a systematic review. J Manip Physiol Ther. 2013;36(7):450–9. 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https://openalex.org/W1990767774	http://www.scirp.org/journal/PaperDownload.aspx?paperID=52258	English	null	Experimental Investigation of Intake Diesel Aerosol Fuel Homogeneous Charge Compression Ignition (HCCI) Engine Combustion and Emissions	Energy and power engineering	2,014	cc-by	8,174	Medhat Elkelawy Department of Mechanical Power Engineering, Tanta University, Tanta, Egypt Email: medhatelkelawy@f-eng.tanta.edu.eg Received 26 September 2014; revised 25 October 2014; accepted 15 November 201 Copyright © 2014 by author and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). h // /l /b / / Energy and Power Engineering, 2014, 6, 513-526 Published Online December 2014 in SciRes. http://www.scirp.org/journal/epe http://dx.doi.org/10.4236/epe.2014.614045 Energy and Power Engineering, 2014, 6, 513-526 Published Online December 2014 in SciRes. http://www.scirp.org/journal/epe http://dx.doi.org/10.4236/epe.2014.614045 Energy and Power Engineering, 2014, 6, 513-526 Published Online December 2014 in SciRes. http://www.scirp.org/journal/epe http://dx.doi.org/10.4236/epe.2014.614045 How to cite this paper: Elkelawy, M. (2014) Experimental Investigation of Intake Diesel Aerosol Fuel Homogeneous Charge Compression Ignition (HCCI) Engine Combustion and Emissions. Energy and Power Engineering, 6, 513-526. http://dx.doi.org/10.4236/epe.2014.614045 1. Introduction Homogeneous charge compression ignition (HCCI) technology offers great potential benefits on low NOx and particular matter (PM) emissions and high thermal efficiency. However, it does have it is unique challenge [1]- [4], particularly the homogenization of fuel, air, and recycled burnt gases prior to ignition, control of ignition and combustion timing, heat release rates, it is a narrow operating window, and high HC and CO emissions. From HCCI engine principal it is important to realize mixture formation and the avoidance of fuel-wall interac- tions to achieve high fuel efficiency, reduce HC and PM emissions, and prevent oil dilution [5]. Regarding the mixture formation and homogenization of fuel and air, two main categories, namely the external and the internal mixture formation can be distinguished. External mixture formation is the simplest way of achieving a homogeneous in-cylinder mixture [6] [7]. This method is also known as port injection or fumigation. The turbulence created by the intake flow supports further homogenization. Because the air-fuel mixture is exposed to the whole time, temperature and pressure develop- ment, the port injection would belong to the early homogenization concepts [8]. A drawback of this strategy is that injection timing cannot be used to influence the start of ignition. Heavy fuels with low volatility results in poor evaporation, increased wall impingement increased of HC and CO emissions, and increase fuel consump- tion and fuel dilution [9]. However, this kind of the fuel injection is mainly attractive for gaseous and liquid fu- els with high volatility. In the case of internal mixture formation, the fuel is directly injected into the cylinder [7]. There are two strategies used for this method, i.e., early and late injection timing [4]. Early injection is mostly used for HCCI heavy fuel applications, in which a long ignition delay with low temperatures homogenizes the air-fuel mixture [10]. A significant part or even the whole amount of the fuel is injected before top dead center. In the case of heavy fuel injection, poor fuel volatility and low air density inside the cylinder may result in considerable wall wetting. For those cases, the new and highly flexible injection systems should be designed, which are used to make adaptation to the variation of combustion-chamber geometry as well as in-cylinder pressure and tempera- ture during the injection. g j Some researchers have already spent much effort on developing low penetration injectors and minimizing wall impingement [4] [11]. 1. Introduction A suitable injection system must have a high degree of flexibility in order to allow adaptation of the injection strategy, according to the varying boundary conditions during injection [12] [13]. High-pressure injection designed with a large number of small nozzle holes is generally used in order to increase spray disintegration, and to include the complete cylinder charge in the mixture formation process while avoid- ing wall film formation. A further adaptation of the spray penetration can be realized by splitting the injection event into several pulses with different durations [14]. In-cylinder injection HCCI engines, the short pulse dura- tion leads to less momentum of the liquid fuel resulting in reduced penetration. The low gas density at the start of injection requires short pulses with reduced injection velocities and relatively long time intervals between the pulses [15] [16]. As the piston moves up, charge density and temperature in the cylinder increase and penetra- tion is reduced. The pulse durations can be prolonged, while the time intervals between subsequent pulses are shortened. At the end of the pulsed injection the distance between nozzle and piston reduces significantly and the mass injected per pulse must be reduced again in order to prevent fuel deposition on the piston [17] [18]. The recent trend for all published diesel HCCI research involves mixture preparation using the ideal condi- tions of HCCI combustion regimes, referred to as external mixture formation [19]. By introducing the fuel ex- ternally to the combustion chamber one can use the turbulence generated by the intake process to create a ho- mogeneous charge regardless of engine conditions. This eliminates the need of fuel system changes, which are necessary for the internal mixture formation method. In this method, the combustion system remains fully opti- mized while running in HCCI combustion mode with nearly ideal mixture preparation. Present experimental investigation is aimed to develop diesel fuel cavitation methodology in the diesel injec- tor nozzle for preparing a fully homogeneous mixture of diesel and air. This research explores the fuel mixture preparation as well as the combustion and emission characteristics of HCCI using an external mixing device “diesel vaporizer chamber”, which requires less engine fuel system modifications in the present DI engine in order to convert it into HCCI engine. Abstract External mixture formation (PFI) of a diesel fuel aerosol has employed to investigate the diesel HCCI engine combustion and emissions characteristics. The key to the external mixture formation with diesel fuel is the proper fuel/air mixture preparation. A proposed intake diesel fuel aerosol system mainly consists of a small chamber, in which the diesel fuel is fully vaporized by means of fuel cavitation inside the diesel injector nozzle. Nozzle cavitation is mainly affected by the injec- tion pressure and the fuel system temperature. Results obtained reveal that the proposed method determines the possibility of producing a complete homogeneous fuel/air mixture, which can be applied to the diesel HCCI engine. With this method, the combustion and emission behavior were entirely optimized and the engine is capable of running in HCCI combustion mode with nearly ideal mixture preparation. In the present investigation, a methodology for the HCCI combustion mode of the diesel aerosol/air mixtures based on the fuel cavitation inside the injector nozzle pa- rameters (such as the injection pressure and the fuel system temperature where fuel premixed ratio, NOx, CO, CO2, and HC emissions) have analyzed. Based on the engine performance and emis- sions characteristics the fuel injection pressure and the fuel system temperature have optimized to produce a suitable fuel premixed ratio and the perfect fuel/air mixture homogeneity at differ- ent engine operating conditions. The optimal injection pressure ranges between 150 - 200 bars, while the fuel system temperature lies within 175˚C - 200˚C. Loops of exhaust gas recirculation (EGR) are used to extend the engine load by controlling the combustion phasing. HCCI, Cavitation, External Mixture Formation, Diesel Injector Nozzle, EGR HCCI, Cavitation, External Mixture Formation, Diesel Injector Nozzle, EGR *Zero emission Diesel HCCI engine. M. Elkelawy 2. Engine and Experimental Setup In the present study, experimental activities have conducted in a heavy-duty commercial single-cylinder diesel engine to investigate the basic performance characteristics of diesel HCCI engines combustion and emissions. The experimental setup facilities will specifically address the operating parameters of the diesel HCCI engines such as the control of ignition timing, heat release rate, emissions over a wide range of loads, speeds, different percentage of EGR rate, different port fuel injection pressures, and temperatures of both of fuel and charged air. Moreover, the test engine was adjusted in our work to run in HCCI mode over a wide range of operating condi- In the present study, experimental activities have conducted in a heavy-duty commercial single-cylinder diesel engine to investigate the basic performance characteristics of diesel HCCI engines combustion and emissions. p y, p y y g y engine to investigate the basic performance characteristics of diesel HCCI engines combustion and emissions. The experimental setup facilities will specifically address the operating parameters of the diesel HCCI engines such as the control of ignition timing, heat release rate, emissions over a wide range of loads, speeds, different percentage of EGR rate, different port fuel injection pressures, and temperatures of both of fuel and charged air. Moreover, the test engine was adjusted in our work to run in HCCI mode over a wide range of operating condi- tions to analyze the engine performance and emissions. This engine was equipped to handle intake heating and fuel additives in addition to the possibility of changing the engine loads at fixed speed and vice versa. Test engine used in this study was naturally aspirated, four-stroke, and water-cooled. Fuel/air homogenous mixture was formed during suction and compression strokes, in which the fuel supply mode was changed from the direct fuel injection to port fuel supply. Table 1 gives the specifications of the engine and Figure 1 illu- strates the schematic diagram of the engine experimental setup. The in-cylinder gas pressure was measured with a Kistlerpiezo-electric pressure transducer installed in the cylinder head, and the pressure transducer was con- nected with a commercial data acquisition card through a Kistler charge amplifier. In-cylinder gas pressure is then correlated to crankshaft angular position using an electro-magnetic sensor mounted on the engine crank- shaft. Four thermocouples were used to measure the temperature of intake air, exhaust gas, coolant water, and lubricating oil. 1. Introduction Diesel vapor produced by this device also contains very small diesel drop- lets in the form of diesel-like aerosol fuel, which will easily mix with air to form either fully or partly homoge- neous mixture. Due to the adequate mixing in the vaporization chamber, formation of mixture of air and partly vaporized fuel aerosol takes place. Various experiments were performed to investigate HCCI combustion using different EGR conditions and engine loads. 514 M. Elkelawy 2. Engine and Experimental Setup A further thermocouple was operated by a feedback control system to adjust the intake air and fuel system temperature. The exhaust emissions concentrations such as NOx, CO, UHC, and CO2 were measured with NHA-500 exhaust gas analyzer which has an accessory of lambda sensor for the measurement of the excess air coefficient (λ). The engine speed and load have controlled by an eddy current dynamometer. Before the in- take manifold, a large stable surge tank was equipped with electric heater operating according to a feedback system being used to control the air charge temperature. 515 Table 1.Test Engine specification. Bore × Stroke/Piston Shape 130 mm × 120 mm/ω Shape Displacement 1.592 L Connecting Rod Length/Crank Radius Ratio 3.742 Compression Ratio 15.1 Fuel Supply Mode Port fuel Supply Intake Valve Open/ Intake Valve Close 12˚ BTDC/38˚ ABDC Exhaust Valve Open/ Exhaust Valve Close 55˚ BBDC/17˚ ATDC Max. Power 20.22 KW at 2200 RPM Figure 1. Schematic diagrams of the engine experimental devices. Table 1.Test Engine specification. Bore × Stroke/Piston Shape 130 mm × 120 mm/ω Shape Displacement 1.592 L Connecting Rod Length/Crank Radius Ratio 3.742 Compression Ratio 15.1 Fuel Supply Mode Port fuel Supply Intake Valve Open/ Intake Valve Close 12˚ BTDC/38˚ ABDC Exhaust Valve Open/ Exhaust Valve Close 55˚ BBDC/17˚ ATDC Max. Power 20.22 KW at 2200 RPM Bore × Stroke/Piston Shape Displacement Connecting Rod Length/Crank Radius Ratio Compression Ratio Fuel Supply Mode Intake Valve Open/ Intake Valve Close Exhaust Valve Open/ Exhaust Valve Close Max. Power Figure 1. Schematic diagrams of the engine experimental devices. Figure 1. Schematic diagrams of the engine experimental devices. 515 M. Elkelawy Homogeneous charge preparation is the most important role in achieving diesel HCCI combustion. Low vola- tility of diesel is the main hurdle in forming a homogeneous mixture of fuel and air, especially when injected in- side the engine manifold. In the present investigation, a diesel vaporization chamber is built up for this purpose. The construction of the diesel vaporization chamber is shown in Figure 1 (part 1). The diesel vaporization chamber has two inputs and two outputs streams. The input streams are assembled for the air and diesel fuel lines, in which both quantities can be controlled and measured. The fuel and air lines ware facilitated with con- trollable heaters to adjust the fuel system temperature. 2.1. Test-Rig Assembly, Calibration It is widely known that, the calibration process is a comparison between measurements which known in magni- tude or correctness made with a certain device and another measurement made in a similar way as possible with a second device. Relatively, the error locates in the results must be within a certain range, which makes the re- sults obtained reliable. Herein, comparing the measured values from the different flow meters of our test rig with the calculated values from the exhaust gas analyzer data will introduce the type of error presented. Generally, measurements have two types of errors, systematic error and the random error. Random errors are not accepted in our measurement applications, it is caused by inherently unpredictable fluctuations in the readings of a mea- surement apparatus or in the experimenter’s interpretation of the instrumental reading. Also, systematic errors are biases in measurement where the mean of many separate measurements differs significantly from the actual value of the measured attribute. In the following we will implement a new method for checking the device reliability for our test-rig assembly. We will calculate the air/fuel ration as a function of the exhaust gas percentages, and compare those results with the measured air/fuel ratio at the same conditions. The calculated air/fuel ratio is called spindt AFR [20]. The spindt AFR calculation method used to deduce the operating air-fuel ratio of the fuel flow rate, fuel characteris- tics, and emissions was introduced by Spindt for conventional fuels [21] [22]. Figure 2 represents the comparison between the calculated and the measured air/fuel ration at different engine loads and fixed engine speed of 1200 RPM with 298 K fuel system temperature. It is clear from Figure 2 that, for the engine operation layout when increasing the engine loads from 0 to 20 HP at the same engine speed the both calculated and the measured air/fuel ratio decreased with increasing the load. Comparing the results shown we can conclude that, our measured acquired results from the flow meters as well as the emissions analyzer lo- cated in an acceptable tolerance range of results fitting analysis. From the shown figure it is clear that, both of the curves at different conditions show a responsible agreement in the increasing and the decreasing trend of both air/fuel ratio. 2. Engine and Experimental Setup The surfaces of the whole fuel system are thermally insu- lated to fix the temperature for all the system operating conditions. Fuel injector sprays the pressurized and heated fuel into the heated air inside the diesel vaporization chamber. The diesel fuel is fully vaporized by means of fuel cavitation inside the diesel injector nozzle. Nozzle cavitation is mainly affected by injection pres- sure and the fuel system temperature. Hot air supplied to the vaporization chamber is mixed with the diesel va- por homogeneously. This mixture is mixed with the EGR and fresh air and supplied to engine cylinder through the charge mixing chamber (part 2 in Figure 1) located in the inlet manifold. A part of the exhaust gas is cooled and recirculated back into the combustion chamber to control the combus- tion phasing and extend the engine loads. The use of EGR reduces the in-cylinder maximum temperature and thereby reducing NOx emissions significantly. The exhaust gas from the engine cylinder is, however, highly pulsating in nature and thus preventing the accurate flow measurement. Therefore, a damper is inserted in the circuit to reduce the exhaust gas pulsations. A flow manometer, mounted downstream of the damper, is used to measure the flow rate of EGR across a calibrated digital reader. The fuel employed was commercial diesel fuel with characteristics shown in Table 2. The fuel is supplied with a set of high pressure pump along with a pre-calibrated flow meters and high accuracy digital balance (±0.001 gram absolute error). The mass flow rate of air was measured with a high accuracy digital flow meter. The atmospheric temperature in the engine test lab is usually maintained constant at 27˚C. The intake air tem- perature was automatically controlled using an electric intake air heater. The coolant temperature is a very im- portant parameter that needs to be controlled during HCCI experiments. For this purpose, the coolant water was kept constant at 85˚C during our experimental activities. 2.1. Test-Rig Assembly, Calibration We can also conclude that, the emission analyzer results of the components of the exhaust are reliable enough for our experimental work accuracy at different conditions and different measurement techniques. 516 M. Elkelawy Table 2. Specifications of test fuel. Cetane Number 54 Density 0.813 g/cm3 Vapor Pressure 40 mm Hg Surface Tension 0.02012 N/m Dynamic Viscosity 3.87 × 10 - 4 Pa·S Boiling Temperature 280˚C (95%) (A/F) Stoich. 14.5 Figure 2. Comparison between calculated and measured Air/Fuel ratio at different engine load, engine speeds 1200 RPM. Table 2. Specifications of test fuel. Figure 2. Comparison between calculated and measured Air/Fuel ratio at different engine load, engine speeds 1200 RPM. 2.2. Study of the Fuel Mixture Preparation 2.2. Study of the Fuel Mixture Preparation Homogeneous charge preparation is the first challenge for the application of the HCCI combustion mode. For port fuel injection (PFI), the injection pressure (Pinj), injection temperature (Tinj), and the physical properties of the fuel play important roles in governing mixture formation characteristics. In this experiment, diesel fuel aerosol was produced inside the injector nozzle due to the occurrence of cavitaion inside the diesel injector noz- zle [23] [24]. The injected diesel into a vaporization chamber remains at a temperature equal the fuel line tem- perature (fuel system temperature). However, inside the fuel vaporization chamber a part of the injected liquid diesel is converted into a diesel vapor then entering the engine, the rest of the fuel is returned back to the tank in liquid state. But the returning liquid fuel may not vaporize completely in every cycle, because of not reaching the vaporization point of the diesel in some conditions. So, the amount of the fuel sucked into the cylinder could not be directly calculated from the fuel consumption, according to electronic balance shown in Figure 1. y calculated from the fuel consumption, according to electronic balance shown in Figure 1. In order to estimate the vaporization rate of the injected diesel fuel, the fuel premixed ratio (FPR), in this work, is defined using the Equations 1 and 2 as the following: FPR vapor fuel m m =   (1) FPR vapor fuel m m =   (1) Where, vapor m is the diesel fuel consumption in the form of aerosol fuel, which can be calculated from the elec- tronic balances reading of fuel m and the liquid m return as can be seen in Figure 1. Where, vapor m is the diesel fuel consumption in the form of aerosol fuel, which can be calculated from the elec- tronic balances reading of fuel m and the liquid m return as can be seen in Figure 1. Return vapor fuel liquid m m m = −    (2) (2) Return vapor fuel liquid m m m = −    517 M. 2.2. Study of the Fuel Mixture Preparation Elkelawy To study the effect of injection pressure and temperate on the production of diesel vapor from the vaporiza- tion chamber and its effect on the fuel premixed ratio (FPR) the engine speed is fixed at 1200 RPM and the fuel system temperature and injection pressure (Tinj and Pinj) have been investigated in the range of 25 to 225˚C, and 75 to 250 bars, respectively. Figure 3 illustrates the percentage of fuel premixed ratio at different engine load at fixed engine speed 1200 RPM and different injection pressure when the fuel system temperature kept constant at 25˚C. As clear in Figure 3 that by increasing the injection pressure the fuel premixed ratio increases at both low load conditions and high load conditions. While, the effect of fuel premixed ratio increment is more pronounced between Pinj is between 75 and 200 bars. Furthermore, for the same engine load by increasing the pressure be- tween the mentioned values the fuel premixed ratio is increased rapidly. Never less, increasing the injection pressure more than 200 bars will add more loads on the tested engine pump without remarkable improvement in the overall fuel premixed ratio for the whole system. In low load conditions, the amount of fuel injected is very small in each cycle, while, in high load conditions, the amount of fuel injected is larger. At the same injection pressure, if the amount of the fuel injected increases, the cone angle, penetration of the spray, the number of droplets, and the droplet diameter will increase. The result matches well the results we obtained from our simu- lation work presented before in the previous publication [24]. By increasing the injection pressure slightly the amount of vapor present in the system and the fuel premixed ratio will take to increase until reach a certain val- ue then it will keep on an almost constant manner. To retain our results and provide more details of the operating range for the system optimal injection pressure and injection temperature we will select the values of injection pressures between 75 and 200 bar to study the effect of varying system temperature on the fuel premixed ratio at fixed engine speed 1200 RPM. As can be seen in Figure 4 we can conclude from the results that at the same load conditions, intake temperature and injection pressure are important factors for FPR. 2.2. Study of the Fuel Mixture Preparation Moreover the increasing value of the fuel premixed ratio in our case did not increase randomly. The optimal operating conditions for our case will locate in the pressure range between 75 and 200 bars, not more than these values, and the temperature effect on the fuel premixed ratio is more pro- nounced as the temperature increased until 225˚C. 3. Diesel HCCI Engine Emissions 3. Diesel HCCI Engine Emissions Homogeneous Charge Compression ignition (HCCI) is an advanced combustion technology being considered Homogeneous Charge Compression ignition (HCCI) is an advanced combustion technology being considered Homogeneous Charge Compression ignition (HCCI) is an advanced combustion technology being considered 518 eous Charge Compression ignition (HCCI) is an advanced combustion technology being considered Figure 3. Fuel premixed ratio with different injection pressure at different engine loads, engine speed 1200 RPM, and 25˚C fuel system temperature. Figure 3. Fuel premixed ratio with different injection pressure at different engine loads, engine speed 1200 RPM, and 25˚C fuel system temperature. Figure 3. Fuel premixed ratio with different injection pressure at different engine loads, engine speed 1200 RPM, and 25˚C fuel system temperature. 518 M. Elkelawy Figure 4. Fuel premixed ratio at different injection pressure and 1200 RPM with different fuel injection temperature (fuel system temperatures). for use in internal combustion (IC) engines to improve fuel economy and reduce NOx and soot emissions. In the CI engine the NO is formed in very hot zones closer to stoichiometric conditions and soot is formed in the fuel Figure 4. Fuel premixed ratio at different injection pressure and 1200 RPM with different fuel injection temperature (fuel system temperatures). Figure 4. Fuel premixed ratio at different injection pressure and 1200 RPM with different fuel injection temperature (fuel system temperatures). for use in internal combustion (IC) engines to improve fuel economy and reduce NOx and soot emissions. In the CI engine, the NOx is formed in very hot zones closer to stoichiometric conditions and soot is formed in the fuel rich regions. The in-cylinder average air/fuel ratio is globally always lean, but locally the combustion process is not. This means that there is a large potential to reduce emissions of NOx and PM by simply mixing fuel and air before combustion take place. In the present study, a homogeneous mixture of the diesel fuel and air was formed by using our technique for the vaporization of diesel in the intake manifold (external mixture formation) to achieve HCCI combustion. In HCCI mode, the shorter combustion duration and lower combustion temperature lead to lower NOx and soot emissions, but higher unburned hydrocarbon (HC) and carbon monoxide (CO) emis- sions compared with standard diesel engines. The experimental results show that the NOx concentration in the exhaust pipe does not exceeding 10 ppm in all of our engine conditions. 3. Diesel HCCI Engine Emissions The power output of the HCCI engine is also limited since the combustion can become unstable and knock-like cylinder pressure oscillations occur as the mixture approaches stoichiometric. Figure 4. Fuel premixed ratio at different injection pressure and 1200 RPM with different fuel injection temperature (fuel system temperatures). for use in internal combustion (IC) engines to improve fuel economy and reduce NOx and soot emissions. In the CI engine, the NOx is formed in very hot zones closer to stoichiometric conditions and soot is formed in the fuel rich regions. The in-cylinder average air/fuel ratio is globally always lean, but locally the combustion process is not. This means that there is a large potential to reduce emissions of NOx and PM by simply mixing fuel and air before combustion take place. In the present study, a homogeneous mixture of the diesel fuel and air was formed by using our technique for the vaporization of diesel in the intake manifold (external mixture formation) to achieve HCCI combustion. In HCCI mode, the shorter combustion duration and lower combustion temperature lead to lower NOx and soot emissions, but higher unburned hydrocarbon (HC) and carbon monoxide (CO) emis- sions compared with standard diesel engines. The experimental results show that the NOx concentration in the exhaust pipe does not exceeding 10 ppm in all of our engine conditions. The power output of the HCCI engine is also limited since the combustion can become unstable and knock-like cylinder pressure oscillations occur as the mixture approaches stoichiometric. From the previous section we already performed an optimization for the range of operating injection pressure and temperature. The analysis of the engine emissions will be performed in the same pre-defined range in order to fasten our results upon the engine operating range. We will study the effect of our proposed technique on the emissions of the HCCI engine at different operating conditions of fuel temperature and injection pressure with different engine loads. Herein we aim to obtain a desired operating condition, in terms of different engine load condition and several options of the fuel injection temperature and pressure are available from our experimental results and the emission analysis performed. 3.1. HCCI Engine CO2 Emissions Figure 5 shows oxides of Carbon (CO2) emissions as a function of injection pressure and temperature for vari- ous engine loads as well as a constant speed of engine 1200 RPM. CO2 emissions were almost independent of the injection pressure as it is shown a constant manner of variation with different injection pressure. It exponen- tially increased and almost stayed constant between 0.8%, and 2.5% (percentage by volume from exhaust gas emission) as the pressure increased. It is obvious that at high engine loads the presence of CO2 is more pro- nounced especially as the injection pressure increased to the range of 150 to 200 bars which means the combus- tion of fuel is better at higher injection pressure. However, with a fixed engine load 1.8 BMEP and 1200 RPM the increasing of the CO2 emissions is slightly different in high injection pressure conditions 150 bars, and 200 519 M. Elkelawy bars. Although 75 bars were the lowest tested pressure, but the rate of CO2 formation at that pressure with in- creasing temperature is very highly increased when the temperature located between 25˚C until 150˚C while it stay constant from 150˚C to 225˚C. This result indicates that CO2 emissions are more a function of the engine load and the diesel fuel injection temperature rather than engine speed or injection pressure in an HCCI engine. bars. Although 75 bars were the lowest tested pressure, but the rate of CO2 formation at that pressure with in- creasing temperature is very highly increased when the temperature located between 25˚C until 150˚C while it stay constant from 150˚C to 225˚C. This result indicates that CO2 emissions are more a function of the engine load and the diesel fuel injection temperature rather than engine speed or injection pressure in an HCCI engine. 3.2. HCCI Engine HC Emissions In Figure 6, hydrocarbon HC emissions increased in a 2nd order polynomial manner with injection pressure vari- ation. As the engine speed is constant and the injection pressure increased then the amount of HC emission will be decreased. While, HC emission increased comparatively with increased engine load. Figure 6 also shows hydrocarbon (HC) emission as a function of fuel injection temperature for various injection pressure at fixed en- gine speed and load, 1200 RPM, and 1.8 BMEP, respectively. 3.1. HCCI Engine CO2 Emissions HC was about 150 - 130 ppm and it decreased in a 2nd order polynomial manner when the injection temperature increased with the same injection pressure. Higher fuel injection pressure showed faster decrease in HC. The concentrations of HC represent the importance of fuel vaporization in the HCCI engine combustion improvement. The results indicated that raising the fuel tempera- ture and the injection pressure, especially in the high load case will enhance the combustion in the engine as it re- duces the HC presence in the engine emissions. It can be observed that the engine operated with diesel vapor-air mixture exhibits a significant reduction in HC at all loads. However, mixture is available inside the cylinder as a homogeneous mixture is completely supplied to the engine. Hence there is an absolutely free combustion from the liquid fraction of fuel pockets with increased temperature and pressure for the same load and speed. 3.3. HCCI Engine CO Emissions In Figure 7, it is clear that by increasing the injection pressure at the same engine load the CO emission de- creased dramatically. Furthermore, the decreasing manner of the CO is almost constant in all cases examined. Also, the tested highest engine load was recorded the lowest values of CO emissions for the same value of injec- tion pressure and engine speed. However, increasing the fuel temperature at the same injection pressure with the fixed engine load will decrease the amount of CO emissions formed as a result of the combustion process. For the same examined fuel system injection temperature the highest pressure applied is recorded the lowest CO emissions in the test cases. It is widely known that, the CO emissions are results of hydrocarbon incomplete combustion. CO emissions are one of the major setbacks of HCCI engine. One major factor, which contributes to higher CO emissions, is low temperature combustion due to lean HCCI engine mixture. As a conclusion from these results, by increasing the injection pressure and injection temperature the mixture preparation for the HCCI engine will be enhanced. The investigation has shown that a diesel engine can run on a homogeneous fuel/air mixture that is generated externally in a fuel vaporizer chamber. The emissions results are very good Figure 5. Variation of CO2 emission as a function of fuel temperature and injection pressure at engine speed 1200 RPM and different load condition. Figure 5. 3.1. HCCI Engine CO2 Emissions Variation of CO2 emission as a function of fuel temperature and injection pressure at engine speed 1200 RPM and different load condition. 520 520 M. Elkelawy Figure 6. Variation of HC emission as a function of fuel temperature and injection pressure at engine speed 1200 RPM and different load condition. Figure 6. Variation of HC emission as a function of fuel temperature and injection pressure at engine speed 1200 RPM and different load condition. Figure 7. Variation of CO emission as a function of fuel temperature and injection pressure at engine speed 1200 RPM and different load condition. describing the exact chart for the engine working map As a remarkable engine results show there is almost zero Figure 6. Variation of HC emission as a function of fuel temperature and injection pressure at engine speed 1200 RPM and different load condition. Figure 7. Variation of CO emission as a function of fuel temperature and injection pressure at engine speed 1200 RPM and different load condition. describing the exact chart for the engine working map. As a remarkable engine results show, there is almost zero NOX emission have recorded during the operation of our achieved method of the diesel HCCI engine. Figure 7. Variation of CO emission as a function of fuel temperature and injection pressure at engine speed 1200 RPM and different load condition. d ibi h h f h i ki A k bl i l h h i l Figure 7. Variation of CO emission as a function of fuel temperature and injection pressure at engine speed 1200 RPM and different load condition. describing the exact chart for the engine working map. As a remarkable engine results show, there is almost zero NOX emission have recorded during the operation of our achieved method of the diesel HCCI engine. describing the exact chart for the engine working map. As a remarkable engine results show, there is almost zero NOX emission have recorded during the operation of our achieved method of the diesel HCCI engine. 4. Monitoring of Diesel HCCI Engine Combustion 4. Monitoring of Diesel HCCI Engine Combustion The present section presents some of the different cases and operating conditions for comparing engine perfor- mance data variations in order to monitor the engine performance and operation. The following engine data analysis will be introduced in order to be as a guide or an operating map for the future researchers working on this issue. Thus, the engine raw data available about the single mode pure diesel HCCI engine is very little, or in another word there are no data for the operating chart of diesel HCCI engines. The engine experimental data are collected simultaneously with the recording of engine performance data at an instant. It has noted that the cha- racteristics of the diesel HCCI engine operating conditions are reflected in the experimental data analysis. Con- versely, this means that information about the engine performance variables can provide an insight into the en- gine conditions and confirm whether these are normal or otherwise the engine operation is not going smoothly. Cylinder pressure and engine performance analysis procedures detect abnormal combustion or engine emissions 521 M. Elkelawy conditions providing early warning of developing problems whether from incorrect settings or component mal- function. The diagnostic procedures for studying engine operating characteristics and engine performance have developed and calculated by aid of a code achieved to calculate the most important features of engine operation at different conditions. By the interpretation of the information collected developing faults can be identified well before they may be visible through the performance data or physical inspection. Evaluation of information from the analysis also allows recommendations for adjustments to diesel fuel evaporation, and engine settings, and for planning of an overhaul of injectors and engine units. In-cylinder pressure data analysis is the most effective way to analyze the engine combustion behavior be- cause in-cylinder pressure history directly influences the power output, combustion characteristics and emissions from an engine. Figures 8 show the measured cylinder pressure data versus crank angle at 200 bars injection pressure, fuel system temperature 150˚C, and 1200 RPM, at different engine load. This figure clearly shows the occurrence of auto-ignition (during HCCI combustion) in all studied cases. Rise in slope of pressure curve vis- à-vis zero load curve shows the normal start of combustion. As the mixture becomes richer, higher load condi- tions, start of combustion shifts towards BTDC side. It happens due to the early start of combustion during the compression stroke. 4. Monitoring of Diesel HCCI Engine Combustion For brake mean effective pressure less than 1.8 bars at no EGR, the combustion starts well before TDC. It can be noted from the representing results that the trends of the maximum pressure are more ob- vious increase in higher load condition. From Figure 8, it can be seen that the rate of pressure rise increases with increasing engine load which leads knock like combustion at 2.2 bars IMEP. It happens mainly because of higher rate of combustion due to charge enrichment. When charge becomes richer, it favors earlier start of combustion due to dominance of cold combustion chemistry of the diesel fuel. Higher fuel quantity (higher load condition) is responsible for higher in-cylinder pressure and earlier start of combustion. Apparent heat release analysis is another characterization tool for HCCI combustion. It is calculated from the acquired cylinder pressure data using “zero dimensional heat release model” [25]. The HCCI heat release pattern is different from conventional combustion modes due to occurrence of the combined phenomenon of simulta- neous ignition of homogeneous mixture using compression (SI and CI). Higher rate of apparent heat release in HCCI combustion creates problems in controlling the combustion rates and affects safety and structural integrity of the engine. The apparent heat release for varying the engine load without EGR effect is shown in Figure 9. The recorded results of pure diesel HCCI combustion process exhibit a two stage heat release model. The rate of heat release analysis indicates that the start of the first stage of the combustion varies little with the engine load, while the secondary stage changes significantly. In higher load conditions, the HCCI engine combustion takes place very fast with a high rate of heat release and steep pressure, which may lead to be excessive noise or even engine damage by knock like combustion. 522 Figure 8.The in-cylinder pressure data at fixed injection pressure of 200 bars and different engine load, fuel system temperature 150˚C, engine speed 1200 RPM. Figure 8.The in-cylinder pressure data at fixed injection pressure of 200 bars and different engine load, fuel system temperature 150˚C, engine speed 1200 RPM. M. Elkelawy Here, early combustion of the charge is controlled by using EGR, which retards the start of combustion sig- nificantly Figure 10. Peak cylinder pressure also decreases with increasing EGR due to reduction in rate of combustion at lower in-cylinder temperature. 4. Monitoring of Diesel HCCI Engine Combustion Figure 11. The in-cylinder heat released at different engine load and EGR percentage a fixed injection pressure of 200 bars, fuel system temperature 150˚C, and engine speed 1200 RPM. apparent heat release and its crank angle position. In all three cases, the apparent heat release curve possesses two peaks, one for low temperature heat release region and the other one for high temperature heat release re- gion. As the EGR percentage increase time delay between first and main heat release, is attributed to the “Nega- tive Temperature Coefficient (NTC) regime”, which is located between the two heat release stages is increased. 4. Monitoring of Diesel HCCI Engine Combustion That observation can be obtained when comparing the recorded engine pressure data in Figure 8 with the data recorded in Figure 10 at 2.2 bars IMEP. In all three EGR conditions (20%, 35%, and 45%), peak cylinder pressure occurs near TDC which shift away from TDC with increasing EGR. It happens because of delayed combustion due to mixture dilution by recirculated exhaust gas. As EGR rate increases, level of dilution also increases, increasing the delay period, which pushes the peak of the pressure curve towards the ATDC side. As the EGR rate increases, delayed combustion is observed for richer mixtures thereby affecting the HCCI combustion positively. Figure 11 shows the effect of engine load and EGR on the Figure 9. The in-cylinder heat energy released at different engine load and fixed injection pressure of 200 bars, fuel system temper- ature 150˚C, and engine speed 1200 RPM. Figure 10. The in-cylinder pressure data at different engine load and EGR percentage at fixed injection pressure of 200 bars, fuel system, temperature 150˚C, and engine speed 1200 RPM. Figure 9. The in-cylinder heat energy released at different engine load and fixed injection pressure of 200 bars, fuel system temper- ature 150˚C, and engine speed 1200 RPM. Figure 10. The in-cylinder pressure data at different engine load and EGR percentage at fixed injection pressure of 200 bars, fuel system, temperature 150˚C, and engine speed 1200 RPM. M. Elkelawy Figure 11. The in-cylinder heat released at different engine load and EGR percentage a fixed injection pressure of 200 bars, fuel system temperature 150˚C, and engine speed 1200 RPM. apparent heat release and its crank angle position. In all three cases, the apparent heat release curve possesses two peaks, one for low temperature heat release region and the other one for high temperature heat release re- gion. As the EGR percentage increase time delay between first and main heat release, is attributed to the “Nega- tive Temperature Coefficient (NTC) regime”, which is located between the two heat release stages is increased. 5 S d C l i Figure 11. The in-cylinder heat released at different engine load and EGR percentage a fixed injection pressure of 200 bars, fuel system temperature 150˚C, and engine speed 1200 RPM. Figure 11. The in-cylinder heat released at different engine load and EGR percentage a fixed injection pressure of 200 bars, fuel system temperature 150˚C, and engine speed 1200 RPM. References [1] Elkelawy, M., Yu-Sheng, Z., Alm El-Din, H. and Yu, J.-Z. (2008) Challenging and Future of Homogeneous Charge Compression Ignition Engines; an Advanced and Novel Concepts Review. Journal of Power and Energy Systems, 2, 1108-1119. http://dx.doi.org/10.1299/jpes.2.1108 [2] Yao, M., Zheng, Z. and Liu, H. 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Summary and Conclusions In the present study, combustion and emission characteristics of a homogeneous air-diesel fuel mixture HCCI engine with external mixture formation were investigated at different engine loads and EGR percentage. The in- dicated results show noticeable improvement in the charge homogeneity if the external mixture preparation technique has used. The formed diesel fuel aerosol inside the diesel vaporization chamber proved successful in achieving diesel vapor by means of cavitation principal. The diesel vaporization process basically is begun in- side the injector wholes by means of cavitation phenomena and continues to produce a fully vaporized diesel vapor aerosol inside a small chamber namely a diesel vaporization chamber. Fuel cavitation inside the diesel in- jection nozzle is mainly affected by the injection pressure and the fuel system temperature. The engine experiments were performed at different mixture strengths to study the engine load range until the rich mixture combustion is limited by the knock-like combustion. As the engine load increased, peak in-cylinder pressure and rate of heat release increase rapidly due to higher rates of combustion and start of combustion shifts to happen before top dead center (BTDC). However, the combustion process of the diesel HCCI is highly sensi- tive to fuel/air homogeneity and EGR percentage. Results obtained reveal that the proposed method determines the possibility of producing a complete homogeneous fuel/air mixture, which can be applied to the diesel HCCI engine. With this method, the combustion and emission behavior were entirely optimized and the engine is ca- pable of running in HCCI combustion mode with nearly ideal mixture preparation. In the present investigation, a methodology for the HCCI combustion mode of the diesel aerosol/air mixtures based on the fuel cavitation inside the injector nozzle parameters (such as the injection pressure and the fuel system temperature where fuel premixed ratio, NOx, CO, CO2, and HC emissions) have analyzed. Based on the engine performance and emissions characteristics the fuel injection pressure and the fuel system temperature have optimized to produce a suitable fuel premixed ratio and a perfect fuel/air mixture homogeneity at different engine operating conditions. The optimal injection pressure ranges between 150 - 200 bars, while the fuel sys- tem temperature lies within 175˚C - 200˚C. 524 M. Elkelawy [21] Bresenham, D., Reisel, J. and Neusen, K. (1998) Spindt Air-Fuel Ratio Method Generalization for Oxygenated Fuels. SAE Technical Paper 982054. http://dx.doi.org/10.4271/982054 [20] Spindt, R.S. (1965) Air-Fuel Ratios from Exhaust Gas Analysis. 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https://openalex.org/W3176750366	https://vestnik.mgimo.ru/jour/article/download/1937/1496	Russian	null	The Public Mission of Archives	Vestnik MGIMO-universiteta	2,012	cc-by	3,570	Общественная миссия архивов А.Н. Артизов, А.В. Серегин Беседа директора Дирекции информационно-издательских программ МГИМО(У) МИД России А.В. Серегина (А.С.) с руководителем Федерального архив- ного агентства А.Н. Артизовым (А.А.) если подчеркну, что сегодняшние архивы способ- ствуют решению задач социально-экономическо- го продвижения страны, развитию гражданско- го общества и эффективному государственному управлению, обеспечивают научное познание прошлого. А.С.: Андрей Николаевич, уровень развития архивного дела в нашей стране влияет на процесс исторического познания, а оно, в свою очередь, обеспечивает устойчивое развитие обществен- ного сознания, способствует самоидентификации современного социума, проходящего через горни- ло кардинальных общественно-политических перемен. Для историков работа в архиве – хлеб насущный. Что сегодня представляют собой оте- чественные архивы, справляются ли они со своей государственной и общественной миссией? р Государственные и муниципальные архивы России ежегодно выполняют миллионы запросов граждан. Большинство этих запросов мы испол- няем бесплатно, поскольку они связаны с под- тверждением трудового стажа граждан, размером заработной платы, то есть выдачей тех сведений, которые необходимы для начисления пенсий. Все больше к нам стало поступать запросов, которые выполняются за плату. Среди них запросы иму- щественного характера, генеалогические о состав- лении родословной. Количественное увеличение последних, кстати, свидетельствует, что мы пере- стаем быть «Иванами, не помнящими родства». у р щ А.А.: В обыденном сознании довольно часто можно встретить различные мнения об архивах. Например, что это всего лишь хранилища, куда списывают старые бумаги. Или, наоборот, они яв- ляются режимными учреждениями со строгим доступом, куда приходят избранные, которые имеют право искать необходимые им докумен- тальные свидетельства. Такие взгляды во многом обусловлены господствовавшей в советское вре- мя недооценкой архивов, культивировавшим- ся «классовым подходом», когда не поощрялось стремление людей поименно знать своих пред- ков: а вдруг среди них окажутся дворяне, купцы, представители других непролетарских сословий? Даже в эпоху перестройки тогдашний начальник Главархива СССР на вопрос о доступе в архивы имел обыкновение отвечать: «У нас архивы от- крыты с 9 утра до 6 часов вечера». б б Но, конечно, архивы – это прежде всего хра- нилища национальной памяти, как бы застыв- шей на архивных полках и одновременно живой, способной серьезно повлиять на общественные настроения, будучи введенной в научный оборот. В российских архивах сосредоточен уникальный материал, грамотное использование которого способно дать отпор разнообразным мифам и фальсификациям вокруг истории нашей страны. Как раз самой действенной мерой борьбы с та- кими фальсификациями является сложившаяся в современной России свобода доступа к архи- вным документам. 2012 – год Российской истории 2012 – год Российской истории Артизов Андрей Николаевич – д.и.н., руководитель Федерального архивного агентства. E-mail: vestnik@mgimo.ru Серегин Александр Васильевич – к.культур.н.,директор Дирекции информационно-издательских про- грамм МГИМО(У) МИД России. E-mail: vestnik@mgimo.ru Общественная миссия архивов ф А.С.: В последнее время порой приходится сталкиваться со спекуляциями, запускаемыми на общественный уровень, что-де архивы сворачива- ют рассекречивание и чуть ли не закрываются. Что можно сказать по поводу якобы закрытия архивов? А.А.: К сожалению, иногда ради «красного словца» подогревают такие слухи и некоторые работники архивов. Что можно сказать по этому поводу? Красноречивее всего говорят цифры. у Итак, в 2009 г. в федеральных архивах было рассекречено более 4 тыс. дел, через год – уже 11 тыс., а в 2011 г. – 37 с половиной тыс. архивных дел полностью и порядка 500 дел частично. За- мечу, что почти 35 тыс. дел из них рассекречено в соответствии с планом деятельности Межведом- ственной комиссии по защите государственной тайны. Ну о каком закрытии или сворачивании архивной работы после этого можно говорить? Ведь значительный рост объемов работы обеспе- чен как раз интенсификацией сотрудничества и взаимодействия аппаратов Росархива и Межве- домственной комиссии. А.С.: Проблем в архивных учреждениях пре- достаточно: и низкая заработная плата архиви- стов, и старение коллективов, поскольку выпуск- ники соответствующих профессиональных вузов плохо идут в архивы, предпочитая работать в организациях, где более высокие оклады, и многое, многое другое… Устойчивая динамика роста образовалась не на пустом месте. Она четко прописана в пяти- летнем плане нашей совместной с Межведом- ственной комиссией работы. Плановая основа позволила нам перейти от преимущественно те- матического рассекречивания к последователь- ному рассекречиванию комплексов архивных фондов. Здесь, полагаю, нужно сделать некото- рые пояснения, раскрывающие суть такого пере- хода. В недавнем прошлом работа по рассекре- чиванию действительно строилась в основном на тематических подборках. Например, собира- лись и готовились документы по теме «холод- ная война» и объединялись в соответствующий сборник. Под этот сборник рассекречивались конкретные документы из различных фондов и дел. Однако, будучи вырванными из контекста принятия различными органами власти тех или иных решений, они не в полном объеме удовлет- воряли исследовательский интерес исторической науки. Вы, наверное, согласитесь, что историка интересует не только определенный документ, но и политическая атмосфера, в которой он при- нимался, уровень его обсуждения и конечно же А.А.: Все, что перечислено в вопросе, к со- жалению, является правдой. Вообще любое раз- витие сопровождают проблемы. А у нас ряд про- блем, и среди них низкий уровень оплаты труда в бюджетной сфере – мы получили в наследст- во. Ежегодно на постоянное хранение в архивы поступает приблизительно 1,5 млн дел. График передачи дел из Архива Президента Российской Федерации утверждается на высшем уровне и, понятно, контролируется с особой ответствен- ностью. Общественная миссия архивов Сегодня существует широкая возможность знакомиться с подлинниками доку- ментов в наших читальных залах, на документаль- у Гражданское общество с разделением собст- венности и власти, приоритетом прав и свобод человека, открытая экономика в глобальном мире неминуемо поднимают значение документальной информации, а значит, архивов. Не преувеличу, Артизов Андрей Николаевич – д.и.н., руководитель Федерального архивного агентства. E-mail: vestnik@mgimo.ru Серегин Александр Васильевич – к.культур.н.,директор Дирекции информационно-издательских про- грамм МГИМО(У) МИД России. E-mail: vestnik@mgimo.ru 92 А.Н. Артизов, А.В. Серегин ных выставках, в Интернете на общеотраслевом портале «Архивы России». Соглашусь с вами, что, наверное, большинство серьезных научных работ основывается на изучении подлинных документов в читальных залах. В год они принимают около 100 тыс. пользователей, в том числе и из-за рубежа. За последние пять лет архивные учреждения Рос- сии выпустили в свет более 300 документальных сборников, посвященных досоветской, советской и современной истории. Начиная с 1990-х гг. происходит старение коллективов. Более половины работников ар- хивов – старше 50 лет. Сложилось тревожное несоответствие между обновляемой материаль- ной базой архивов и их кадровым потенциалом. Больше того, наряду с увеличением объемов, так сказать традиционной работы, перед архивами появились новые задачи по внедрению совре- менных информационных технологий и приему на хранение электронных документов. Со сво- ей стороны мы прикладываем огромные усилия, чтобы соответствовать новым вызовам времени. Но одних наших усилий явно недостаточно. Мы ожидаем государственной помощи в решении этих проблем, иначе они перерастут в угрозу ин- формационной безопасности России. р р Российские архивы – это огромнейшее хо- зяйство, в котором занято более 14 тыс. человек. Это 15 федеральных государственных архивов, 233 региональных государственных архивных центра и 2357 муниципальных архивов. Но это еще не все. 21 орган федеральной исполнительной власти и организация осуществляют депозитар- ное хранение документов с последующей переда- чей в госархивы. 126 тыс. архивов организаций, в большинстве частных, обеспечивают временное хранение документов. Есть также десятки спе- циализированных частных кампаний, которые оказывают различные услуги в сфере архивного дела. Архивный фонд России (без ведомственных архивов) насчитывает порядка 246 млн дел (во всех без исключения архивах страны сосредото- чено около 464 млн дел), 4,3 процента которых являются секретными. Это сотни тысяч дел, порой имеющих принципиальное значение для правиль- ного понимания событий прошлого. Конечно, се- рьезное значение мы придаем рассекречиванию архивов. Здесь, на наш взгляд, сделано немало. На- зову лишь одну цифру, в которой сфокусировалась вся проведенная работа: к настоящему времени рассекречено более 10 млн дел. Общественная миссия архивов Не хочу скрывать, что обработка та- ких огромных объемов документов требует, по меньшей мере, стабильности кадрового состава архивной службы. Чтобы ее поддерживать, нуж- на достойная зарплата. Ее нет, соответственно отсутствует и стабильность (среднемесячная заработная плата по федеральным архивам в прошлом году составила 21,7 тыс. рублей, в этом году выйдем примерно на 24 тыс. рублей, что для Москвы и Санкт-Петербурга, конечно, недостаточно). 93 2012 – Год российской истории А.А.: В современном информационном мире влияние печатно-издательского контента на массовое сознание необратимо перемещается на периферию, сужается значимость телевизион- ного вещания, особенно для молодого поколения. Напротив, все более возрастает роль Интерне- та. Повседневная практика свидетельствует об этой перемене. Дневная аудитория российского Интернета в возрастном сегменте от 12 до 34 лет превысила телевизионную и продолжает расти. точки зрения и аргументы политиков и государ- ственных деятелей, вовлеченных в подготовку и принятие решения. р р Как ученый, я не погрешу перед истиной, если скажу, что историческое комментирование, введение в научный оборот новых документов без подобного сопровождения теряет в своей объективности, а значит, и в качестве. Уже в рам- ках плана на 2006– 2010 гг. федеральные архивы начали работу по рассекречиванию комплексов (фондов) документов. В новом пятилетнем плане этот подход закреплен. В прошлом году были рассекречены в общей сложности 893 дела поста- новлений Совета Народных Комиссаров – Сове- та Министров СССР за 1941–1956 гг., почти 5 тыс. протоколов заседаний Президиума и Политбюро ЦК КПСС с 1957 по 1971 г., 1,5 тыс. дел аппарата ЦК КПСС с 1966 по 1968 г. Поэтому широкое представительство архивов в Интернете совершенно необходимо. От нас этого ждут общественность, все потребители архивной информации. В этой связи вспоминаю случай, ког- да на официальном сайте Росархива были разме- щены рассекреченные материалы по Катынской трагедии. Сайт не выдержал наплыва посетителей и, как принято говорить у программистов, «завис», не справился с одномоментным желанием мил- лионов ознакомиться с документами. Спрогно- зировать такой взрыв интереса к историческим свидетельствам мы просто не смогли, и, наверное, никто бы не смог. Лишь спустя какое-то время бесперебойная работа сайта была восстановлена, а для себя мы сделали необходимые выводы. Особый интерес, и не только для историков, представляют планируемые к рассекречиванию документы из фондов Сталина, Ежова, Ждано- ва, Молотова, Микояна, Чичерина. Это более 2 тыс. дел и 1,5 тыс. документов. Мы отдаем себе отчет, что историческая наука и общественность ждут их рассекречивания, что эти документы могут стать дополнительными источниками в понимании трагических и тяжелейших страниц нашего прошлого. Общественная миссия архивов В рамках мероприятий федеральной целевой программы «Культура России (2012 – 2018 гг.)» мы, в частности, намечаем создать мощный автома- тизированный комплекс справочно-поисковых средств к основной массе архивных документов. Это позволит, с одной стороны, обеспечить пра- ва граждан на доступ к архивной информации, улучшить качество обслуживания пользователей, с другой – повысить эффективность деятельности самих архивов, сократить издержки на хранение, учет и поиск информации, необходимой для ис- полнения запросов органов власти, организаций и граждан. р Мы намерены и дальше последовательно рас- секречивать архивные документы, обеспечивая к ним широкий доступ. С другой стороны, при проведении рассекречивания нельзя забывать о соблюдении требований законодательства о госу- дарственной тайне и иных охраняемых законам тайнах. Баланс в соблюдении этих интересов – об- щемировая практика. Возьмем, к примеру, Англию. Сравнительно недавно там истек срок секретности документов, связанных с пребыванием в Лондо- не прилетевшего туда накануне войны Рудольфа Гесса. Что и говорить, материалы его допросов представляют огромный интерес для понимания причин Второй мировой войны и политики ан- глийского руководства, которая, прямо скажем, не всегда согласовывалась с декларируемыми им нормами морали и нравственности. Историческое сообщество всего мира с нетерпением ждало рас- секречивания этих материалов, надеясь, что они прольют свет на существующие темные пятна. Но англичане продлили срок секретности. Вероятно, они не захотели наносить ущерб своему междуна- родному авторитету и своей безопасности. р Планируется продолжить перевод в элек- тронный формат описей федеральных и реги- ональных архивов, с тем чтобы довести долю оцифрованных заголовков дел до 65%, провести оцифровку наиболее востребованных фондов и каталогов федеральных архивов; создать коллек- ции качественных электронных образов докумен- тов Архивного фонда Российской Федерации в общем объеме около 5 млн листов, отражающих важнейшие события отечественной истории, и разместить их на портале «Архивы России» и на сайтах архивных учреждений. Вообще вопросы доступа к архивным документам мы намерены решать с акцентом в сторону замещения превали- рующих сегодня форм публичного представления архивных документов посредством выставок и документальных публикаций на бумажном носи- теле электронными экспозициями с размещением в Интернете. р у р у А.С.: Работа по сохранению и популяриза- ции исторического наследия России является, пожалуй, одной из важнейших задач архивной службы в целом. И конечно же, особое значение сегодня приобретает работа в Интернете. От ее правильного и успешного решения во многом зависит устойчивое развитие исторической на- уки страны, объективное и правдивое толкова- ние сложнейших исторических событий. Какое место в работе российских архивов занимает интернет-пространство и каким вы видите его дальнейшее развитие? Общественная миссия архивов Каким образом будет производиться отбор и оцифровка исторических документов для обеспечения доступа к ним через телеком- муникационные сети? у А.А.: Частично на эти вопросы я уже от- ветил. Добавлю, что информатизация отрасли проходит в соответствии с принимаемыми на федеральном уровне и в регионах программами информатизации архивных учреждений. Такая программа Росархивом принята в 2011 г., с ней можно ознакомиться на официальном сайте агентства. р К сожалению, не отвечает молодежным запросам и наш отраслевой портал «Архивы России», хотя для профессионалов он является вполне надежным историческим интернет-ре- сурсом, который уже сегодня дает уникальные возможности для поиска архивной информации. Основное развитие портала, конечно, еще впе- реди. Попробую пофантазировать. Мне лично будущее портала видится в сочетании возмож- ностей массмедийного средства и площадки для интерактивного общения и дискуссий с обратной связью, чего, кстати, не хватает телевидению. Как массмедийное средство портал мог бы взять на себя задачи размещения: б Полный переход к автоматизированным технологиям поиска архивной информации возможен, пожалуй, только после перевода в электронный вид всего научно-справочного аппарата к хранящимся в архивах документам. Этот процесс трудоемкий и весьма затратный. Представьте себе, что на возведение комплекса зданий площадью 50 тыс. квадратных метров для архива Великой Отечественной войны нужно ориентировочно до 5 млрд рублей (его строительство начнется в Подольске в рамках федеральной программы «Культура России»). Так вот, на сплошную оцифровку планируемых к перемещению в этот архив дел средств потре- буется в шесть раз больше. – во-первых, электронных образов архивных документов с их обязательным научным коммен- тарием (как это сделано в отношении коллекции документов о Нюрнбергском процессе); – во-первых, электронных образов архивных документов с их обязательным научным коммен- тарием (как это сделано в отношении коллекции документов о Нюрнбергском процессе); у – во-вторых, текстов, опубликованных на бу- маге сборников архивных документов; у – в-третьих, виртуальных архивных экскур- сий, экспозиций и выставок. Как интерактивная площадка портал мог бы регулярно работать с посетителями, интересую- щимися прошлым и желающими прояснить для себя степень правдивости и добросовестности источников и тех или иных исторических иссле- дований. Для таких посетителей, помимо функци- онирующего в интерактивном режиме форума портала, должна быть предоставлена возможность прямого общения с «дежурным специалистом портала» в режиме реального времени. К кон- кретному историческому событию портал мог бы готовить соответствующие лекции специалистов, проводить «веб-семинары и дискуссионные сто- у р Преобразования последних лет позволили архивам значительно нарастить свой информа- ционный потенциал. Однако позитивные пере- мены проходят на фоне длительного недофи- нансирования. В одночасье улучшить ситуацию не получится. Общественная миссия архивов Кстати говоря, умелое и широкое использова- ние Интернета – весьма действенное средство для борьбы с разного рода фальсификациями прой- денного Россией исторического пути, с мифоло- гизированными представлениями о прошлом. И мы им пока плохо пользуемся. Если говорить 94 А.Н. Артизов, А.В. Серегин лы». В федеральных архивах работают 23 доктора наук и 143 кандидата наук. Это весьма мощные научные силы. Мало какой научный центр рас- полагает такими квалифицированными специа- листами, знания и умение которых мы, наверное, еще не научились правильно использовать. честно, то возможности отечественной истори- ческой науки в информационном пространстве резко ограничены тиражами печатных изданий и телевизионной сеткой вещания, которая к тому же не позволяет реагировать на антироссийские исторические фальсификации в оперативном режиме и лишена обратной связи. Негативное воздействие такого положения сказывается на умонастроениях молодежи, которая утратила интерес к печатной продукции и предпочитает проводить время у компьютера. честно, то возможности отечественной истори- ческой науки в информационном пространстве резко ограничены тиражами печатных изданий и телевизионной сеткой вещания, которая к тому же не позволяет реагировать на антироссийские исторические фальсификации в оперативном режиме и лишена обратной связи. Негативное воздействие такого положения сказывается на умонастроениях молодежи, которая утратила интерес к печатной продукции и предпочитает проводить время у компьютера. у А.С.: Должен заметить, что ваше видение будущего портала почти полностью согласуется с его проработкой, которую вела Президентская Комиссия по противодействию попыткам фаль- сификации истории в ущерб интересам России по созданию в стране исторического сайта в сети Интернет. Недавним Указом Президента она упразднена. Как вы относитесь к такому решению? у А что сегодня в Рунете? Имеются десятки русскоязычных сайтов, в той или иной степени освещающих историческую проблематику. На- иболее значимый из них – «Руниверс», который информационно насыщен за счет сканирован- ных книг и собраний документов из библиотек. Как хранилище материалов этот сайт представ- ляет определенный интерес для специалистов- историков. Но он не пользуется популярностью у молодежи. Все другие сайты грешат бессистем- ностью выкладываемых материалов, наличием огромного числа ошибочных и недостоверных сведений, перегруженностью рекламой и наце- ленностью на сенсационность. Зато такие сайты часто посещает молодежь, не имея доступной альтернативы для формирования объективной точки зрения. р А.А.: Высказываю на этот счет свою гра- жданскую позицию: надеюсь, что с водой не выплеснут ребенка. А.С.: По своему опыту знаю, что российские архивы все больше обращаются в своей работе к современным технологиям обработки и достав- ки информации. Как проходит информатиза- ция архивной отрасли? Когда архивы смогут перейти на автоматизированные технологии поиска? 2012 – Год российской истории 2012 – Год российской истории В приоритетном порядке осуществляется оцифровка научно-справочного аппарата. Из до- кументальных массивов в первую очередь будут переведены в электронный формат и представ- лены в сети Интернет документы, включенные в Государственный свод уникальных документов Архивного фонда Российской Федерации. Также будут оцифрованы отдельные фонды советской эпохи из ГАРФ, РГАСПИ, РГАЛИ, РГВА, РГАЭ. Продолжится пополнение коллекции фотодоку- ментов периода Великой Отечественной войны на сайте «Победа» и создание виртуальных эк- спозиций архивных документов, посвященных памятным датам отечественной и мировой исто- рии. В 2012 г. планируется представить в сети Интернет выставки к 1150-летию российской государственности, 400-летию окончания Сму- ты XVI – XVII вв. и к 100-летию со дня рожде- ния выдающегося конструктора космической техники Б.Е. Чертока. А.С.: Как историк, доктор наук, вы много за- нимались проблемами отечественной истории ХХ в. Что, по-вашему, нужно сделать для того, чтобы появились учебники, достоверно отража- ющие советский период истории России? В какой степени архивы сотрудничают с образователь- ными учреждениями? у р А.С.: Создание добротного учебника, при всех других необходимых на то условиях, требует вре- менной дистанции от описываемых событий. Как говорится, прошлое должно устояться. В России сравнительно недавно произошла революция. Гуманитарная наука еще не выработала срок, ко- торый объективно необходим для того, чтобы об- новить систему идеологического и исторического сопровождения общественной жизни страны, ча- стью которой конечно же являются учебники. На- кал страстей в ходе общественной дискуссии на эту тему свидетельствует о том, насколько болезненно и остро мы проходим путь собственной идентифи- кации на новом витке общественно-исторического развития. Учебник – это слепок переживаемого страной исторического момента. Он не способен быть лучше или хуже этого момента, и его невоз- можно назначить «хорошим» даже из самых вы- соких кабинетов. р Значительным событием стало недавнее размещение на портале «Архивы России» про- граммного комплекса «Центральный фондовый каталог». В него включена информация о более чем 73 тыс. фондов 12 федеральных архивов. Пользователям предоставлена возможность лю- бого контекстного поиска по названиям и датам фондов. Намечено дополнить данный ресурс сведениями о фондах региональных архивов. Вместе с тем понятно нетерпение общест- венности, которая хочет объективных, качест- венных учебников. Такие учебники – важнейшее средство выполнения государством своей обра- зовательной функции. С учебниками связывается нравственное и духовное становление молодежи, которая наиболее не защищена от негативного влияния фальсифицированных и недобросо- вестных исторических продуктов. Я считаю, что каждый гуманитарий на своем месте должен помогать созданию новых учебников. Приме- нительно к архивам эта помощь – обеспечение доступа к основным документам советской эпохи, систематическая публикация таких документов. Общественная миссия архивов Вот почему в настоящее время из 186,2 млн дел государственных и муниципаль- ных архивов в различные базы данных, позволя- ющие осуществлять автоматизированный поиск, внесено лишь 17,5 млн дел. Но положительные изменения начались, и их, надеюсь, не остано- вить. 95 2012 – Год российской истории Хотя вынужден напомнить о том, на что ранее уже обращал внимание – между количеством и качест- вом доступных и опубликованных исторических источников и глубиной познания прошлого нет автоматической связи. А.С.: Российские государственные архивы уже два года участвуют в многотомном про- должающемся издании «Великая Победа». На сегодняшний день в свет вышло 8 томов. Наш Университет готовит к выходу в текущем году еще три тома. Между МГИМО(У) и Росархивом наладилось весьма плодотворное взаимодейст- вие. Каким вы видите его дальнейшее развитие? р А.А.: Приятно сознавать, что архивная служба приняла непосредственное участие в большой работе по созданию этого труда, став- шего победителем и лауреатом многих россий- ских и ряда международных конкурсов. Мы, конечно, продолжим совместную работу над многотомником и постараемся решить все сто- ящие перед нами задачи, как это было и раньше. В период с 1992 по 2010 г. архивы подготовили и издали примерно тысячу документальных сбор- ников и трудов по самым разнообразным исто- рическим сюжетам. В их числе такие фундамен- тальные серийные публикации, как «Политические партии России», «История сталинского ГУЛАГА», «Трагедия советской деревни», «Россия. ХХ век», «История создания и развития оборонно-промыш- ленного комплекса России и СССР», «Культура и власть. От Сталина до Горбачева», «Голод в СССР». А добротных учебников как не было, так пока и нет. Что касается расширения научного взаи- модействия между Росархивом и МГИМО(У), то, учитывая серьезные языковые возможности Университета, целесообразно пойти по пути сов- местной работы в изучении архивных докумен- тов на иностранных языках. Это, в первую оче- редь, документы Германии и других государств, перемещенные в ходе Второй мировой войны на территорию нашей страны и хранящиеся ныне в Российском государственном военном архиве. Некоторые из этих документов – предмет пре- тензий с целью их возможного возвращения. Такие документы обязательно должны пройти научную экспертизу, в которой, без сомнения, полезно участие ученых Университета. р у Что же касается работы с образовательными учреждениями, то архивисты никогда не забывали о ней. Об этом я говорил в своем выступлении на Первом Всероссийском съезде учителей истории в прошлом году. у у Вообще сфера научного сотрудничества безгранична. Нужны только желание, силы и средства. р у Artizov A.N., Seregin A.V. Public Mission of Archives. у Artizov A.N., Seregin A.V. Public Mission of Archives.
https://openalex.org/W3023019337	http://eudl.eu/pdf/10.4108/icst.tridentcom.2015.259876	English	null	OneLab Tutorial: A Single Portal to Heterogeneous Testbeds	null	2,015	cc-by	1,034	1. MOTIVATION Large-scale experimentation in varying types of environments is difficult to achieve, and until recently, experimenters have had to either construct their own platforms or rely on simulation, emulation, or analysis to gain results. However, these methods largely do not provide adequate or verified results. There lacked a viable model for the federation of large-scale testbeds that reconciled the challenges posed by how to provide a single entry point to access heterogeneous and distributed resources, and how to federate these resources that are under the control of multiple authorities. Efforts such as the FIRE1 initiative in Europe and GENI2 in the United States have worked to develop such a model, and the OneLab3 experimental facility, which came online in 2014, realizes this model, making a set of world-class testbeds freely available to researchers through a unique credential for each user and a common set of tools. OneLab provides a large- scale facility for rapid and remote testing that produces exhaustive results, at no charge to the experimenter. We allow users to deploy innovative experiments across our federated platforms that include the embedded object testbeds of FIT IoT-Lab4, the cognitive radio testbed of FIT CorteXlab5, the wireless testbeds of NITOS-Lab6, and the internet overlay testbeds of PlanetLab Europe (PLE)7, which together provide thousands of nodes for experimentation. The OneLab portal allows single-entry point access to these platforms, and provides users with unique credentials. This is made possible through the adoption of Slice- based Federation Architecture (SFA), an API for authentication and authorization that was conceptualized by the GENI initiative in the US, where each authority authenticates users and authorizes access to the resources; and MySlice8, the portal technology that we have developed to federate heterogeneous resources. The tutorial will walk the attendees through the process of creating an account, reserving resources, accessing the gateway server and running a basic experiment from the console. Users will be able to sign up and we will authorize their accounts on the spot, so that they may immediately access portal resources and deploy the experiments described below. We will launch three basic experiments on three of our different platforms: FIT IoT- Lab, NITOS, and PLE; so as to demonstrate the entire process of testing through the portal. The first experiment on FIT IoT-Lab will teach participants how to navigate the testbed tools. 1 http://www.ict-fire.eu/home.html. 2 http://www.geni.net/. 3 S. Fdida, T. Friedman, T. Parmentelat. “OneLab: An Open Federated Facility for Experimentally Driven Future Internet Research”. In Tania Tronco, ed., New Network Architectures: The Path to the Future Internet, Studies in Computational Intelligence. Springer Verlag, vol. 297, pp. 141-152, 2010; http://onelab.eu/. 4 http://www.iot-lab.info/. 5 http://www.cortexlab.fr/. 6 http://fit-nitos.fr/. 7 http://planet-lab.eu/. 8 http://www.myslice.info/ Loic Baron, Radomir Klacza, Mohammed Yasin Rahman, Ciro Scognamiglio, Nina Kurose, Timur Friedman, Serge Fdida UPMC Sorbonne Universités 4 Place Jussieu 75005 Paris, France firstname.lastname@lip6.fr Loic Baron, Radomir Klacza, Mohammed Yasin Rahman, Ciro Scognamiglio, Nina Kurose, Timur Friedman, Serge Fdida UPMC Sorbonne Universités 4 Place Jussieu 75005 Paris, France firstname.lastname@lip6.fr deploying experiments across our federated platforms. We will open with a short explanation of our experimental facility, its testbeds, and the ways in which OneLab reconciled the technical challenges of federating: how SFA allows for the federation of resources under multiple authorities and how MySlice provides a user-friendly interface displaying resources in web format. Following this overview, we will show attendees how to deploy an experiment on one of our FIT IoT-Lab testbeds through the OneLab portal. The projected outcome of this tutorial is that through its completion, users will finish with an account on the OneLab portal and the knowledge on how to access and deploy experiments on dozens of world-class testbeds. 2. AIMS The focus of our tutorial will be on providing attendees with hands-on experience in navigating the OneLab portal and Lastly, the experiment on PLE will provide each user with various initscripts describing different experiments. They will use these to configure their reserved PLE resources, deploy experiments, and view the output of each experiment. Users who are already familiar with PlanetLab will see how to deploy experiments through the new polyvalent OneLab platform. 1. MOTIVATION They will deploy their experiment, and then interact with running nodes by reading sensor values and sending radio packets. The purpose of this experiment will be to allow all attendees to have an introductory experience on navigating the OneLab portal and reserving resources, as they will both book IoT-Lab resources and view results remotely. The second experiment will allow users to reserve NITOS nodes and use the OMF (cOntrol and Management Framework) framework to control the experiment. This will give users the opportunity to learn how to use the OMF tool for experiment management and control. TRIDENTCOM 2015, June 24-25, Vancouver, Canada Copyright © 2015 ICST DOI 10.4108/icst.tridentcom.2015.259876 2 http://www.geni.net/. 2.1 Target Audience Our tutorial is targeted towards all users, coming from beginner to advanced backgrounds in experimental platforms. The OneLab facility is completely open to researchers, students, and scientific, academic, or industrial innovators. Those who are interested in IoT testing, wireless testing, or testing on internet-overlaid 4 http://www.iot-lab.info/. 6 http://fit-nitos.fr/. 7 http://planet-lab.eu/. 8 8 http://www.myslice.info/ TRIDENTCOM 2015, June 24-25, Vancouver, Canada Copyright © 2015 ICST DOI 10.4108/icst.tridentcom.2015.259876 platforms will all take a particular interest in our tutorial experiments, as will those interested in learning OMF as a tool. The IoT-Lab experiments will particularly interest those dedicated to the WSN community, and especially to people who are looking for an accurate scientific tool to support the design, development, tuning, and experimentation of real large-scale sensor network applications. All of our platforms are generic, open and flexible, meaning that a user can deploy his or her applications without any kind of restrictions on the programming language, the programming model, or the OS. It may be used by people working on the physical layer, the MAC layer, or any other layer of the OSI model, or on a more global application. 3. DURATION AND SPEAKERS The tutorial session will last two hours. A list of speakers is as follows: Loic Baron, the lead engineer of the OneLab team, will present the OneLab federation and guide the participants through using the portal. He will be assisted by an engineer from FIT IoT-Lab to
https://openalex.org/W2900736441	https://boundaryvalueproblems.springeropen.com/track/pdf/10.1186/s13661-018-1090-z.pdf	English	null	An exact bifurcation diagram for a reaction–diffusion equation arising in population dynamics	Boundary value problems	2,018	cc-by	9,803	© The Author(s) 2018. 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, pro- vided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Abstract We analyze the positive solutions to –v = λv(1 – v); 0, ∂v ∂η + γ √λv = 0; ∂0, where 0 = (0,1) or is a bounded domain in Rn, n = 2,3, with smooth boundary and \|0\| = 1, and λ, γ are positive parameters. Such steady state equations arise in population dynamics encapsulating assumptions regarding the patch/matrix interfaces such as patch preference and movement behavior. In this paper, we will discuss the exact bifurcation diagram and stability properties for such a steady state model. MSC: 35J25 MSC: 35J25 Keywords: Mathematical biology; Reaction–diﬀusion model; Bifurcation; Stability ( 2018) 2018:170 Goddard II et al. Boundary Value Problems https://doi.org/10.1186/s13661-018-1090-z An exact bifurcation diagram for a reaction–diffusion equation arising in population dynamics Jerome Goddard II1* , Quinn A. Morris2, Stephen B. Robinson3 and Ratnasingham Shivaji4 *Correspondence: jgoddard@aum.edu 1Department of Mathematics & Computer Science, Auburn University Montgomery, Montgomery, USA Full list of author information is available at the end of the article R ES EA RCH Open Access 1 Introduction Habitat fragmentation creates landscape-level spatial heterogeneity which inﬂuences the population dynamics of the resident species. Of particular interest, fragmentation often leads to declines in abundance of the species as the fragmented landscape becomes more susceptible to edge eﬀects between the remnant habitat patches and the lower quality human-modiﬁed “matrix” surrounding these focal patches [1–3]. Studies of movement behavior in response to diﬀerent habitat edge conditions clearly demonstrate that the com- position of the matrix can inﬂuence emigration rates, patterns of movement, and within- patch distributions of a species (e.g., [4–6]). Movement behavior has been shown to be very species-speciﬁc [7], even in the same fragmented habitat. Even though the task of connecting the wealth of empirical information available about individual movement and mortality in response to matrix composition to predictions about patch-level persistence is indeed formidable, the reaction–diﬀusion framework and its underlying random walk models have seen some success in addressing this connection [8]. The reaction–diﬀusion framework’s major strength is that the model’s dynamics can be analyzed mathematically, providing important patch-level predictions of population Goddard II et al. Boundary Value Problems ( 2018) 2018 Goddard II et al. Boundary Value Problems Page 2 of 17 ( 2018) 2018:170 ( 2018) 2018:170 persistence, leading to its wide adoption by ecologists ([9, 10], and [11]). This framework is also ideally suited to handle fragmentation and edge-mediated eﬀects as the partial dif- ferential equation(s) involved require explicit deﬁnition of edge behavior via boundary conditions ([10] and [2]). In [12], the authors formalize a framework to facilitate the connection between small- scale movement and patch-level predictions of persistence through a mechanistic model based on reaction–diﬀusion equations. The model is capable of incorporating essential information about edge-mediated eﬀects such as patch preference, movement behavior (e.g., emigration rates, patterns of movement, and within-patch distributions), and matrix- induced mortality at the patch/matrix interface. The authors then mathematically analyze the model’s predictions of persistence with a general logistic-type growth term. In particu- lar, the focus of [12] is to provide bounds on demographic attributes and patch size in order for the model to predict persistence of a species in a given patch based on assumptions on the patch/matrix interface and to explore their sensitivity to demographic attributes both in the patch and the matrix, as well as patch size and geometry. 1 Introduction The purpose of this present work is to provide an exact description of the bifurcation curve of positive steady states to their model when the growth term is logistic. In the following subsections, we will brieﬂy summarize the modeling framework and boundary condition derivation given in [12] and present our main results. We provide the proof of these results in Sect. 2. In Sect. 3, for the case n = 1, we provide an alternative proof of our results in the case = (0,1) via a quadrature method and discuss the evolution of the bifurcation curves as a model parameter varies. We discuss biological implications of our results in Sect. 4. Fi- nally, in Appendix 1, we provide the derivation of the boundary condition focused on in this paper, and in Appendix 2, we provide results on certain eigenvalue problems that we employ in the proof of our main result. 1.1 Modeling framework We consider a patch 0 in Rn when n = 1,2, or 3 with \|0\| = 1, where \|0\| = ⎧ ⎪⎪⎨ ⎪⎪⎩ length of 0; n = 1, area of 0; n = 2, volume of 0; n = 3. We assume here that the boundary of 0 (denoted by ∂0) is smooth. Now, we deﬁne the focal patch as = {ℓx\|x ∈0} yielding \|\| = ⎧ ⎪⎪⎨ ⎪⎪⎩ ℓ; n = 1, ℓ2; n = 2, ℓ3; n = 3, where ℓis a positive parameter representing the patch size (see [10]). In this way, we are able to separate the combined eﬀects of patch size (ℓ) and patch geometry (geometry of 0) on population persistence. In the model, u(t,x) represents the density of a theoreti- Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Page 3 of 17 Value Problems ( 2018) 2018:170 Page 3 of 1 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Goddard II et al. Boundary Value Problems Page 3 of 17 ( 2018) 2018:170 cal population inhabiting . Here, the variable t represents time and x represents spatial location within . The model is then as follows: ⎧ ⎪⎪⎨ ⎪⎪⎩ ut = Du + ru(1 – u K ); t > 0,x ∈, u(0,x) = u0(x); x ∈, D ∂u ∂η + √S0D0 κ u = 0; t > 0,x ∈∂, (1) (1) where the parameter D is the diﬀusion rate inside the patch, D0 is the diﬀusion rate in the matrix surrounding , r is the patch intrinsic growth rate, S0 is the death rate in the matrix, and κ is a parameter encapsulating assumptions regarding the patch/matrix inter- face such as patch preference and movement behavior. Also, ∂u ∂η represents the outward normal derivative of u, r is the intrinsic growth rate of the population inside , K is the carrying capacity, and u0 is the initial distribution of population density in the patch. The parameters D, D0, S0, r, K, and κ are always positive. Note that the boundary condition in (1) is derived in Appendix 1. The interface scenarios listed in [12] correspond to certain values of the parameter κ as originally derived in [13]. 1.1 Modeling framework Recall that in the random walk model, organisms are assumed to move the step size x with probability p every t units of time. The diﬀusion rate is then obtained by taking parabolic limits in such a way that D = lim x,t→0+ px2 t (2) (2) is constant (see, for example, [9, 14], and [10]). Table 1 taken directly from [12] lists each scenario along with their κ-value, name, biological interpretation, and selected references for each scenario. Note that α will denote the probability that an organism remains in the patch upon reaching the patch/matrix interface. Table 1 Listing of interface scenarios with descriptions and selected references from [12] Scenario name Scenario description κ References Continuous density Organisms move between the patch and the matrix with equal probability. Step sizes and movement probabilities are equal in the patch and the matrix. 1 [15] Type I Discontinuous density (DD) Organisms modify their movement behavior at the patch/matrix interface and would have a probability α of remaining in or leaving diﬀerent from 50%. Step sizes diﬀer between the patch and the matrix, whereas movement probabilities are equal. α 1–α D0 D [8, 16] Type II Discontinuous density (DD) Organisms modify their movement behavior at the patch/matrix interface and would have a probability α of remaining in or leaving diﬀerent from 50%. Step sizes are equal between the patch and the matrix but movement probabilities are diﬀerent. α 1–α D0 D [8, 16] Type III Discontinuous density (DD) Organisms remain in with probability α diﬀerent from 50%. Movement probabilities and step sizes are the same between the patch and the matrix. α 1–α [17, 18] Page 4 of 17 Goddard II et al. Boundary Value Problems ( 2018) 2018: Goddard II et al. Boundary Value Problems Table 2 Values of γ for each of the interface scenarios. DD denotes discontinuous density as described in Table 1. See [12] Table 2 Values of γ for each of the interface scenarios. DD denotes discontinuous density as described in Table 1. 1.1 Modeling framework See [12] Interface scenario γ -value Continuous density S0D0 rD Type I DD 1–α α √ S0 √r Type II DD 1–α α √ S0D √ rD0 Type III DD 1–α α S0D0 rD Now, applying the change of variables ˜t = rt, ˜x = x ℓ, v = u K , and λ = rℓ2 D , (1) reduces to (after dropping the tilde): ⎧ ⎪⎪⎨ ⎪⎪⎩ vt = v + λv(1 – v); t > 0,x ∈0, v(0,x) = v0(x); x ∈0, ∂v ∂η + γ √ λv = 0; t > 0,x ∈∂0 (3) ⎧ ⎪⎪⎨ ⎪⎪⎩ vt = v + λv(1 – v); t > 0,x ∈0, v(0,x) = v0(x); x ∈0, ∂v ∂η + γ √ λv = 0; t > 0,x ∈∂0 (3) (3) with steady state equation with steady state equation with steady state equation ⎧ ⎨ ⎩ –v = λv(1 – v); 0, ∂v ∂η + γ √ λv = 0; ∂0, (4) (4) where λ is unitless and \|0\| = 1. The value of the unitless parameter γ is given in Table 2 listed by interface scenario. Theorem 1 Given any γ > 0: Theorem 1 Given any γ > 0: (a) If λ > λ1(γ ), then the trivial solution of (4) is unstable and there exists a unique positive solution vλ to (4) which is globally asymptotically stable. Furthermore, ∥vλ∥∞→0+ as λ →λ1(γ )+ and ∥vλ∥∞→1 as λ →∞; (b) If 0 < λ ≤λ1(γ ), then the trivial solution of (4) is globally asymptotically stable and there is no positive solution to (4). (b) If 0 < λ ≤λ1(γ ), then the trivial solution of (4) is globally asymptotically stable and there is no positive solution to (4). Note that λ1(γ ) →0 as γ →0+. Theorem 1 is illustrated in Fig. 1. We prove our results via the method of sub-super solutions and the principle of linearized stability. 1.2 Statement of the main result In this paper, we study existence, nonexistence, uniqueness results, and stability proper- ties for (4). The following deﬁnitions of stability and instability presented here come from Lyapunov stability, which is deﬁned with respect to initial perturbations (see, for example, [19]). A solution vs(x) of (4) is said to be stable if for every ϵ > 0 there exists δ > 0 such that ∥v(t,·) – vs∥∞< ϵ for t > 0 whenever ∥v0 – vs∥∞< δ, where v(t,x) is the solution of (3). If, in addition, ∥v(t,·) – vs∥∞→0 as t →∞, then vs is said to be asymptotically stable. In the case that this holds for all initial functions v(0,x), then vs is said to be globally asymptoti- cally stable. The steady state vs is said to be unstable if it is not stable. Finally, vs(x) is said to be an isolated steady state if there exists a neighborhood Nvs of vs in C() such that vs is the only steady state solution in Nvs. To precisely state our results, we ﬁrst consider the eigenvalue problem ⎧ ⎨ ⎩ –w = λw; 0, ∂w ∂η + γ √ λw = 0; ∂0. (5) (5) It follows that (5) has a principal eigenvalue λ1(γ ) > 0 (see Appendix 2). Taking w as an eigenfunction such that ∥w∥∞= 1 and w is nonnegative, we ﬁrst note that by the maximum principle w > 0; 0. Now, if w(x0) = 0 on ∂0, then by Hopf’s lemma we must have ∂w ∂η \|x0 < 0 and thus ( ∂w ∂η + γ √ λw)\|x0 ̸= 0. Hence, w > 0 in 0. Now, we establish the following. It follows that (5) has a principal eigenvalue λ1(γ ) > 0 (see Appendix 2). Taking w as an eigenfunction such that ∥w∥∞= 1 and w is nonnegative, we ﬁrst note that by the maximum principle w > 0; 0. Now, if w(x0) = 0 on ∂0, then by Hopf’s lemma we must have ∂w ∂η \|x0 < 0 and thus ( ∂w ∂η + γ √ λw)\|x0 ̸= 0. Hence, w > 0 in 0. Now, we establish the following. Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Goddard II et al. Boundary Value Problems Page 5 of 17 ( 2018) 2018:170 Figure 1 An illustration of the bifurcation curve for (4) as established in Theorem 1. 1.2 Statement of the main result Bifurcation curve (λ vs ∥u∥∞) of the positive solutions u of (4) as shown in Theorem 1 Figure 1 An illustration of the bifurcation curve for (4) as established in Theorem 1. Bifurcation curve (λ vs ∥u∥∞) of the positive solutions u of (4) as shown in Theorem 1 2 Proof of Theorem 1 In this section, we provide a proof of our main results given in Theorem 1. Proof Let λ and γ be ﬁxed, and let σ1 be the principal eigenvalue and φ > 0 in 0 be the corresponding eigenfunction such that ∥φ∥∞= 1 to the eigenvalue problem: Proof Let λ and γ be ﬁxed, and let σ1 be the principal eigenvalue and φ > 0 in 0 be the corresponding eigenfunction such that ∥φ∥∞= 1 to the eigenvalue problem: ⎧ ⎨ ⎩ –φ – λφ = σφ; 0, ∂φ ∂η + γ √ λφ = 0; ∂0. (6) (6) Note that (6) is a linearization of (4) about the trivial solution. We also recall below the principle of linearized stability (see Lemma 1) given in [20, Theorem 1.1] (but also see [10, pp. 147–148] for example). Let δ1 be the principal eigenvalue of ⎧ ⎨ ⎩ –φ – λφ = δφ; 0, ∂φ ∂η + γ √ λφ = δφ; ∂0 (7) (7) with corresponding normalized eigenfunction φ(x) > 0; 0. See Appendix 2 for justiﬁca- tion of the existence of the principal eigenvalues of (6) and (7). with corresponding normalized eigenfunction φ(x) > 0; 0. See Appendix 2 for justiﬁca- tion of the existence of the principal eigenvalues of (6) and (7). Lemma 1 Let δ1 be the principal eigenvalue of (7). Then the following hold: Lemma 1 Let δ1 be the principal eigenvalue of (7). Then the following hold: a) If δ1 ≥0, then the trivial solution of (4) is stable. (a) If δ1 ≥0, then the trivial solution of (4) is stable. (a) If δ1 ≥0, then the trivial solution of (4) is stable. (b) If δ1 < 0, then the trivial solution of (4) is unstable. (b) If δ1 < 0, then the trivial solution of (4) is unstable. (b) If δ1 < 0, then the trivial solution of (4) is unstable. Note that by Lemma 5 in Appendix 2, we have that sign(δ1(λ,γ )) = sign(σ1(λ,γ )). Thus, it suﬃces to only consider the sign of σ1(λ,γ ). Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Page 6 of 17 Goddard II et al. Boundary Value Problems To prove (a), let λ > λ1(γ ). 2 Proof of Theorem 1 Page 7 of 17 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Figure 2 An illustration of the bifurcation curve for (8). Bifurcation curve (λ vs ∥u∥∞) of the positive solutions u of (8) Figure 2 An illustration of the bifurcation curve for (8). Bifurcation curve (λ vs ∥u∥∞) of the positive solutions u of (8) Also, to prove the property ∥vλ∥∞→1 as λ →∞, we note that the Dirichlet boundary value problem ⎧ ⎨ ⎩ –ψ = λψ(1 – ψ); 0, ψ = 0; ∂0, ⎧ ⎨ ⎩ –ψ = λψ(1 – ψ); 0, ψ = 0; ∂0, (8) (8) has a unique positive solution ψλ for λ > λD 1 with ∥ψλ∥∞< 1 and ∥ψλ∥∞→1– as λ →∞, where λD 1 > 0 is the principal eigenvalue of has a unique positive solution ψλ for λ > λD 1 with ∥ψλ∥∞< 1 and ∥ψλ∥∞→1– as λ →∞, where λD 1 > 0 is the principal eigenvalue of ⎧ ⎨ ⎩ –w = λw; 0, w = 0; ∂0. ⎧ ⎨ ⎩ –w = λw; 0, w = 0; ∂0. (9) (9) See Fig. 2 for an illustration of the structure of positive solutions of (8). See Fig. 2 for an illustration of the structure of positive solutions of (8). Since ∂ψ ∂η < 0 in 0, clearly ψλ is a subsolution to (4) for λ ≫1, and since w = 1 is a supersolution to (4) for λ > 0, we must have vλ ∈[ψλ,1] and hence ∥vλ∥∞→1– as λ →∞. Now, to show the stability properties of vλ, recall that we have ψ = m1φ is a strict subso- lution for all m1 ∈(0,– σ1 λ ) and Z ≡M is a strict supersolution for all M > 1. This implies that φ < vλ < Z and φ can be made arbitrarily small and Z can be made arbitrarily large. This fact combined with a result such as Theorem 6.7 of Chap. 5 in [19] immediately shows that vλ is globally asymptotically stable, proving (a). To prove (b), we ﬁrst show the nonexistence of a positive solution of (4) when λ ≤λ1(γ ) (which by Lemma 4 in Appendix 2 implies σ1 ≥0). 2 Proof of Theorem 1 (By Lemma 4 in Appendix 2, we have that σ1(λ,γ ) < 0 and hence the zero solution is unstable.) Next we will show the existence of a positive solution vλ with the property that ∥vλ∥∞→0+ as λ →λ1(γ )+. Let ψ := mφ for m > 0 to be chosen later with φ the principal eigenfunction of (6) corresponding to σ1(λ,γ ). Then we have –ψ – λψ(1 – ψ) = m[σ1 + λmφ]φ; 0, and ∂ψ ∂η = m∂φ ∂η = –mγ √ λφ = –γ √ λψ; ∂0. Hence ψ = m1φ with any m1 ∈(0,– σ1 λ ) is a strict subsolution of (4) (since ∥φ∥∞= 1), and ψ = m2φ with m2 = – σ1 λ[min0 φ] m2 = – σ1 λ[min0 φ] is a supersolution of (4). Clearly m2 > m1, and hence by the method of sub-super solutions (see [21]), (4) has a positive solution vλ such that m1φ < vλ ≤m2φ for λ > λ1(γ ). Note here that when λ →λ1(γ )+, [–σ1] →0+ while min0 φ ̸→0 since it approaches the eigenfunc- tion corresponding to the principal eigenvalue λ1(γ ) of (5), which we discussed earlier. Thus, m1 →0+ and m2 →0+, and, in particular, ∥vλ∥∞→0+ as λ →λ1(γ )+. Next, we show that this positive solution is, in fact, unique. To see this, we assume that (4) has two positive solutions v1 and v2. Without loss of generality, we can assume that v2 is the maximal positive solution (since w = 1 is a global supersolution, this maximal positive solution must exist when a positive solution exists), and hence v2 ≥v1 in 0. Supposing v1 and v2 are distinct, by integration by parts (Green’s second identity), we obtain 0 (v2)v1 – (v1)v2 dx = ∂0 ∂v2 ∂η v1 – ∂v1 ∂η v2 ds = ∂0 (–γ √ λv2)v1 – (–γ √ λv1)v2 ds = 0, while 0 (v2)v1 – (v1)v2 dx = 0 –λv2(1 – v2) v1 + λv1(1 – v1) v2 dx = 0 λv1v2(v2 – v1)dx > 0. = 0 λv1v2(v2 – v1)dx This is a contradiction, and hence v1 ≡v2 and (4) has a unique positive solution vλ for λ > λ1(γ ). This is a contradiction, and hence v1 ≡v2 and (4) has a unique positive solution vλ for λ > λ1(γ ). 2 Proof of Theorem 1 Assume to the contrary that v is a positive solution of (4), then by Green’s second identity, we obtain 0 (v)φ – (φ)v dx = ∂0 (–γ √ λv)φ – (–γ √ λφ)v ds while 0 (v)φ – (φ)v dx = 0 –λv(1 – v) φ + σ1φv + λφv dx = 0 λv2φ + σ1φv dx = 0 λv2φ + σ1φv dx > 0. Goddard II et al. Boundary Value Problems Page 8 of 17 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 ( 2018) 2018:170 Hence, we have a contradiction and therefore (4) has no positive solution when λ ≤λ1(γ ). Finally, since λ ≤λ1(γ ) implies σ1(λ,γ ) ≥0, the trivial solution of (4) is stable. But since there is no positive solution of (4) for λ ≤λ1(γ ), the trivial solution must be globally asymptotically stable. Hence, Theorem 1 is proven. □ Hence, we have a contradiction and therefore (4) has no positive solution when λ ≤λ1(γ ). Finally, since λ ≤λ1(γ ) implies σ1(λ,γ ) ≥0, the trivial solution of (4) is stable. But since there is no positive solution of (4) for λ ≤λ1(γ ), the trivial solution must be globally asymptotically stable. Hence, Theorem 1 is proven. □ 3 One-dimensional problem 4 and 5, respectively. Note that when γ →0+, we approach the Neumann boundary condition case, and when γ →∞, we approach the Dirichlet boundary condition case. 3 One-dimensional problem Boundary Value Problems ( 2018) 2018:170 Goddard II et al. Boundary Value Problems Page 9 of 17 ( 2018) 2018:170 and hence, setting t →1 2, we obtain and hence, setting t →1 2, we obtain √ λ = √ 2 ρ q ds F(ρ) – F(s) . √ λ = √ 2 ρ q ds F(ρ) – F(s) . Now the boundary conditions require that ρ and q satisfy Now the boundary conditions require that ρ and q satisfy Now the boundary conditions require that ρ and q satisfy F(ρ) = 2F(q) + γ 2q2 2 . (12) F(ρ) = 2F(q) + γ 2q2 2 . (12) Note that given ρ ∈(0,1), there exists unique q = q(ρ) ∈(0,ρ) satisfying (12), and we can show that Note that given ρ ∈(0,1), there exists unique q = q(ρ) ∈(0,ρ) satisfying (12), and we can show that G(ρ) = √ 2 ρ q(ρ) ds F(ρ) – F(s) G(ρ) = √ 2 ρ q(ρ) ds F(ρ) – F(s) ell-deﬁned and continuous on (0,1). is well-deﬁned and continuous on (0,1). is well-deﬁned and continuous on (0,1). Further, given ρ ∈(0,1), for λ satisfying Further, given ρ ∈(0,1), for λ satisfying √ λ = G(ρ) = √ 2 ρ q(ρ) ds F(ρ) – F(s) , (13) (13) (10) has a positive solution of the form given in Fig. 3 deﬁned by (10) has a positive solution of the form given in Fig. 3 deﬁned by u(t) q(ρ) ds F(ρ) – F(s) = √ 2λt; t ∈ 0, 1 2 . u(t) q(ρ) ds F(ρ) – F(s) = √ 2λt; t ∈ 0, 1 2 . Hence (13) describes the bifurcation diagram for positive solutions of (10). Using Mathe- matica computation, we provide below this bifurcation diagram for several values of γ . In particular, we illustrate the evolution of the bifurcation diagram as γ →0+ and γ →∞in Figs. 4 and 5, respectively. Note that when γ →0+, we approach the Neumann boundary condition case, and when γ →∞, we approach the Dirichlet boundary condition case. Hence (13) describes the bifurcation diagram for positive solutions of (10). Using Mathe- matica computation, we provide below this bifurcation diagram for several values of γ . In particular, we illustrate the evolution of the bifurcation diagram as γ →0+ and γ →∞in Figs. 3 One-dimensional problem n the case 0 = (0,1), equation (4) reduces to the two-point boundary value problem In the case 0 = (0,1), equation (4) reduces to the two-point boundary value problem ⎧ ⎪⎪⎨ ⎪⎪⎩ –v′′ = λv(1 – v);(0,1), v′(0) = γ √ λv(0), v′(1) = –γ √ λv(1). ⎧ ⎪⎪⎨ ⎪⎪⎩ –v′′ = λv(1 – v);(0,1), v′(0) = γ √ λv(0), v′(1) = –γ √ λv(1). (10) (10) From Theorem 1, (10) has a unique positive solution when λ > λ1(γ ) and no positive solution when λ < λ1(γ ), where λ1(γ ) is the principal eigenvalue of ⎧ ⎪⎪⎨ ⎪⎪⎩ –v′′ = λv;(0,1), v′(0) = γ √ λv(0), v′(1) = –γ √ λv(1). (11) ⎧ ⎪⎪⎨ ⎪⎪⎩ –v′′ = λv;(0,1), v′(0) = γ √ λv(0), v′(1) = –γ √ λv(1). (11) A straightforward calculation will show that λ1(γ ) = 4( π 2 –tan–1( 1 γ ))2. Note that as γ →0+, λ1(γ ) →0 and as γ →∞, λ1(γ ) →π2 = λD 1 . A straightforward calculation will show that λ1(γ ) = 4( π 2 –tan–1( 1 γ ))2. Note that as γ →0+, λ1(γ ) →0 and as γ →∞, λ1(γ ) →π2 = λD 1 . We now use the quadrature method introduced by Laetsch in [22] and further extended in [23–26], and [27]. Suppose that u is a positive solution with u( 1 2) = ρ (say) and u(0) = q (say). Note that since (10) is autonomous, the solution must be symmetric about t = 1 2 and take the form shown in Fig. 3. Multiplying the diﬀerential equation in (10) by u′ and integrating yields u′(t) = 2λ F(ρ) – F u(t) ; t ∈ 0, 1 2 , u′(t) = 2λ F(ρ) – F u(t) ; t ∈ 0, 1 2 , where F(z) = z 0 s(1 – s)dt. Further integration yields where F(z) = z 0 s(1 – s)dt. Further integration yields u(t) q ds F(ρ) – F(s) = √ 2λt; t ∈ 0, 1 2 , n Figure 3 Shape of positive solutions to (10). Illustration of the typical shape of a positive solution to (10) ems ( 2018) 2018:170 Page 9 of 17 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Goddard II et al. 4 Biological implications of our results These model results give important predictions on population persistence at the patch level based solely on demographic parameters, e.g., patch diﬀusion rate and intrinsic growth rate, as well as matrix diﬀusion rate and death rate. We note that our analysis covers all four of the interface scenarios listed in Table 1. Although the exact deﬁnition of Figure 4 Bifurcation curves for (10) as γ →∞. The curves correspond (from left to right) to γ = 1, γ = 10, and γ = 100. Note that as γ →∞, the bifurcation curves approach π2, which is the ﬁrst eigenvalue of the Dirichlet problem Figure 4 Bifurcation curves for (10) as γ →∞. The curves correspond (from left to right) to γ = 1, γ = 10, and γ = 100. Note that as γ →∞, the bifurcation curves approach π2, which is the ﬁrst eigenvalue of the Dirichlet problem Page 10 of 17 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Figure 5 Bifurcation curves for (10) as γ →0+. The curves correspond (from left to right) to γ = 0.1, γ = 0.5, and γ = 1. Note that as γ →0+, the bifurcation curves approach 0, which is the ﬁrst eigenvalue of the Neumann problem Figure 5 Bifurcation curves for (10) as γ →0+. The curves correspond (from left to right) to γ = 0.1, γ = 0.5, and γ = 1. Note that as γ →0+, the bifurcation curves approach 0, which is the ﬁrst eigenvalue of the Neumann problem γ depends upon the interface scenario given (see Table 2), the qualitative behavior of the persistence of the organism is the same as γ varies. Notice that the principal eigenvalue σ1(λ,γ ) of (6) plays a crucial role in determining the dynamics of the model. In fact, it represents the fastest possible growth rate for the linear growth model corresponding to (3) (see [3] or [10]). As indicated in Theorem 1 (and the proof therein), when λ ≤λ1(γ ) we have that σ1(λ,γ ) ≥0, and the only nonnegative steady state of (3) is the trivial one, u ≡0. In this case, the model predicts extinction for any nonnegative initial population density proﬁle. In fact, loses due to mortality in the matrix outpace the reproductive rate in the patch. Appendix 1: Derivation of the boundary condition in (1) Here, we summarize the derivation of the boundary condition in (1) from [12]. Their ap- proach combines the ideas from [8] and [3] under the patch/matrix interface conditions given in these respective papers. The boundary condition in (1), namely D∂u ∂η + √S0D0 κ u = 0; t > 0,x ∈∂, (14) D∂u ∂η + √S0D0 κ u = 0; t > 0,x ∈∂, (14) allows modeling of the eﬀects of movement behavior changes in response to the patch/ matrix interface and hostility of the matrix surrounding the patch. To see this, [12] com- bines the approach of modeling the eﬀects of a hostile matrix in [15] with the interface conditions given in [8]. Following the derivation in [15], let us consider a one-dimensional patch = (0,ℓ) (ℓ> 0 denotes the patch size) surrounded by an inﬁnite “sea” of hostile territory. The population density w is subject to the following growth law exterior to : allows modeling of the eﬀects of movement behavior changes in response to the patch/ matrix interface and hostility of the matrix surrounding the patch. To see this, [12] com- bines the approach of modeling the eﬀects of a hostile matrix in [15] with the interface conditions given in [8]. Following the derivation in [15], let us consider a one-dimensional patch = (0,ℓ) (ℓ> 0 denotes the patch size) surrounded by an inﬁnite “sea” of hostile territory. The population density w is subject to the following growth law exterior to : wt = D0wxx – S0w, (15) (15) wt = D0wxx – S0w, where D0 is a positive parameter representing the diﬀusion rate and S0 is a positive pa- rameter representing the death rate of the organism in the matrix. Continuity of ﬂux is a natural condition that will imply all organisms leaving the patch will enter the matrix and organisms leaving the matrix will enter the patch (see [8]). Thus, no organisms are introduced or lost at the interface. However, a discontinuity is introduced in the density at the patch/matrix interface to account for changes in movement behavior. Now, let D be the diﬀusion rate inside and follow the random walk derivation given in [8] to yield the interface conditions: where D0 is a positive parameter representing the diﬀusion rate and S0 is a positive pa- rameter representing the death rate of the organism in the matrix. 4 Biological implications of our results Thus, the theoretical organism cannot colonize the patch, and any remnant population in the patch will become extinct. However, when λ > λ1(γ ), we have that σ1(λ,γ ) < 0 and (3) admits a unique steady state that is positive in such that all positive initial population density proﬁles will propagate to this steady state over time. In this case, the global nature of the stability of the positive steady state gives a fairly strong notion of persistence of the species. The patch is large enough in this case to shield a suﬃcient proportion of the popu- lation from mortality induced by the hostile matrix. This prediction leads to a formula for minimum patch size of the population given as ℓ∗(γ ) = D r λ1(γ ). Note that this formula can be numerically estimated and depends upon parameters in the patch (diﬀusion rate and intrinsic growth rate), parameters in the matrix via γ (diﬀusion rate and death rate), and the geometry of the patch 0. This notion of a minimum patch size agrees with the well-known notion of a minimum core area (in the case of n = 2) requirement. Note that λ1(γ ) can be viewed as a quantiﬁca- tion of the loss of the population due to interactions with the hostile matrix where γ en- capsulates parameters regarding the hostile matrix. Also, it is easy to see that λ1(γ ) →λD 1 as γ →∞, and this reveals an important model prediction of the existence of a maximum possible eﬀect of population loss due to the hostile matrix. Patches with a lethal matrix can still guarantee a prediction of persistence as long as the patch size is larger than D r λD 1 , where the maximum eﬀect of the lethal matrix on the population is quantiﬁed in λD 1 . This minimum patch size approaches inﬁnity if either (1) the patch diﬀusion rate is arbitrarily large, since a large diﬀusion rate ensures that a very high proportion of the population will encounter loss at the patch/matrix interface, or (2) the intrinsic growth rate is arbitrarily small, which for a ﬁxed patch diﬀusion rate will imply that the population is not able to recover the loss associated with interaction with hostile matrix. Page 11 of 17 ( 2018) 2018:170 Goddard II et al. Boundary Value Problems Appendix 1: Derivation of the boundary condition in (1) Continuity of ﬂux is a natural condition that will imply all organisms leaving the patch will enter the matrix and organisms leaving the matrix will enter the patch (see [8]). Thus, no organisms are introduced or lost at the interface. However, a discontinuity is introduced in the density at the patch/matrix interface to account for changes in movement behavior. Now, let D be the diﬀusion rate inside and follow the random walk derivation given in [8] to yield the interface conditions: D∂u ∂η = D0 ∂w ∂η0 ; t > 0,x ∈{0,ℓ}, (16) u = κw; t > 0,x ∈{0,ℓ}, (17) D∂u ∂η = D0 ∂w ∂η0 ; t > 0,x ∈{0,ℓ}, (16) t 0 ∈{0 ℓ} (17) (16) (17) u = κw; t > 0,x ∈{0,ℓ}, (17) where κ is a positive, unitless parameter, η is the outward normal direction for the patch, and η0 is the inward normal direction for the matrix. The only steady state solution to (15) S where κ is a positive, unitless parameter, η is the outward normal direction for the patch, and η0 is the inward normal direction for the matrix. The only steady state solution to (15) which is nonnegative and bounded for x < 0 is of the form w(x) = C1e S0 D0 x; x ≤0, where C1 ≥0 is a constant (see [15]). Hence, applying the interface conditions (16) and (17) to this solution yields –Dux(t,0) + √S0D0 κ u(t,0) = 0; t > 0. (18) (18) A similar argument for x = ℓalso yields A similar argument for x = ℓalso yields Dux(t,ℓ) + √S0D0 κ u(t,ℓ) = 0; t > 0, (19) (19) or equivalently, D∂u ∂η + √S0D0 κ u = 0; t > 0,x ∈{0,ℓ}. (20) (20) Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Page 12 of 17 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 For domains in higher dimensions with arbitrary boundary shapes, easily extending such a derivation is not possible. Appendix 1: Derivation of the boundary condition in (1) In this paper, we make the assumption that (2) is a reasonable approximation of the boundary behavior of an organism, where the parameter S0 can be interpreted as a death rate in the matrix, D0 as the diﬀusion rate in the matrix, and κ as a measure of the discontinuous “jump” in density at the patch/matrix interface. Appendix 2: Results for eigenvalue problems (5), (6), and (7) First we consider the eigenvalue problem ⎧ ⎨ ⎩ –z = λz; 0, ∂z ∂η = μz; ∂0 (21) ⎧ ⎨ ⎩ –z = λz; 0, ∂z ∂η = μz; ∂0 (21) for given μ ∈R. We recall the following result from [28]. for given μ ∈R. We recall the following result from [28]. Lemma 2 For each μ ∈R, (21) has a principal eigenvalue ¯λ1(μ), and the eigencurve ¯λ1(μ) ⊂R2 is Lipschitz continuous, strictly decreasing, and concave. Furthermore, ¯λ1(0) = 0 and the eigenfunction associated with any point on ¯λ1(μ) is strictly positive in 0. We now state and prove a result regarding the limiting value of ¯λ1(μ) as μ →–∞. Fig- ure 6 illustrates Lemma 3. Lemma 3 ¯λ1(μ) →λD 1 as μ →–∞, where λD 1 is the principal eigenvalue of (9). Proof We note that, for any μ ∈R, we may characterize ¯λ1(μ) by ¯λ1(μ) = min u∈H1(0)\{0} 0 \|∇u\|2 dx – μ ∂0 u2 ds 0 u2 dx . (22) (22) Let λD 1 be the principal eigenvalue of (9) with corresponding normalized eigenfunction φD 1 chosen such that 0 φD 1 = 1. Testing (22) with u = 1 and u = φD 1 shows that ¯λ1(μ) ≤–μ\|∂0\| \|0\| ¯λ1(μ) ≤–μ\|∂0\| \|0\| and ¯λ1(μ) ≤λD 1 , ¯λ1(μ) ≤λD 1 , ¯λ1(μ) ≤λD 1 , the Figure 6 Plot of μ vs ¯λ1(μ). The curve illustrates the fact that ¯λ1(μ) →λD 1 as μ →–∞ Figure 6 Plot of μ vs ¯λ1(μ). The curve illustrates the fact that ¯λ1(μ) →λD 1 as μ →–∞ ms ( 2018) 2018:170 Page 13 of 17 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 ( 2018) 2018:170 Goddard II et al. Boundary Value Problems Page 13 of 17 respectively. Taking a sequence μn →–∞such that the corresponding eigenfunctions un, without loss of generality, satisfy 0 u2 n dx = 1, we observe that ¯λ1(μn) = 0 \|∇un\|2 dx – μn ∂0 u2 n ds. Since μn < 0, we have 0 = ¯λ1(0) < ¯λ1(μn) < λD 1 . By Lemma 2, limμ→–∞¯λ1(μ) = ¯λ1(–∞) ≤λD 1 for some ¯λ1(–∞) ∈R. Without loss of generality, we may assume –μn ∂0 u2 n ds →α ≥0, and thus ∂0 u2 n →0. Appendix 2: Results for eigenvalue problems (5), (6), and (7) 0 Since {un} is bounded in H1(0), we may select a subsequence so that un ⇀u in H1(0), un →u in L2(0) and in L2(∂0). It follows that 0 u2 dx = 1 and ∂0 u2 ds = 0, and hence u ∈H1 0(0). By the weak lower semicontinuity of 0 \|∇u\|2 dx, we get that By the weak lower semicontinuity of 0 \|∇u\|2 dx, we get that 0 \|∇u\|2 dx + α ≤liminf n→∞ 0 \|∇un\|dx – μn ∂0 u2 n ds = ¯λ1(–∞) ≤λD 1 . But by Poincare’s inequality, we have λD 1 ≤ 0 \|∇u\|2 dx, and hence we must have α = 0 and ¯λ1(–∞) = λD 1 . Furthermore, 0 \|∇u\|2 dx = λD 1 , and thus, without loss of generality, u = φD 1 . Moreover, limn→∞ 0 \|∇un\|2 dx = 0 \|∇u\|2 dx, and hence un →u = φD 1 in H1(). □ But by Poincare’s inequality, we have λD 1 ≤ 0 \|∇u\|2 dx, and hence we must have α = 0 and ¯λ1(–∞) = λD 1 . Furthermore, 0 \|∇u\|2 dx = λD 1 , and thus, without loss of generality, u = φD 1 . Moreover, limn→∞ 0 \|∇un\|2 dx = 0 \|∇u\|2 dx, and hence un →u = φD 1 in H1(). □ Next, we consider the eigenvalue problem (5), namely Next, we consider the eigenvalue problem (5), namely Next, we consider the eigenvalue problem (5), namely ⎧ ⎨ ⎩ –w = λw; 0, ∂w ∂η + γ √ λw = 0; ∂0 (23) (23) for given γ > 0. It is easy to see that the principal eigenvalue λ1(γ ) of (23) is nothing but the y-coordinate of the intersection of the curves ¯λ1(μ) and μ2 γ 2 (see Fig. 7). It is also straight- forward to show that λ1(γ ) is an increasing function of γ , λ1(γ ) →λD 1 as γ →∞, and λ1(γ ) →0 as γ →0 (see Fig. 8). Next, we consider the eigenvalue problem (6), namely, for given λ > 0 and γ > 0, for given γ > 0. It is easy to see that the principal eigenvalue λ1(γ ) of (23) is nothing but the y-coordinate of the intersection of the curves ¯λ1(μ) and μ2 γ 2 (see Fig. 7). Appendix 2: Results for eigenvalue problems (5), (6), and (7) It is also straight- forward to show that λ1(γ ) is an increasing function of γ , λ1(γ ) →λD 1 as γ →∞, and λ1(γ ) →0 as γ →0 (see Fig. 8). Next, we consider the eigenvalue problem (6), namely, for given λ > 0 and γ > 0, ⎧ ⎨ ⎩ –ψ – λψ = σψ; 0, ∂ψ ∂η + γ √ λψ = 0; ∂0. (24) (24) Once again, it is easy to see that the principal eigenvalue σ1(λ,γ ) of (24) exists and must satisfy Once again, it is easy to see that the principal eigenvalue σ1(λ,γ ) of (24) exists and must satisfy λ + σ1(λ,γ ) = ¯λ1(– √ λγ ) (25) λ + σ1(λ,γ ) = ¯λ1(– √ λγ ) (25) (see Fig. 9). Furthermore, the following result holds. Lemma 4 If λ < λ1(γ ), then σ1(λ,γ ) > 0, and if λ = λ1(γ ), then σ1(λ,γ ) = 0. Also, if λ > λ1(γ ), then σ1(λ,γ ) < 0. Proof Note that if λ < λ1(γ ) then – √ λγ > – λ1(γ )γ . Now, μ2 γ 2 < λ1(μ) for – λ1(γ )γ < μ < 0 since μ2 γ 2 is a convex function, while λ1(μ) is a concave function (see Fig. 9). But Page 14 of 17 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 ( 2018) 2018:170 Figure 7 Illustration of the existence of λ1(γ ). The plot illustrates the existence of λ1(γ ) Figure 8 Illustration of monotonicity of λ1(γ ) when γ2 > γ1. The plot illustrates that λ1(γ ) is an increasing function. Note that γ2 > γ1 Figure 9 Illustration of the existence of σ1(λ,γ ). The plot illustrates the existence of σ1(λ,γ ) λ = (– √ λγ )2 γ 2 . Hence, taking μ = – √ λγ , we obtain λ < ¯λ1(– √ λγ ) (26) Figure 7 Illustration of the existence of λ1(γ ). The plot illustrates the existence of λ1(γ ) Figure 7 Illustration of the existence of λ1(γ ). The plot illustrates the existence of λ1(γ ) Figure 8 Illustration of monotonicity of λ1(γ ) when γ2 > γ1. The plot illustrates that λ1(γ ) is an increasing function. Note that γ2 > γ1 hen Figure 8 Illustration of monotonicity of λ1(γ ) when γ2 > γ1. Appendix 2: Results for eigenvalue problems (5), (6), and (7) The plot illustrates that λ1(γ ) is an increasing function. Note that γ2 > γ1 Figure 9 Illustration of the existence of σ1(λ,γ ). The plot illustrates the existence of σ1(λ,γ ) The Figure 9 Illustration of the existence of σ1(λ,γ ). The plot illustrates the existence of σ1(λ,γ ) λ = (– √ λγ )2 γ 2 . Hence, taking μ = – √ λγ , we obtain λ = (– √ λγ )2 γ 2 . Hence, taking μ = – √ λγ , we obtain λ = (– √ λγ )2 γ 2 . Hence, taking μ = – √ λγ , we obtain λ = (– √ λγ )2 γ 2 . Hence, taking μ = – √ λγ , we obtain λ < ¯λ1(– √ λγ ) λ < ¯λ1(– √ λγ ) (26) (26) and by (25) we have σ1(λ,γ ) > 0. A similar argument for the case when λ > λ1(γ ) gives that σ1(λ,γ ) < 0. Note that λ = λ1(γ ) implies that we have equality in (26), and thus, σ1(λ,γ ) = 0. □ and by (25) we have σ1(λ,γ ) > 0. A similar argument for the case when λ > λ1(γ ) gives that σ1(λ,γ ) < 0. Note that λ = λ1(γ ) implies that we have equality in (26), and thus, σ1(λ,γ ) = 0. □ Now, for ﬁxed λ and γ , we consider the eigenvalue problem Now, for ﬁxed λ and γ , we consider the eigenvalue problem ⎧ ⎨ ⎩ –φ – λφ = δφ; 0, ∂φ ∂η + γ √ λφ = δφ; ∂0. (27) (27) (27) Page 15 of 17 Goddard II et al. Boundary Value Problems ( 2018) 2018:170 Figure 10 Illustration of the existence of ˜δ1(λ,γ ). The plot illustrates the existence of ˜δ1(λ,γ ) Figure 10 Illustration of the existence of ˜δ1(λ,γ ). The plot illustrates the existence of ˜δ1(λ,γ ) γ ). Figure 10 Illustration of the existence of ˜δ1(λ,γ ). Author details ut o deta s 1Department of Mathematics & Computer Science, Auburn University Montgomery, Montgomery, USA. 2Department of Mathematics & Statistics, Swarthmore College, Swarthmore, USA. 3Department of Mathematics & Statistics, Wake Forest University, Winston-Salem, USA. 4Department of Mathematics & Statistics, University of North Carolina Greensboro, Greensboro, USA. Received: 21 March 2018 Accepted: 1 November 2018 Received: 21 March 2018 Accepted: 1 November 2018 Competing interests Competing interests The authors declare that they have no competing interests. Funding Funding This work was supported in part by the National Science Foundation via grant DMS-1516560 for the ﬁrst author and grant DMS-1516519 for the last author. Authors’ contributions Authors’ contributions The authors contributed equally to the writing of this paper. The authors read and approved the ﬁnal manuscript. The authors contributed equally to the writing of this paper. 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Wiley, Chichester (2003) p. 411. Wiley, Chichester (2003) 11. Holmes, E.E., Lewis, M.A., Banks, R.R.V.: Partial diﬀerential equations in ecology: spatial interactions and population dynamics. Ecology 75(1), 17–29 (1994) 11. Holmes, E.E., Lewis, M.A., Banks, R.R.V.: Partial diﬀerential equations in ecology: spatial interactions and population dynamics. Ecology 75(1), 17–29 (1994) dynamics. Ecology 75(1), 17–29 (1994) 12. Cronin, J.T., Goddard, J. II, Shivaji, R.: Eﬀects of patch matrix and individual movement response on population i t t th t h l l P i t (2017) 12. Cronin, J.T., Goddard, J. II, Shivaji, R.: Eﬀects of patch matrix and individual movement response on population persistence at the patch-level. Preprint (2017) 12. Cronin, J.T., Goddard, J. II, Shivaji, R.: Eﬀects of patch matrix and individual movement response on population persistence at the patch-level. Preprint (2017) 12. Cronin, J.T., Goddard, J. II, Shivaji, R.: Eﬀects of patch matrix a persistence at the patch-level. Preprint (2017) p p p 13. Maciel, G., Lutscher, F.: How individual movement response to habitat edges aﬀects population persistence and 13. Maciel, G., Lutscher, F.: How individual movement response to habitat edges aﬀects population persistence and 13. Maciel, G., Lutscher, F.: How individual movement response to habitat edges aﬀects population persistence and spatial spread. Am. Nat. 182(1), 42–52 (2013) 13. Maciel, G., Lutscher, F.: How individual movement response 13. Maciel, G., Lutscher, F.: How individual movem spatial spread. Am. Nat. 182(1), 42–52 (2013) p g p p p spatial spread. Am. Nat. 182(1), 42–52 (2013) spatial spread. Am. Nat. 182(1), 42–52 (2013) p p 14. Appendix 2: Results for eigenvalue problems (5), (6), and (7) Boundary Value Problems ( 2018) 2018:170 Acknowledgements The authors would like to thank to the anonymous reviewers whose suggestions greatly improved this manuscript. Funding This work was supported in part by the National Science Foundation via grant DMS-1516560 for the ﬁrst author and grant DMS-1516519 for the last author. Availability of data and materials Availability of data and materials Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. a ab ty o data a d ate a s Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. Appendix 2: Results for eigenvalue problems (5), (6), and (7) The plot illustrates the existence of ˜δ1(λ,γ ) Notice that letting ˜δ = δ – √ λγ implies that (27) becomes Notice that letting ˜δ = δ – √ λγ implies that (27) becomes ⎧ ⎨ ⎩ –φ = (λ + √ λγ + ˜δ)φ; 0, ∂φ ∂η = ˜δφ; ∂0 (28) (28) and the principal eigenvalue ˜δ1(λ,γ ) is nothing but the x-coordinate of the intersection of the curves ¯λ1(˜δ) and ˜δ + (λ + √ λγ ) (see Fig. 10). Hence the principal eigenvalue δ1(λ,γ ) of (27) exists and is given by and the principal eigenvalue ˜δ1(λ,γ ) is nothing but the x-coordinate of the intersection of the curves ¯λ1(˜δ) and ˜δ + (λ + √ λγ ) (see Fig. 10). Hence the principal eigenvalue δ1(λ,γ ) of (27) exists and is given by δ1(λ,γ ) = ˜δ1(λ,γ ) + √ λγ . (29) (29) We next establish a relationship between the signs of δ1(λ,γ ) and σ1(λ,γ ) in the follow- ing result. Lemma 5 sign(δ1(λ,γ )) = sign(σ1(λ,γ )). Lemma 5 sign(δ1(λ,γ )) = sign(σ1(λ,γ )). Proof Let φ1 and φ2 be corresponding positive eigenfunctions in (27) and (28). Then, by Green’s second identity, we have that 0 (φ1)φ2 – (φ2)φ1 dx = ∂0 ∂φ1 ∂η φ2 – ∂φ2 ∂η φ1 ds, (30) (30) which implies which implies δ1(λ,γ ) – σ1(δ,γ ) 0 φ1φ2 dx = –δ1(λ,γ ) ∂0 φ2φ1 ds. (31) (31) Now, it immediately follows that σ1(λ,γ ) = 0 if and only if δ1(λ,γ ) = 0, and if δ1(λ,γ ) ̸= 0, then we have Now, it immediately follows that σ1(λ,γ ) = 0 if and only if δ1(λ,γ ) = 0, and if δ1(λ,γ ) ̸= 0, then we have σ1(λ,γ ) – δ1(λ,γ ) δ1(λ,γ ) > 0. (32) σ1(λ,γ ) – δ1(λ,γ ) δ1(λ,γ ) > 0. (32) Thus, if δ1(λ,γ ) > 0, then we must have that σ1(λ,γ ) > δ1(λ,γ ) > 0, and if δ1(λ,γ ) < 0, then σ1(λ,γ ) < δ1(λ,γ ) < 0. Hence the result. □ Finally, combining Lemmas 4 and 5, the following lemma immediately follows. Lemma 6 If λ < λ1(γ ), then δ1(λ,γ ) > 0. Also, if λ > λ1(γ ), then δ1(λ,γ ) < 0. Page 16 of 17 Goddard II et al. 25. Goddard, J. II, Morris, Q., Son, B., Shivaji, R.: Bifurcation curves for singular and nonsingular problems with nonlinear boundary conditions. Electron. J. Diﬀer. Equ. 2018, 26 (2018) 26. Goddard, J. II, Price, J., Shivaji, R.: Analysis of steady states for classes of reaction–diﬀusion equations with U-shaped density dependent dispersal on the boundary (2017, in press) 27. Miciano, A.R., Shivaji, R.: Multiple positive solutions for a class of semipositone Neumann two point boundary value problems. J. Math. Anal. Appl. 178(1), 102–115 (1993) 28. Rivas, M.A., Robinson, S.: Eigencurves for linear elliptic equations. European Journal ESAIM (to appear) Goddard II et al. Boundary Value Problems ( 2018) 2018:170 References Anuradha, V., Maya, C., Shivaji, R.: Positive solutions for a class of nonlinear boundary value problems with b b d d h l l 23. Anuradha, V., Maya, C., Shivaji, R.: Positive solutions for a class of nonlinear boundary value problems with Neumann–Robin boundary conditions. J. Math. Anal. Appl. 236(1 y pp ( ), ( ) 24. Goddard, J. II, Lee, E.K., Shivaji, R.: Population models with nonlinear boundary conditions. Electron. J. Diﬀer. Equ. Conf. 19, 135–149 (2010) 24. Goddard, J. II, Lee, E.K., Shivaji, R.: Population models with nonlinear boundary conditions. Electron. J. Diﬀer. Equ. Conf. 19, 135–149 (2010) Page 17 of 17 Page 17 of 17 density dependent dispersal on the boundary (2017, in press) j problems. J. Math. Anal. Appl. 178(1), 102–115 (1993)
https://openalex.org/W4206722619	https://www.thno.org/v12p1419.pdf	English	null	Oral delivery of protein and peptide drugs: from non-specific formulation approaches to intestinal cell targeting strategies	Theranostics	2,022	cc-by	17,507	Abstract The past few years has witnessed a booming market of protein and peptide drugs, owing to their superior efficiency and biocompatibility. Parenteral route is the most commonly employed method for protein and peptide drugs administration. However, short plasma half-life protein and peptide drugs requires repetitive injections and results in poor patient compliance. Oral delivery is a promising alternative but hindered by harsh gastrointestinal environment and defensive intestinal epithelial barriers. Therefore, designing suitable oral delivery systems for peptide and protein drugs has been a persistent challenge. This review summarizes the main challenges for oral protein and peptide drugs delivery and highlights the advanced formulation strategies to improve their oral bioavailability. More importantly, major intestinal cell types and available targeting receptors are introduced along with the potential strategies to target these cell types. We also described the multifunctional biomaterials which can be used to prepare oral carrier systems as well as to modulate the mucosal immune response. Understanding the emerging delivery strategies and challenges for protein and peptide drugs will surely inspire the production of promising oral delivery systems that serves therapeutic needs in clinical settings. Key words: Protein and peptide drug; Oral delivery system; Physical and biochemical barrier; Intestinal mucosa; Intestinal cell targeting; Oral bioavailability. Theranostics 2022; 12(3): 1419-1439. doi: 10.7150/thno.61747 2022; 12(3): 1419-1439. doi: 10.7150/thno.61747 Received: 2021.04.17; Accepted: 2021.11.20; Published: 2022.01.01 Received: 2021.04.17; Accepted: 2021.11.20; Published: 2022.01.01 Oral delivery of protein and peptide drugs: from non-specific formulation approaches to intestinal cell targeting strategies Guanyu Chen1, Weirong Kang2, Wanqiong Li1, Shaomeng Chen1, Yanfeng Gao1 1. School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Shenzhen, China. 2. Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China. 1. School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Shenzhen, China. 2. Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The Univers  Corresponding author: Prof. Yanfeng Gao, School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Shenzhen 518107, China. Phone: +86 020 84723750; fax: +86 020 84723750. E-mail: gaoyf29@mail.sysu.edu.cn  Corresponding author: Prof. Yanfeng Gao, School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Shenzhen 4723750; fax: +86 020 84723750. E-mail: gaoyf29@mail.sysu.edu.cn © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativec ee http://ivyspring.com/terms for full terms and conditions. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2021.04.17; Accepted: 2021.11.20; Published: 2022.01.01 Key words: Protein and peptide drug; Oral delivery system; Physical and biochemical barrier; Intestinal mucosa; Intestinal ce targeting; Oral bioavailability. 1419 1419 Theranostics 2022, Vol. 12, Issue 3 Ivyspring International Publisher Ivyspring International Publisher Introduction Moreover, trypsin and α-chymotrypsin are the major proteolytic enzymes in the intestinal lumen [9]. Figure 2 shows the mucus layer covering GIT epithelial membrane is considered as the first physical barrier. Mucin is the main component which is a highly glucosylated glycoprotein. The backbone consists repeating sequences of serine, proline and threonine residues. The O-linked oligosaccharide side chains are generally terminated in L- fructose, sulfonic acid or sialic acid. Therefore, the intestinal mucus layer shows negatively charged [10, 11]. Second physical barrier, the layer of epithelial cells connecting with tight junctions, which forming a seal wall for the drug permeation [12]. Furthermore, PPDs being metabolized by the enterocytes cytochrome P450 3A4 (CYP3A4) enzyme and being pumped out via P-gp efflux protein, as well as the post-absorptive clearance are other involving barriers for oral drug delivery [13]. In this review, we summarize major barriers for oral delivery of PPDs, and the state-of-the-art formulation approaches for promoting the oral bioavailability of PPDs. Intestinal cell targeting strategies are presented with an emphasis on examples that showed great potential for clinical applications. Additionally, multifunctional biomaterials which can be used to prepare oral carrier systems as well as to modulate the mucosal immune response are also discussed. In this review, we summarize major barriers for oral delivery of PPDs, and the state-of-the-art formulation approaches for promoting the oral bioavailability of PPDs. Intestinal cell targeting strategies are presented with an emphasis on examples that showed great potential for clinical applications. Additionally, multifunctional biomaterials which can be used to prepare oral carrier systems as well as to modulate the mucosal immune response are also discussed. Introduction Though parenteral administration is the most commonly employed administration route for PPDs, it often associates with poor patient compliance [3]. Compared to parental administration, oral drug delivery routes are advantageous in terms of patient compliance, safety, long-term dosing and manufacturing costs. Further, oral administration is used for both local and systemic delivery of a wide range of drug molecules, from small molecules to biomacromolecules [4]. However, oral delivery of macromolecules (such as PPDs) is particularly challenging due to their physicochemical properties and the involving barriers in the gastrointestinal tract (GIT) [5]. The major strategies to deliver PPDs orally Enormous efforts have been made over the past few decades to realize the therapeutic efficacy of protein and peptide drugs (PPDs). Owing to their excellent specificity and biocompatibility, PPDs can achieve ideal therapeutic effects at relatively low doses [1]. Since the isolation of insulin in 1922, the use of PPDs as therapeutic agents has been considered as an attractive approach to combat various diseases (Figure 1). Recent developments in the biotechnology and pharmaceutical sciences have made it possible to produce potential therapeutic PPDs in commercial quantities [2]. By far, over 240 PPDs has been approved by FDA and a variety of potential drug candidates in clinical trials. https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1420 with improved the therapeutic efficacy can be categorized into non-targeting and targeting delivery, including chemical modification and drug delivery systems for PPDs to avoid enzymatic degradation and reduce off-target drug distribution. Targeting different GIT area can be achieved by exploiting its physiological features and combining the PPDs with suitable drug formulations [6]. Moreover, the presence of numerous types of intestinal cells, such as enterocytes, M cells, goblet cells and Paneth cells interspersed throughout the GIT provides various targets and allows for the design of a broad array of passive or active targeting delivery systems. from the carrier systems and pass on their way to the target receptors within the harsh intestinal environment. Ingested PPDs first encounter digestive enzymes in our oral cavity, including amylase and lipase in the saliva [7]. The second enzymatic barrier is the intensive acidic environment and the presence of pepsin and cathepsin that degrades most of the PPDs in our stomach [8]. Gastric pH might alter the ionization of the PPDs causing change of structure or function of the drug. Physical and biochemical barriers and mechanism of intestinal drug absorption The absorption of orally administered PPDs from the GIT into the systemic circulation is limited by various factors. These include the release of drugs Figure 1. Milestones in the development of oral delivery of PPDs. https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1421 Figure 2. Biochemical and physical barriers for oral drug delivery, and the structure of intestinal mucosa with major intestinal cell types. Figure 3. A diagram of transport pathways of protein and peptide compounds over the intestinal mucosal epithelial membrane. The two major mechanism of drugs permeate through the intestinal mucosa are the passive diffusion via the transcellular or paracellular pathway (Figure 3), and the carrier-mediated transport including active transport and facilitated diffusion [14]. The permeation mechanism for a particular drug depends on its physiochemical properties such as hydrophilic drugs tend to penetrate over epithelium via paracellular pathway [17], and the hydrogen-bonding capability of the drugs dictated by the number of hydrogen bond donors and acceptors usually no more 10 and 5, respectively [18]. Carrier-mediated transport is energy dependent, and has notable features of substrate specificity and 1421 Theranostics 2022, Vol. 12, Issue 3 Figure 2. Biochemical and physical barriers for oral drug delivery, and the structure of intestinal mucosa with major intestinal cell types. Figure 2. Biochemical and physical barriers for oral drug delivery, and the structure of intestinal mucosa with major intestinal cell types. Figure 2. Biochemical and physical barriers for oral drug delivery, and the structure of intestinal mucosa with major intestinal cell types. Figure 3. A diagram of transport pathways of protein and peptide compounds over the intestinal mucosal epithelial membrane. Figure 3. A diagram of transport pathways of protein and peptide compounds over the intestinal mucosal epithelial membrane. The two major mechanism of drugs permeate through the intestinal mucosa are the passive diffusion via the transcellular or paracellular pathway (Figure 3), and the carrier-mediated transport including active transport and facilitated diffusion [14]. The permeation mechanism for a particular drug depends on its physiochemical properties such as molar mass, polarity, lipophilicity and hydrophilicity [15, 16]. Lipophilic, non-ionized form of drugs generally have higher permeability, while the ionized, hydrophilic drugs tend to penetrate over epithelium via paracellular pathway [17], and the hydrogen-bonding capability of the drugs dictated by the number of hydrogen bond donors and acceptors usually no more 10 and 5, respectively [18]. Physical and biochemical barriers and mechanism of intestinal drug absorption Carrier-mediated transport is energy dependent, and has notable features of substrate specificity and saturability. It requires the interaction of drugs with a protein carrier often in the apical side of the intestinal membrane [19]. https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1422 Cationization Cationic drugs are more permeable over the intestinal mucosa compared with anionic drugs, it is due to the negatively charged glycoproteins and glycosphingolipids on the intestinal cell membrane [23]. Hence, formulating a cationic drug is postulated to elevate the drug permeability. However, peptide cationization may lead to increased immunogenicity, which will result in faster removal of the drug from the body and hence loss of activity. Moreover, its non-specific targeting in terms of tissue uptake, and potential toxicity found in the kidney and liver limits its therapeutic clinical use [23]. Lipidization p [ ] Studies have showed that PPDs can be cationized by chemical conjugation demonstrated efficient intracellular delivery via adsorptive-mediated endocytosis. Futami et al. demonstrated the negatively charged mammalian cell membrane consisting glycoproteins and glycosphingolipids, cationization of these proteins elevated their ability for intestinal drug permeation [24]. Moreover, the recent developed sophisticated protein chemistry, controlled chemical modifications, such as substitutions, PEGylation and acylation, could significantly reduce side effects. Strategies to avoid protein misfolding and aggregation during storage are benefit in protein fibrillation. This in turn to prevent unforeseen side effects in drug delivery. [25]. Thus, cationization has proven to be a great tool for oral PPDs delivery. Rapidly and completely transported drugs are generally lipophilic and distribute readily into the epithelial cell membranes of GIT [20]. The overall polarity of a drug molecule can be reduced by adding a non-polar or removal of a polar group to increase the lipophilicity, which leads to a higher concentration gradient for facilitating the diffusion of drugs over the intestinal mucosa. However, lipidization can reduce the water solubility of original drug. A typical drawback of lipidization is reduced receptor affinity [21]. y One example is the leu-enkephalin peptide which is chemically modified by a reversible aqueous lipidization method with a dimethylmaleic anhydride analog. This resultant drug was stable in various pH phosphate buffers and showed greater stability against enzymatic degradation. The study demonstrated the lipidization may be an enabling strategy which can be used to enhance oral absorption [22]. Nobex Corporation added a hydrophilic PEG chain (protection from enzymatic degradation) and a lipophilic alkyl chain to insulin for oral administration. Phase III results announced that it failed to meet the target endpoint, and recent iterations of PEG conjugation technique which include C10 and bile salts, presumably to promote peptide drug permeation. C10 elevates intestinal membrane fluidity via interaction with protein and lipids on the membrane, and it permeate over through both transcellular and paracellular pathways. However, Sakai et al. reported that high concentrations of C10 (>50 mM) could lead to significant cytotoxicity to Caco-2 cells, thus limiting the use of this technique [29]. Additionally, it has been reported that lipidized drug inhibits the P-gp efflux pump. This strategy is particular suitable for Biopharmaceutical Classification System (BCS) class IV drugs that were reported to be easily effluxed by P-gp transporter [22]. Chemical modification The oral bioavailability of PPDs is often hampered by their physicochemical characteristics, such as hydrophilicity, large molecular weight and sensitivity to enzymes and pH. To alter the physiochemical properties of PPDs, chemical modifications strategies, including lipidization, cationization, PEGylation and prodrug formation have been applied. PEGylation Generally, PEGylation is the covalent attachment of polyethylene glycol (PEG) to PPDs and elevate their half-lives due to steric hindrance against proteolytic enzymes. The increase in the molecular mass can improve both pharmacokinetic and pharmacodynamic properties of PPDs [26]. However, PEG may lead to size enlargement, increased viscosity, or reduce cell affinity and limits the biological activity. Moreover, the non-biodegradable PEG materials might trigger adverse effects [27]. g gg Minimol et al. have developed a PEGylated starch acetate nanoparticulate system for oral insulin delivery. An amphiphilic polymeric derivative was obtained by PEG conjugating with starch acetate, subsequently incubated with drug solution at the critical micelle concentration, forming self-aggregated drug loaded PEGylated nanoparticles. These self-aggregated nanoparticles showed only 32 nm in size allowing large surface area of the particles to contact with the intestinal mucosa. Moreover, the nanoparticles with great intestinal mucosal bioadhesion further promoted the drug permeation over the intestine [28]. https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1423 The commonly used absorption enhancers are surfactants, fatty acids, chelators, glycerides, bile salts, salicylates, chitosan and cholesterol. They normally increase the solubility and bioadhesion of the drug or drug carrier system which allows more drug amount to be retained at the absorption site and resulting in greater drug oral bioavailability [41]. However, it was found some absorption enhancers such as claudins, EDTA, sodium cholate, sodium dodecyl sulfate may cause the disruption of membrane integrity and systemic toxicity. The constant tight junction opening can cause mucosal damage and may also transport toxic molecules across the intestinal membrane [80]. Sadekar et al. have developed an oral form of camptothecin by co-delivering with cationic, amine-terminated dendrimer, which is a promising intestinal mucosal penetration enhancer, drug solubilizers for oral drug delivery. The results showed camptothecin solubilization in gastric fluid and significantly enhanced oral drug absorption without opening the tight junction [38]. Prodrug formation A prodrug is a chemical derivative of a main drug, it usually has greater stability, solubility, lipophilicity and intestinal permeability. It converts to an active drug in vivo usually undergoes transformation either by a chemical or an enzymatic reaction. Esterification of hydroxyl, amino acid, or carboxylic acid containing drugs can increase lipophilicity, thus improve intestinal drug permeation [29, 30]. However, the highly lipid-soluble drugs may bind to plasma protein, and limit free drugs in the plasma. Especially for PPDs, modification of PPDs maybe diminishes their specific receptors binding, since the plasma protein may occupy certain portion of the available PPDs. In some cases, during its activation stage, the prodrug might consume a vital cell constituent leading to its depletion. Peptide cyclization and unnatural amino acids substitution Cyclic cell-penetrating peptides (CPPs) show high stability and demonstrate great potential for the intracellular delivery [31]. Cyclization normally improves the stability by removing exposed C and N termini of the peptides, which are susceptible to enzymatic cleavage. Verdine et al. [32] and Clark et al. [33] have proved that peptide cyclization by adding a lipophilic linker and enhanced oral absorption and drug stability. Our research team has previously developed a cyclic peptide C25 with disulfide bond by using a phage display technology targeting immune checkpoint LAG-3, and the cyclic peptide showed great stability and in vivo antitumor activity [34]. Besides, our group previously had modified L-peptides to D-peptides [35, 36]. As L-peptides are susceptible to enzymatic degradation, lead to shorter half-lives. Thus, chemically modified to D-peptides have brought greater stability within GIT and systemic circulation. Sodium N-[8-(2-hydroxybenzoyl)amino]caprylat e (SNAC) is a promising absorption enhancer can enhance passive permeation of polar charged drug molecules through the intestinal epithelium. This is noteworthy in view of the very low tendency of a polar drug to permeate over the lipophilic intestinal epithelial membrane [42]. Several PPDs including calcitonin, insulin and heparin were conjugated with SNAC to promote the intestinal drug permeation [43]. Semaglutide utilized this technique is in clinical trials, that has shown protection against gastric enzymes and enhanced hydrophobicity to promote the peptide drug permeate over the intestine. Additionally, SNAC has not been reported to be associated with significant disruption of the tight junctions, change in membrane fluidity, thus the low toxicity is beneficial for later clinical studies [42]. Another effective permeation enhancer, 8-(N-2- hydroxy-5-chloro-benzoyl)-amino-caprylic acid) (5-CNAC) is the leading examples of Eligen® technology from Emisphere. It was reported that 5-CNAC can deliver macromolecules (> 150 kDa), enhances transcellular absorption without disrupting intestinal integrity. Karsdal et al. incorporated 5-CNAC with calcitonin for oral administration. 5-CNAC interacts with calcitonin forming an insoluble entity at low pH in stomach, once it reaches small intestine at higher pH, the complex dissolves and facilitates intestinal drug uptake, resulting in systemic exposure of intact peptide [44]. Currently there are ongoing trials for oral Eligen®- calcitonin for the treatment of osteoporosis [45]. Moreover, Novo Nordisk’s oral semaglutide which now has been marketed as tablet. Oral form of semaglutides, as Absorption enhancers Absorption enhancers are usually one of a varied class of chemical moieties, they are used to improve drug absorption by facilitating intestinal cells permeation [37, 38]. Generally, absorption enhancers alter the structural integrity of the epithelium or by simply promoting drug diffusion across the intestinal mucosa [39]. The associated mechanisms of action which include: changing membrane fluidity or mucus viscosity, and/or opening tight junctions, generally governed by passive diffusion and modeled by Fick’s first law of diffusion [39, 40]. https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1424 glucagon-like peptide-1 (GLP-1) analogues, also utilises Emisphere Technologies’ proprietary Eligen® Technology [46]. The mucolytic agent modified nanoparticles exhibited free Brownian motion and facilitate drug permeation over intestinal mucosa. In diabetic rats, the mucolytic agent modified nanoparticles generated a prominent hypoglycemic response and showed an bioavailability of 2.8-fold higher than that of unmodified nanoparticles. [55]. While mucus-penetrating strategies continue to be extensively investigated, the efficacy and safety have not yet been validated in large clinical trials. Microparticulate carrier systems Microparticles (size varying 1-100 µm) with high surface to volume ratio and greater intimate contact of the drugs with the intestinal epithelial layer, prolong gastric resident time, thus lead to higher drug absorption and oral bioavailability [61]. For example, microparticles have shown that encapsulation of PPDs for oral administration and achieved a sustained biological activity. Surface modification of microparticles can be achieved by conjugation, coating or crosslinking. For example, collagen microparticles modified by photochemical crosslinking [62], and silk fibroin coated polylactide-co-glycolide acid (PLGA) and alginate microparticles have been used to further prolong the release of the peptide drug [63]. Onishi et al. have Proteolytic enzyme inhibitor Direct inhibiting proteolytic enzyme by using an enzyme inhibitor is another way to circumvent intestinal enzyme activities. Proteolytic enzyme inhibitors such as aprotinin (inhibitor of trypsin and chymotrypsin), leupeptin (inhibitor for plasmin, trypsin, papain), chicken ovomucoid (trypsin inhibitor) and FK448 (chymotrypsin inhibitor). These proteolytic enzyme inhibitors are usually co-formulated with PPDs to prevent enzymatic degradation in intestinal mucosa. However, it was also reported that the safety of using enzyme inhibitors is a major concern. The excess use of this excipients may restrict certain therapeutic effects or trigger undesirable pharmacological activities [50, 51]. The most clinically advanced enzyme inhibition example is an oral insulin formulation known as ORMD-0801 consisting soybean trypsin inhibitor and a chelating agent that scavenges calcium. This treatment showed a significant 24.4% reduction in the frequencies of glucose readings >200 mg/dL, and a significant mean 16.6% decrease in glucose AUC [52]. M l Direct inhibiting proteolytic enzyme by using an enzyme inhibitor is another way to circumvent intestinal enzyme activities. Proteolytic enzyme inhibitors such as aprotinin (inhibitor of trypsin and chymotrypsin), leupeptin (inhibitor for plasmin, trypsin, papain), chicken ovomucoid (trypsin inhibitor) and FK448 (chymotrypsin inhibitor). These proteolytic enzyme inhibitors are usually co-formulated with PPDs to prevent enzymatic degradation in intestinal mucosa. However, it was also reported that the safety of using enzyme inhibitors is a major concern. The excess use of this excipients may restrict certain therapeutic effects or trigger undesirable pharmacological activities [50, 51]. Drug carrier systems The most clinically advanced enzyme inhibition example is an oral insulin formulation known as ORMD-0801 consisting soybean trypsin inhibitor and a chelating agent that scavenges calcium. This treatment showed a significant 24.4% reduction in the frequencies of glucose readings >200 mg/dL, and a significant mean 16.6% decrease in glucose AUC [52]. Modulation of pH PPDs are usually formulated with enteric coating to prevent their degradation in the acidic environment. Once the enteric coating reaches the intestine, the increase in pH leads in dissolution of the coating and release the drugs, as was illustrated for an oral calcitonin form that has been tested in clinical trials [47]. Moreover, Intestinal and pancreatic enzymes are also able to degrade PPDs in the neutral to basic environment in the small intestine. The use of citric acid in the oral PPDs formulation results in a decrease in pH, inhibiting degradation by the peptidases. Lei et al. have demonstrated that co-administration of citric acid reduced the activity of intestinal tryptic enzymes and resulted in higher oral bioavailability of calcitonin [48]. However, the major concern is the distortion of physiological pH. Other limitations involve the long-term drug stability and the incompatibility upon dilution [49]. Cell-penetrating peptides Cell-penetrating peptides (CPPs) are usually derived from viruses that are efficient at cell entry or membrane translocation, non-viral proteins or smaller molecules normally interact with membrane glycosaminoglycans, promoting PPDs to enter intestinal epithelial cells via endocytic pathways. However, the use of CPPs to elevate the oral bioavailability of PDDs has not yet been validated in the clinic [56]. Recently, CPPs such as HIV-1 Tat, penetration and oligoarginine are commonly used for oral delivery of various drugs [57, 58]. Kamei et al. have used oligoarginine as a CPP to elevate the oral bioavailability of the peptide drug, leuprolide, the results found that leuprolide-oligoarginine conjugate attached to cell-surface proteoglycans and subsequently permeate over the ileal epithelial membrane via endocytosis pathway [59]. However, inherent limitations were involved, including poor stability, toxicity and endosomal entrapment. To overcome this limitation, the enteric capsules can be used to avoid acidic and enzymatic degradation, thus promoting stability, and the sustain drug release of the CPPs modified formulation lower the toxicity of the CPPs to the intestinal mucosa [60]. Mucolytic agents Mucolytic agents, also called mucus penetrating agents, which are able to facilitate the permeation of the drugs across the mucus barrier and elevate oral bioavailability of PPDs [53]. In the reported preclinical studies, the use of PEG allows to promote mucus penetration [54]. Liu et al. have developed a novel self-assembled nanoparticle composed of insulin and trimethyl chitosan, and a dissociable mucolytic agent. https://www.thno.org 1425 Theranostics 2022, Vol. 12, Issue 3 developed enteric-coated chitosan-4-thio-butyl- amidine conjugate microparticles for oral delivery of calcitonin [64]. Yu et al. have developed a glucose-responsive microsphere that could be used as an efficient insulin carrier for oral delivery, and resulted in sustained hypoglycemic effect [65]. Several other new microparticulate systems have been developed recently. Such as temperature-responsive microspheres, dynamic hydrogel microspheres and glucose-responsive microspheres. However, the general limitations involve the polymer/drug miscibility, excipients compatibility for the system as well as the physical and chemical instability upon storage [66]. developed enteric-coated chitosan-4-thio-butyl- amidine conjugate microparticles for oral delivery of calcitonin [64]. Yu et al. have developed a glucose-responsive microsphere that could be used as an efficient insulin carrier for oral delivery, and resulted in sustained hypoglycemic effect [65]. Several other new microparticulate systems have been developed recently. Such as temperature-responsive microspheres, dynamic hydrogel microspheres and glucose-responsive microspheres. However, the general limitations involve the polymer/drug miscibility, excipients compatibility for the system as well as the physical and chemical instability upon storage [66]. permeation than larger particles [72]. During the process of endocytosis, the plasma membrane invaginates and pinches off to form enclosed vesicles and enter systemic circulation. Additionally, reducing the versicle size results in larger surface area, thus enhancing dissolution rate and solubility of PPDs However, limitations of nanoparticulate carrier systems are associated with limited drug loading and high particle aggregation due to thermodynamic instability, and scale-up difficulty for manufacturing [73]. Fan et al. have synthesized deoxycholic acid-conjugated chitosan, and loaded with the insulin into deoxycholic acid-modified nanoparticles (DNPs). It can overcome multiple intestinal barriers, internalized Caco-2 cells via apical sodium-dependent bile acid transporter (ASBT)-mediated endocytosis, and promoted the intracellular trafficking and basolateral release of insulin [74]. Lee et al. developed a dual ligand functionalized pluronic-based nanoparticle for oral delivery of insulin. Chitosan and zonula occludins toxin (ZOT)-derived, tight junction opening peptide were conjugated to nanoparticles to increase the intestinal permeation of loaded insulin through the paracellular pathway [75]. Hydrogels Hydrogels generally contain water phase, a crosslinked polymer and a drug component. Usually they can respond to environmental changes to alter network structure, mechanical strength and swelling manner [67, 68]. Generally, hydrogels remain insoluble even imbibe great amounts of biological fluids, therefore they appear to stabilize the embedded PPDs, protecting the PPDs from degradation in the harsh GI environment [69]. In addition, the PPD loaded hydrogel is able to prolong retention time within specific gut regions thus elevate the drug absorption. However, hydrogels for oral delivery of PPDs have not made significant progress towards the clinical trials [68, 70]. O’Neill et al. have developed whey protein hydrogels encapsulating riboflavin. The dried microbeads hydrogel showed great resistance to GI degradation, underwent swelling and sustained release drug in GIT [71]. Our team has previously developed a hydrogel using various mucoadhesive polymers to deliver glutathione. This polymeric hydrogel has shown great benefit for promoting the stability and bioavailability of the peptide drug [67]. However, the main limitation of oral hydrogel is the physical and/or chemical instability issues, fast hydrogel disintegration may occur while it contacts with large amount of gut fluid after oral administration [69]. Gold nanoparticle technology Many publications have proposed the potential of gold nanoparticles (GNPs) for biomedical applications. The small size and multi-valence arrangement around the gold core elevates the capacity to improve drug biodistribution and hence effectiveness and safety [77]. However, the GNPs that has entered clinical trials is CYT-6091 (Aurimune) is the only GNPs that have entered clinical trial currently. They are gold core particles incorporating TNF-α (a cytokine) and showed a particle size of 27 nm approximately. Studies demonstrated that incorporating TNF-α onto the gold platform improved systemic tolerability. In phase I studies, the safety profile showed the GNPs were well tolerated for patients with advanced cancer [78]. Ultrasmall GNPs, with size of only 2–3 nm, have also showed great potential in a wide variety of therapeutic applications. It was demonstrated that ultrasmall GNPs with size around 2 nm have a relatively longer Mucolytic agents Our research team has also developed a PLGA based double emulsion nanoparticles for delivering glutathione. This nanoparticulate delivery system was able to elevate the drug retention time on mucosa, avoiding enzymatic degradation and promotes the transmucosal permeation of glutathione. However, the safety and biocompatibility of the polymeric materials and applicability of scaling up in manufacturing still remain a challenge [76]. Nanoparticulate carrier systems Nanoparticulate carrier systems, usually with particle size of less than 1 µm, such as polymeric or lipid nanoparticles, nanoemulsions and niosomes for oral drug delivery are of interest owing to the great benefit in promoting drug stability, provide a sustained drug release profile and elevate drug absorption over intestinal wall. In general, smaller particles of less than 500 nm are usually undergoes endocytosis and shows greater intestinal drug https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1426 promote drug permeation through physiological barriers. In general, ionic liquids interact with various hydrophilic and hydrophobic amino acids of a protein through an intricate balance of hydrogen bonds, disulfide bonds, ionic interactions and hydrophobic effects. When mix with water or body fluid, a more complex interplay between ions occurs, which can result in formation of microemulsions or micelles [86]. Williams et al. developed an ionic liquid-based formulation for oral delivery of insulin, and the system showed high drug loading, a better access to the intestinal absorptive surface and prevented enzymatic degradation. Molecular dynamics simulation studies have shown that ionic liquids can remove water from the surface of enzymes to the same extent as polar organic solvents like acetonitrile. However, the safety issue is the major concern and the bulking care is essential particularly during handling and transport [87]. Banerjee et al. have developed an ionic liquid-based oral formulation of insulin. This biocompatible delivery system has good long-term stability and facilitates intestinal absorption via paracellular uptake through the opening of tight junctions, results in promising insulin oral bioavailability. Thus, ionic liquids present an unprecedented and under-explored therapeutic opportunity with immense potentials for oral delivery of PPDs [87]. plasma half-life, improved tissue penetration compared with larger counterparts. Furthermore, ultrasmall GNPs offer a particularly high surface/volume ratio, which leads to greater dose-efficiency and all these indicated that it is a promising drug delivery vehicle for PPDs [79]. Liposomes Liposomes are generally composed of one or more phospholipid membrane bilayers surrounding aqueous inner phase with sizes from 15 nm to 10 µm [48] (Figure 4A). Momoh et al. and Erel et al. have developed nano-encapsulated mucin and chitosan nanoparticles in W/O microemulsion for oral insulin delivery. They showed high entrapment efficiency and stability, sustained drug release and elevated intestinal permeation [83, 84]. In recent years, self- microemulsifying drug delivery systems (SMEDDS), which emulsify spontaneously when exposed to the GIT fluid have been receiving increased attention. However, the low drug loading and the amounts of surfactant/co-surfactants used are limiting its application. Our group has previously developed a bicontinuous microemulsion for oral delivery of beta-carotene, which is a peptide drugs with very poor solubility. The optimized oral microemulsion promotes the stability and allows solubilizing beta-carotene, is a promising basis for further development as a functional beverage, as well as an oral delivery system for poor solubility peptide drugs [85]. ( g ) Liposomes can be divided into six types based on their size and structures as shown in Figure 4B. Lipophilic drugs are embedded in the phospholipid layers while hydrophilic molecules are encapsulated in the aqueous inner core. This nature of liposomes that can carry both water soluble and lipid soluble drugs is called amphiphilic [88]. Suzuki et al. have prepared a chondroitin sulfate-g-glycocholic acid- coated liposomes for oral exendin-4 (Ex-4) delivery. The long term pharmacodynamic effects, of daily oral exendin-4 loaded liposomes (300 μg/kg) were better than daily subcutaneous administration of Ex-4 solution (20 μg/kg) over 4 weeks [89]. Wang et al. have used bovine serum albumin (BSA) adsorbed to cationic liposomes (CLs) to form protein corona liposomes (PcCLs) for oral delivery of insulin. The results showed great intestinal permeation, led to an increase of drug oral bioavailability and hypoglycemic effect [90]. Our group has previously developed a deformable liposome to encapsulate catechin, which is a peptide drug extracted from Microemulsion Microemulsion is an isotropic, transparent and thermodynamically stable system which consists of water, oil and surfactant, usually with a co-surfactant. Droplet size is normally less than 200 nm. Structurally, they are divided into three phases: water-in-oil (W/O), oil-in-water (O/W) and bicontinuous microemulsion [80]. Surfactants with a hydrophilic lipophilic balance (HLB) value greater than 12 are hydrophilic and predominantly forming O/W emulsions, while surfactants with HLB values less than 12 are favor in formation of W/O emulsion. Surfactants generally lower the surface tension to promote the drug solubility and opening tight junctions momentarily to enhance drug permeability. Moreover, surfactants having HLB greater than 20 usually require the addition of co-surfactants. However, some surfactants may cause some degree of toxicity, thus the amount of surfactant used requires careful consideration. Other limitations include the disintegration of the system due to dilution in the gut, and in vivo instability below the critical micelle concentration [81, 82]. Ingestible self-orienting system An ingestible self-orienting system is a recent invented device that physically inserts a drug-loaded millipost through the GI mucosa with promising bioavailability. Inspired by the self-orienting leopard tortoise, Abramson et al. have developed an ingestible self-orienting millimeter-scale applicator (SOMA) that tends to position itself to engage with GIT, designed to resist external forces such as fluid flow, peristaltic motion upon reaching a stable point on the GIT wall. It then deploys milliposts fabricated from drugs directly through the intestinal mucosa while avoiding perforation. Figure 5 demonstrates the device positions to the stomach lining, orients its injection of the drug payload toward the GIT wall [96]. This SOMA device has demonstrated promising efficacy to deliver insulin orally and could be used to deliver other PPDs orally. However, the drawback involves the deliverable dose is constrained by the formulation, volume and stability of the millipost. By increasing the size of millipost can elevate drug loading but might compromise the intestinal mucosa and trigger perforation risk. Furthermore, the long-term chronic effects brought by daily gastric injections shall be evaluated. Still, the SOMA represents a great platform for oral delivery of PPDs [97]. Ionic liquid Ionic liquids as low melting salts with melting point <100°C, often formulated to enhance the dissolution of poorly soluble drugs, as well as to https://www.thno.org 1427 Theranostics 2022, Vol. 12, Issue 3 green tea leaf, that can be easily undergo hydrolysis. The developed liposomes demonstrated great protection for the peptide drugs and elevated the bioavailability significantly [91]. However, the major limitations involve poor stability, drug leakage of liposomes and short shelf life. The intact liposomes are difficult to permeate over the lipophilic intestinal epithelium, thus lower the oral bioavailability, especially for BCS class Ⅲ drug [92]. based microneedle device for oral insulin delivery. The microneedle capsule was designed to dissolve at pH levels encountered in the small intestine. The results showed the insulin levels instantly increased and the blood glucose was reduced within 30 min, with an oral bioavailability of over 10% [95]. green tea leaf, that can be easily undergo hydrolysis. The developed liposomes demonstrated great protection for the peptide drugs and elevated the bioavailability significantly [91]. However, the major limitations involve poor stability, drug leakage of liposomes and short shelf life. The intact liposomes are difficult to permeate over the lipophilic intestinal epithelium, thus lower the oral bioavailability, especially for BCS class Ⅲ drug [92]. Gastrointestinal Permeation Enhancement Technology (GIPET®) GIPET® is an oral solid dose technology can effectively increase oral absorption of a variety of low permeability PPDs. This strategy focuses on the use of medium chain fatty acid or its variants coupled with salts, resulting in greater hydrophobicity and penetration characteristics that open epithelial tight junction [103]. This technology is low cost and safe, which has great advanced to the clinic. GIPET® consists three major enteric coated formats. GIPET® I, is an enteric coated tablet with drug in selected weight ratios. GIPET® II, is a microemulsion form Transient Permeation Enhancer® (TPE®) Intestinal patches consist polymeric matrix embedding drugs, usually with a stabilizer. They can adhere to the intestinal wall and positioning the drugs directly to the intestinal epithelium, and meanwhile protecting the drugs from local enzymatic degradation [98]. Recently, Banerjee et al. have fabricated an insulin loaded mucoadhesive oral patches integrated with iontophoretic circuit and surgically placed in the intestine. It was found the iontophoresis could disrupt the tight junctions of intestinal epithelium and facilitate insulin transport via paracellular pathway, without impairment of the intestinal mucosa. However, clinical proof of oral patch technology has not yet been forthcoming. However, the limiting drug loading and stability issue upon storage shall be considered [98, 99]. Our research team has previously developed a mucoadhesive polymers‐based patch as a carrier system for delivery of glutathione. Various mucoadhesive biomimetic polymers were screen and the mucoadhesive patch was prepared using a simple casting method, and without using other unnecessary excipients. The optimal mucoadhesive patch has shown great potential for oral delivery of glutathione and other PPDs [100]. TPE® had been used for oral delivery of octreotide. TPE® is an oily suspension of octreotide that consists a permeation enhancer that can transiently modify the integrity of intestinal epithelium by opening the tight junction. It also consists polysorbate-80, allow to alter the thickness of intestinal mucus, thus further promote the intestinal drug uptake. Moreover, several peptides have been incorporated into TPE® including teriparatide, leuprolide, insulin and octreotide. However, a main concern in application of TPE®, the intestinal tight junction opening that cause toxicity, or the use of food emulsifiers or other excipients might initiate autoimmune disease [101, 102]. Currently, Phase I studies of octreotide capsules resulted in an oral bioavailability of about 0.7% and primary endpoints were achieved in two Phase III studies. The oral octreotide dose required to achieve these endpoints was over 200 times that of the 0.1 mg subcutaneous injection, which demonstrated a big achievement of this promising oral form [101]. Biodegradable microneedle-based delivery system The inherent attractiveness of microneedle-based delivery strategy demonstrates the great suitability for various PPDs delivery, even with large molecular weight [93]. Prausnitz et al. have utilized microneedle technology for oral drug delivery. They placed a 0.5-cm2 drug loaded microneedle patch onto the arms connected to a base, and called this device a luminal unfolding microneedle injector (LUMI). Once the oral administered device reached the intestine, the polymeric material holding the spring was dissolved, led to actuation that pushed the LUMI out, pressing the microneedle patches against the intestinal wall, allowing the drugs directly penetrate the intestinal epithelium. The Rani Therapeutics company has developed a related technology that deployed oral microneedles that has been carried out in a clinical trial currently, using octreotide as a model drug. Moreover, up to 0.3 mg of drug can be loaded into LUMI, which is sufficient for many potent PPDs [94]. Recently, it has been reported the preclinical studies of two oral microneedle devices, a poly(methacrylic acid-co-ethyl acrylate) and PEG Figure 4. A) Basic liposome structure. B) Different model membranes of liposomes. SUVs: small unilamellar vesicles; LUVs: large unilamellar vesicles; MLVs: multilamellar vesicles; MVVs: multivesicular vesicles; OLVs: oligolamellar vesicles; GUVs: giant unilamellar vesicles. Figure 4. A) Basic liposome structure. B) Different model membranes of liposomes. SUVs: small unilamellar vesicles; LUVs: large unilamellar vesicles; MLVs: multilamellar vesicles; MVVs: multivesicular vesicles; OLVs: oligolamellar vesicles; GUVs: giant unilamellar vesicles. https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1428 Figure 5. The ingestible self-orienting millimeter-scale applicator after oral administration, and the device could autonomously position itself to the intestinal mucosa. (Adapted with permission from [97], copyright 2021.) orienting millimeter-scale applicator after oral administration, and the device could autonomously position itself to the intestinal mucosa. (Ad opyright 2021.) Figure 5. The ingestible self-orienting millimeter-scale applicator after oral administration, and the device could autonomously position itself to the intestinal mucosa. (Adapted with permission from [97], copyright 2021.) Figure 5. The ingestible self-orienting millimeter-scale applicator after oral administration, and the device could autonomously position itself to the intestinal mucosa. (Adapted with permission from [97] copyright 2021 ) Formulation technology with combinational strategies The following are some of the drug delivery technologies that utilize combinational strategies mentioned above (Figure 6), in order to advance and accelerate the oral absorption of PPDs. These are the successful examples with combinational strategies that are either in preclinical stage or at ongoing clinical settings are summarized below. https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1429 encapsulated within an enteric coated gel capsule. GIPET® III, consists of drugs with fatty acid derivatives within an enteric coated gel capsule. Currently, the Phase I and II studies have shown the safety profile of the three formats given on a repeated basis [104]. In addition, permeation enhancer C10, have been incorporated to increase intestinal membrane fluidity and promote transcellular drug transport. Moreover, another feature of GIPET® promotes the oral bioavailability of drugs may relate to inhibition of P-gp efflux [103]. studies, including phase III oral calcitonin and phase I oral leuprolide. The results from multiple preclinical as well as early and late-stage clinical studies have demonstrated the promising applicability of PeptelligenceTM to the oral delivery of PPDs [107]. ThioMatrix™ technology Thiolated mucoadhesive polymers (thiomers) that are capable of forming covalent bonds with intestinal mucus glycoproteins via thiol/disulfide exchange reactions. Thus, thiomers modified delivery system enhances the intestinal mucoadhesion, prolongs the retention in GIT and lead to higher oral bioavailability. In addition, thiomers also exhibit enzyme inhibitory, permeation enhancing and efflux pump inhibitory properties. However, thiomers are rather unstable in formulation form as they are subject of thiol oxidation at pH ≥ 5 unless sealed under inert conditions. Therefore, the use of pre-activated thiol groups might be an interesting approach to enhance its stability [108]. ThioMatrix™ GmbH (Vienna, Austria) uses thiomers incorporates with reduced glutathione, to enhance oral delivery of hydrophilic macromolecules based on inhibition of protein tyrosine phosphatase by thiol groups. The results demonstrated the thiomeric mucoadhesive, permeation enhancing, and efflux pump inhibition properties were promising, thus lays a great platform for oral delivery of PPDs [103]. Peptelligence technology PeptelligenceTM is a highly developed, clinically proven platform technology that enables the oral delivery of PPDs. It protects PPDs from acid hydrolysis, enzymatic degradation, and also enhances paracellular transport [105]. Enteris’s Peptelligence technology focuses on two main strategies, the first is a permeation enhancer, which opens tight junctions and facilitates paracellular transport. Second is a pH-lowering agent, lowering the local pH of the intestinal fluids in order to reduce protease activity. Additionally, the coating of the organic acid granules forms a thin barrier that prevents PPDs from acid degradation in the stomach [106]. This technology was initially developed by Unigene and then Enteris Biopharma (Boonton, NJ, USA). Enteris has demonstrated positive results in several clinical Figure 6. The overview of main formulation strategies for oral delivery of PPDs, including chemical modification, addition of effective agents, drug carrier systems and medical devices. Figure 6. The overview of main formulation strategies for oral delivery of PPDs, including chemical modification, addition of effective ag devices Figure 6. The overview of main formulation strategies for oral delivery of PPDs, including chemical modification, addition of effective agents, drug carrier systems and medical devices. Figure 6. The overview of main formulation strategies for oral delivery of PPDs, including chemical modification, addition of effective agents, drug carrier systems and medical d i https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1430 acidic degradation in the stomach and protecting the drugs from the bile salts [114]. Nano Inclusion technology Oral sCT (Ostora™) is built around coated citric acid vesicles in a Eudragit®-coated capsule, and currently has completed Phase III, indicating it is a clinically advanced oral peptide format. Briefly, it uses lauroyl carnitine chloride as the permeation enhancer to promote intestinal drug permeation, and citric acid as a pH lowering agent, lowering pH to reduce protease activity, as well as encapsulating within a Eudgradit® capsule to prevent the drugs from acidic degradation in the stomach [111]. There are other platforms with clinical trial data: TPE (Chiasma), POD™ (Oramed), Eligen® (Emisphere), IN-105 (Biocon) and GIPET (Merrion). What stands out about these formulations is their simplicity compared with highly complex delivery constructs [111, 112]. This technology allows to solubilize potent molecules that have minimal solubility at biological pH for oral delivery [117]. Midatech’ MidaSolve project, MTX110, utilizes the MidaSolve nanosaccharide inclusion technology to solubilize panobinostat, allowing it to be orally administered via a micro-catheters system. Therefore, this technology focuses on promoting drug solubility, meanwhile the delivery system also elevates the oral drug bioavailability as well as to facilitate the drug to cross the blood-brain-barrier. The initial Phase I study showed promising safety profile in patients. Phase II trial of safety, tolerability, recommended dose and efficacy in 19 patients are under investigation. The study endpoint is expected to be patient survival after 12 months [118]. Q-Sphera™ technology Transferrin (Tf) is an endogenous serum protein that transports iron to cells expressing the Transferrin receptor (TfR) through TfR-mediated endocytosis. Studies have applied Tf to prepare drug carrier system to deliver PPDs, genes and poor soluble drugs to the target tissues including intestinal epithelium and blood brain barriers that abundantly express Tf receptors [109]. Melanie et al. generated and expressed functionally active colony-stimulating factor (G-CSF) as a recombinant fusion protein incorporated with Tf to evaluate the function of Tf as a carrier for oral delivery of G-CSF. The results demonstrated that the Tf moiety of the fusion protein not only promoted the drug permeation over the GI epithelium, but also protected the drug from enzymatic degradation [110]. Therefore, it demonstrates that a Tf-based recombinant fusion protein technology is a promising approach for future development of orally active PPDs. Q-Sphera™ technology is a novel platform to individually print narrow size distribution particles of approximate 30 μm to generate predictable pharmacokinetic profile. This micro-piezo technology was developed by the MidaTech [115]. Midatech’s Q-Sphera technology focuses on long acting injectables using proprietary piezo printing technology that encapsulates PPDs into polymeric microparticles with precision properties. The piezo printing process regulates the internal pH inside microparticles and reduces the likelihood of protein destruction. Additionally, the Q-Sphere technique does not use surfactants, toxic solvents or biphasic mixtures, providing a promising safety profile of the technique. An example of Midatech’s Q-Sphera has utilized an advanced 3D printing technology to fabricate a PLGA microparticle depot system. It is low cost and environmentally friendly, with an efficient high yield production and scalable manufacture [116]. Targeting intestinal cell for oral PPDs delivery Protein/Peptide Conditions or diseases Delivery approach ClinicalTrials.gov identifier Homeopathic antibodies to the TLR3 FYW peptide (TAO1) ●Common Cold Impregnation of pre-made tablets NCT01651715 (Phase I/ Phase II) Anti-CD3 monoclonal antibody ●Chronic Hepatitis C Neutralize stomach pH for enhancing stability of the Mab with Omeprazole NCT01459419 (Phase II) ● Nonalcoholic Steatohepatitis NCT01205087 (Phase II) Insulin ●Diabetes Mellitus, Type 1 pH sensitive Capsules NCT02580877 (Phase II); NCT00419562 (Phase III); NCT02535715 (Phase II); ●Diabetes Mellitus, Type 2 pH sensitive Capsules and enzyme inhibition NCT02954601 (Phase II); NCT01889667 (Phase II); ●Brittle Type I Diabetes Mellitus pH sensitive Capsules and enzyme inhibition NCT00867594 (Phase II) ●Nonalcoholic Steatohepatitis pH sensitive Capsules and enzyme inhibition NCT04616014 (Phase II); ●Diabetes Hepatic directed vesicles NCT00814294 ((Phase II/Phase III)); NCT00521378 ●Diabetes Mellitus, Type 1 Insulin modification and enhanced osmosis NCT01035801 (Phase I) ●Diabetes Mellitus, Type 2 Insulin modification and enhanced osmosis NCT03392961 (Phase I); NCT03430856 ((Phase II/Phase III) ●Insulin-Dependent ●Diabetes Mellitus Nanoparticle encapsulation and permeability enhancement NCT01120912 (Phase I); NCT01973920 (Phase II); NCT01772251 (Phase I/ Phase II) Glucagon like peptide-1 Analogue ●Diabetes Permeation enhancer NCT02094521 (Phase I) Leuprolide ●Endometriosis Permeation enhancer, pH modulator and enzyme inhibitor NCT05096065 (Phase II) Salmon calcitonin ●Osteopenia Antiproteolysis and absorption enhancement NCT01292187 (Phase II); NCT00959764 (Phase III) Acyline ●Contraception Gastrointestinal permeation enhancement NCT00603187 (Phase I/ Phase II) Dolcanatide ●Constipation Chemical modification NCT01983306 (Phase II) Parathyroid hormone (1-34) ●Hypoparathyroidism Permeation enhancers and enzyme inhibitor NCT02152228 (Phase II) Cyclosporine A (CSA) ●Ulcerative Colitis Emulsion (Oil-in-water) NCT01033305 (Phase II) Table 1. Examples of formulation strategies of oral insulin with advantages and disadvantages. Oral formulation techniques Advantage Disadvantage References Liposomes (e.g. HDV-1) Superior mucus-penetrating capability; Excellent intestinal epithelial absorption. Poor stability [89, 121, 122] Microemulsion Improve the encapsulation efficiency. Large particle size may exist [89, 121, 122] Nanoparticulate carrier system (e.g. Oshadi oral insulin) High insulin loading; Promoted insulin intestinal permeation. Complex preparation process; May lead to cytotoxicity [123] Hydrogels Great stability, rapid response rate; High elasticity, and good biocompatibility. Lack controlled release manner under different pH [89, 121, 122] Hydrogels and Cell-penetrating peptides Controlled release manner; Permeation stimulatory effect Stability issue within GIT [123] Microparticulate High encapsulation efficiency Large particle size leads to poor absorption [89, 121, 122] Absorption enhancers (e.g. ORMD-0801, IN-105, Oshadi oral insulin) Protecting against enzymatic degradation; Improving drug absorption Risk of infections. [124-126] pH sensitive enteric coating (e.g. ORMD-0801, Capsulin) Protect the drug from pepsin hydrolysis; Sustained released and greater drug absorption. Oleotec™ and Soctec™ gastro-retentive technology Oramed is a carrier system used for oral delivery insulin and GLP-1, which was developed by the Oramed Pharmaceuticals. Ormade’s oral insulin is available as ORMD-0801, it allows to protect drug from enzymatic degradation and elevate the intestinal permeation of insulin. Ormades oral insulin was investigated for both type I and type II diabetes. It is currently under phase II clinical trial for oral insulin delivery and phase I trial for oral GLP-I delivery (NCT02535715) [113]. Oleotec™ and Soctec™ gastro-retentive technologies were introduced by the Skyepharma. This strategy mainly focuses on promoting the drugs being absorbed in the stomach. Briefly, the technique prolongs the retention of the drugs within the stomach, and gradually releasing the encapsulated drug without being degraded by the acidic environment [119]. Upon oral administrated the formulated dosage, the delivery system encapsulating drug was activated by GIT fluid. The polymer gradually swelled and enlarged 8 and 10 times in size, which guaranteeing its preservation in the stomach Orasome is a polymer-based liposome for oral delivery of insulin and human growth factor, which was introduced by the Endorex Corporation. This formulation allows to protect the loaded PPDs from https://www.thno.org 1431 Theranostics 2022, Vol. 12, Issue 3 identified and characterized with different surface receptors which could be potential targets for oral PPDs delivery. The therapeutic applications of most PPDs largely depend on receptor-mediated endocytosis, and the relative affinity to these receptors are crucial. Therefore, targeting these stimulating endocytosis receptors on intestinal cell surface has drawn great attention for delivery of PPDs. For this purpose, surface modified drug delivery systems or ligand-grafted drugs are required. In the following sections, the use of ligands for targeting the major receptor of different types of intestinal cells will be discussed (Figure 7). even after 6 – 8 hours of gastric emptying and released drugs in a sustained manner [120]. The Accordion Pill™ is a typical gastro retentive formulation composed of polymeric films. It has a planar structure with multi-layer folded to an accordion shape, and encapsulated within a capsule. Upon reaching the stomach, the capsule dissolves, the Accordion Pill™ unfolds and allows to retain within the stomach for up to 12 hours [119]. Targeting intestinal cell for oral PPDs delivery A variety of intestinal cell types has been A variety of intestinal cell types has been in Table 1. Examples of formulation strategies of oral insulin with advantages and disadvantages. Oral formulation techniques Advantage Disadvantage References Liposomes (e.g. HDV-1) Superior mucus-penetrating capability; Excellent intestinal epithelial absorption. Poor stability [89, 121, 122] Microemulsion Improve the encapsulation efficiency. Large particle size may exist [89, 121, 122] Nanoparticulate carrier system (e.g. Oshadi oral insulin) High insulin loading; Promoted insulin intestinal permeation. Complex preparation process; May lead to cytotoxicity [123] Hydrogels Great stability, rapid response rate; High elasticity, and good biocompatibility. Lack controlled release manner under different pH [89, 121, 122] Hydrogels and Cell-penetrating peptides Controlled release manner; Permeation stimulatory effect Stability issue within GIT [123] Microparticulate High encapsulation efficiency Large particle size leads to poor absorption [89, 121, 122] Absorption enhancers (e.g. ORMD-0801, IN-105, Oshadi oral insulin) Protecting against enzymatic degradation; Improving drug absorption Risk of infections. [124-126] pH sensitive enteric coating (e.g. ORMD-0801, Capsulin) Protect the drug from pepsin hydrolysis; Sustained released and greater drug absorption. Difficulties in oral administration for infants or younger children. [124, 127] Insulin modification (e.g. IN-105) Protect drug from enzyme and acid degradation; Controlled release manner Identify suitable modification sites. Bioactivity may be reduced after modification. [126, 128] Table 2. Current clinical status of major PPDs for oral administration. Targeting intestinal cell for oral PPDs delivery had developed an EGP peptide which targeted the heparan sulfate proteoglycans on the intestinal enterocytes. The EGP modified nanoparticles promoted the enterocytes uptake involving caveolae-mediated endocytosis and avoided lysosomal entrapment, thus facilitated the direct apical-to-basolateral transcytosis. Oral administrated insulin EGP NPs generated a strong hypoglycemic response on diabetic rats with 10.2-fold increase in bioavailability compared with free insulin [136]. Further, the stability of peptides can be improved by simple modification, such as terminal blocking and insertion of D-amino acids [137]. We have previously identified a PD-1/PD-L1 blocking D-peptide by using a liquid-phase phage display screening method, and it showed proteolysis- resistance and great stability in vivo, which is remarkably beneficial for its oral delivery [70]. Arginine–glycine–aspartic acid (RGD) is widely used ligands to target integrin αvβ3 receptors, which are Targeting intestinal cell for oral PPDs delivery Difficulties in oral administration for infants or younger children. [124, 127] Insulin modification (e.g. IN-105) Protect drug from enzyme and acid degradation; Controlled release manner Identify suitable modification sites. Bioactivity may be reduced after modification. [126, 128] Table 1. Examples of formulation strategies of oral insulin with advantages and disadvantages. Table 2. Current clinical status of major PPDs for oral administration. Protein/Peptide Conditions or diseases Delivery approach ClinicalTrials.gov identifier Homeopathic antibodies to the TLR3 FYW peptide (TAO1) ●Common Cold Impregnation of pre-made tablets NCT01651715 (Phase I/ Phase II) Anti-CD3 monoclonal antibody ●Chronic Hepatitis C Neutralize stomach pH for enhancing stability of the Mab with Omeprazole NCT01459419 (Phase II) ● Nonalcoholic Steatohepatitis NCT01205087 (Phase II) Insulin ●Diabetes Mellitus, Type 1 pH sensitive Capsules NCT02580877 (Phase II); NCT00419562 (Phase III); NCT02535715 (Phase II); ●Diabetes Mellitus, Type 2 pH sensitive Capsules and enzyme inhibition NCT02954601 (Phase II); NCT01889667 (Phase II); ●Brittle Type I Diabetes Mellitus pH sensitive Capsules and enzyme inhibition NCT00867594 (Phase II) ●Nonalcoholic Steatohepatitis pH sensitive Capsules and enzyme inhibition NCT04616014 (Phase II); ●Diabetes Hepatic directed vesicles NCT00814294 ((Phase II/Phase III)); NCT00521378 ●Diabetes Mellitus, Type 1 Insulin modification and enhanced osmosis NCT01035801 (Phase I) ●Diabetes Mellitus, Type 2 Insulin modification and enhanced osmosis NCT03392961 (Phase I); NCT03430856 ((Phase II/Phase III) ●Insulin-Dependent ●Diabetes Mellitus Nanoparticle encapsulation and permeability enhancement NCT01120912 (Phase I); NCT01973920 (Phase II); NCT01772251 (Phase I/ Phase II) Glucagon like peptide-1 Analogue ●Diabetes Permeation enhancer NCT02094521 (Phase I) Leuprolide ●Endometriosis Permeation enhancer, pH modulator and enzyme inhibitor NCT05096065 (Phase II) Salmon calcitonin ●Osteopenia Antiproteolysis and absorption enhancement NCT01292187 (Phase II); NCT00959764 (Phase III) Acyline ●Contraception Gastrointestinal permeation enhancement NCT00603187 (Phase I/ Phase II) Dolcanatide ●Constipation Chemical modification NCT01983306 (Phase II) Parathyroid hormone (1-34) ●Hypoparathyroidism Permeation enhancers and enzyme inhibitor NCT02152228 (Phase II) Cyclosporine A (CSA) ●Ulcerative Colitis Emulsion (Oil-in-water) NCT01033305 (Phase II) Table 2. Current clinical status of major PPDs for oral administration. https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1432 Figure 7. The overview of the intestinal cells-targeting strategies with the major cell types and the associated main receptors for oral delivery of PPDs. [133]. Owing to the abundant glycosylated proteins and lipids on intestinal enterocyte cells, lectins have great potential to promote cellular uptake of PPDs via specific binding [134]. Peptides are particularly suitable as ligands because they are small, ease in synthesis and typically nonimmunogenic [135]. Zheng et al. Enterocyte targeting Enterocytes are hyperpolarized epithelial cells with a columnar shape. They are the most prevalent cell type and are often targeted for the oral PPDs delivery. Several receptors have been reported to be expressing on the apical surface of enterocytes. Ligands, including vitamins, proteins, monoclonal antibody fragments and oligopeptides are often used for enterocyte targeting [129, 130]. Vitamins are commonly used ligands to decorate delivery systems for targeting specific intestinal cell receptors. Since they are very stable, safe with easy tunability. Vitamin B12 and biotin (vitamin B7) has been used for intestinal enterocyte targeting and showed promising results. Folic acid (vitamin B9) and thiamine have also been used as ligands for oral targeted delivery [131]. Folic acid which enters enterocytes via a pH- and sodium ion-dependent pathway has been reported as efficient enterocyte- targeted ligands for the delivery of insulin and vancomycin [132]. Li et al. used folic acid as a targeting ligand that grafted on nanoparticles to target the proton-couple folate transporter expressed on intestinal enterocytes, it mediated the endocytosis and facilitated the permeation over the intestinal mucosa https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1433 transmembrane glycoproteins overexpressed in intestinal Caco-2 cell line [138]. epithelial cells and are responsible for producing mucins [149]. So far, very few proven goblet cells targeting ligands were reported. Jin et al. have developed a trimethyl chitosan chloride (TMC) nanoparticle modified with CSK peptide for oral insulin delivery. The results showed the CSK peptide has significant effect on promoting drug permeation over intestinal epithelium, and the insulin loaded CSK modified nanoparticles produced a better hypoglycemic effect, with a 1.5-fold higher insulin oral bioavailability compared with drug solution [150]. Previously, our research group had developed gemcitabine loaded CSK-TMC conjugates that significantly enhanced the drug uptake in mucus-producing cells due to the goblet cells targeting ability, and vastly elevated the oral drug bioavailability of 5.4-fold compared with plain drug solution [151]. Moreover, a study demonstrated that wheat germ agglutinin (WGA) can bind to E-cadherin, which is also presented on mucus- secreting goblet cells. Interestingly, WGA modified nanocrystals were able to invade villi of goblet cells and reach lamina propria by transcytosis. The WGA modified nanocrystals showed an increased oral bioavailability of 17.5- and 2.4-folds higher than that of coarse crystals and plain drug, respectively [152]. Therefore, the development of E-cadherin-targeting drug delivery systems also can be an alternative strategy for intestinal goblet cell targeting. Dendritic cell targeting Dendritic cells (DCs) play a key role in protective immunity against pathogens [153]. Intestinal DCs are a small subset of DCs that consist a large network in the intestinal immune system. DCs are distributed throughout the GIT, including the lamina propria of the intestine, mesenteric lymph nodes and Peyer’s patches [154]. It has been reported that mannose, Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), integrins, neonata Fc receptors and scavenger receptors are the main endocytic receptors that expressed on the surface of DCs. Due to this variety of receptors, DCs have the ability to recognize various surrounding signals and induce immune responses [155, 156]. Among the receptors, mannose receptors are the most popular receptor presented on the surface of DCs that offer the great potential for PPDs to target DCs. TLRs and CLRs were also proved as receptors to mediate cellular endocytosis. However, Enterocyte targeting Considering the large size and large number of goblet cells presented in the small intestine, thus it is worth investigating more specific targeting endocytosis- mediated receptors/transporters on goblet cells, as well as the more of the particular ligands targeting to them. Microfold cell targeting Microfold cell (M cells) are one type of intestinal epithelial cells mainly located in the epithelium of Peyer’s patches. Various types of cargo can be transported across M cells, such as antigens, bacteria, viruses and particles. Without passing through lysosomes, the cargo can avoid the lysosomal degradation which is a major benefit for transcytosis of the cargo. Furthermore, the undeveloped microvilli and glycocalyx structures of M cells allow the cargo to permeate over easier compared with the enterocytes [139]. Owing to the above-mentioned features, M cell demonstrates as a promising target for oral PPDs delivery. The reported M cell targeting molecules include plant lectins, outer membrane bacterial proteins, and monoclonal antibodies. They were conjugated to the surface of delivery systems in order to improve the intestinal absorption of PPDs [140-143]. Glycoprotein 2 (GP2) is a highly expressed transcytotic receptor found on the intestinal M cells [144]. FimH is an Escherichia coli-derived protein that showed GP2-targeting property. Fan et al. have developed a mucosal vaccine FimH-chitosan-pVP1 which exhibited great M cell-targeting capability, and this vaccine loaded FimH modified delivery system drastically uptakes by intestinal M cell, and promotes the dendritic cells maturation via TLR4-dependent signaling pathway [145]. Lectin has been reported as another ligand to target M cells [146]. Styrene maleic acid (SMA) nanomicelles were used to deliver epirubicin orally, which showed an increase of drug uptake without interrupting the intestinal membrane integrity. The SMA-micelles increased 2-fold drug accumulation in liver and spleen, and 6-fold and 15-fold higher accumulation in the lung and tumor, respectively. Additionally, SMA micelles showed colocalization with M cells and accumulation in Peyer's patches, which together confirms the M-cell mediated uptake and transport of SMA micelles [147]. Moreover, ulex europaeus agglutinin-1 (UEA-1) is a fucose-specific lectin with an affinity for glycoproteins presented on M cells. UEA-1 modified carrier systems have demonstrated preferable uptake by M cells in mouse model, but their targeting ability in human M cells has not been clarified in clinical trials [147, 148]. However, very limited number of ligands that could specifically bind to M cells were reported, especially to the transcytosis receptors. Enteroendocrine cell targeting Paneth cells usually assist in maintaining the microbiome and are located at the crypts of intestinal villi. They have a longer survival time (up to 60 days) compared with enterocytes [165], suggesting their potential of being a good target for drug delivery. Toll-like receptor 9 (TLR9), is found to be expressed in Paneth cells, it recognizes bacterial DNA consisting unmethylated cytidine-phosphate-guanosine (CpG) dinucleotides. A study has reported that the oral delivery of oligonucleotides consisting a CpG sequence (CpG-ODNs) led to Paneth cell degranulation [166]. Rumio et al. further studied the various TLRs presented on Paneth cells by orally delivering TLR agonists, and the results showed that the TLR3 agonist polyinosinic-polycytidylic acid also led to Paneth cell degranulation [166]. However, these receptors are located in the endosome but not cell surface. Moreover, there is no published research that has exploit PDDs delivery by targeting Paneth cell and therefore the associated mechanisms underlying Paneth cell function are still unknown. Enteroendocrine cells (EECs) are epithelial cells scattered throughout the whole GIT, which account for about 1% of the total intestinal cells [159]. EECs constitute the largest endocrine system in our bodies, with over twenty different hormones that are secreted from intestinal EECs. Gut hormones physiologically regulate multiple biological effects, including intestinal motility and forming physical barrier for drug permeation. The apical membrane of enteroendocrine L and K cells expresses several receptors called G protein-coupled receptors (GPCRs), such as GPR40, GPR41, GPR43, GPR119 and GPR120. These receptors could be bound by dietary ligands such as carbohydrates, proteins, and lipids. These nutrients often stimulate the receptors and lead to secretion of enteroendocrine hormones [160, 161]. So far, very limited studies have focused in EEC targeting in oral drug delivery. Nagatake et al. reported that EECs expressed a tight junction membrane protein, claudin-4 (Cld4). Orally administered luminal antigens targeting Cld4 were found to be taken up by Cld4+ cells, indicating that Cld4-mediated transport can be a potential pathway for targeting delivery of PPDs. In addition, it was found that orally administered luminal antigens were taken up by the Cld4+ EECs, raising the possibility that EECs may also play a role in initiation of mucosal immunity [162]. Shrestha et al. introduced a lipid-based nanoparticle which can act as endogenous ligands stimulating the release of GLP-1 via lipid-sensing pathways in enteroendocrine L cells [163]. This study demonstrated that great potential of L cell targeting for treating GI disorders. Enteroendocrine cell targeting Xu et al. have developed an innovative oral nanosystem to increase GLP-1 production and promote the oral absorption of peptides. The results showed the nanosystem triggered endogenous secretion of GLP-1 and increased its oral bioavailability by 4%. The nanosystem synergizes its own biological effect with the encapsulated peptide drug leading to a significant Goblet cell targeting Goblet cells make up to 16% of the total intestinal https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1434 they also function as receptors to deliver activating signals to DCs via stimulating intracellular signaling cascades [157]. Therefore, DCs targeting strategy is ideal for oral vaccine delivery. Ramakrishna et al. have successfully targeted the mannose receptor expressed on DCs using a fully human mannose receptor-specific antibody, B11, as a cargo to deliver human chorionic gonadotropin hormone. The results demonstrated B11 has great targeting capability toward DCs, and that mannose receptors and TLRs contribute towards activation and maturation of DCs by a mechanism that may be driven by a combination of peptide antigens and adjuvants [158]. improvement of insulin resistance and glucose tolerance. This formulation strategy represents a promising approach for oral PPDs delivery in incretin-based diabetes treatment [121]. Another study by Xu et al, the team has developed and compared different fatty acid-targeted nanocarriers and evaluated the L cell stimulation induced by the nanocarriers in vitro and in vivo. The results showed the DSPE-PEG2000 modified lipid-based nanocarriers had increased oral bioavailability of endogenous GLP-1 up to 8-fold in normoglycemic mice, and strengthened its biological effect [164]. Conclusions and future perspectives The development of successful treatments involves not only the discovery of new therapies but also their adequate delivery to their targets. In this context, oral delivery of PPDs remains being a highly challenging endeavor. If we take into account that the first attempt to administer insulin orally was carried out in the 1920s and that, so far, there are very limited marketed oral formulations containing such large molecules, this enterprise may seem rather disappointing (Table 1). Various approaches have been developed for oral delivery of PPDs, including chemical modification on PPDs, co-administration with absorption enhancers and utilization of drug carriers or medical devices. Drug delivery systems targeted to various intestinal cell types are one of the most exploited strategies in the oral PPDs delivery. Although oral PPDs formulation approaches confer some significant advantages, more research is required given that the transition of these approaches from the bench to the bedside is associated with many challenges. This is partly caused by the physicochemical properties of PPDs with complex intrinsic nature, which can even lead to immunogenic reactions, and partly by GIT barriers, which related to enzyme secretion and physiology that are unavoidable. As an intestinal immune adjuvant, most carbohydrates are widely found in plants, bacteria, and yeast. They are safe, low toxic, biodegradable, with high adjuvancy, thus, the carbohydrate-based adjuvants are promising candidates [170]. Among all the carbohydrate-based materials, β-glucan is an excellent biomaterial to be used as drug carrier system with M-cell targeting property, as well as acting as an immunomodulating agent. β-glucan particles are spherical empty and highly purified cell walls of Saccharomyces cerevisiae and can be loaded with many classes of PPDs [171]. In addition, β-glucans were predominantly observed in the Peyer’s patches of the small intestine, suggesting that β-glucans are mainly taken up by intestine through M cells, which demonstrated its intestinal M cell-targeting property. Moreover, it was found that the oral administrated β-glucans particles were initially taken up by intestinal M cells, subsequently taken up by macrophages. These macrophages had expressed the β-glucan receptor Dectin-1, suggesting that it was taken up into the macrophages by phagocytosis through this receptor [172]. These receptors recognize β-glucans particles and alert the host phagocytic immune cells and release of pro-inflammatory cytokines/chemokines. After maturation and migration of these antigen-presenting cells, T and B cell responses are also initiated [171, 173]. Biomaterial with intestinal immune modulating function The Peyer’s patches of the small intestine play a central role in the intestinal immune system. The M cells of the Peyer’s patches actively take up high-molecular weight components and are highly active in the process of phagocytosis and transcytosis. Thus, their main role is to take up luminal bacteria or large particles, transfer to DCs in the M-cell pocket for initiation of mucosal immune responses, and contribute to the homeostasis of the intestinal immune system [167]. Moreover, as one of the main characters of M cells is that very limited lysosomes are presented within the cells, which lead to very low lysosomal enzyme degradation to the transporting cargo. Thus, the peptide antigens or other intact particles taken up by M cells are more easily transferred directly to DCs in the M-cell pockets or underneath the M-cells, and https://www.thno.org 1435 Theranostics 2022, Vol. 12, Issue 3 subsequently triggers immune-related activities, and modulating immune responses [168]. C-type lectins, etc.), subsequently activate macrophages, DCs, NK cells, T lymphocytes or B lymphocytes, promoting the production of immune-related molecules, such as cytokines, antibodies, etc [169, 174]. Recently, polysaccharides have caught scientists’ attention, and many studies were employed polysaccharides as components of nanomaterials for modulation of the immune system. For example, mannan (α-MOS) can induce immune response by binding to CLRs (such as CD206) and TLRs. Haddadi et al. conjugated α-MOS with PLGA, the result showed the delivery system modified by α-MOS could promote phenotypic and functional maturation of DCs [169]. The new generation of polymeric biomaterials, which should be adaptive, complex, and intelligent. Thus, generally cover two main characteristics. First, biocompatible materials with controllable rigidity and functionality, forming polymeric biomaterials which can be widely used in oral drug delivery, tissue engineering, etc. Second, the biomaterials often “encoded” with information which could be read by proteins on intestinal cell surface, contributing significantly to intestinal cell-cell/cell-matrix communication. This information role of biomaterials can be demonstrated not only as intestinal cell targeting reagents but also as immune adjuvants in GIT [169]. Conclusions and future perspectives Ma X, Williams RO. Polymeric nanomedicines for poorly soluble drugs in oral delivery systems: an update. Int J Pharm Investig. 2018; 48: 61-75. 5. Aguzzi C, Cerezo P, Viseras C, Caramella C. Use of clays as drug delivery systems: possibilities and limitations. Appl Clay Sci. 2007; 36: 22-36. y y 6. Ritschel W. Microemulsions for improved peptide absorption from the gastrointestinal tract. 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Membrane mucins of the intestine at a glance. J Cell Sci. 2020; 133: jcs240929. j 11. Bansil R, Turner BS. The biology of mucus: composition, synthesis and organization. Adv Drug Deliv Rev. 2018; 124: 3-15. 12. Odenwald MA, Turner JR. The intestinal epithelial barrier: a therapeutic target? Nat Rev Gastroenterol Hepatol. 2017; 14: 9-21. y g p Lastly, even though multiple intestinal cells targeting delivery systems showed great potentials for oral delivery of PPDs, and several formulations are currently in advanced clinical trials, and disruptive novel technologies questioning previously established ideas have been proposed (Table 2). However, moving the applications from benchtop to bedside is still the biggest challenge, considering the cost and complexity of to accommodate the growing pool of PPDs. To help with the clinical transition of these approaches, standardization of preclinical parameters and procedures, integrative technology designs considering translational aspects, and knowledge sharing. Preclinical in vitro and in vivo studies could be performed under uniform conditions to enable accurate comparisons of various approaches. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (U20A20369, 81822043), the "Pearl River Talent Plan" Innovation and Entrepreneurship Team Project of Guangdong Province (2019ZT08Y464), the Shenzhen Science and Technology Program (KQTD20190929 173853397), the 67th batch funding from Postdoctoral Science Foundation of China (75110-41090012) and the Shenzhen Science and Technology Program (Grant No. GXWD20201231165807008). 23. Prado HJ, Matulewicz MC. Cationization of polysaccharides: a path to greener derivatives with many industrial applications. Eur Polym J. 2014; 52: 53-75. 24. 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Conclusions and future perspectives Therefore, β-glucan is a biomaterial not just suitable to fabricate drug carrier system, also has intestinal M cell-targeting capability as well as immune modulating property. Advanced delivery systems with novel biocompatible material and potential ligands, have demonstrated great potential in targeting different intestinal cells. However, limited numbers of receptors and ligands are available. In-depth understandings of the GIT biology in the molecular level are crucial for the discovery of new potential new receptor-ligand pairs. Based on the nature of disease and PPDs, single or combined receptor-ligand pairs could be used for intestinal cell targeting in future applications. Additionally, the cellular uptake pathways of oral drug delivery systems have not been comprehensively understood, and that poses gaps in Moreover, there are many other carbohydrate- based biomaterials, which have great intestinal bioadhesion, intestinal cell targeting properties, as well as intestinal immune modulating properties. There are many literatures reported that carbohydrates act as adjuvants through binding to specific innate immune receptors (e.g., TLRs, NOD2, https://www.thno.org Theranostics 2022, Vol. 12, Issue 3 1436 knowledge regarding the interaction of PPDs delivery systems with the GI barriers and the dynamics PPDs metabolism. Further, as in most of the studies described herein, sustainable and tunable drug release for PPDs is still a challenge. The development of novel biocompatible materials with stimuli- responsive ability could be a potential solution. As a crucial type of biomaterial, we consider carbohydrates not only as matter or a structural component but also as information or signaling molecules. Although most of the discussed applications are still far from clinical use, carbohydrates deserve to be developed into next-generation biomaterials for oral drug delivery systems with great potential. knowledge regarding the interaction of PPDs delivery systems with the GI barriers and the dynamics PPDs metabolism. Further, as in most of the studies described herein, sustainable and tunable drug release for PPDs is still a challenge. The development of novel biocompatible materials with stimuli- responsive ability could be a potential solution. As a crucial type of biomaterial, we consider carbohydrates not only as matter or a structural component but also as information or signaling molecules. 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https://openalex.org/W4379988157	https://digital.csic.es/bitstream/10261/330352/1/ijerph-20-06085-v2.pdf	English	null	NextGEM: Next-Generation Integrated Sensing and Analytical System for Monitoring and Assessing Radiofrequency Electromagnetic Field Exposure and Health	International journal of environmental research and public health/International journal of environmental research and public health	2,023	cc-by	12,414	Citation: Petroulakis, N.; Mattsson, M.-O.; Chatziadam, P.; Simko, M.; Gavrielides, A.; Yiorkas, A.M.; Zeni, O.; Scarﬁ, M.R.; Soudah, E.; Otin, R.; et al. NextGEM: Next-Generation Integrated Sensing and Analytical System for Monitoring and Assessing Radiofrequency Electromagnetic Field Exposure and Health. Int. J. Environ. Res. Public Health 2023, 20, 6085. https://doi.org/10.3390/ ijerph20126085 Academic Editors: Michele Guida, Martin Röösli and Paul B. Tchounwou Received: 2 March 2023 Revised: 11 April 2023 Accepted: 18 May 2023 Published: 8 June 2023 7 Sphynx Analytics Limited, Nicosia 2012, Cyprus 8 Research Group Smart Sensor Systems, The Hague University of Applied Sciences, 2628 AL Delft, The Netherlands 8 Research Group Smart Sensor Systems, The Hague University of Applied Sciences, lf h h l d Citation: Petroulakis, N.; Mattsson, M.-O.; Chatziadam, P.; Simko, M.; Gavrielides, A.; Yiorkas, A.M.; Zeni, O.; Scarﬁ, M.R.; Soudah, E.; Otin, R.; et al. NextGEM: Next-Generation Integrated Sensing and Analytical System for Monitoring and Assessing Radiofrequency Electromagnetic Field Exposure and Health. Int. J. Environ. Res. Public Health 2023, 20, 6085. International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health j p NextGEM: Next-Generation Integrated Sensing and Analytical System for Monitoring and Assessing Radiofrequency Electromagnetic Field Exposure and Health Nikolaos Petroulakis 1,* , Mats-Olof Mattsson 2,* , Panos Chatziadam 1 , Myrtill Simko 2 , Andreas Gavrielides 3 , Andrianos M. Yiorkas 3, Olga Zeni 4 , Maria Rosaria Scarﬁ4 , Eduardo Soudah 5 , Ruben Otin 5 , Fulvio Schettino 6 , Marco Donald Migliore 6 , Andreas Miaoudakis 7, George Spanoudakis 7 , John Bolte 8,9 , Erdal Korkmaz 8, Vasileios Theodorou 10 , Eleni Zarogianni 10 , Susanna Lagorio 11 , Mauro Biffoni 11 , Andrea Schiavoni 12 , Mauro Renato Boldi 12, Yuri Feldman 13 , Igal Bilik 13,14 , Anna Laromaine 15 , Martí Gich 15 , Marco Spirito 16, Maryse Ledent 17 , Seppe Segers 17 , Francisco Vargas 18, Loek Colussi 19, Mathieu Pruppers 9, Dan Baaken 20 and Anna Bogdanova 21 1 Institute of Computer Science, Foundation for Research and Technology-Hellas (FORTH-ICS), 70013 Heraklion, Greece 2 SciProof International AB, 83158 Ostersund, Sweden 3 2 SciProof International AB, 83158 Ostersund, Sweden 3 eBOS Technologies Limited, Nicosia 2322, Cyprus 4 Institute for Electromagnetic Sensing of the Environment, Consiglio Nazionale delle Ricerche (CNR-IREA), 80124 Napoli, Italy 5 International Centre for Numerical Methods in Engineering (CIMNE), 08034 Barcelona, Spain 6 6 Department of Electrical and Computer Science Engineering, University of Cassino and Southern Lazio, 03043 Cassino, Italy 7 https://doi.org/10.3390/ ijerph20126085 9 Centre for Sustainability, Environment and Health, National Institute for Public Health and the Environment (RIVM) 3720 BA Bil h Th N h l d 9 Centre for Sustainability, Environment and Health, National Institute for Public Health and the Environment (RIVM), 3720 BA Bilthoven, The Netherlands 10 Intracom Telecom, 19002 Peania, Greece 11 Italian National Institute of Health, 00161 Rome, Italy 11 Italian National Institute of Health, 00161 Rome, Italy 12 Telecom Italia Spa, 20123 Milan, Italy 13 Department of Applied Physics, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 14 14 Department of Electrical and Computer Engineering, Ben Gurion University of the Negev, 14 Department of Electrical and Computer Engineering, Ben Gurion University of the Negev, B Sh 8410501 I l 14 Department of Electrical and Computer Engineering, Ben Gurion University of the Negev, Beer Sheva 8410501, Israel 15 Institut de Ciència de Materials de Barcelona, Consejo Superior de Investigaciones Cientíﬁcas (ICMAB-CSIC), 08193 Barcelona, Spain 16 Department of Microelectronics, Delft University of Technology, 2628 CN Delft, The Netherlands 19 Dutch Authority for Digital Infrastructure, 9700 AL Groningen, The Netherlands 20 Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany Institute of Veterinary Physiology, University of Zurich, 80 21 Institute of Veterinary Physiology, University of Zurich, 8006 Zurich, Switzerland * Correspondence: npetro@ics.forth.gr (N.P.); mats-olof.mattsson@sciproof-international.se (M.-O.M.) Abstract: The evolution of emerging technologies that use Radio Frequency Electromagnetic Field (RF-EMF) has increased the interest of the scientiﬁc community and society regarding the possible adverse effects on human health and the environment. This article provides NextGEM’s vision to assure safety for EU citizens when employing existing and future EMF-based telecommunication technologies. This is accomplished by generating relevant knowledge that ascertains appropriate prevention and control/actuation actions regarding RF-EMF exposure in residential, public, and occupational settings. Fulﬁlling this vision, NextGEM commits to the need for a healthy living and working environment under safe RF-EMF exposure conditions that can be trusted by people and be in line with the regulations and laws developed by public authorities. NextGEM provides a framework for generating health-relevant scientiﬁc knowledge and data on new scenarios of exposure to RF-EMF in multiple frequency bands and developing and validating tools for evidence-based risk 1. Introduction While emerging technologies that use radiofrequency electromagnetic ﬁelds (RF-EMF, 100 kHz–300 GHz), particularly in telecommunications, are vital for modern life, there is an increasing consideration of the possible adverse effects on human health and the environment, which may be potentially exacerbated by aggregation of different types of RF-EMF signals. Some concerned citizen groups perceive ﬁfth-generation telecommuni- cation systems (5G; 5G New Radio; 5G NR) as a more signiﬁcant threat to public health than previous-generation systems. The exposure guidelines and standards issued by the International Commission for Non-Ionizing Radiation Protection (ICNIRP) and the Interna- tional Committee on Electromagnetic Safety of the Institution of Electrical and Electronic Engineers (ICES-IEEE) are set to prevent the occurrence of such adverse effects. These guidelines are based on comprehensive reviews of the relevant scientiﬁc literature, provid- ing exposure reference levels and basic restrictions for workers and the general population; the latter includes an additional safety factor to account for vulnerable groups. Regarding occupational exposures, the European Union (EU) follows the ICNIRP guidelines [1] (Di- rective 2013/35/EU [2]), which are in force in all Member States. As for exposure to the general public, the EU published a recommendation (1999/519/EC [3]) for exposures to EMF (0 Hz to 300 GHz), with limits derived from the ICNIRP 1998 guidelines [1]. However, due to the non-binding nature of the recommendation and the different types of EU legisla- tion [4], the related policies vary across European countries [5]. In addition, the exposure limits established by the ICNIRP are based on a thermal threshold. However, there is a debate about the adequacy of these limits and the need to include non-thermal effects for which the results are currently inconclusive. The limits are based on solid and conclusive evidence, while non-thermal effects are not conclusive and not widely accepted by the scientiﬁc community. Considering all the above, the adoption of new telecommunication technologies such as 5G requires extensive investigations regarding the potential causal effects between RF-EMF and health to address the following needs: • Updated appraisal of the scientiﬁc evidence regarding possible links between RF-EMF and health effects for (i) the general public, (ii) workers, and (iii) vulnerable groups to aid public authorities in implementing evidence-based policies, risk assessment, and communication. • Quality criteria, standards, and methodologies (i) to assess RF-EMF exposure and health effects, and (ii) for biological investigations fulﬁlling the 3R (Replace, Reduce, and Reﬁne) standards. 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/). https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2023, 20, 6085. https://doi.org/10.3390/ijerph20126085 Int. J. Environ. Res. Public Health 2023, 20, 6085 2 of 18 assessment. Finally, NextGEM’s Innovation and Knowledge Hub (NIKH) will offer a standardized way for European regulatory authorities and the scientiﬁc community to store and assess project outcomes and provide access to ﬁndable, accessible, interoperable, and reusable (FAIR) data. assessment. Finally, NextGEM’s Innovation and Knowledge Hub (NIKH) will offer a standardized way for European regulatory authorities and the scientiﬁc community to store and assess project outcomes and provide access to ﬁndable, accessible, interoperable, and reusable (FAIR) data. Keywords: radio frequency (RF); electromagnetic ﬁeld (EMF); communication engineering and systems telecommunications; biological effects; occupational health; public and environmental health 1. Introduction • Understanding interaction mechanisms between RF-EMF and biological systems, including combined exposures with other agents and multiple signals. • Adequate scientiﬁc communication to improve awareness among authorities, employ- ers, and citizens, counteracting RF-EMF misinformation. The NextGEM project will provide a framework (see Figure 1 for an overview of the NextGEM approach) for generating health-relevant scientiﬁc knowledge and data on new scenarios of exposure to RF-EMF in multiple frequency bands (“NextGEM: Next-Generation Integrated Sensing and Analytical System for Monitoring and Assessing Radiofrequency Electromagnetic Field Exposure and Health” is a 4-year research and innovation project (1 July 2022–30 June 2026 funded under Horizon Europe GA 101057527, www.nextgem.eu The NextGEM project will provide a framework (see Figure 1 for an overview of the NextGEM approach) for generating health-relevant scientiﬁc knowledge and data on new scenarios of exposure to RF-EMF in multiple frequency bands (“NextGEM: Next-Generation Integrated Sensing and Analytical System for Monitoring and Assessing Radiofrequency Electromagnetic Field Exposure and Health” is a 4-year research and innovation project (1 July 2022–30 June 2026 funded under Horizon Europe GA 101057527, www.nextgem.eu Int. J. Environ. Res. Public Health 2023, 20, 6085 3 of 18 3 of 18 (accessed on 1 March 2023)). A variety of experimental studies will be carried out at different levels of non-thermal exposures to assess the occurrence of potential effects of RF-EMF alone or in combination with other agents during short- and long-term exposures. Based on the literature reviews, including papers published by other consortia and the work carried out in the project, an exploratory risk quantiﬁcation, in the case of causal effects of RF-EMF alone or in co-exposure, will be performed. NextGEM will develop and validate tools for evidence-based risk assessment. NextGEM will also implement the Innovation and Knowledge Hub (NIKH) for RF-EMF and Health, offering a standardized way for European regulatory authorities and the scientiﬁc community to store and assess project outcomes and provide access to FAIR data. All the experimental results from NextGEM will be available in the knowledge base of NIKH platform that will be integrated in the NextGEM website along with scientiﬁc results (public deliverables, publications, guidelines, policy recommendations) and other project results such as brochures, ﬂyers, video clips etc. The ﬁnal goal is to promote a healthy living and working environment, especially for vulnerable groups, under safe RF-EMF exposure conditions, trusted by people, and in line with the regulations and laws issued by public authorities. 1. Introduction Carcinogenicity Multiple RF-EMF exposure 2G, 3G, 4G 5G Reproduction and developmental effects Cancer related effects Human effects Combined exposure (EMF with physical and chemical agents) Public Awareness Risk Assessment Causal Links EMF & Health EXPOSURES EFFECTS Biological effects Citizens Vulnerable Authorities Regulators STAKEHOLDERS Monitoring Monitoring Figure 1. NextGEM approach. The vision of the NextGEM project to assure safety for EU citizens when employing existing and future EMF-based telecommunication technologies is presented in this article and organized as follows: Section 2 focuses on the related activities and NextGEM’s advancement of existing knowledge. Next, Section 3 presents NextGEM’s methodology under speciﬁc directions. Section 4 presents the identiﬁed case studies to evaluate the investigated research and developed directions. Finally, Section 5 concludes this article. Carcinogenicity Multiple RF-EMF exposure 2G, 3G, 4G 5G Reproduction and developmental effects Cancer related effects Human effects Combined exposure (EMF with physical and chemical agents) Public Awareness Risk Assessment Causal Links EMF & Health EXPOSURES EFFECTS Biological effects Citizens Vulnerable Authorities Regulators STAKEHOLDERS Monitoring Monitoring Figure 1. NextGEM approach. Figure 1. NextGEM approach. The vision of the NextGEM project to assure safety for EU citizens when employing existing and future EMF-based telecommunication technologies is presented in this article and organized as follows: Section 2 focuses on the related activities and NextGEM’s advancement of existing knowledge. Next, Section 3 presents NextGEM’s methodology under speciﬁc directions. Section 4 presents the identiﬁed case studies to evaluate the investigated research and developed directions. Finally, Section 5 concludes this article. The vision of the NextGEM project to assure safety for EU citizens when employing existing and future EMF-based telecommunication technologies is presented in this article and organized as follows: Section 2 focuses on the related activities and NextGEM’s advancement of existing knowledge. Next, Section 3 presents NextGEM’s methodology under speciﬁc directions. Section 4 presents the identiﬁed case studies to evaluate the investigated research and developed directions. Finally, Section 5 concludes this article. 2.1. 5G Exposure Patterns 5G New Radio (NR) differs from earlier generations of wireless technologies (e.g., frame duration, bandwidth, and power allocation) in its use of massive multiple input multiple output (Ma-MIMO) beam-forming and in the use of millimeter waves (i.e., FR2) to support multiple usage scenarios [6,7]. The current deterministic approaches in exposure distribution for passive antennas used in earlier generations of mobile communication are different from the actual exposure to Ma-MIMO 5G antennas. Ma-MIMO antenna technology has introduced a degree of freedom in the management of exposure distribution. Based on that, the possibility to shape and direct emissions only in the direction where service is required allows optimization of the coverage and power usage with a consequent reduction of total exposure. Under these conditions, the exposure is present and distributed per the requested services. This makes power distribution estimation more challenging, necessitating creative developments of statistical features suitable for power distribution estimates in time and space. NextGEM accomplishes necessary and much sought-after improvements in ﬁeld-level assessment in real scenarios by developing a maximum power extrapolation technique and a suitable statistical model in both single user (SU) MIMO and multiple user (MU) MIMO cases. 2. Related Activities NextGEM performs several activities, including measurements and modeling of 5G exposure patterns (i.e., FR1: frequency band 1 (below 7.125 GHz); FR2: frequency band 2 (above 24.250 GHz)) with the respective RF-EMF modeling tools. In addition, the physics of the processes initiated by exposure will be studied and experimental in vitro and in vivo and human studies will be carried out. Thus, NextGEM evaluates possible causal risks by conducting umbrella reviews of published systematic reviews and meta-analyses and performing a health risk assessment. The ambition of NextGEM with regard to other related works is presented in the next subsections. 4 of 18 Int. J. Environ. Res. Public Health 2023, 20, 6085 2.2. RF-EMF Modelling Tools RF-EMF modelling tools are able to compute the ﬁelds generated by radiating sources; however, they may exhibit limitations in modeling capabilities, availability, and computa- tional performance. For instance, ﬁnite-difference time-domain (FDTD) tools require box- like discretization of the geometry, which can induce modeling errors on generally curved geometries. There are also techniques that allow the management of surfaces not aligned to the grid. Considering the changing exposure landscape, it is urgent to develop a framework of open-source tools to ﬁll the gaps not covered by the commercial software by adding new multi-physics models (high-performance computing (HPC)-platform-compatible) to improve accuracy and detail. NextGEM will develop next-generation, open-source computational tools for RF- EMF environmental levels and body-absorbed energy measurements. New numerical algorithms will be tested and validated to improve numerical efﬁciency and accuracy. New interfaces (multiple wave planes with human-body geometry and a full-wave EM solver) and capabilities (coupling EM with other physical models) will be developed, further advancing RF-EMF modeling in complex environments. These new algorithms will be implemented in ERMES [8]. ERMES is an open-source ﬁnite element code in the frequency domain that implements in C++ a simpliﬁed version of the weighted regularized Maxwell equation method. This ﬁnite element formulation produces well-conditioned matrices which can be solved efﬁciently with low-memory-consuming iterative methods. Furthermore, thanks to the null kernel of its differential operator, it can operate distinctly in quasi-static and high-frequency regimens. 2.4. In Vitro and In Vivo Investigations A large amount of in vitro and in vivo studies are available on the effects of 2G–4G RF-EMF [15,16], while 5G has not yet been investigated. In the current published literature, in most cases, no effect was detected, and when present, it was due to a temperature increase. However, some studies suggested that non-thermal exposure can affect a variety of biological functions, including the expression of several genes [15], although these ﬁndings have not been conﬁrmed in other independent investigations [17]. Overall, results are often contradictory, and well-performed mechanistic studies, using an intervention in biological processes investigating speciﬁc biochemical and molecular events and molecules, are needed [18]. The majority of the studies were performed using one single frequency and not considering co-exposures using different RF-EMFs or other agents, which could reveal possible synergistic, additive or protective effects of different factors. Furthermore, a substantial part of the studies suffers from severe quality deﬁciencies [16]; therefore, these studies have limited use for risk assessment. For a better understanding of the biological effects and molecular mechanisms of 3G, 4G, and 5G RF-EMF exposure in realistic scenarios, including combined exposure (with other agents) and multiple exposure (with different RF-EMF frequencies and signals), in vitro (cell lines and primary human cells), in vivo (C. elegans), and ex vivo (human RBC and lymphocytes) studies will be conducted based on harmonized exposure and experimental protocols between the different groups. Such studies focusing on complex exposure conditions have not been previously performed and go signiﬁcantly beyond the state of the art (SoTA). The focus is on effects related to carcinogenesis and reproductive toxicity and Hb conformation in human RBC. Different -omics approaches will also be used to identify genes and proteins as potential exposure marker candidates. Candidate genes identiﬁed will be further investigated in an ex vivo exposure study on lymphocytes and in an exploratory human study. The obtained results will be the foundation for hazard and risk assessments within the NextGEM project, allowing these activities to be performed more accurately. 2.3. Physics of the Processes Initiated by Exposure of Living Cells to RF-EMF Dielectric dispersion is a dissipative (i.e., thermal) phenomenon. However, the non- thermal interaction of RF-EMF with biological systems may be based on dielectric disper- sion as the permittivity decreases with increasing frequency [9]. For RF-EMF non-thermal effects of mobile communication carrier frequencies, the targets may include fast mobility of amino acids, lipids, and nucleotides, as well as proton hopping (3G/4G operation frequen- cies) or water (5G) [10]. As 3G–5G RF-EMFs have a minor effect on the dipole or charged groups’ mobility, it is possible that the envelope of the modulated carrier frequencies affects the cells. Speciﬁcally, in this case, the action of the signal is expected on the organelles, membranes, and nuclei due to the slow migration of ions or deformation of ion clouds at the membrane surfaces, or polarization of cellular membranes [11]. A progressive increase in carrier frequencies used for telecommunication results in the corresponding reduction of the penetration depth through the human body. Although human skin penetration Int. J. Environ. Res. Public Health 2023, 20, 6085 5 of 18 5 of 18 depth at 3G–5G operation frequencies (0.5–60 GHz) affects only the skin dermis layer [12], blood may respond to the RF-EMF when passing through the dermal microvasculature and thus transducing the signal to the peripheral tissues [13]. Indeed, previous studies indicate non-thermal effects of acute exposure to RF-EMF on erythroid precursors and red blood cells (RBCs) [14]. Signiﬁcant knowledge advancement regarding the possible impact of RF-EMF on (i) hemoglobin function, (ii) the state of hydration shells and free water, and (iii) membrane properties will be accomplished in NextGEM. Frequency depen- dencies, response kinetics, and duration, as well as tissue-speciﬁcity, will be studied. Such a mechanistic understanding is crucial to understanding if non-thermal RF-EMF effects are possible and, if so, under which circumstances. 2.6. Umbrella Review of Neoplasia Risks from RF-EMF Exposure in Humans The methodology of systematic reviews (SR) of environmental health topics, including the harmonization of the currently disparate approaches for assessing the quality and weight of evidence, is undergoing major improvements. Consensus-building efforts have recently led to the publication of a set of “Recommendations for the conduct of systematic reviews in toxicology and environmental health research (COSTER)” [22]. Several SRs of cancer hazards from exposure to RF-EMF are in progress, promoted by the WHO [23] and other entities [24], including a planned re-evaluation of the carcinogenicity of RF-EMF by the IARC [25]. However, no epidemiological study has investigated cancer risks and how they relate to individual integrated exposure from multiple RF-EMF sources. To provide the integrated exposure and carcinogenesis data for evidence-based risk assessment, NextGEM will perform umbrella reviews of SRs and meta-analyses of human observational studies of neoplasm risks from exposure to RF-EMF. Umbrella reviews can provide the highest quality of evidence if performed and interpreted correctly. This novel approach to RF-EMF health effects studies goes signiﬁcantly beyond the state of the art. The available SRs will be compared in terms of transparency, ﬁdelity to predeﬁned protocols, utility in answering the scientiﬁc question(s) of interest, and credibility of their ﬁndings, using reference standards and expert judgment. The utility and credibility of an SR depends on the adequacy of the tools used to assess the most likely sources of bias in a given body of evidence and on the soundness of the conceptual framework used to appraise and integrate the relevant evidence streams. NextGEM possesses the methodological and subject-matter expertise required for a critical summary of ﬁndings from SRs of epidemiological and experimental studies of the effect of RF-EMF exposure on cancer and reproductive outcomes and will focus on these hazards [26,27]. 2.7. Health Risk Assessment A risk assessment needs to include exposure assessment, hazard identiﬁcation, and char- acterization, including knowledge about the mode of action that underpins the risk as- sessment process. Although many institutions perform risk assessment [28], there is no standard procedure for evidence-based risk assessment. The European Commission sug- gests a “Weight of evidence” (WoE) approach [29] where evidence from epidemiological and experimental human in vivo, in vitro, and in silico studies are integrated for an overall risk assessment. Challenges for RF-EMF risk assessment include that risk assessment generally takes exposure guidelines into account, where primarily acute effects are the only ones considered. Risk assessment for exposure situations characterized by long-term and combined exposures and/or co-exposures with other agents do not exist. For the ﬁrst time, integrated risk assessment protocols will be provided, including exposure situations characterized by long-term exposures, combined exposures to different RF-EMFs, and co-exposures with other agents. NextGEM will ensure this by pronounced stakeholder involvement, integrating novel exposure and dosimetry methodologies for RF-EMF exposure assessment, and identifying health-relevant markers in high-quality experimental bio-effects studies. A unique risk assessment tool will be developed in collaboration with and for use by different stakeholders and validated in case studies. 2.5. Human Studies The micronucleus (MN) test can investigate human genetic damages on, e.g., exfoliated buccal cells. Several studies applied this technique to explore the potential adverse effects of mobile phone RF-EMF, but the results were contradictory [19,20]. Most of the studies have several shortcomings that preclude ﬁrm conclusions [21]. Furthermore, the lack of knowledge about the characteristics of MN and other nuclear abnormalities in oral cells also limits the interpretation of the results. Results bringing the knowledge signiﬁcantly beyond SoTA regarding genetic damage due to RF-EMF exposure in humans will be obtained from studies on MN formation and gene expression in exfoliated buccal cells from heavy and light mobile phone users. Different techniques will be applied for the deﬁnition of both groups to prevent the limitations identiﬁed in previous studies as much as possible. A pilot study on healthy volunteers will also be conducted (double-blind) after exposure to 5G carrier frequencies and modulation envelopes to detect potential acute effects on physiological cell parameters in human blood cells (RBC, lymphocytes). In this pilot study, a container set up to reduce the levels of environmental electromagnetic ﬁelds (from the Int. J. Environ. Res. Public Health 2023, 20, 6085 6 of 18 6 of 18 building and outside) located in ISSeP-Liège (Belgium) will be used where on the right side of the container is the test room for the volunteers and on the left side is the technical equipment, including the test instruments and the computer that manages the exposures. 2.6. Umbrella Review of Neoplasia Risks from RF-EMF Exposure in Human 3. Methodology 3.1. The NextGEM Project Methodological Approach 3.1. The NextGEM Project Methodological Approach The NextGEM framework can efﬁciently integrate all technical enablers of the project and encompass real-time monitoring and historical and experimental data. • Sensing and data source: Data for RF-EMF exposure assessment will be collected from the literature and/or experimentally measured in different real-life scenarios • Sensing and data source: Data for RF-EMF exposure assessment will be collected from the literature and/or experimentally measured in different real-life scenarios Int. J. Environ. Res. Public Health 2023, 20, 6085 7 of 18 7 of 18 through the case studies conceived and developed during the project. As regards the new generation communication networks, novel measurement paradigms aim to be developed together with wearable sensing devices suitable for the 5G mm wave band. through the case studies conceived and developed during the project. As regards the new generation communication networks, novel measurement paradigms aim to be developed together with wearable sensing devices suitable for the 5G mm wave band. • Analytics and experimentation: The combination of multiple approaches from real- life case scenarios, exposure assessment, and umbrella reviews of epidemiological data in combination with in vitro, ex vivo, and in vivo experiments elucidates possible biological and health effects of RF-EMF interactions and their underlying mechanisms. g y g • Applications, tools, and services: NextGEM will provide novel exposure assessment devices and protocols as well as risk assessment tools, integrated into the NIKH, representing the main key exploitable outcomes. • Applications, tools, and services: NextGEM will provide novel exposure assessment devices and protocols as well as risk assessment tools, integrated into the NIKH, representing the main key exploitable outcomes. 3.2. RF-EMF Exposure Assessment Modeling and Measurements Public Health 2023, 20, 6085 8 of 18 8 of 18 average power pattern (NAPP), which seems particularly suitable to compare different antenna technologies and scenarios, and is easy to integrate with stochastic models of user distributions, as well as with ray-tracing algorithms. Channel power measurements will be performed with a spectrum analyzer, whereas measurements in the code domain will be performed with a signal analyzer with 5G NR decoding capabilities. Both directive and omnidirectional antennas will be used. Both line of sight (LOS) and non-LOS will be tested with one or more user devices. RF-EMF exposure paradigms and self-monitoring: 5G NR technology makes inten- sive use of MIMO technologies, and the user’s exposure is related to the EM coupling in the body. For these reasons, a body-worn portable exposure meter can mimic real scenarios obtaining reliable data to monitor RF-EMF exposure. The NextGEM project will develop multiple body-wearable portable exposure meters to be employed in real-life use-case sce- narios. To achieve this novel wearable sensing device, the current sensing node, operating at 27.5 GHz, will be redesigned using planar antennas with plastic lenses (easier to integrate into a wearable device), providing higher spatial selectivity. The newly developed sensor will be tested in the ADome TU Delft characterization facility, allowing the identiﬁcation of the critical electrical speciﬁcation of the sensor and adequate design of the sensor-carrying vest with full spatial coverage. The single sensors and the vest’s ﬁrst prototypes will be tested in the TU Delft Green Village. In this campus location, 5G FR2 bands are licensed to the university for real use-case testing scenarios. The devices and the vest will then be fully characterized inside this test location with known excitation signals for being employed in the measurement campaign. 3.2. RF-EMF Exposure Assessment Modeling and Measurements The RF-EMF exposure assessment includes both modeling and measurements of the experimental setup (dosimetry), RF-EMF levels inside and outside the human body, and environmental RF-EMF levels in indoor and outdoor scenarios (Figure 2). Simulations FR1 & FR2 Measurements FR1 & FR2 Body Inside & Outside the body Dosimetry during bio experiments Dosimetry for bio experiments Environment Bio Experiments Outside the body Indoor & Outdoor Field in Scenarios Indoor & Outdoor Field in Scenarios Exposure Distribution & Statistics Figure 2. Multi-scale approach to RF-EMF exposure assessment. Inside & Outside the body Bio Experiments Figure 2. Multi-scale approach to RF-EMF exposure assessment. Multi-Scale Model-Based Assessment of RF-EMF Exposure in the Human Body: The scope of this activity is a multiscale approach to RF-EMF exposure assessment by simulation and/or measurements from the small (cells and small organisms) to medium (human body) and large scale (cities, buildings). Initially, the unperturbed RF-EMF distri- bution generated by the radiating source will be calculated (or measured) at every point of interest in the indoor/outdoor environment (city, workplace, school, home). This informa- tion will be used as input to the RF-EMF software tool for the exposure conditions in the experiments to calculate the ﬁelds outside/inside a human body. The various simulations and experiments will take place in parallel and will be used bidirectionally to improve computational models and experimental conclusions to compare the results. The RF-EMF software will be used with exposure source properties, the radiated region’s CAD model, the human body’s electromagnetic properties, and the human body’s CAD model. Exposure Compliance Assessment and Environmental Protection: RF-EMF expo- sure assessment is particularly challenging in 5G due to its high ﬂexibility with respect to the antenna technologies employed, such as beamforming capabilities and/or different MIMO implementations, as well as its sophisticated energy-efﬁcient signaling structure. NextGEM will develop novel measurement techniques for different antenna technologies (such as continuous beamforming, a grid of beams, SU-MIMO, MU-MIMO, and Ma-MIMO, based on maximum power extrapolation (MPE)). MPE techniques have been proposed and successfully applied in past-generation cellular systems but require a generalization from a “passive” determination of what is on air in a certain moment to an “active” measurement forcing the system under test to assume the most suitable conﬁguration. In NextGEM, novel statistical models will be developed for realistic exposure assessment in both indoor and outdoor scenarios measurements. A possible approach [30] relies on the normalized Int. J. Environ. Res. 3.3. Experimental Studies Genomic instability will be evaluated by micronucleus (MN) and Comet assays. Transcriptomics and epigenetic tests will be performed on selected conditions based on the outcomes of the above-mentioned tests. Two conditions will be investigated on human neuroblastoma cells (apoptosis, cell cycle progression) to test multiple RF-EMF signals: (a) 1800 MHz GSM, 1950 MHz UMTS, and 2.4 GHz WiFi signals and (b) 3.5 GHz and 2.4 GHz WiFi. To address the effect of combined exposures, 4G exposure will be performed in combination with menadione (human neuroblastoma cells) and 5G exposure in combination with UV-B light (human keratinocytes). Biochemical and Biophysical mechanisms of EMF responses Cancer and reproduction-related endpoints In vitro experiments on Neuroblastoma cells and keratinocytes Cytogenetic tests, gene expression (Omics), epigenetic tests Ex vivo experiments on Lymphocytes from human donors Humans Analyses of the expression of genes of interest by RT-qPCR in buccal cells from heavy/light mobile phone users Identification of genes of interest Modelling: type of signal, exposure duration Identification of exposure condition of interest relating to the mechanisms, if any In vitro experiments on RBC/lymphocytes Reproduction: in vivo on C. elegans Humans: Analyses of RBC of people exposed to the condition of interest Water, amino acids and protein (Hb) interactions Experimental complexity Exposure systems set up and dosimetry : 3,5 GHz and 26 GHz - Co-exposure with other EMF signals, chemicals, physical agents In vivo experiments on C. elegans In vivo experiments on C. elegans H2O Figure 3. Overall strategy for biological investigations. Cytogenetic tests, gene expression (Omics), epigenetic tests Humans Analyses of the expression of genes of interest by RT-qPCR in buccal cells from heavy/light mobile phone users Figure 3. Overall strategy for biological investigations. In vivo studies on C. elegans: C. elegans will be employed as an in vivo simple model to evaluate cancer-related endpoints and perform genetic screening. Furthermore, repro- duction in the parental and second generation will be also assayed. The exposure system is similar to the one adopted for in vitro studies. Oxidative stress and genetic screening will be evaluated on wild-type C. elegans (N2) in vitro with ﬂuorescent markers. For reproduction assays, synchronized nematodes will be exposed at different developmental stages—eggs and L1 to L4 larval stages. Growth, egg and organism development, and motility parame- ters will be assessed by imaging processing. Gene expression will be assayed as in in vitro and ex vivo experiments. 3.3. Experimental Studies NextGEM aims to collect robust and reliable data by combining different experimental approaches: in vitro studies on human cell lines, in vivo studies on the model organism Caenorhabditis elegans (C. elegans), and ex vivo exposure studies on human cells (lym- phocytes). Certain in vitro studies will be carried out at more than one lab for replication purposes. Cell models used for experimental studies have been chosen because of the existence of standardized and widely recognized assays (buccal epithelium cells, lym- phocytes, neuroblastoma cells), appropriateness for studies of the poorly penetrating 5G signals (keratinocytes), and relative simplicity in the organization and lack of morpholog- ical variation (RBC), as can be seen in Figure 3. The experimental studies in NextGEM focus on cancer-related endpoints, reproduction and development, and ﬁnally on different biophysical and biochemical mechanisms, as will be described below. All experiments are going to be performed taking into consideration environmental factors such as noise, vibrations, and background magnetic ﬁelds and will eliminate these factors. In vitro studies on mammalian cell lines: Human neuroblastoma and keratinocyte cell models will be used to investigate the effect of exposure to 4G and 5G signals, given alone, as multiple frequencies, and combined with other agents, to evaluate cancer-related endpoints. Numerical and experimental dosimetry techniques will be employed to design and characterize new exposure systems and/or optimize existing ones and to calculate the dosimetry parameters (speciﬁc absorption rate (SAR), electric ﬁeld, power density) in the sample. The design of the applicator device will be driven by efﬁciency and SAR uniformity criteria, ensuring the control of environmental (temperature, humidity, CO2) parameters according to the recommendations for good laboratory practice (GLP). For dose homogeneity (numerically evaluated through the variation coefﬁcient, i.e., the ratio between standard deviation and average SAR), different solutions have to be adopted in the sub- mmWave band [31] and in the lower frequency band [32]. Several solutions (waveguides, transverse EM cell, radial transmission line, wire patch cell, reverberating chambers, etc.) will be considered. Short (1 to 24 h) and long (3 weeks) exposure durations will be applied, according to the biological systems used and the endpoints measured, at different SAR values, to investigate the effect of 4G (1950 MHz) and 5G (3.5–26.5 GHz) signals. Oxidative stress, apoptosis, and cell cycle progression will be analyzed (ﬂow cytometry, Int. J. Environ. Res. Public Health 2023, 20, 6085 9 of 18 9 of 18 molecular biology techniques). 3.3. Experimental Studies Ex vivo studies on lymphocytes from human donors: Lymphocytes from human donors will be exposed to 5G signals (3.5 GHz), alone and in combination with other agents, to analyze the expression of a selection of cancer-related genes. The exposure system and dosimetry is similar to the one adopted for in vitro studies on keratinocytes. Ex vivo exposure will be performed on lymphocytes collected from healthy human donors, with the same exposure conditions adopted for the short-term in vitro studies. Combined ex- posures will be carried out on lymphocytes with ethyl methanesulfonate (EMS)/menadione genotoxicants. The comet assay will be applied to evaluate the induction of DNA damage. Gene expression and epigenetic tests will be applied to selected conditions based on the Comet assay results. Biological effects of RF-EMF exposure in humans: Buccal cells from self-reported heavy and light mobile phone users will be used to investigate the expression of genes identiﬁed in in vitro and ex vivo studies. Self-reported mobile phone users will be selected Int. J. Environ. Res. Public Health 2023, 20, 6085 10 of 18 10 of 18 based on the amount of time calling with the mobile phone at the ear level. Information will be obtained related, e.g., to the type of phone used in the last 10 years and the behavior when calling, to estimate SAR values at the inner cheek level (dosimetry modeling). Moreover, actual measurements are envisaged to conﬁrm the classiﬁcation in both groups. Buccal cells will be taken from heavy and light mobile phone users following established methods for cell collection [33]. Reverse transcription-quantitative polymerase chain reaction (RT- qPCR) will be performed on a selection of genes, as deﬁned in in vitro studies with the genomics tool. g Effects of RF-EMF exposure on human RBCs: In a ﬁrst study, blood samples will be taken from healthy volunteers following exposures based on the conditions deﬁned elsewhere in the project (5G carrier frequencies (3G/4G/5G) and modulation envelope). The system will be an evaluation board based on software deﬁned radio (SDR) technology with a maximum frequency of 6 GHz and a minimum quadrature sampling of 8 bits. The output frequency of the multiplier will be emitted in the FR2 band with a modulation scheme as deﬁned in an earlier task. The available output emission will be ampliﬁed via a high-frequency ampliﬁer and connected to a directional antenna whose gain will be set according to the required level. 3.3. Experimental Studies A second SDR will be used in reception to ensure the monitoring of the experiment. In a second exploratory study, healthy volunteers (15 males and 15 females aged 18–25 years) will participate in two double-blind sessions, one real and one sham (random) exposure (45 min). In samples taken after exposure, blood count, RBC indices, redox state, and NO level, CO-oximetry, RBC aggregability and morphology will be assessed. Biophysical and Biochemical mechanisms of RF-EMF exposure in blood and RBC: The RF-EMF (4G and 5G) interaction with human RBCs will be studied, in which the possi- ble molecular, sub-cellular/cellular targets of the exposure, and the RF-EMF parameters (repetition frequency, slopes, on-off cycles, etc.) will be identiﬁed. A vector signal generator interconnected to an exposure setup equipped with a temperature controller and pumps to introduce ﬂow conditions will be used to expose RBC suspensions. A microﬂuidic unit will be developed by using a radiofrequency generator and a directional plasmonic phase modulator adapted to 26 GHz. Both systems will allow multiple exposures to RF-EMF signals. A special system will be developed by combining a TEM line for the RF-EMF exposure with a high-performance network analyzer for real-time assessment of dielectric parameters. The responses of cellular hemoglobin (Hb) and membranes will be evaluated using dielectric spectroscopy to monitor changes in protein structure and its surface charge through the changes in free and bound water dynamics. Aggregability, deformability, membrane stability, hydration, and cell morphology will be analyzed using conventional techniques (sedimentation rate, microscopy, the changes in RBC indices) and the methodology available at the partners’ sites (ektacytometry, microﬂuidic approaches, ﬂow cytometry). 3.4. Causal Links between RF-EMF Exposure Level and Duration and Potential Health Effects The methods for evaluating and integrating the evidence on environment and health topics are transitioning from an “expert-based narrative” to a “systematic” review. SRs of aetiological studies are regarded as the best-practice method for evaluating evidence used in decision-making [34]. Complexity is an inherent feature of environmental health hazard and risk assessments, and methodological advances in all evidence streams relevant to hazard/risk assessments (toxicology, epidemiology, and exposure science) are increasing the challenges for evidence assessment and integration. Umbrella review of human cancer hazards from exposure to near-ﬁeld and far-ﬁeld sources of RF-EMF: WHO is undertaking an updated assessment of health hazards from exposure to RF-EMF, including 10 systematic reviews of experimental and observational studies on six major topics (cancer, adverse reproductive outcomes, cognitive impairment, symptoms, oxidative stress, and heat-related effects) [35]. There have already been pub- lished detailed systematic review protocols for the effect of RF-EMF on different topics, Int. J. Environ. Res. Public Health 2023, 20, 6085 11 of 18 11 of 18 such as [36,37], which will be considered in NextGEM for setting out research questions and methodology. Cancer is the most relevant potential hazard of RF-EMF at exposure levels below current international guidelines. If RF-EMF exposure increased the risk of cancer, this would have serious public health consequences and require population-level preven- tive strategies, including a revision of the threshold-based limitation principle currently applied to non-ionizing exposure in the radiofrequency range [38]. Neoplastic diseases are also the most investigated potential adverse effect of prolonged exposure to RF-EMF from mobile communication, accounting for about 56% of all related epidemiological studies (n = 327) up to 2021, as indexed in the specialized literature database EMF-Portal (https://www.EMF-portal.org/en (accessed on 1 March 2023)). NextGEM’s consortium includes cutting-edge methodological and subject-matter expertise in umbrella reviews and meta-analyses required for a critical summary of ﬁndings from SRs of epidemiological studies on the potential carcinogenicity of RF-EMF [23], and will focus on this outcome. NextGEM will compile the available evidence on RF-EMF and cancer risk in humans by performing an umbrella review of relevant narrative or quantitative reviews published in the last decade. Umbrella reviews, deﬁned as the systematic collection and evaluation of information taken from multiple systematic reviews and meta-analyses, have the potential to provide the highest quality of evidence, graded according to the criteria described by Papatheodorou [39]. 3.4. Causal Links between RF-EMF Exposure Level and Duration and Potential Health Effects Development and validation of a Risk Assessment model to assist evidence- informed decision making on RF-EMF exposure: The basic existing assessments of risks associated with RF-EMF exposure need to be improved, and this is a capital objective of NextGEM. To this aim, relevant data from an exposure assessment, in vitro and in vivo experiments, the level of evidence resulting from the umbrella reviews of human observational studies, the evidence pertaining to experimental studies of carcinogenic effects of RF-EMF in animal and cells models, and the results of the human observational studies conducted within NextGEM will be integrated. Common exposure protocols will be deﬁned at the early stages of this project to ensure the harmonized data collection required for a robust risk assessment. Based on stakeholder (industry, insurers, regulators, and consumers) input, require- ments for output information criteria will be identiﬁed. Data underpinning the different stages of risk assessment from different lines of evidence will be obtained from activities and assessed for their appropriateness for hazard identiﬁcation and characterization, expo- sure assessment, risk characterization, and uncertainty analysis. The ﬁnal product is an evidence-based integrative risk assessment. Any lack or shortcoming of data will feed back to activities for completion. The models and output parameters identiﬁed in this task will be submitted to a dedicated case study for the ﬁrst round of sensitivity and performance testing, which, after reﬁnement, will be iterated to involve all case studies. 3.5. Development of NextGEM Innovation and Knowledge Hub The implementation of NextGEM Innovation and Knowledge Hub (NIKH) is a practi- cal realization and integration of scientiﬁc components. It will monitor, store, share and access EMF exposure and biological data to ensure compliance with safety standards, minimize exposure levels in set environments and contexts, and increase citizens’ aware- ness on EMF information and research [40]. To enable the validation and exploitation, a benchmarked proof-of-concept reference platform will be developed. The development of NIKH includes four different stages: (i) designing the platform to store the innovations and research outputs produced within NextGEM, (ii) including external scientiﬁc knowledge from previous research or through synergies with projects funded under other clusters and pillars of Horizon Europe, or other EU programs, (iii) offering a link to RF-EMF stakehold- ers, and (iv) enabling security, trustworthiness, and General Data Protection Regulation (GDPR) compliance (Figure 4). 12 of 18 Int. J. Environ. Res. Public Health 2023, 20, 6085 Public authorities, regulators, policymakers, government bodies, ministries, environmental bodies, legal bodies, citizen groups, workers, communities NextGEM Innovation & Knowledge Hub News Awareness Results In vitro Heath Risk Assessment Systematic Reviews Scientific Knowledge External Knowledge NextGEM Innovation EMF-Portal.org Decision making Causal Link EMF and Health? Scientific Data EMF Data Bio Data Analysis Modeling Security, trustworthiness and GDPR compliance Data transfer Data Processing Data storage Data Management Stakeholders Knowledge Base & Observatory Analytics and Experimentation Processes Bio and Data Ethics Applications tools and services Data Ingestion Orchestration & Management Application Portal Mobile Application Dashboard Health Risk Assessment Tool Experimental Results Ex-vivo Human Studies In vivo EU Health Data Space Other HE Projects Epimiological Studies Umbrella Reviews Risk Assessment Models Environmental Health Assessment Policy recommendations Practical Guidelines Figure 4. NextGEM innovation and knowledge hub (NIKH). Stakeholders Figure 4. NextGEM innovation and knowledge hub (NIKH). Platform design for collecting research outputs: The NIKH will coordinate the pro- cesses for translating analytics and experimentation model descriptions to deployed jobs over the end-to-end system from data management and analytics components all the way to end-user applications and services. NIKH aims to offer template services, embodying NextGEM’s logic for diverse research scenarios, managing execution planning (explicit and automatically decided), and handling interactions between NextGEM components while conﬁguring their seamless integration. g g g Collection and storing of data within NextGEM and from other projects, activities, and studies: NextGEM will provide a vast amount of research outputs. 4. Case Studies NextGEM results will be applied and validated through three case studies, covering different groups of geographical and socio-economic conditions, as well as vulnerable people exposed to different signals (i.e., multiple RF-EMF exposure, including 4G and 5G signals, focused on FR1 or FR2): (1) potential effects of indoor levels of RF-EMF exposure of vulnerable people on reproduction and development, (2) optimised outdoor urban planning and 5G design architecture and investigations for public awareness of cancer-related health hazards, and (3) bealth effects of exposure to RF-EMF in indoor and outdoor environments. 4.1. Case Study 1—Potential Effects of Indoor Levels of RF-EMF Exposure of Vulnerable People on Reproduction and Development The COVID-19 pandemic has increased the general public’s indoor exposure to RF- EMF radiated by multiple sources, such as wireless personal communication devices (Wi-Fi, Bluetooth) or other applications (security scanners, smart meters, and medical equipment) in conjunction with ambient environmental exposure. The effect of RF-EMF exposure on the health of vulnerable people, such as pregnant women or children, has not been studied sufﬁciently. In addition, the potential exposure to RF-EMF and other physical or chemical agents has increased concerns regarding fertility and children’s development. The main scope of NextGEM in Case Study 1 (Figure 5) is to investigate potential RF-EMF effects on vulnerable people by analyzing the existing literature and developing an exposure modeling approach to establish possible thresholds for safe/unsafe situations of single and multiple exposures and the potential risk on fertility and children’s development. C. elegans as model organism Acute/Chronic Exposure C. elegans Computational Model Eggs, L1 to L4 (72h life cycle) and F2 generation DNA Damage Numerical Simulation Simulated internal field levels and distribution Correlation Computational model of pregnancy dosimetry (incl. mother, fetus, child) Level of exposeure (threshold level) loading channel control channels phenotyping fine neuronal pattern Optical modalities: high-magnification, fluorescence Figure 5. Case Study 1—Potential effects of indoor levels of RF-EMF exposure of vulnerable people on reproduction and development. Figure 5. Case Study 1—Potential effects of indoor levels of RF-EMF exposure of vulnerable people on reproduction and development. To this aim, the review of scientiﬁc evidence on RF-EMF and adverse reproductive outcomes will be undertaken. Appraisal of the evidence regarding potential hazards of RF-EMF exposure for pregnant women and children (health agencies, risk assessors) is an important and often overlooked issue for the community. 3.5. Development of NextGEM Innovation and Knowledge Hub The NextGEM workﬂow includes setting up the RF-EMF data platform, integrating existing data from available public and reviewed sources, and integrating new EMF-relevant data generated within the project. Thus, the stored data will be accessible from the NextGEM dashboard as soon as the primary obligations related to scientiﬁc publishing, etc., are fulﬁlled. NextGEM will furthermore make the data collection accessible and expandable for users after project end, which needs a long-term commitment regarding database management. Connection with the RF-EMF Stakeholders: In the multimodal environment, NIKH will provide a fully conﬁgurable NextGEM dashboard for public authorities, the scientiﬁc community, and various citizen and societal groups. A graphical web-based front-end dash- board will be developed to facilitate the visualization of data from RF-EMF measurements, analytical models, bio-experiment results, and risk assessment together with practical guidelines, tools, and applications, thus supporting public authorities and regulators with scientiﬁc evidence to implement exposure directives and improve their RA, management and communication. Security, trustworthiness and GDPR compliance: To assure NextGEM operation from a cybersecurity perspective, as well as to provide evidence for its GDPR compli- ance, the SANL Security Assurance Platform (SAP) will be employed [41]. Following a model-driven approach, the SAP will be tailored to the NIKH. The real-time operational compliance of the platform to security, privacy, and GDPR requirements will be provided in an evidence-based manner through the integration of the SAP, which will enable hybrid assessments based on vulnerability analysis, penetration testing, and continuous monitor- ing of the NextGEM assets. This establishment and operation of continuous security and privacy assurance checks for the NIKH and its security and privacy control mechanisms will ensure the security and privacy posture of it. 13 of 18 Int. J. Environ. Res. Public Health 2023, 20, 6085 13 of 18 4. Case Studies Therefore, the ﬁeld distribution will be simulated numerically inside the body of fetuses, pregnant women, and children (ERMES software). In addition, C. elegans will be evaluated as a simple in vivo organism fulﬁlling 3R requirements. The collection of electromagnetic property values of C. elegans tissues and their growth medium will be obtained from own measurements and the literature. The ex- posure of C. elegans to different RF-EMF frequencies and modulation patterns, along with simultaneous numerical simulations, will be carried out to obtain the ﬁeld distribution in critical parts of the body (eggs, reproductive system). The potential cross-generational health effects on reproduction and development will be investigated experimentally during the life cycle of C. elegans after RF-EMF exposure. The results of both numerical simulations and C. elegans experiments performed will be evaluated separately and fed into the models. Int. J. Environ. Res. Public Health 2023, 20, 6085 14 of 18 14 of 18 4.2. Case Study 2—Optimised Outdoor Urban Planning and 5G Design Architecture and Investigations for Public Awareness on Cancer-Related Health Hazards 4.2. Case Study 2—Optimised Outdoor Urban Planning and 5G Design Architecture and Investigations for Public Awareness on Cancer-Related Health Hazards 5G is considered the key technology for enabling a wide range of application scenarios and the effective spreading of the smart city concept. In total, 50 billion devices are foreseen in such a scenario by 2023, with a connection density of 1 million/km2 (https://www. accenture.com/_acnmedia/PDF-146/Accenture-5G-WP-US.pdf (accessed on 1 March 2023) spread over different outdoor and indoor locations, from underground to sky coverage for unmanned aerial vehicle (UAV) applications. Moreover, the use of Ma-MIMO antennas and higher frequency bands has completely changed the network’s coverage and management requirements. RF-EMF will only be present when and where it is needed. In parallel to measurements, computations will be performed on simulated and real scenarios to analyze the effect of Ma-MIMO antennas and transmitter positions on ﬁeld distribution. However, there are concerns that this technology increases the risks of carcinogenesis. The scope of this case study (Figure 6) is to assess the urban planning and exposure management for 5G NR location design architecture and to examine the possibility of cell site distribution in an urban environment to optimize the exposure and to analyze ﬁeld distribution over the territory due to Ma-MIMO antennas. 4. Case Studies Definition of realistic exposure conditions Input/ Advice to Guidelines Regulatory and Standardiz ation Bodies Cancer-related responses (with / without combined exposures) Systematic reviews on cancer Computations performed on simulated and real scenarios Elevation Beamforming Azimuth Beamforming Figure 6. Case Study 2—Optimized outdoor urban planning and 5G design architecture and investi- gations for public awareness on cancer-related health hazards. Figure 6. Case Study 2—Optimized outdoor urban planning and 5G design architecture and investi- gations for public awareness on cancer-related health hazards. Real standard measurement periods, in compliance with ICNIRP guidelines, and long-term (hours to days) acquisitions will be carried out to evaluate 5G RF-EMF exposure by resorting to maximum power extrapolation (MPE) techniques applied to the ﬁeld level measured in real trafﬁc and environmental conditions, both in line-of-sight and non-line- of-sight conditions. Statistical models will be developed and validated in real scenarios to consider such variability in time and space of the distribution, coverage, and design antenna mapping. In this respect, the measurement procedures for coverage quality evaluation and exposure assessment need to be rethought and reviewed to implement effective and efﬁcient measurement methodologies for validating developed models in optimal planning. Urban planning, based on ray-tracing methodologies, will be performed on simulated and real scenarios to analyze transmitter positioning on ﬁeld distribution and levels. The RF- EMF ﬁeld distribution over scenarios due to the new technologies (e.g., 5G and Ma-MIMO) and such statistical models will be evaluated in terms of the exposure characterization for different purposes, such as new methodologies for exposure assessment. p p g p In addition, biological investigations will be carried out in vitro (neuroblastoma cells and human keratinocytes) and ex vivo (human lymphocytes) to gain information on potential health risks under realistic exposure conditions in 5G frequencies as used in existing 5G design architectures. Selected realistic conditions will be tested to investigate biological responses in the presence and absence of other agents (combined exposures). An umbrella review of epidemiological studies of cancer risks in relation to near-ﬁeld and far-ﬁeld RF-EMF exposure and risk assessment approaches on cancer will be performed. 4.3. Case Study 3—Health Effects of Exposure to RF-EMF in Indoor and Outdoor Environments The 5G system is expected to enable smart industries by providing massive and reliable high-bandwidth communication links and creating high-bandwidth campus environments supporting high data rate connectivity. 5G wireless networks will employ higher frequency bands to support such connectivity demands. In such scenarios, the RF-EMF exposure Int. J. Environ. Res. Public Health 2023, 20, 6085 15 of 18 15 of 18 in speciﬁc locations will be highly dynamic in time and space, based on the user’s data request and spatial movement in a given area. The passive RF-EMF exposure of individuals will then vary considerably compared to 4G/LTE cases. Users with different demands in the same workplace are exposed to various sources of RF-EMF. However, the adoption of the high-frequency band, and usage of beam steering devices, in both indoor and outdoor locations (e.g., campus, industrial) requires the development of novel measurement devices and deeper analysis to evaluate the potential health risks or symptoms due to the directional RF-EMF exposure in high frequencies (FR2, above 26 GHz). The main scope of this case study (Figure 7) is to assess the exposure of 5G signals in the FR2 mmWave bands according to personalized demands in indoor and outdoor environments and studies of possible biological effects and their mechanisms on RBC. Sensor characterization for spatial response Portable blood test set Double-blinded trials with healthy volunteers Markers of blood response to the EMF exposure ? Wearable sensors Developments for in vitro and ex vivo RBCs testing Sensors and setup tested with 5G FR2 Application to case scenario 2 campus Industrial Cross validation Figure 7. Case Study 3—Health effects of exposure to RF-EMF in indoor and outdoor environments. Figure 7. Case Study 3—Health effects of exposure to RF-EMF in indoor and outdoor environments. To achieve this goal, NextGEM will conduct an extensive literature review on the effects of RF-EMF biophysical mechanisms and on RF-EMF assessment sensing technology. The indoor and outdoor exposures will be modeled using the ERMES ray-tracing tools, including the provision of a ﬁrst-order model including scattering from the environment to computed received power. This case study will deﬁne the indoor (i.e., industrial environ- ment) and outdoor university campus environment with multiple FR2 antenna modules with steering capabilities covering the deﬁned area to provide a higher data rate. This includes identifying signal test cases/scenarios to identify low-, medium-, and high-body RF-EMF exposure. 4.3. Case Study 3—Health Effects of Exposure to RF-EMF in Indoor and Outdoor Environments p The assessment of real-life exposure will be measured with wearable vest sensors. The sensing element will provide the required sensitivity to accurately sample the average energy received in the selected case study, operating in the FR2 mm wave bands and capable of covering the entire-body RF-EMF exposure (i.e., distributed sensors). The number and the location of the developed sensors on the vest will provide entire-body RF-EMF exposure. The testing of measurement systems will be carried out in the TU Delft lab and the outdoor measurement in the IoT Green Village 5G in the Delft Campus with the participation of volunteer students. In addition, the possible biophysical and biochemical mechanisms of mmWave exposure (FR2) will be investigated on RBC to identify the fundamental mode of action of acute (minutes to hours) exposure and to identify the signal features (pattern, intensity, duration) of importance. If the in vitro experiments show effects of the exposure found in the case study, a double-blind experiment on volunteers will be performed to investigate the possibility of blood effects. 4.4. Expected Outcomes from Case Studies It is expected that the case studies will provide different outcomes to the scientiﬁc society, citizens, public authorities and the regulator. More speciﬁcally, Case Study 1 can upgrade simulation tools and models (useful for exposure assessors) which the scien- Int. J. Environ. Res. Public Health 2023, 20, 6085 16 of 18 tiﬁc community can apply in further research on RF-EMF exposure. In addition, health standards will be updated regarding the potential effects of different frequencies (health agencies, risk assessors) and, ﬁnally, the outcome of the research will generate the basis for practical guidelines. The expected outcome of Case Study 2 will include the antenna optimization mapping criteria (telecom operators) and updating or verifying RF-EMF limits (regulators). It will also increase knowledge and public awareness regarding the biological responses and 5G frequency exposures. The expected outcome of Case Study 3 will include the assessment of actual exposure to 5G frequency bands in everyday activities on the campus. Finally, the identiﬁcation of a potential correlation between 5G RF-EMF exposure parameters and RBC effects (health agencies, risk assessors) will be useful to increase the knowledge and awareness of citizens regarding potential health issues and 5G. 5. Conclusions The ambitious vision of NextGEM was presented in this work through the description of the speciﬁc activities and related methodologies. The multidisciplinary expertise of the NextGEM partnership spanning over telecommunication engineering, cell biology, human studies and epidemiology, will be exploited during the four-year project to guarantee the fulﬁllment of its main objectives. Relevant scientiﬁc knowledge and data on new scenarios of the next generation of RF-EMF, on the effects and interaction with biological materials relevant to human health, will be generated in the project. Together with the current epidemiological knowledge on cancer and reproductive outcomes, this knowledge will be integrated into an evidence-based risk assessment for use by different stakeholders. The development of the NextGEM Innovation and Knowledge Hub for EMF and Health will ensure the accessibility of the project results to the scientiﬁc community, the public, and the European regulators and improve awareness. Finally, NextGEM results will be validated in three case studies to ensure the secure and safe living of vulnerable citizens and workers. Author Contributions: Conceptualization, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B.; methodology, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B.; software, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., E.S., R.O., A.M., G.S., V.T., E.Z.; validation, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B.; investigation, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B.; writing—original draft preparation, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B..; writing—review and editing, N.P., M.-O.M., P.C., M.S. Author Contributions: Conceptualization, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B.; methodology, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B.; software, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., E.S., R.O., A.M., G.S., V.T., E.Z.; validation, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B.; investigation, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B.; writing—original draft preparation, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B..; writing—review and editing, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B.; visualization, N.P., M.-O.M., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., J.B., E.K., V.T., A.S., Y.F., A.L., M.G., M.S. (Marco Spirito), S.S., A.B.; supervision, N.P., M.-O.M.; project administration, N.P., M.-O.M., P.C.; funding acquisition, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B. as contributors to the funded Horizon Europe Project NextGEM. All authors have read and agreed to the published version of the manuscript. References 1. ICNIRP. Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic ﬁelds (up to 300 GHz). Health Phys. 1998, 74, 494–521. 2. European Union. Directive No. 2013/35/EU, of 26 June 2013, on the Minimum Health and Safety Require-Ments Regarding the Exposure of Workers to the Risks Arising from Physical Agents (Electromagnetic Fields). Off. J. Eur. Union 2013, L179, 1–21. Available online: http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32013L0035&from=PT (accessed on 1 March 2023). 3. 1999/519/EC: Council Recommendation of 12 July 1999 on the Limitation of Exposure of the General Public to Electromagnetic Fields (0 Hz to 300 GHz). Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=celex:31999H0519 (accessed on 1 March 2023). 4. Types of Legislation, European Commission. Available online: https://european-union.europa.eu/institutions-law-budget/law/ types-legislation_en (accessed on 1 March 2023 ). 5. Stam, R. Comparison of International Policies on Electromagnetic Fields: Power Frequency and Radiofrequency Fields; National Institute for Public Health and the Environment (RIVM): Bilthoven, Utrecht, 2018. 6. IEC/IEEE 62209-1528-2020; Measurement Procedure for the Assessment of Speciﬁc Absorption Rate of Human Exposure to Radiofrequency Fields from Hand-Held and Body-Mounted Wireless Communication Devices. International Organization for Standardization (ISO): Geneva, Switzerland, 2020. 7. IEC/TR 62669:2019; Case Studies Supporting IEC 62232—Determination of EMF Field Strength, Power Density and SAR in the Vicinity of Radiocommunication Base Stations for the Purpose of Evaluating Human Exposure. International Electrotechnical Commission: Geneva, Switzerland, 2019. 8. Otin, R. ERMES: A nodal-based ﬁnite element code for electromagnetic simulations in frequency domain. Comput. Phys. Commun. 2013, 184, 2588–2595. [CrossRef] 9. Schwan , H.P. Electrical properties of tissue and cell suspensions. Adv. Biol. Med. Phys. 1957, 5, 147–209 10. Raicu, V.; Feldman, Y. (Eds.) Dielectric Relaxation in Biological Systems: Physical Principles, Methods, and Applications; Oxford University Press: New York, NY, USA, 2015. 11. Asami, K. Characterization of heterogeneous systems by dielectric spectroscopy. Prog. Polym. Sci. 2002, 27, 1617–1659. [CrossRef] 11. Asami, K. Characterization of heterogeneous systems by dielectric spectroscopy. Prog. Polym. Sci. 2002, 27, 1617–1659. [CrossRef] 12. Stücker, M.; Struk, A.; Altmeyer, P.; Herde, M.; Baumgärtl, H.; Lübbers, D.W. The cutaneous uptake of atmospheric oxygen g y yg pp y p y 13. Darvishi, M.; Mashati, P.; Kala, S.; Paridar, M.; Takhviji, V.; Ebrahimi, H.; Zibara, K.; Khosravi, A. Electromagnetic radiation: A new charming actor in hematopoiesis? Expert Rev. Hematol. 2021, 14, 47–58. [CrossRef] [PubMed] 14. Mousavy, S.J.; Riazi, G.H.; Kamarei, M.; Aliakbarian, H.; Sattarahmady, N.; Shariﬁzadeh, A.; Safarian, S.; Ahmad, F.; Moosavi-Movahedi, A.A. 5. Conclusions (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B.; visualization, N.P., M.-O.M., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., J.B., E.K., V.T., A.S., Y.F., A.L., M.G., M.S. (Marco Spirito), S.S., A.B.; supervision, N.P., M.-O.M.; project administration, N.P., M.-O.M., P.C.; funding acquisition, N.P., M.-O.M., P.C., M.S. (Myrtill Simko), A.G., A.M.Y., O.Z., M.R.S., E.S., R.O., F.S., M.D.M., A.M., G.S., J.B., E.K., V.T., E.Z., S.L., M.B., A.S., M.R.B., Y.F., I.B., A.L., M.G., M.S. (Marco Spirito), M.L., S.S., M.P., F.V., L.C., D.B., A.B. as contributors to the funded Horizon Europe Project NextGEM. All authors have read and agreed to the published version of the manuscript. Int. J. Environ. Res. Public Health 2023, 20, 6085 17 of 18 Funding: This work is part of the European Union’s Horizon Europe research and innovation program under grant agreement No. 101057527 (NextGEM). Funded by the European Union. Views and opinions expressed are however those of the authors only and do not necessarily reﬂect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them. ICMAB-CSIC and CIMNE have also been partially supported by the Spanish Ministry of Science and Innovation, through the Severo Ochoa Programme for Centres of Excellence in R&D (CEX2019–000917-S) and (CEX2018-000797-S), respectively. Funding: This work is part of the European Union’s Horizon Europe research and innovation program under grant agreement No. 101057527 (NextGEM). Funded by the European Union. Views and opinions expressed are however those of the authors only and do not necessarily reﬂect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them. ICMAB-CSIC and CIMNE have also been partially supported by the Spanish Ministry of Science and Innovation, through the Severo Ochoa Programme for Centres of Excellence in R&D (CEX2019–000917-S) and (CEX2018-000797-S), respectively. 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https://openalex.org/W4289260877	https://www.nature.com/articles/s42004-022-00769-2.pdf	English	null	Surface Modifications of eight-electron Pd/Ag Superatomic Alloys	Research Square (Research Square)	2,022	cc-by	11,156	1 Department of Chemistry, National Dong Hwa University, No. 1, Sec. 2, Da Hsueh Rd. Shoufeng, Hualien 97401, Taiwan, ROC. 2 Department of Chemistry, Veer Surendra Sai University of Technology, Burla, Sambalpur, Odisha 768018, India. 3 Department of Chemistry, C. V. Raman Gobal University, Bidya Nagar, Bhubaneswar, Odisha 752054, India. 4 Univ Rennes, CNRS, ISCR-UMR 6226, F-35000 Rennes, France. ✉email: jeanyves.saillard@univ-rennes1.fr; chenwei@mail.ndhu.edu.tw Surface modiﬁcations of eight-electron palladium silver superatomic alloys Subrat Kumar Barik1,2,3, Chih-Yuan Chen1, Tzu-Hao Chiu1, Yu-Rong Ni1, Franck Gam4, Isaac Chantrenne4, Samia Kahlal4, Jean-Yves Saillard 4✉& C. W. Liu 1✉ https://doi.org/10.1038/s42004 022 00769 2 OPEN Surface modiﬁcations of eight-electron palladium silver superatomic alloys Subrat Kumar Barik1,2,3, Chih-Yuan Chen1, Tzu-Hao Chiu1, Yu-Rong Ni1, Franck Gam4, Isaac Chantrenne4, Samia Kahlal4, Jean-Yves Saillard 4✉& C. W. Liu 1✉ p // g/ / Atomically precise thiolate-protected coinage metal nanoclusters and their alloys are far more numerous than their selenium congeners, the synthesis of which remains extremely challenging. Herein, we report the synthesis of a series of atomically deﬁned dithiophosph(in) ate protected eight-electron superatomic palladium silver nanoalloys [PdAg20{S2PR2}12], 2a–c (where R = OiPr, a; OiBu, b; Ph, c) via ligand exchange and/or co-reduction methods. The ligand exchange reaction on [PdAg20{S2P(OnPr)2}12], 1, with [NH4{Se2PR2}12] (where R = OiPr, or OnPr) leads to the formation of [PdAg20{Se2P(OiPr)2}12] (3) and [PdAg20{- Se2P(OnPr)2}12] (4), respectively. Solid state structures of 2a, 2b, 3 and 4 unravel different PdAg20 metal frameworks from their parent cluster, originating from the different distribu- tions of the eight-capping silver(I) atoms around a Pd@Ag12 centered icosahedron with C2, D3, Th and Th symmetries, respectively. Surprisingly ambient temperature crystallization of the reaction product 3 obtained by the ligand exchange reaction on 1 has resulted in the co- crystallization of two isomers in the unit cell with overall T (3a) and C3 (3b) symmetries, respectively. To our knowledge, this is the ﬁrst ever characterized isomeric pair among the selenolate-protected NCs. Density functional theory (DFT) studies further rationalize the preferred geometrical isomerism of the PdAg20 core. 1 Department of Chemistry, National Dong Hwa University, No. 1, Sec. 2, Da Hsueh Rd. Shoufeng, Hualien 97401, Taiwan, ROC. 2 Department of Chemistry, Veer Surendra Sai University of Technology, Burla, Sambalpur, Odisha 768018, India. 3 Department of Chemistry, C. V. Raman Gobal University, Bidya Nagar, Bhubaneswar, Odisha 752054, India. 4 Univ Rennes, CNRS, ISCR-UMR 6226, F-35000 Rennes, France. ✉email: jeanyves.saillard@univ-rennes1.fr; chenwei@mail.ndhu.edu.tw COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem 1 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 [Ag8(X){Se2P(OiPr)2}6]+ (X = H, Cl or Br)30, [Ag11(µ9-Se) (µ3-X)3{Se2P(OiPr)2}6] (X = I31, Br32), [Ag11(µ9-I)(µ3-I)3{Se2P (OiPr)2}6]+ 33, [Ag12(μ5-X)2{Se2P(OEt)2}10] (X = Br, I)34 [Ag20{Se2P(OiPr)2}12]20, [Ag21{Se2P(OEt)2}12]+ 20, [PtAg20{Se2- P(OR)2}12] (R = iPr or nPr)21 and [AuAg20{Se2P(OEt)2}12]+ 20. In fact the latter species, fabricated via a ligand exchange method, is the ﬁrst structurally characterized alloy NC entirely covered by a Se ligand shell. We have produced several Pt/Pd doped Ag NCs of late21,35,36, among them the M21 core metallic system pre- dominates (See Supplementary Table S1). Results and discussion h d h Synthesis and characterization of [PdAg20{S2PR2}12], R = OiPr (2a), R = OiBu (2b), R = Ph (2c). Beforehand, we have synthe- sized and structurally characterized the thermodynamically stable alloy [PdAg20{S2P(OnPr)2}12] (1). This cluster can be formally regarded as a centered icosahedral [Pd@Ag12]4+ 8-electron superatomic core (with 1S2 1P6 1D0 superatomic conﬁguration) passivated by an outer shell made of eight Ag+ and twelve monoanionic dithiophosphate (dtp) ions in such a way the complete PdAg20 metallic kernel is lowered to ideal C2 symmetry and the whole NC to C136. NC 1 is robust, yet the liability of its protecting ligands tempted us to study its ligand exchange (LE) behavior. As shown in the Fig. 1, the treatment of 1 with 12 equivalents of NH4[S2P(OiPr)2] at −20 °C in tetrahydrofuran (THF) led to the formation of [PdAg20{S2P(OiPr)2}12] (2a) in 70% yield within an hour. There was no obvious color change observed when NH4[S2P(OiPr)2] was added to the brown red solution of 1 in THF during the course of reaction, thus the progress of reaction was monitored by thin layer chromatography (TLC). Alternatively, the same compound was produced more conveniently by direct archetypal one pot synthetic method in moderate yield (41 %) (See Experimental methods). In parallel to the synthesis of 2a, compound [PdAg20{S2P(OiBu)2}12] (2b) was synthesized via co-reduction method in 40% yield. Compound [PdAg20{S2P(OPh)2}12] (2c) was synthesized via the LE method in 65% yield (Experimental methods). All NCs (2a–c) have been characterized by positive ion mode electrospray ionization mass p p The initial attempt in the synthesis of a selenolate protected cluster, namely Au25(SeC8H17)18−, was made in 2011 by Negeshi and his coworkers via the reduction of an Au(I) selenolate complex by NaBH423. Subsequently, the same group also syn- thesized Au38(SeC12H25)24 from Au38(SR)24 via a ligand exchange (LE) method24. Zhu et al. demonstrated that selenophenolate- protected Au1825 and Au2511 NCs exhibit different optical prop- erties from those of their thiolate homologs. Later, studies on Au24(ER)20 (E = Se or S) NCs unraveled different structures and optical properties between both families of chalcogenolates26. In 2013, Pradeep and his co-workers reported the ﬁrst atomically precise silver NC protected by selenolates, Ag44(SePh)30, which revealed similar properties as its thiolate counterpart27. More recently, Bootharaju et al. reported a Cd-doped silver NC pro- tected by selenophenolates, namely Cd12Ag32(SePh)36, which exhibits rare near-infrared (NIR) photoluminescence at ∼1020 nm18. Surface modiﬁcations of eight-electron palladium silver superatomic alloys Subrat Kumar Barik1,2,3, Chih-Yuan Chen1, Tzu-Hao Chiu1, Yu-Rong Ni1, Franck Gam4, Isaac Chantrenne4, Samia Kahlal4, Jean-Yves Saillard 4✉& C. W. Liu 1✉ https://doi.org/10.1038/s42004 022 00769 2 OPEN Thus, we intended to outspread our approach in the development of dsep-protected PdAg20 alloy NCs. Amid the several synthetic methods available, the ligand exchange method37–40 is one of most fruitful strategies to yield molecularly pure NCs stabilized by dsep ligands. Herein we report the isolation of a series of 8-electron superatomic, dichalcogenolate-protected PdAg20 alloy NCs that include a pair of selenolate-protected isomers. O O ver the last decades, the chemistry of atomically and structurally precise Au and Ag nanoclusters (NCs) and their alloys have gained a broad attention in modern science owing to their potential applications in catalysis, optoe- lectronics, electrochemical studies, chemical sensing, biomedicine and chiral cluster syntheses1–9. Size focusing synthesis in com- bination with atomic precision studied by X-ray crystallography sets apart these ultra-small sub-nanometer size NCs from their colloidal analogs for achieving aforementioned properties. To date, hundreds of atomically deﬁned ligand-protected coinage metal NCs and their alloys have been synthesized. In this regard, the more common protecting ligands employed to isolate dif- ferent NCs are thiols, phosphines, alkynes, hydrides or their combinations1,10–16. In contrast, structurally precise Au and Ag clusters co-protected by selenols are much rarer, with approxi- mately twenty reported examples17–22. In order to illuminate the effects of surface functionalization of nanoparticles, recent reports have demonstrated the fabrication of several stable functional nanomaterials by using selenolates in the place of thiolates as protecting ligands. Unlike in thiolate-protected NCs, true struc- tural isomerism, which is an interesting feature for ﬁne tuning many properties, has not been reported so far for selenolate- protected NCs. Thus, the syntheses of selenolate-protected NCs are of paramount importance. Results and discussion h d h As part of our research efforts in the synthesis of selenolate- protected Ag clusters, we have isolated a series of diselenopho- sphate (dsep) protected mono- and bimetallic silver clusters such as [Ag7(H){Se2P(OiPr)2}6]28, [Ag10(Se){Se2P(OiPr)2}8]29, g. 1 Synthesized compounds. Synthesis of dichalcogenolate protected Pd-Ag alloy NCs 2a, 3 and 4 via ligand exchange reaction. COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem 2 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Table 1 Spectroscopic data of compounds for 1, 2(a–c), 3 and 4. Compound 31P NMR(ppm) UV–vis(nm) Emission (nm)a ESI-MS (m/z, M+) 1 104.9 384, 436 748 4930.8 2a 101.66 384, 436 741 4931.15 2b 103.85 384, 436 732 5267.64 2c 63.11 419, 486 669 2735.76b 3 68.16 410, 501 712 6055.77 4 73.59 408, 498 702 6056.02 aPhotoluminescence recorded in 2-Methyl tetrahydrofuran at 77 K. bESI-MS peak corresponds to [M + 2Ag]2+ peak. Fig. 2 Mass spectrometry. ESI-MS (Positive mode) of [2a + Ag]+. Insets: experimental (black) and simulated (red) mass spectra. Table 1 Spectroscopic data of compounds for 1, 2(a–c), 3 and 4. Compound 31P NMR(ppm) UV–vis(nm) Emission (nm)a ESI-MS (m/z, M+) 1 104.9 384, 436 748 4930.8 2a 101.66 384, 436 741 4931.15 2b 103.85 384, 436 732 5267.64 2c 63.11 419, 486 669 2735.76b 3 68.16 410, 501 712 6055.77 4 73.59 408, 498 702 6056.02 aPhotoluminescence recorded in 2-Methyl tetrahydrofuran at 77 K. bESI-MS peak corresponds to [M + 2Ag]2+ peak. (Calcd. 2735.8916), respectively. In order to elucidate the structure of these nanoalloys (2a–c), single crystals X-ray diffraction studies were undertaken. We were successful to crystallize 2a and 2b. The details of their X-ray structural analysis were discussed below. All of our attempts to crystallize 2c were failed. Table 1 Spectroscopic data of compounds for 1, 2(a–c), 3 and 4. of our attempts to crystallize 2c were failed. Single crystals of suitable quality for X-ray diffraction for 2a and 2b were grown by crystallization from diffusion of hexane into a concentrated dichloromethane solution at −4 °C within couple of weeks. Surprisingly, the resulting solid-state structures unveil different conﬁguration of the outer shell which protects the 8-electron [Pd@Ag12]4+ core, as illustrated in Fig. 3 compared to that of 1. In particular, the arrangements of the 8 Ag+ capping atoms around the centered icosahedral core differs from that in 1, as one can see in Fig. 3 (from Fig. 3b→3d→3f). Results and discussion h d h Upon the replacement in 1 of the dithiophosphates ligands having linear alkyl chain (n-propyl) with their branched derivatives (di-isopropyl dithiophosphates), the 31P{1H} NMR spectrum in CDCl3 displays a signal shift from 104.9 ppm to 101.66 ppm at room temperature (Supplementary Fig. S3). The 1H NMR spectrum of 2a in CDCl3 shows two set of signals with a multiplet ranged at δ = 4.97–4.85 ppm (corresponding to the –OCH groups) and a doublet ranged at δ = 1.35–1.33 ppm (corresponding to –(CH3)2), in an integration ratio of 1:6 which is clearly attributed to the iPr groups (Supplementary Data 1, Fig. S2). [ g21{ 2 ( )2}12] The inner icosahedral Pd@Ag12 cores of 2a and 2b are very similar to that of 1. The Pd-Ag radial bond distances average 2.755 Å, 2.767 Å for 2a and 2b, respectively (2.757 Å in 1) and the peripheral Agico-Agico and Agico-Agcap bond distances in 2a and 2b are also fairly similar to those of 1 (Table 2). Thus, the 8-electron nanoalloys 1, 2a and 2b whose compositions differ only by the nature of their alkyl substituents, can be considered as pseudo-isomers. The presence of different arrangements of their outer shells is likely the result of the slightly different steric factors of their alkyl chains in 1 and 2a. The ESI mass spectrum (positive-ion mode) was recorded to identify the molecular formula of 2a. The spectrum reveals two prominent bands corresponding to [2a + Ag]+ at m/z 4931.15 (calcd. 4930.97), and [2a + Ag+H]2+ at m/z 2465.59 (calcd. 2465.95). Their simulated isotopic distributions are in good agreement with the experimental results (Fig. 2). The position of the molecular ion peak in 2a matches exactly with its parent NC 1, signifying the retention metal atomicity upon LE. Moreover, the UV–vis absorption spectrum of 2a features the same absorption pattern (384, 436 nm) as its parent NC 1. Thus, from the above spectroscopic data obtained in solution state, one would presume that the structure of 2a is the same as 1. [PdAg20{Se2P(OiPr)2}12], (3) and its 3(Pn) solid-state structure. After successful isolation of the 2a–c NCs, it was indeed obvious to attempt the synthesis of their diselenophosphate (dsep) protected analogs via LE reactions. Compound 1 was treated with NH4[Se2P(OiPr)2] at −20 °C in THF (Fig. 1). Results and discussion h d h Whereas in both 1 and 2a NCs the PdAg20 skeleton adopts pseudo-C2 symmetry; that of 2b the PdAg20 skeleton adopts pseudo-D3 symmetry (Supplementary Fig. S4). The twelve dtp ligands in 2a are equally distributed on both sides of the pesudo-C2 axis (Supplementary Fig. S4). These dtp ligands are coordinated to both icosahedral and capping silver atoms (Agico and Agcap, respectively) in ﬁve different binding modes bimetallic biconnectivity (η2: µ1, µ1), bimetallic triconnectivity (η2: µ2, µ1), trimetallic triconnectivity (η3: µ2, µ1), trimetallic tetraconnectivity (η3: µ2, µ2) and tetrametallic tetraconnectivity (η4: µ2, µ2) (Supplementary Fig. S5) in a ratio of 1:1:7:1:2. Further the seven dtp ligands with trimetallic triconnectivity (η3: µ2, µ1), differ in the coordination to different combination of Agcap and Agico atoms except for a couple of dtp ligands (red box, in Supplementary Fig. S5). As in any 8-electron dtp- or dsep-protected M21 NC characterized so far, the eight Ag+ capping atoms in 2a lie in a nearly planar AgSe3 coordination mode, making locally stable 16-electron metal centers. With the 12 protecting ligands around the PdAg20 metal skeleton (Fig. 3c), the entire molecular symmetry of 2a is C1. On the other hand, the total twelve dtp ligands in 2b are distributed in three spherical rows around the pseudo-C3 axis in 3:6:3 ratios (Supplementary Fig. S4). They bind to both capping and icosahedral silver atoms only through two coordination patterns that are trimetallic triconnectivity and trimetallic tetraconnectiv- ity (Supplementary Fig. S6), in such a way the whole NC ideal symmetry is reduced to C3. A similar C3 arrangement has been described in the related 8-electron NC [Ag21{S2P(OiPr)2}12]+ 41. Photoluminescence recorded in 2-Methyl tetrahydrofuran at 77 K. bESI-MS peak corresponds to [M + 2Ag]2+ peak. Fig. 2 Mass spectrometry. ESI-MS (Positive mode) of [2a + Ag]+. Insets: experimental (black) and simulated (red) mass spectra. Fig. 2 Mass spectrometry. ESI-MS (Positive mode) of [2a + Ag]+. Insets: experimental (black) and simulated (red) mass spectra. spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. ESI mass spectra of 2a–c have been provided in Table 1, Fig. 2 and Supplementary Figs. S1 and S2. 31P{1H} and 1H NMR spectra of 2a–c have been provided in Table 1 and Supplementary Data 1, Figs. 1–5. COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 ARTICLE Fig. 3 Molecular structures of 1, 2a and 2b. a, b Total structure of 1 (propoxy groups omitted for clarity) and its Pd@Ag20 metallic core with C2 symmetry, respectively. c, d Total structure of 2a (isopropoxy groups omitted for clarity) and its Pd@Ag20 metallic core with C2 symmetry, respectively36. e, f Total structure of 2c (isobutoxy groups omitted for clarity) and its Pd@Ag20 metallic core with D3 symmetry, respectively. (color code. Pd: orange; Agico: pink, Agcap: green; S: yellow; P: sky blue). Fig. 3 Molecular structures of 1, 2a and 2b. a, b Total structure of 1 (propoxy groups omitted for clarity) and its Pd@Ag20 metallic core with C2 symmetry, respectively. c, d Total structure of 2a (isopropoxy groups omitted for clarity) and its Pd@Ag20 metallic core with C2 symmetry, respectively36. e, f Total structure of 2c (isobutoxy groups omitted for clarity) and its Pd@Ag20 metallic core with D3 symmetry, respectively. (color code. Pd: orange; Agico: pink, Agcap: green; S: yellow; P: sky blue). Table 2 Selected structural metrics (average (top line) and ranges (bottom line)) of compounds for 1, 2a-b, 3, 3a-b and 4. ctural metrics (average (top line) and ranges (bottom line)) of compounds for 1, 2a-b, 3, 3a-b and 4. Table 2 Selected structural metrics (average (top line) and ranges (bottom line)) of compounds for 1, 2a-b, 3, 3a-b and 4. Entry Agico-Agico Agico-Agcap Agico-E Agcap-E E-E 1 2.897 2.971 2.687 2.540 3.414 2.827-2.987 2.856-3.346 2.471-3.047 2.480-2.726 2.772-2.747 2a 2.896(7) 3.020(7) 2.605(14) 2.542(19) 3.399 2.807(1)−2.997(1) 2.863(1)−3.287(1) 2.486(4)−2.933(4) 2.472(5)−2.651(4) 3.304-3.456 2b 2.893(6) 3.014(5) 2.542(2) 2.506(1) 3.400 (3) 2.829(8)−2.930(1) 2.847(9)−3.204(7) 2.480(3)−2.590(2) 2.330(1)−2.590(3) 3.380(4)−3.430(2) 3 (Pn) 2.902(11) 2.949(100) 2.683(10) 2.614(15) 3.688 2.845(2)−2.969(2) 2.899(2)−3.005(2) 2.666(3)−2.696(3) 2.595(3)−2.639(3) 3.655-3.704 3(P31c) 3a 2.901(9) 2.950(8) 2.668(7) 2.607(10) 3.665 2.841(2)−2.957(3) 2.901(3)−2.989(2) 2.662(4)−2.674(3) 2.600(3)−2.617(3) 3.646-3.691 3b 2.896(10) 2.965(9) 2.739(8) 2.607(12) 3.668 2.845(3)−2.945(2) 2.880(3)−3.123(3) 2.628(3)−3.104(4) 2.576(3)−2.673(3) 3.653-3.694 4 2.893(2) 2.949(2) 2.668(2) 2.618(2) 3.698 2.833(2)−2.961(2) 2.902(2)−3.009(2) 2.630(2)−2.699(2) 2.607(2)−2.637(2) 3.677-3.721 Table 2 Selected structural metrics (average (top line) and ranges (bottom line)) of compounds change, however we believe the change in the ligand environment plays a major role for the immediate color change. Note that, when we performed ligand exchange reaction onto 1 with n-propyl dithiophosphate surface ligand by iso-propyl dithiophosphate ligand, then there was no remarkable color changes observed, even though reaction culminated in the formation of altered metal core. Results and discussion h d h The reaction pro- ceeded immediately as the color of the reaction mixture altered from brown red to purple which indicates replacement of surface dtp ligands by dsep ligands (Supplementary Fig. S7). The possibility of partial replacement of ligands cannot be excluded, however we have never encountered the partial replacement when we intend to produce dsep protected NCs from their dtp siblings via LE method20–22,28–34. In particular, most of the cases these reactions are associated with alteration of the metallic cores20–22,28–34. The change in metallic core is certainly a reason for the distinct color p Similarly, the 31P{1H} NMR spectrum displays one type of resonance for 2b (δ = 103.8 ppm) and for 2c (δ = 63.1 ppm). The 1H NMR spectra of 2b and 2c displayed three and two types of resonances, corresponding to iso-butoxy and phenyl groups, respectively. Further, the positive ion mode ESI mass spectra of 2b and 2c show prominent bands corresponding to [M + Ag]+ at m/z 5267.64 (Calcd. 5267.62) and [M + 2Ag]2+ at m/z 2735.7615 MMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem 3 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 a Total structure of [PdAg20{Se2P(OiPr)2}12] (3(Pn)) (isopropoxy groups omitted for clarity), b Illustration of the Pd@Ag20 metallic core in 3(Pn) with Th symmetry, c A view of the Pd@Ag12 centered icosahedron core with its 8 capping Ag atoms, d The centered Pd@Ag12 icosahedron inscribed in Ag8 cube (color code. Pd: orange red; Agico: pink, Agcap: green; Se: light orange; P: light blue). shown in Fig. 4 and exhibits a Pd-centered icosahedral Ag12 core inscribed in a cube made up of 8 capping Ag atoms, in such a manner that the entire Pd@Ag12@Ag8 framework attains ideal Th symmetry. The whole metal kernel is protected by 12 dsep ligands situated on the 12 edges of the cube, in such a way that the whole NC ideal symmetry is reduced to T. The detailed molecular structure of 3(Pn) is identical to that of 3a, discussed in the next section below. PdAg20 framework of Th symmetry, is shown in Fig. 4b–d. Their twelve dsep ligands display trimetallic triconnectivity (η3: µ2, µ1) bridging pattern with two capping Ag atoms (Agcap) and one icosahedral Ag atom (Agico), reducing the whole ideal NC sym- metry to T. The molecular structure of 3b (C3 symmetry) is similar to that of 2b (see above) and [Ag21{S2P(OiPr)2}12]+ 41. The differences in the positions of the outer capping Ag atoms in 3a and 3b, and their possible interchange pathway, is illustrated in Supplementary Fig. S9. To the best of our knowledge clusters 3a and 3b constitute the ﬁrst pair of true isomers within the family of Se-protected NCs certiﬁed by X-ray crystallography. The Pd-Agico average distances in 3a and 3b are equivalent (2.758(10) Å and 2.754(10) Å, respectively), as well as their average Agico-Agico distance (2.901(9) Å and 2.896(10) Å, respectively). Thus, the structure of the Pd@Ag12 core in 3a and 3b is quite independent from the conﬁguration of the outer sphere (Table 2). The average Agico@Se distance in both 3a and 3b are larger than the Agcap@Se distances. The Se···Se bite dis- tances in 3a and 3b are fairly similar (Table 2) and slightly shorter than those observed in [Ag21{Se2P(OEt)2}12]20 (3.697 Å) and [AuAg20{Se2P(OEt)2}12]20 (3.697 Å). It should be also mentioned that the molecularly pure as- synthesized NC 2a also was used as starting precursor for LE reaction in order to produce 3. Therefore, the reaction of 2a with NH4[Se2P(OiPr)2] at −20 °C in THF was performed (Fig. 1). COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 The positive ion ESI mass spectrum of reaction mixture shows a prominent band or molecular ion peak at m/z = 6055.77 (calcd. 6055.23) corresponding to [PdAg20{Se2P(OiPr)2}12 + Ag]+ (Sup- plementary Fig. S8). extremely bad quality crystals. Nevertheless, diffusion of hexane into a saturated acetone solution of the reaction mixture kept at −10 °C yielded proper single crystals within a week. The crystals of 3 obtained from this low temperature crystallization were dissolved in d6-acetone and subjected to 31P{1H} and 1HH NMR studies. The 31P{1H} NMR spectrum at ambient temperature shows a single resonance at δ = 68.16 ppm (161.9 MHz, d6- acetone) ﬂanked with two set of satellites (1JP-Se = 604.51 and 710.40 Hz) (Supplementary Data 1, Fig. 6). 1H NMR spectrum shows characteristic signals of isopropyl ligands of di-isopropyl diselenophosphates (Supplementary Data 1, Fig. S7). extremely bad quality crystals. Nevertheless, diffusion of hexane into a saturated acetone solution of the reaction mixture kept at −10 °C yielded proper single crystals within a week. The crystals of 3 obtained from this low temperature crystallization were dissolved in d6-acetone and subjected to 31P{1H} and 1HH NMR studies. The 31P{1H} NMR spectrum at ambient temperature shows a single resonance at δ = 68.16 ppm (161.9 MHz, d6- acetone) ﬂanked with two set of satellites (1JP-Se = 604.51 and 710.40 Hz) (Supplementary Data 1, Fig. 6). 1H NMR spectrum shows characteristic signals of isopropyl ligands of di-isopropyl diselenophosphates (Supplementary Data 1, Fig. S7). p y g In order to determine its molecular structure, much effort was devoted to obtain suitable crystals for single crystal X-ray diffraction. Several sets of crystallization with varied solvent combinations and in mutable temperatures ended up producing Single crystals obtained were subjected to the X-ray diffraction study. Their analysis reveals that 3 crystallize in Pn space group. Its solid-state structure is labeled 3(Pn) in the following. It is COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem 4 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Fig. 4 Molecular structure of 3(Pn). a Total structure of [PdAg20{Se2P(OiPr)2}12] (3(Pn)) (isopropoxy groups omitted for clarity), b Illustration of the Pd@Ag20 metallic core in 3(Pn) with Th symmetry, c A view of the Pd@Ag12 centered icosahedron core with its 8 capping Ag atoms, d The centered Pd@Ag12 icosahedron inscribed in Ag8 cube (color code. Pd: orange red; Agico: pink, Agcap: green; Se: light orange; P: light blue). Fig. 4 Molecular structure of 3(Pn). COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Likewise, in the transformation from 1 to 3 the solution undergoes an instant color change from brown to purple. The work up of the reaction mixture was done immediately. The 31P{1H} NMR spectrum of the reaction mixture exclusively shows single resonance at δ = 68.16 ppm in d6-acetone, the same resonance as observed in 31P{1H} NMR spectrum of 3. Thus, the transformation from 2a to 3 was conﬁrmed by 31P{1H} NMR spectroscopy. The solid-state structure of [PdAg20{Se2P(OiPr)2}12], 3(P31c). Single-crystals of 3 could also be obtained by slow diffusion in hexane into the saturated acetone solution of reaction mixture at ambient temperature. Their X-ray analysis reveals that in such conditions 3 crystallizes in the P31c space group. This solid-state phase is labeled 3(P31c) in the following. It reveals an isomeric NC pair of [PdAg20{Se2P(OiPr)2}12] clusters (3a and 3b), co- crystallized in the unit cell in a 1:1 ratio, with T and C3 pseudo- symmetry, respectively (Fig. 5)42. The molecular structure of 3a (T symmetry) is the same as that of 3(Pn), as well as that of the previously reported isoelectronic monocationic [MAg20{- Se2P(R)2}12]q+ (M = Ag or Au; R = OEt; q = 1: M = Pt; R = Oi/ nPr; q = 0)20,21. The structural metrics of 3a and 3(Pn), are similar (Table 2). Their icosahedral Pd@Ag20 core embedded within a cuboid made of eight capping Ag atoms, resulting in a The two isomers assemble in a layer-by-layer mode. Each layer consists of pure 3a(T) or 3b(C3) (Fig. 6a). The T and C3 layer are alternately stacked along the [001] direction (Fig. 6b). The three-fold rotational axes of 3a and 3b are parallel to the c axis of the trigonal lattice. Finally, it is worth mentioning at this point that the isomer selectivity of the low temperature crystallization (3(Pn), T isomer) facilitates its further spectro- scopic characterizations. [PdAg20{Se2P(OnPr)2}12], (4). Given the synthesis of 2a-3 via ligand-exchange-induced structure transformation route it is indeed inevitable not to synthesize another normal propyl alkyl chain analog. Note that the precedence of structurally precise selenium protected alloy clusters is awfully inadequate. Thus, as shown in Fig. 1, we have endeavored the ligand COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem 5 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Fig. 5 The molecular structure of 3a and 3b. a The two co-crystallized structures of [PdAg20{Se2P(OiPr)2}12], 3a (left) and 3b (right). COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Isopropoxy groups have been omitted for better clarity. b The Th Pd@Ag20 metallic core of 3a. c The C3 Pd@Ag20 metallic core of 3b. (color code. Pd: orange; Agico: blue, Agcap: pink; Se: light orange; P: dark green). Fig. 5 The molecular structure of 3a and 3b. a The two co-crystallized structures of [PdAg20{Se2P(OiPr)2}12], 3a (left) and 3b (right). Isopropoxy groups have been omitted for better clarity. b The Th Pd@Ag20 metallic core of 3a. c The C3 Pd@Ag20 metallic core of 3b. (color code. Pd: orange; Agico: blue, Agcap: pink; Se: light orange; P: dark green). Fig. 6 Illustration of packing diagrams of the co-crystallized NCs 3a and 3b with dsep ligands omitted for clarity. a View down the b axis. b View down the c axis (color code: T layers are in blue and C3 layers in pink.). Fig. 6 Illustration of packing diagrams of the co-crystallized NCs 3a and 3b with dsep ligands omitted for clarity. a View down the b axis. b View down the c axis (color code: T layers are in blue and C3 layers in pink.). propyl diselenophosphate ligands (Supplementary Data 1, Fig. S9). The ESI mass (positive-ion mode) spectrum shows a prominent band owing to [4 + Ag]+ at m/z = 6056.02 (calcd. 6056.49), and its simulated isotopic distribution is in good agreement with the experimental one (Supplementary Fig. S10). Based on these spectroscopic evidences, the mole- cular structure of 4 should adopt the same T arrangement as that of 3a. This is conﬁrmed by the solid state structure of 4 obtained from single-crystal X-ray diffraction (Fig. 4 and Supplementary Fig. S11). Its structural metrics are similar to those of 3(Pn) and 3a (Table 2). Interestingly, 4 crystallize as a racemate in the P21 space group. replacement of dithiophosphates on 1 by diselenophosphates with linear alkyl chain (n-propyl). The reaction leads to the formation of [PdAg20{Se2P(OnPr)2}12] (4) in 75 % yield. Its 31P{1H} spectrum in CDCl3 displays a signal at δ = 73.59 ppm ﬂanked with two set of satellites (1JP-Se = 604.51 and 710.40 Hz) at room temperature (Supplementary Data 1, Fig. 8). COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 red-shifted to their alkyl relatives. The change from alkyl to phenyl of the dtp substituents can alter the photoluminescence intensity. Compounds 1, 2a, and 2b show photoluminescence in their solution state at 77 K. Their emission maxima in 2-methyl tetrahydrofuran (MeTHF) occur at λmax = 748 nm, 741 nm and λmax = 732 nm, respectively (Fig. 7 and Supple- mentary Figs. S12 and S13). Cluster 2c is also emissive in solution state at 77 K. Its emission maximum appears at 669 nm in MeTHF (Fig. 7 and Supplementary Fig. S13) which is blue shifted to its parent cluster 1. Optical properties of the title NCs. It is interesting to note that the side chain in dithiophosph(in)ate ligands can lead to the variance of photoluminescence properties. The differed alkyl chains such as n-propyl (1), i-propyl (2a), i-butyl (2b), have least variance and look reddish while the phenyl derivative (2c) which was only obtained by ligand exchange appear to be orange to the naked eye. The UV–Vis spectra of 1, 2a and 2b show similar broad optical absorption bands at 384 and 436 nm, the latter band being intense (Table 1, Fig. 7). On the other hand, the phenyl derivative 2c features different absorption bands (419 and 486 nm) where the former is found to be more intense (Fig. 7). The absorption bands in 2c are The UV–vis spectra feature two major broad absorption bands for 3 (λmax = 410 and 501 nm) and 4 (λmax = 408 and 498 nm) (Supplementary Fig. S14) which are red shifted with respect to those observed in their parent cluster 1 (λmax = 384 and 436 nm) (Fig. 8a). Cluster 3 and 4 displays photoluminescence in solution at 77 K where the emission maximum in 2-methyl tetrahydro- furan occurs at 712 and 702 nm, respectively which are slightly blue shifted with respect to those of their dtp analogs 1 or 2a (Fig. 8a and Supplementary Fig. S15). The time resolved photoluminescence spectra (77 K) of 2a–c, 3 and 4 exhibits a single exponential decay curve (Fig. 8b, c and Supplementary Figs. S16–18). The observed emission lifetimes (τ) of the dithiophosphate analogs 2a-b (2a: τ = 235.3 µs and 2b: τ = 198.8 µs) are comparatively longer than their diselenopho- sphate counterparts (3: τ = 82.8 µs and 4: τ = 82.1 µs). COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 The 1H{31P} NMR spectrum of 4 in CDCl3 reveals three set of signals with multiplets ranged at δ = 4.03–4.02 ppm (corresponds to –OCH2 group), δ = 1.78–1.70 ppm (corresponds to –CH2) and at δ = 0.95–0.92 ppm (corresponds to –CH3) in an integration ratio of 1:1:1.5, which is clearly attributed to nPr group of di- COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem 6 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Calculations on the T, C3 and C136 structures of [M@Ag20{dtp/dsep}12]±q indicated also a small energy differences between these structures, in parti- cular between T and C3, independently from the nature of M. Calculations on the simpliﬁed model [PdAg20{Se2PH2}12] with a slightly different basis set as previously35 found the T isomer to be slightly more stable, both in total energy (ΔE = 3.7 kcal/ mol) and free energy (ΔG = 0.2 kcal/mol), this last value being not signiﬁcantly different from zero. Calculations on the less simpliﬁed model [PdAg20{Se2P(OMe)2}12] found similar results with ΔE = 3.7 kcal/mol and ΔG = 2.7 kcal/mol. Although calculations on the real clusters 3a and 3b were not performed owing to their large size, these results conﬁrm our previous ﬁnding that the T and C3 structures are close in energy, with the T isomer tending to be slightly more favored in the case of diselenolate ligands. The very small computed energy difference between the two isomers is fully consistent with their observation as co-crystallized species. The C1 structure adopted by compound 2a was also calculated in the case of the [PdAg20{Se2PH2}12] model. It was also found less stable than its T isomer (ΔE = 10.3 kcal/mol and ΔG = 4.8 kcal/mol). In the case of the dithiolate model [PdAg20{S2PH2}12], this energy difference is reduced (ΔE = 4.6 kcal/mol and ΔG = 0.0 kcal/mol), in agreement with the observation of 2a. They illustrate close similarities in the bonding situation of the various isomers, in full consistency with their closeness in energy. All these computed species have their three highest occupied orbitals of 1P nature, whereas the 1D level correspond the lowest vacant orbitals. (2M in THF) and other chemicals were purchased from different commercial sources available and were used as received. The ligands and metal precursors used in this work, NH4[E2P(R)] (R = OnPr, OiPr, OiBu, or Ph; E = S or Se)44–46, [Ag(CH3CN)4] PF647 and Pd[S2P(OnR’)2]2 (R’ = OnPr, OiPr or OiBu)48, were synthesized as described in the literature. All the solvents used in this work were distilled under N2 atmosphere. ESI-mass spectra were recorded on a Fison Quattro Bio-Q (Fisons Instruments, VG Biotech, U. K.). Bruker Advance DPX300 FT-NMR spectrometer was used to record NMR spectra that operate at 300 and 400 megahertz (MHz) while recording 1H, 121.49, and 161.9 MHz for 31P and 100 MHz for 13C. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Further the lifetime of that of dithiophosphinate analog 2c is of 60 µs which is shorter compared to its dithiophosphate and diseleno- phosphate relatives. The emission lifetimes in the order of microseconds for NCs 2a–c, 3 and 4 indicate the occurrence of phosphorescence in each case. Fig. 7 Photophysical properties. UV–vis spectrum (left) of 1, 2a, 2b and 2c in 1 ×10−5 M CH2Cl2 and the normalized emission spectrum (right) of 1, 2a, 2b and 2c in MeTHF at 77 K. Computational studies of title NCs. In a recent DFT investigation on the alloying of dichalcogenolate-protected Ag21 species43, we have shown that in the case of 8-electron NCs of the type [MAg20{dtp/dsep}12]±q (M = group 9 to group 12 metal), Fig. 7 Photophysical properties. UV–vis spectrum (left) of 1, 2a, 2b and 2c in 1 ×10−5 M CH2Cl2 and the normalized emission spectrum (right) of 1, 2a, 2b and 2c in MeTHF at 77 K. Fig. 8 Experimental and theoretical photophysical studies of title clusters. a UV–vis spectrum of 1, and 3 in 1 ×10−5 M CH2Cl2 (left) and emission spectrum of 1 and 3 in MeTHF at 77 K (right). b, c Time-resolved photoluminescence spectrum of 2a and 3 at 77 K. d The TD-DFT-simulated UV–vis spectrum of 2a’, 3a’ and 3b’. Fig. 8 Experimental and theoretical photophysical studies of title clusters. a UV–vis spectrum of 1, and 3 in 1 ×10−5 M CH2Cl2 (left) and emission spectrum of 1 and 3 in MeTHF at 77 K (right). b, c Time-resolved photoluminescence spectrum of 2a and 3 at 77 K. d The TD-DFT-simulated UV–vis spectrum of 2a’, 3a’ and 3b’ Fig. 8 Experimental and theoretical photophysical studies of title clusters. a UV–vis spectrum of 1, and 3 in 1 ×10−5 M CH2Cl2 (left) and emission spectrum of 1 and 3 in MeTHF at 77 K (right). b, c Time-resolved photoluminescence spectrum of 2a and 3 at 77 K. d The TD-DFT-simulated UV–vis spectrum of 2a’, 3a’ and 3b’. COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem 7 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 when M is a group 9 or 10 metal, it strongly prefers occupying the center of the icosahedron, i.e., [M@Ag20{dtp/dsep}12]±q. The reason lies in the involvement of the nd(M) valence orbitals in the metal-metal bonding through their stabilization by the vacant superatomic 1D shell. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Residual solvent protons were used as a reference (δ, ppm, CDCl3, 7.26). 31P{1H} NMR spectra were referenced to external 85% H3PO4 at δ 0.00. UV–Visible absorption spectra were measured on a Perkin Elmer Lambda 750 spectrophotometer using quartz cells with path length of 1 cm. The elemental analyses were done using a Perkin-Elmer 2400 CHN analyzer. Photoluminescence spectra and lifetime measurements were carried out using an Edinburgh FLS920 ﬂuorescence spectrometer. Synthesis of [PdAg20{S2P(OiPr)2}12] (2a) Ligand exchange method. In an oven-dried Schlenk tube, [PdAg20{S2P(OnPr)2}12], 1 (0.100 g, 0.0207 mmol) was dissolved in THF (5 mL) and was placed at −20 °C for 15 min. Then twelve equivalents of NH4[S2P(OiPr)2] (0.057 g, 0.2484 mmol) were added to the solution. The reaction mixture was stirred for 1 h in the same temperature. The solvent was dried under vacuum and the obtained residue was extracted with hexane (3×5 mL) and ﬁltered to remove decomposed impurities from ligand. In this reaction NH4[S2P(OnPr)2] has been isolated as byproduct. The hexane solution was passed through Al2O3 column followed by ether. The brown-red solution obtained was dried which yielded (0.070 g, 70.17% based on Pd) [PdAg20{- S2P(OiPr)2}12] (2a). p TD-DFT calculations on [PdAg20{S2PH2}12] (C1) and [PdAg20{Se2PH2}12] (T and C3), as models for 2a(2a’), 3a(3a’) and 3b(3b’), provided the simulated UV–Vis spectra shown in Fig. 8d. They are in good agreement with their experimental counterparts (Fig. 8d). The low-energy band is of 1P →1D nature and a comparison of Figs. 8a and 8d let to suggest that the T isomer of 3 might be the dominant species in solution. a) Direct method. In an oven-dried Schlenk ﬂask, [Ag(CH3CN)4](PF6) (0.5 g, 1.2 mmol) was suspended in THF (15 mL). To this NH4[S2P(OiPr)2] (0.14 g, 0.6 mmol), and [Pd{S2P(OiPr)2}] (0.020 g, 0.0626 mmol) were added sequentially. Then the reaction ﬂask was placed in a low temperature bath at −20 °C for 15 min. LiBH4 ∙THF (0.2 mL, 0.8 mmol) was added slowly via syringe to the reaction mixture and the orange colored solution immedi- ately turned to black after the LiBH4 ∙THF addition. The reaction was aged 24 h at the same temperature. The solvent was evaporated under vacuum. In order to eliminate decomposed impurities from ligand, the residue was thoroughly washed with deionized water and was subse- quently extracted in CH2Cl2. The resulting CH2Cl2 solution was dried and then the residue was further dissolved in hexane. Conclusions I In summary, we have isolated and characterized a series of dtp- and dsep-protected 8-electron superatomic Pd doped silver NCs, of which several were structurally characterized. These structurally precise NCs feature a Pd-centered Ag12 icosahedron capped by 8 silver(I) atoms and 12 dichalcogenolate ligands with metallic PdM20 frameworks of ideal C2, Th, and D3 sym- metries, respectively, which reduce to C1, T, and C3, respec- tively, when the 12 ligands are considered. Selenium-ligand exchange on 1 (C1 symmetry) induced the formation of a pair of [PdAg20{Se2P(OiPr)2}12] structural isomers that are T-sym- metric (3a, T symmetry) and 3b (C3 symmetry). To our knowledge this is the ﬁrst ever reported isomeric pair of selenium-protected NCs existed in a unit cell. In fact, these are the rare evidence of structurally characterized alloy cluster completely covered by Se shell. Overall this work demonstrates that the ligand exchange synthetic method indeed provides insights to the development of both pseudo and true structural isomeric alloy NCs. 2a. ESI-MS(m/z) [M + Ag]+ calcd. for C72H168Ag21O24P12PdS24, 4930.97; found, 4931.15; 1H NMR (22 °C, 300 MHz, CDCl3, δ, ppm): 4.97–4.85 (m, 24H, OCH), 1.35–1.33 (d, 144 H, CH3); 31P{1H} NMR (22 °C, 121.49 MHz, CDCl3, δ, ppm): 101.66; UV–vis [λmax in nm, (ε in M−1cm−1)]: 384 (71254), 436 (106058). 2a. ESI-MS(m/z) [M + Ag]+ calcd. for C72H168Ag21O24P12PdS24, 4930.97; found, 4931.15; 1H NMR (22 °C, 300 MHz, CDCl3, δ, ppm): 4.97–4.85 (m, 24H, OCH), 1.35–1.33 (d, 144 H, CH3); 31P{1H} NMR (22 °C, 121.49 MHz, CDCl3, δ, ppm): 101.66; UV–vis [λmax in nm, (ε in M−1cm−1)]: 384 (71254), 436 (106058). COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Then solution in hexane was passed through the Al2O3 column. Then the column was run by hexane/ether (80:20 v/v) mixture which resulted to brown-red solid. The brown-red solid was then re-dissolved in CH2Cl2 and was chromatographed on silica gel TLC plates. Elution with a hexane/CH2Cl2 (40:60 v/v) mixture which yielded molecu- larly pure compound (2a) in 41 % yield (0.124 g based on Pd). Synthesis of [PdAg20{S2PPh2}12] (2c) Synthesis of [PdAg20{S2PPh2}12] (2c) Ligand exchange method. In an oven-dried Schlenk tube, [PdAg20{S2P(OnPr)2}12], 1 (0.025 g, 0.005 mmol) was dissolved in THF (10 mL) and was placed at −20 °C for 5 min. Then K[S2PPh2] (0.019 g, 0.065 mmol) was added to the solution. The resulting reaction mixture was allowed to stir for 2 min at the same temperature. The solvent was then evaporated under vacuum. The obtained residue was extracted with hexane (3 × 5 mL) and ﬁltered to eliminate by-product NH4[S2P(OnPr)2]. The hexane solution was passed through Al2O3 column followed by ether. Then the pure orange compound (2c) obtained was dried which produced 65 % yield (0.017 g based on Pd). [PdAg20{Se2P(O Pr)2}12] (4) in 75% yields (0.022 g based on Pd). 4: ESI-MS(m/z) [M + Ag]+ calcd for C72H168Ag21O24P12PdSe24, 6056.48; found 6056.02; 1H NMR (22 °C, 400 MHz, d6-acetone, δ, ppm): 0.90 (t, 72H, CH3), 1.59 (q, 48H, CH2), 3.85(m, 48, CH2); 31P{1H} NMR (22 °C, 161.9 MHz, d6-acetone, δ, ppm): 73.59 (1JP-Se = 604.51 and 710.40 Hz); UV–vis [λmax in nm, (ε in M−1cm−1)]: 408(95700), 498(68700). [ g20{ 2 ( )2}12] ( ) y ( g ) 4: ESI-MS(m/z) [M + Ag]+ calcd for C72H168Ag21O24P12PdSe24, 6056.48; found 6056.02; 1H NMR (22 °C, 400 MHz, d6-acetone, δ, ppm): 0.90 (t, 72H, CH3), 1.59 (q, 48H, CH2), 3.85(m, 48, CH2); 31P{1H} NMR (22 °C, 161.9 MHz, d6-acetone, δ, ppm): 73.59 (1JP-Se = 604.51 and 710.40 Hz); UV–vis [λmax in nm, (ε in M−1cm−1)]: 408(95700), 498(68700). y g 2c. ESI-MS(m/z) [M + 2Ag]2+ calcd. for C144H120Ag20P12PdS24, 2735.89; found, 2735.76; 1H NMR (22 °C, 300 MHz, CDCl3, δ, ppm). 7.44, 7.85(m,120 H, C6H5); 31P{1H} NMR (22 °C, 121.49 MHz, CDCl3, δ, ppm): 63.14; UV–vis [λmax in nm, (ε in M−1cm−1)]: 419 (44872), 486 (28290). Single crystal X-ray structure determination. Single crystals sui- table for X-ray diffraction analysis of 2a, 2b, 3, (3a and 3b) and 4 were obtained by diffusing hexane into concentrated CH2Cl2 or acetone solution at room and (or) low temperature within one or two weeks. The single crystals were mounted on the tip of glass ﬁber coated in paratone oil, and then frozen. Data were collected on a Bruker APEX II CCD diffractometer using graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 150 K (2a, 3, (3a and 3b)) and 100 K (2b and 4). Synthesis of [PdAg20{S2P(OiBu)2}12] (2b). acetone resulted the wine-red colored solution. Then the solution was then evaporated for further analysis. Suitable single crystals for X-ray diffraction were grown at low and ambient tempera- tures. The crystallization in the ambient temperature revealed a co-crystallization of 3a and 3b with chemical formula [PdAg20{Se2P(OiPr)2}12]. The yield of 3a·3b was 78 % (0.096 g based on Pd). a) Direct method. In an oven-dried Schlenk ﬂask, [Ag(CH3CN)4](PF6) (0.5 g, 1.2 mmol) was suspended in THF (15 mL). To this NH4[S2P(OiBu)2] (0.155 g, 0.6 mmol), and [Pd{S2P(OiBu)2}] (0.030 g, 0.086 mmol) were added one after another. Then the reaction ﬂask was placed in low temperature bath at −20 °C for 15 min. LiBH4 ∙THF (0.2 mL, 0.8 mmol) was added slowly via syringe to the reaction mixture and then the resulting solution was stirred at the same temperature for 24 h. The orange colored solution instantaneously turned to black after the addition of LiBH4 ∙THF. The solvent was dried completely under vacuum. Then the residue was washed thoroughly with deionized water followed by the extraction in CH2Cl2. The resulting CH2Cl2 solution was dried and then the residue was further dissolved in hexane. That solution in hexane was passed through the Al2O3 column. Then the column was run by hexane/ether (80:20 v/v) mixture which resulted to red solid. Moreover the red solid was re-dissolved in CH2Cl2 and was chromatographed on silica gel TLC plates. Elution with a hexane/CH2Cl2 (40:60 v/v) mixture which yielded molecularly pure com- pound (2b) in 40 % yield (0.177 g based on Pd). Compound 2b can also be synthesized via ligand replacement method but the crystalline materials can only be obtained by a co- reduction method. Note that in this metiod, compound 1 was treated with twelve equivalents of NH4[S2P(OiBu)2] in THF at ambient temperature for 10 min. 3a. ESI-MS(m/z) [M + Ag]+ calcd. for C72H168Ag21O24P12PdSe24, 6056.48; found 6055.77; 1H NMR (22 °C, 400 MHz, d6-Acetone, δ, ppm): 5.07–4.95 (q, 24H, OCH), 1.47–1.38 (m, 144H, CH2); 31P{1H} NMR (22 °C, 161. 9 MHz, CDCl3, δ, ppm): 68.16 (JP-Se = 604.51 and 710.40 Hz; UV–vis [λmax in nm, (ε in M−1cm−1)]: 410 (1033762), 501 (690654). Alternative synthesis of 3 or 3a. In a Flame-dried Schlenk tube, [PdAg20{S2P(OiPr)2}12], 2a (0.100 g, 0.0207 mmol) was dissolved in THF (5 mL) and was placed at −20 °C for 15 min. Synthesis of [PdAg20{S2P(OiBu)2}12] (2b). Then twelve equivalents of NH4[Se2P(OiPr)2] (0.080 g, 0.2484 mmol) was added to the solution. The resulting mixture was stirred for 2 min at the same temperature. The solvent was dried under vacuum and residue was then extracted with hexane (3 × 5 mL) and ﬁl- tered to remove decomposed impurities from ligand. In this reaction NH4[S2P(OnPr)2] has been isolated as byproduct. The hexane solution was passed through Al2O3 column followed by acetone which exclusively led to the isolation of compound 3 in 73% yields (0.090 g based on Pd). y g 3 or 3a. ESI-MS(m/z) [M + Ag]+ calcd for C72H168Ag21O24P12PdSe24, 6056.48; found 6055.77; 1H NMR (22 °C, 400 MHz, d6-Acetone, δ, ppm): 5.07–4.95 (q, 24H, OCH), 1.47–1.38 (m, 144H, CH2); 31P{1H} NMR (22 °C, 161. 9 MHz, CDCl3, δ, ppm): 68.16 (JP-Se = 604.51 and 710.40 Hz); UV–vis [λmax in nm, (ε in M−1cm−1)]: 410 (1033762), 501 (690654). y g 3 or 3a. ESI-MS(m/z) [M + Ag]+ calcd for C72H168Ag21O24P12PdSe24, 6056.48; found 6055.77; 1H NMR (22 °C, 400 MHz, d6-Acetone, δ, ppm): 5.07–4.95 (q, 24H, OCH), 1.47–1.38 (m, 144H, CH2); 31P{1H} NMR (22 °C, 161. 9 MHz, CDCl3, δ, ppm): 68.16 (JP-Se = 604.51 and 710.40 Hz); UV–vis [λmax in nm, (ε in M−1cm−1)]: 410 (1033762), 501 (690654). 3 2b. ESI-MS(m/z) [M + Ag]+ calcd. for C96H216Ag21O24P12PdS24, 5267.62; found, 5267.64; 1H NMR (22 °C, 300 MHz, CDCl3, δ, ppm). 3.91 (t, J = 6.76 Hz, 48H), 2.00 (m, J = 6.08 Hz, 24H), 0.94 (d, J = 6.12 Hz, 144H); 31P{1H} NMR (22 °C, 161.9 MHz, CDCl3, δ, ppm); 103.85 UV–vis [λmax in nm, (ε in M−1cm−1)]: 384 (25994), 436 (47813). 2b. ESI-MS(m/z) [M + Ag]+ calcd. for C96H216Ag21O24P12PdS24, 5267.62; found, 5267.64; 1H NMR (22 °C, 300 MHz, CDCl3, δ, ppm). 3.91 (t, J = 6.76 Hz, 48H), 2.00 (m, J = 6.08 Hz, 24H), 0.94 (d, J = 6.12 Hz, 144H); 31P{1H} NMR (22 °C, 161.9 MHz, CDCl3, δ, ppm); 103.85 UV–vis [λmax in nm, (ε in M−1cm−1)]: 384 (25994), 436 (47813). Synthesis of [PdAg20{Se2P(OnPr)2}12] (4). In an oven-dried Schlenk tube, [PdAg20{S2P(OnPr)2}12], 1 (0.025 g, 0.005 mmol) was dissolved in THF (5 mL) and was placed at −20 °C for 15 min. Then twelve equivalents of NH4[Se2P(OnPr)2] (0.021 g, 0.065 mmol) was added to the solution. The resulting mixture was stirred for 2 min in the same temperature. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Experimental methods p Reagents and Instrumentation. The reactions were carried out by using standard Schlenk techniques under N2 atmosphere. LiBH4 COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem 8 Synthesis of [PdAg20{S2PPh2}12] (2c) Absorption corrections for area detector were performed with SADABS49 and the integration of raw data frame was performed with SAINT50. The structure was solved by direct methods and reﬁned by least-squares against F2 using the SHELXL-2018/3 package51,52, incorporated in SHELXTL/PC V6.1453. All non-hydrogen atoms were reﬁned Synthesis of [PdAg20{S2P(OiBu)2}12] (2b). The solvent was evaporated under vacuum and residue was extracted with hexane (3 × 5 mL) and ﬁltered to remove decomposed impurities from ligand. In this reaction NH4[S2P(OnPr)2] has been isolated as byproduct. The hexane solution was passed through Al2O3 col- umn followed by acetone which exclusively led to the isolation of [PdAg20{Se2P(OnPr)2}12] (4) in 75% yields (0.022 g based on Pd). Synthesis of [PdAg20{Se2P(OiPr)2}12] (3, 3a and 3b) Synthesis of [PdAg20{Se2P(OiPr)2}12] (3, 3a and 3b) y [ g20{ 2 ( )2}12] ( , ) Synthesis of 3a and 3b. In a Flame-dried Schlenk tube, [PdAg20{S2P(OnPr)2}12], 1 (0.100 g, 0.0207 mmol) was dissolved in THF (5 mL) and was placed at −20 °C for 15 min. Twelve equivalents of NH4[Se2P(OiPr)2] (0.080 g, 0.2484 mmol) was added to that solution. The resulting mixture was stirred for 2 min in the same temperature. The solvent was dried completely under vacuum and residue was extracted in hexane (3×5 mL) and ﬁltered to remove decomposed impurities from ligand. In this reaction NH4[S2P(OnPr)2] has been isolated as byproduct. The solution was passed through Al2O3 followed by the addition 9 COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 Table 3 Crystallographic data for compounds 2a-b, 3, 3a-b, and 4. Compound 2a 2b 3 3a and 3b 4 Empirical formula C72H168Ag20O24P12PdS24 C96H216Ag20O24P12Pd S24 C72H168Ag20O24P12Pd Se24 C72H168Ag20O24P12PdSe24 C72H168Ag20O24P12PdSe24 Crystal system, space group Triclinic, P1 Hexagonal, P 63 Monoclinic, Pn Trigonal, P31c Monoclinic, P21 a (Å) 15.8599(12) 19.2447(7) 17.9414(11) 22.5252(9) 15.9494(6) b (Å) 18.0946(14) 19.2447(7) 26.7432(15) 22.5252(9) 29.3080(12) c (Å) 29.382(2) 26.1772(12) 18.8174(11) 40.401(2) 16.4792(7) α (°) 82.1487(15) 90 90 90 90 β (°) 77.2002(15) 90 113.2950(10) 90 107.4809(10) γ (°) 66.2229(13) 120 90 120 90 V(Å3) 7514.0(10) 8396.1(7) 8292.8(8) 17752.5(17) 7347.4(5) Z 2 2 2 4 2 ρcalcd, g·cm−3 2.132 2.041 2.382 2.226 2.689 μ, mm−1 3.166 2.841 7.834 7.319 8.842 Temperature, K 150(2) 100(2) 150(2) 150(2) 100(2) θmax, deg./ completeness, % 25.00/95.8 24.999/100 25.00/99.6 24.99/99.9 24.99 / 99.9 Reﬂections collected/unique 44,057/25,382 [R(int) = 0.0237] 58,687/7962 [R(int) = 0.1157] 48,427/22,452 [R(int) = 0.0217] 113,818/ 20,789 [R(int) = 0.0863] 48,724/25,686 [R(int) = 0.0512] Restraints/ parameters 459/1432 582/680 818/1550 712/967 456/1440 R1a, wR2b [I > 2σ(I)] 0.0668,0.1355 0.0798, 0.1909 0.0342, 0.0887 0.1125,0.3046 0.0399,0.0971 R1a, wR2b (all data) 0.0867,0.1511 0.1036, 0.2320 0.0387, 0.0919 0.1291,0.3191 0.0477,0.1046 Absolute structure parameter – 0.40(18) 0.037(9) 0.18(3) 0.327(9) Goodness of ﬁt on F2 1.084 1.102 1.031 1.037 1.023 Largest diff. peak and hole, e/Å 3 1.897 and −1.680 1.686 and −1.393 1.572 and −0.866 2.540 and −2.782 1.750 and −1.625 aR1 ¼ ΣjjFoj jFcjj=ΣjFoj. bwR2 ¼ fΣ½wðF2 o F2 c Þ 2=Σ½wðF2 oÞ2g 1=2 . tallographic data for compounds 2a-b, 3, 3a-b, and 4. ARTICLE ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-022-00769-2 anisotropically. The compound 2a was crystallized in P1 space group; one of the isopropyl groups (C4-C6) was disordered over two positions with the same occupancy. The compound 3 is crystallized in Pn space group and one of the isopropyl groups (C16-C18) was disordered over two positions, reﬁned with the same occupancy; 3a and 3b were co-crystallized in P31c space group where two Se atoms (Se9 and Se14) of the ligands were disordered over two positions with 90% and 10% occupancy and one of the isopropyl groups (C13-C15) was disordered over two positions with the same occupancy. Crystallographic data for compounds 2a-b, 3, 3a-b, and 4 have been listed below in Table 3. The X-ray crystallographic coordinates for structures reported in this Article have been deposited at the Cambridge Crystal- lographic Data Center (CCDC), under deposition number CCDC 1985874 (2a), CCDC 2189558 (2b), CCDC 1985873 (3), CCDC 1985872 (3a-b), and CCDC 1985875 (4). These data can be obtained free of charge from The Cambridge Crystallographic Data Center via www.ccdc.cam.ac.uk/data_request/cif.” The crystallographic information ﬁles for compounds 2a-b, 3, 3a-b, and 4 are provided as Supplementary Data 2. 5. Wang, M. et al. Au25(SG)18 as a ﬂuorescent iodide sensor. Nanoscale 4, 4087–4090 (2012). 6. Murphy, C. J. et al. Photoluminescence-based correlation of semiconductor electric ﬁeld thickness with adsorbate Hammett substituent constants. Adsorption of aniline derivatives onto cadmium selenide. J. Am. Chem. Soc. 112, 8344–8348 (1990). 7. Murray, R. W. Nanoelectrochemistry: metal nanoparticles, nanoelectrodes, and nanopores. Chem. Rev. 108, 2688–2720 (2008). 8. 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[Ag48(C≡CtBu)20(CrO4)7]: An atomically precise silver nanocluster co-protected by inorganic and organic ligands. J. Am. ARTICLE Chem. Soc. 141, 4460–4467 (2019). Computational details. DFT calculations were carried out on simpliﬁed model clusters with the Gaussian 16 package54. The considered ligand simpliﬁcations allow to save a huge amount of CPU time and its validity has been proven in many occasions20–22,36,41,43. The BP86 functional55,56 was used together with the general triple-ζ polarized Def2-TZVP basis set from EMSL Basis Set Exchange Library57,58. All the optimized geometries were ascertained as true minima on the potential energy surface by performing vibrational frequency calculations. The natural atomic orbital (NAO) charges were computed with the NBO 6.0 program59. The UV–visible transitions were calculated on the above-mentioned optimized geometries by means of time- dependent DFT (TD-DFT) calculations, with the CAM-B3LYP functional60 and the LanL2DZ + pol61–63 basis set. The UV–visible spectra were simulated from the computed TD-DFT transitions and their oscillator strengths by using the SWizard code64, each transition being associated with a Gaussian function of half-height width equal to 1000 cm−1. The Cartesian coordinates for computed compounds are provided as Supplementary Data 3. 15. Dhayal, R. S., Van Zyl, W. E. & Liu, C. W. Polyhydrido copper clusters: synthetic advances, structural diversity, and nanocluster-to-nanoparticle conversion. Acc. Chem. Res. 49, 86–95 (2016). 16. Qu, M. et al. Bidentate phosphine-assisted synthesis of an all-alkynyl- protected Ag74 nanocluster. J. Am. Chem. Soc. 139, 12346–12349 (2017). h l l b l d b l l l d ll 17. Kang, X. & Zhu, M. Metal nanoclusters stabilized by selenol ligands. 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They are also from the author (C.W.L.) upon reasonable request. 27. Chakraborty, I. et al. Ag44(SeR)30: A hollow cage silver cluster with selenolate protection. J. Phys. Chem. Lett. 4, 3351–3355 (2013). 28. Liu, C. W. et al. [Ag7(H){E2P(OR)2}6] (E = Se, S): Precursors for the fabrication of silver nanoparticles. Inorg. Chem. 52, 2070–2077 (2013). 29. Zhong, Y.-J. et al. A new synthetic methodology in the preparation of bimetallic chalcogenide clusters via cluster-to-cluster transformations. Molecule 26, 5391–5403 (2021). 30. Liu, C. W. et al. Stable silver(I) hydride complexes supported by diselenophosphate ligands. Inorg. Chem. 49, 468–475 (2010). Received: 18 July 2022; Accepted: 3 November 2022; Received: 18 July 2022; Accepted: 3 November 2022; 31. Liu, C. W. et al. Selenium-centered undecanuclear silver cages surrounded by iodo and dialkyl diselenophosphato ligands: syntheses, structures and photophysical properties. Inorg. Chem. 45, 2335–2340 (2006). 32. Liu, C. W. et al. 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Synthesis of [PdAg20{Se2P(OiPr)2}12] (3, 3a and 3b) 2a 2b 3 3a and 3b 4 a C72H168Ag20O24P12PdS24 C96H216Ag20O24P12Pd S24 C72H168Ag20O24P12Pd Se24 C72H168Ag20O24P12PdSe24 C72H168Ag20O24P12PdSe24 Triclinic, P1 Hexagonal, P 63 Monoclinic, Pn Trigonal, P31c Monoclinic, P21 15.8599(12) 19.2447(7) 17.9414(11) 22.5252(9) 15.9494(6) 18.0946(14) 19.2447(7) 26.7432(15) 22.5252(9) 29.3080(12) 29.382(2) 26.1772(12) 18.8174(11) 40.401(2) 16.4792(7) 82.1487(15) 90 90 90 90 77.2002(15) 90 113.2950(10) 90 107.4809(10) 66.2229(13) 120 90 120 90 7514.0(10) 8396.1(7) 8292.8(8) 17752.5(17) 7347.4(5) 2 2 2 4 2 2.132 2.041 2.382 2.226 2.689 3.166 2.841 7.834 7.319 8.842 150(2) 100(2) 150(2) 150(2) 100(2) % 25.00/95.8 24.999/100 25.00/99.6 24.99/99.9 24.99 / 99.9 e 44,057/25,382 [R(int) = 0.0237] 58,687/7962 [R(int) = 0.1157] 48,427/22,452 [R(int) = 0.0217] 113,818/ 20,789 [R(int) = 0.0863] 48,724/25,686 [R(int) = 0.0512] 459/1432 582/680 818/1550 712/967 456/1440 0.0668,0.1355 0.0798, 0.1909 0.0342, 0.0887 0.1125,0.3046 0.0399,0.0971 0.0867,0.1511 0.1036, 0.2320 0.0387, 0.0919 0.1291,0.3191 0.0477,0.1046 – 0.40(18) 0.037(9) 0.18(3) 0.327(9) on 1.084 1.102 1.031 1.037 1.023 k 1.897 and −1.680 1.686 and −1.393 1.572 and −0.866 2.540 and −2.782 1.750 and −1.625 ΣjFoj. 2 c Þ 2=Σ½wðF2 oÞ2g 1=2 . 3 Crystallographic data for compounds 2a-b, 3, 3a-b, and 4. und 2a 2b 3 3a and 3b 4 al formula C72H168Ag20O24P12PdS24 C96H216Ag20O24P12Pd S24 C72H168Ag20O24P12Pd Se24 C72H168Ag20O24P12PdSe24 C72H168Ag20O24P12PdSe24 system, roup Triclinic, P1 Hexagonal, P 63 Monoclinic, Pn Trigonal, P31c Monoclinic, P21 15.8599(12) 19.2447(7) 17.9414(11) 22.5252(9) 15.9494(6) 18.0946(14) 19.2447(7) 26.7432(15) 22.5252(9) 29.3080(12) 29.382(2) 26.1772(12) 18.8174(11) 40.401(2) 16.4792(7) 82.1487(15) 90 90 90 90 77.2002(15) 90 113.2950(10) 90 107.4809(10) 66.2229(13) 120 90 120 90 7514.0(10) 8396.1(7) 8292.8(8) 17752.5(17) 7347.4(5) 2 2 2 4 2 ·cm−3 2.132 2.041 2.382 2.226 2.689 −1 3.166 2.841 7.834 7.319 8.842 ature, K 150(2) 100(2) 150(2) 150(2) 100(2) eg./ teness, % 25.00/95.8 24.999/100 25.00/99.6 24.99/99.9 24.99 / 99.9 ons d/unique 44,057/25,382 [R(int) = 0.0237] 58,687/7962 [R(int) = 0.1157] 48,427/22,452 [R(int) = 0.0217] 113,818/ 20,789 [R(int) = 0.0863] 48,724/25,686 [R(int) = 0.0512] nts/ ters 459/1432 582/680 818/1550 712/967 456/1440 2b I)] 0.0668,0.1355 0.0798, 0.1909 0.0342, 0.0887 0.1125,0.3046 0.0399,0.0971 2b a) 0.0867,0.1511 0.1036, 0.2320 0.0387, 0.0919 0.1291,0.3191 0.0477,0.1046 e re ter – 0.40(18) 0.037(9) 0.18(3) 0.327(9) ss of ﬁt on 1.084 1.102 1.031 1.037 1.023 diff. peak e, e/Å 3 1.897 and −1.680 1.686 and −1.393 1.572 and −0.866 2.540 and −2.782 1.750 and −1.625 Foj jFcjj=ΣjFoj. Σ½ ðF2 F2Þ 2=Σ½ ðF2Þ g 1=2 COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem 10 COMMUNICATIONS CHEMISTRY \| (2022) 5:151 \| https://doi.org/10.1038/s42004-022-00769-2 \| www.nature.com/commschem References 1. Jin, R. et al. Atomically precise colloidal metal nanoclusters and nanoparticles: fundamentals and opportunities. Chem. Rev. 116, 10346–10413 (2016). 2. Daniel, M.-C. & Astruc, D. Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem. Rev. 104, 293–346 (2004). 3. Luo, J. et al. Catalytic activation of core-shell assembled gold nanoparticles as catalyst for methanol electrooxidation. Catal. Today 77, 127–138 (2002). 4. Yang, X. et al. Gold nanomaterials at work in biomedicine. Chem. Rev. 115, 10410–10488 (2015). 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Novel silver diselenophosphate clusters: structures of Ag10(µ10-Se)[Se2P(OEt)2]8 and {Ag[Se2P(OPri)2]}6. J. Chem. Soc., Dalton Trans. 1974–1979 (2002). C.W.L. conceived and designed the project. S.K.B. synthesized compounds 2a (via ligand exchange method), 3, 3a and 3b. C.-Y.C. synthesized compounds 2a (via direct method), C.W.L. conceived and designed the project. S.K.B. synthesized compounds 2a (via ligand exchange method), 3, 3a and 3b. C.-Y.C. synthesized compounds 2a (via direct method), 2c and 4. Y.-R.N. synthesized compounds 2b. T.-H.C. performed X-ray crystallography. F.G., I.C., S.K. and J.-Y.S. performed DFT calculations. S.K.B., J.-Y.S. and C.W.L. co- wrote the paper with input from all co-authors. All the authors proofread and approved exchange method), 3, 3a and 3b. C.-Y.C. synthesized compounds 2a (via direct method), 2c and 4. Y.-R.N. synthesized compounds 2b. T.-H.C. performed X-ray crystallography. F.G., I.C., S.K. and J.-Y.S. performed DFT calculations. S.K.B., J.-Y.S. and C.W.L. co- 2c and 4. Y.-R.N. synthesized compounds 2b. T.-H.C. performed X-ray crystallography. F.G., I.C., S.K. and J.-Y.S. performed DFT calculations. S.K.B., J.-Y.S. and C.W.L. co- 46. Artem’ev, A. V. et al. Facile atom-economic Synthesis of ammonium diselenophosphinates via three-component reaction of secondary phosphines, elemental selenium, and ammonia. Synthesis 11, 1777–1780 (2010). wrote the paper with input from all co-authors. All the authors proofread and approved the ﬁnal manuscript for submission. 47. Alyl, A. A. M., Walfortn, B. & Lang, H. Z. Crystal structure of tetrakis(acetonitrile)silver(I) tetraﬂuoroborate, [Ag(CH3CN)4][BF4]. Z. Fur Krist. N. Cryst. Struct. 219, 489–491 (2004). p g The authors declare no competing interests. 48. Yordanov, N. D. et al. 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W2088717921.txt	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0052200&type=printable	en		Gut Contents as Direct Indicators for Trophic Relationships in the Cambrian Marine Ecosystem	PloS one	2,012	cc-by	10,873	Gut Contents as Direct Indicators for Trophic Relationships in the Cambrian Marine Ecosystem Jean Vannier* Laboratoire de géologie de Lyon: Terre, Planètes, Environnement, Université de Lyon, Université Lyon 1, Villeurbanne, France Abstract Present-day ecosystems host a huge variety of organisms that interact and transfer mass and energy via a cascade of trophic levels. When and how this complex machinery was established remains largely unknown. Although exceptionally preserved biotas clearly show that Early Cambrian animals had already acquired functionalities that enabled them to exploit a wide range of food resources, there is scant direct evidence concerning their diet and exact trophic relationships. Here I describe the gut contents of Ottoia prolifica, an abundant priapulid worm from the middle Cambrian (Stage 5) Burgess Shale biota. I identify the undigested exoskeletal remains of a wide range of small invertebrates that lived at or near the water sediment interface such as hyolithids, brachiopods, different types of arthropods, polychaetes and wiwaxiids. This set of direct fossil evidence allows the first detailed reconstruction of the diet of a 505-million-year-old animal. Ottoia was a dietary generalist and had no strict feeding regime. It fed on both living individuals and decaying organic matter present in its habitat. The feeding behavior of Ottoia was remarkably simple, reduced to the transit of food through an eversible pharynx and a tubular gut with limited physical breakdown and no storage. The recognition of generalist feeding strategies, exemplified by Ottoia, reveals key-aspects of modern-style trophic complexity in the immediate aftermath of the Cambrian explosion. It also shows that the middle Cambrian ecosystem was already too complex to be understood in terms of simple linear dynamics and unique pathways. Citation: Vannier J (2012) Gut Contents as Direct Indicators for Trophic Relationships in the Cambrian Marine Ecosystem. PLoS ONE 7(12): e52200. doi:10.1371/ journal.pone.0052200 Editor: Andrew A. Farke, Raymond M. Alf Museum of Paleontology, United States of America Received August 11, 2012; Accepted November 12, 2012; Published December 26, 2012 Copyright: ß 2012 Jean Vannier. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The author’s research is supported by ANR (Agence Nationale de la Recherche) grants (ORECO and RALI) and by the European Assemble Project (2010, Sven Lovén Centre for Marine Sciences at Kristineberg, Sweden). ANR website at: www.agence-nationale-recherche.fr, ASSEMBLE (Association of European Marine Biological Laboratories): www.assemblemarine.org. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The author has declared that no competing interests exist. * E-mail: jean.vannier@univ-lyon1.fr skeletal elements from the Cambrian of California, Utah, Canada (Burgess Shale) and Australia [30] may have been produced by arthropods with robust gnathobasic appendages such as Sidneyia [31]. Rare fossil associations [32] and trace fossils [33] have suggested possible hunting or scavenging behaviors but these relationships require quantification. Qualitative and quantitative analyses of the communities from the Burgess Shale [2,34] and the Maotianshan Shale [35–37] have provided detailed information on the diversity of ecological types and the presumed organization of the early and middle Cambrian ecosystems but do not tell us about the exact trophic links between species. Recent theoretical models [38] have predicted strong similarities between the trophic organization of Cambrian food webs and modern ones but lack detailed testing by fossil evidences. By contrast, the analysis of gut contents presented here and exemplified by the priapulid worm Ottoia prolifica from the middle Cambrian Burgess Shale provides direct and detailed evidence for trophic relationships and new insights both into the actual diet and feeding behavior of Cambrian animals. The case of priapulids reveals the potential of a source of information that has long been considered as relatively limited and anecdotal [39,40]. A noticeable exception though is S. Conway Morris’ comprehensive work [39] on the priapulid worms from the Burgess Shale in which the gut contents of Ottoia are first described. This pioneer work is important in that it led to the concept of Ottoia as an iconic Cambrian predator and Introduction The study of exceptionally preserved Cambrian biotas [e.g., Burgess Shale [1,2], Chengjiang [3,4], Sirius Passet [5–7] and Emu Bay Shale [8–10] has led to accurate reconstructions of the anatomy, lifestyles [11–13], visual properties [10], and even behaviors [14,15] of early animals. However, information is lacking concerning their interactions within the food chain and their diet. The functioning of the Cambrian ecosystem has mainly been addressed through a combination of indirect fossil evidence supported by modern analogues [16]. Typically, the feeding types (e.g. predation vs. particle-feeding) and strategies (sediment-eating vs. carnivory) of most Cambrian animals have been inferred from the morphofunctional analysis of their food-gathering apparatuses/limbs [17–20] and digestive systems [21]. The predatory habit of anomalocaridids, for example, is supported by evidence from their frontal appendages, mouth apparatus [22–24] and sophisticated eyes [10], but there is no direct evidence of what organisms they actually preyed upon. Mechanical models using finite element analysis [25] and recent studies of the oral cone [26] contradict the view that anamolocaridids were durophagous predators able to perform strong biting motions and to inflict wounds on hard exoskeletons [24,27,28]. The contents from coprolites [29] provide a degree of trophic resolution but cannot be tied to particular predators although some coprolites composed entirely of crushed PLOS ONE \| www.plosone.org 1 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia eous, siltstones and silty graphitic mudstones, typically with a weathered horizontally-banded appearance; and 2) the slightly younger Raymond Quarry Member, characterized by grey, greenish and brown layered blocky-slaty mudstone [1,2,42–44]. The Ottoia specimens kept in the collections of the National Museum of Natural History, Smithsonian Institution, Washington D.C. (USNM), all come from excavations at Walcott’s original site (the so-called Phyllopod Bed within the Walcott Quarry Member). Those from the Royal Ontario Museum (ROM) collections, Toronto, were collected from both the Raymond and Walcott Quarry Members (RQ, RT and WQ, WT numbers respectively) in successive seasons of excavations and talus picking (RT, WT) between 1975 and 2000 by Royal Ontario Museum parties led by D. Collins. Altogether more than 2,600 specimens of Ottoia prolifica formed the basis of my study. My results also invite reassessment of the function and the complexity of Cambrian marine food webs where animals, for the first time in their history, played a major role in the transfer of mass and energy. The interpretations here also challenge the notion of strict feeding regimes and linear food chain and provide support for a marine trophic web where energy flow circulated via multiple animal interactions and parallel pathways [41], as it does in present-day ecosystems. Materials and Methods Our fossil material comes from two stratigraphic horizons in the middle Cambrian Burgess Shale Member: 1) the Walcott Quarry Member, characterized by fossiliferous, finely laminated, calcar- Figure 1. Count data and composition of the gut contents of Ottoia prolifica, from the middle Cambrian Burgess Shale Formation (Series 3, Stage 5; see [45]). The pie diagrams illustrate differences in the diet of Ottoia from the Raymond Quarry (RQ+RT) and the Walcott (WQ+WT) Quarry. Hyolithids dominate in the gut contents from the Raymond Quarry followed in decreasing order by brachiopods, agnostids, trilobites, bradoriids, ASE (presumed wiwaxiids), SLE (presumed polychaetes) and trilobites. In the Walcott Quarry, three almost equally represented groups (SLE, hyolithids and ASE) are prevalent, followed by bradoriids, trilobites, agnostids and brachiopods. (1) guts containing skeletal fragments and/or undetermined material and a variable proportion of sediment; (2) guts containing skeletal elements or fragments that belong to animal species present in the Burgess Shale biota; (3) guts containing elements that belong to a single species (e.g. only hyolithid skeletal elements). Empty guts generally appear as a colored or reflective strip running axially from the pharynx to the anus. ASE, almond-shape elements (presumed wiwaxiid sclerites); RQ, RT, collection specimens from the Raymond Quarry and talus (Royal Ontario Museum); SLE, setae-like elements (presumed polychaete chaetae); USNM, collection specimens from the National Museum of Natural History, Smithsonian Institution, Washington D.C.; WQ, WT, collection specimens from the Walcott Quarry and talus (Royal Ontario Museum). Raw data in Table S1. doi:10.1371/journal.pone.0052200.g001 PLOS ONE \| www.plosone.org 2 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 2. General morphology of Ottoia prolifica from the middle Cambrian Burgess Shale. A, ROM 61780a, high concentration of complete specimens. B–D, ROM 61759, ROM 61752 and ROM 61757, complete specimens. E, F, ROM 61751 and ROM 61765, details of anterior part. G, ROM 61760, details of introvert bearing curved scalids. H, I, ROM 61769 and ROM 61764, details of posterior part showing bursa and posterior hooks. Abbreviations: a, anus; an, trunk annulation; bu, bursa; gu, gut; in, introvert; m, mouth; ph, posterior hook; pt, pharyngeal teeth; px, pharynx; px(e), everted pharynx; px(i), inverted pharynx; sc, scalid; tr, trunk. Scale bar: 1 cm for A–D and 5 mm for E–I. doi:10.1371/journal.pone.0052200.g002 Figure 3. General morphology of Recent priapulid worms exemplified by Priapulus caudatus collected from near the Kristineberg Marine Station, Gullmarfjord, Sweden, depth ca. 30 m. A, B, general view of a live specimen in sea water and simplified section through body showing major anatomical features. C, section through pharynx (sclerotized pharyngeal teeth in orange; introvert removed. D, F, frontal view of a slightly everted pharynx showing pentagonal pattern of pharyngeal teeth around mouth opening and details of pharyngeal teeth. E, G, scalid rows along bulbous introvert and details of scalids (tip perforated). H–J, feces of Priapulus caudatus filled with compacted undigested material and enclosed by a transparent membrane, bundles of undigested polychaete chaetae and undetermined gut contents (mainly sediment and detritus of various origin). D–G, I, J, are scanning electron micrographs of dessicated specimens. Abbreviations: a, anus; an, trunk annulation; bu, bursa; ca, caudal appendage; cc, coelomic cavity; fc, feces contents; fm, feces membrane; go, gonads; gu, gut; in, introvert; m, mouth; pm, pharyngeal muscles; pt, pharyngeal tooth; px, pharynx; rm, retractor muscle; sc, scalid; sr, scalid row; tr, trunk. Scale bar: 1 cm for A, B; 5 mm for C; 500 mm for D, E, H; 200 mm for F; 100 mm for G; 10 mm for I; 5 mm for J. doi:10.1371/journal.pone.0052200.g003 PLOS ONE \| www.plosone.org 3 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 4. Elemental mapping of the gut of Ottoia prolifica from the middle Cambrian Burgess Shale. The mapping reveals anatomical partitioning of the gut, with elevated C, Fe and P that probably reflects its organic-rich original composition and early diagenetic mineralizations in pyrite, apatite or calcite. A–E, ROM 61758b. A, B, general view under normal and polarized light (white arrow to indicate mapped area). C, D, back scattered image of gut showing patches of carbonaceous film; this film is interpreted as remains of the gut wall, rather than gut contents. E, elemental mapping. Abbreviations: a, anus; cf, carbonaceous film; gc, gut content; gu, gut; m, mouth. Scale bar: 1 cm for A, B; 5 mm for E; 1 mm for C; 20 mm for D. doi:10.1371/journal.pone.0052200.g004 PLOS ONE \| www.plosone.org 4 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia PLOS ONE \| www.plosone.org 5 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 5. Sedimentary ingesta within the gut of Ottoia prolifica from the middle Cambrian Burgess Shale Formation. A–D, USNM 196195, three-dimensionally preserved gut contents, general view and thin section; gut material (C) is easily distinguished from the matrix (D) by its brown colour due to high organic content. Crystals (in white) are not specific to the gut and are observed elsewhere in the matrix though smaller and less concentrated; they are interpreted as sponge spicules [21]. E, F, ROM 61755a, isolated fragment of gut content seen in transverse section. G, H, ROM 61754, gut contents showing small skeletal fragments and undetermined elements embedded in sediment. I, J, ROM 61755a, transverse section through upper part of gut content; the uppermost thin layer possibly represent the gut wall. K, ROM 61755b, thin carbonaceous film overlying gut contents, possibly representing the gut wall. gc, gut contents; se, sediment; sf, skeletal fragment; ue, undetermined element. A–D, courtesy L. Wilson (see also [21]). A, G are light photographs; B and D were taken in transmitted light; E, F, H-K are scanning electron micrographs (K, back-scattered image). Scale bar: 5 mm for A; 2 mm for G; 500 mm for B, H; 100 mm for C, D; 50 mm for K; 10 mm for E, I; 5 mm for F; 2 mm for J. doi:10.1371/journal.pone.0052200.g005 Cambrian System is currently in the process of ratification by the International Union of Geological Sciences (IUGS). The Burgess Shale Formation belongs to Series 3, Stage 5 (see recent provisional chart [45]). For convenience, I maintain usage of ‘‘middle Cambrian’’ for this formation. This research does not involve human participants. I obtained permission to study the Burgess Shale fossil collections from the Royal Ontario Museum (ROM,Toronto) and the Smithsonian National Museum of Natural History (USNM, Washington D.C.) from Jean-Bernard Caron and Douglas Erwin, respectively. The were examined, only a small percentage had preserved gut contents (Fig. 1, Table S1). The recent priapulid Priapulus caudatus was collected from the Gullmar fjord near the Sven Lovén Centre for Marine Sciences at Kristineberg, Sweden and from near The White Sea Biological Station ‘‘Kartesh’’ (WSBS), Russia. Digital photography (with polarizing filters to increase contrast of anatomical features), scanning electron microscopy and Energydispersive X-ray spectroscopy (EDX) analysis were used to study the morphology and chemical composition of the fossil and Recent material. The global chronostratigraphic subdivision of the Table 1. Hyolithid elements in the gut contents of Ottoia prolifica from the Middle Cambrian Burgess Shale: countings and measurements. HYOLITHIDS RQ+RT % WQ+WT Ottoia prolifica with hyolithid conchs 53 100 19 1 conch in gut 33 62.5 10 2 conchs in gut 10 19 7 3 conchs in gut 6 11 4 conchs in gut 3 5 conchs in gut % USNM % ALL % 100 28 100 100 100 53 14 50 57 57 37 8 28.5 25 25 1 5.5 3 11 10 10 5.5 0 0 2 7 5 5 1 2 1 5.5 0 0 2 2 6 conchs in gut 0 0 0 0 1 3.5 1 1 number of hyolithid conchs 88 100 32 100 53 100 173 100 position 1 (anterior) 1 1 2 6.5 4 7.5 7 4 position 2 (mid-anterior) 8 9 3 9.5 6 11.5 17 10 position 3 (mid-posterior) 28 58 8 25 23 43.5 59 34 position 4 (posterior) 51 32 19 59.5 20 37.5 90 52 position 1 (anterior) 1 1 2 6.5 4 7.5 7 4 position 2 (mid-anterior) 8 9 3 9.5 6 11.5 17 10 position 3 (mid-posterior) 28 58 8 25 23 43.5 59 34 orientation of conchs 1 72 82 24 75 37 70 133 77 orientation of conchs 2 16 18 8 25 16 30 40 23 conch length: 0–0.99 mm 1 1 0 0 0 0 1 0.5 conch length: 1–1.99 mm 6 7 5 16 0 0 11 6.5 conch length: 2–2.99 mm 8 9 4 13 4 8 16 9.5 conch length: 3–3.99 mm 17 19.5 6 19.5 12 23 35 20.5 conch length: 4–4.99 mm 15 17 8 26 10 19 33 19.5 conch length: 5–5.99 mm 20 23 6 19.5 12 23 38 22.5 conch length: 6–6.99 mm 8 9 1 3 11 21 20 11.5 conch length: 7–7.99 mm 4 4.5 1 3 1 2 6 3.5 conch length: 8–8.99 mm 5 6 0 0 0 0 5 3 conch length: 9–9.99 mm 2 2 0 0 0 0 2 1 conch length: 10–10.99 mm 0 0 0 0 2 4 2 1 RQ, RT, WQ, WT: collections of the Royal Ontario Museum, Toronto, Raymond Quarry and talus, Walcott Quarry and talus, respectively. USNM, collections of the Smithsonian National Museum of Natural History, Washington D.C. Raw data in Table S1. orientation of conchs 1 = conch apex pointing upwards within the gut of Ottoia; orientation of conchs 2 = conch apex pointing downwards. doi:10.1371/journal.pone.0052200.t001 PLOS ONE \| www.plosone.org 6 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Table 2. Brachiopod elements in the gut contents of Ottoia prolifica from the Middle Cambrian Burgess Shale: countings and measurements. BRACHIOPODS RQ+RT % WQ+WT % USNM % ALL % Ottoia prolifica with brachiopods 15 100 2 100 1 100 18 100 with Micromitra burgessensis 10 67 1 50 0 0 11 61 with Diraphora bellicostata 1 7 0 0 1 100 2 11 with undet. brachiopods 4 26 1 50 0 0 5 18 number of brachiopod valves 18 100 2 100 1 100 21 100 position 1 (anterior) 0 0 0 0 0 0 0 0 position 2 (mid-anterior) 1 5.5 0 0 1 100 2 9.5 position 3 (mid-posterior) 6 33.5 0 0 0 0 6 28.5 position 4 (posterior) 11 61 2 100 0 0 13 62 number of measured valves 18 100 2 100 1 100 21 100 valve width: 0–0.99 mm 1 5.5 1 50 0 0 2 9,5 valve width: 1–1.99 mm 6 33.5 1 50 0 0 7 33.5 valve width: 2–2.99 mm 6 33.5 0 0 0 0 6 28.5 valve width: 3–3.99 mm 3 16.5 0 0 0 0 3 14 valve width: 4–4.99 mm 0 0 0 0 1 100 1 5 valve width: 5–5.99 mm 2 11 0 0 0 0 2 9.5 valve width: 6–6.99 mm 0 0 0 0 0 0 0 0 RQ, RT, WQ, WT: collections of the Royal Ontario Museum, Toronto, Raymond Quarry and talus, Walcott Quarry and talus, respectively. USNM, collections of the Smithsonian National Museum of Natural History, Washington D.C. Raw data in Table S1. doi:10.1371/journal.pone.0052200.t002 pointed conch, an operculum and a pair of curved appendages called helens; Figs. 6A–H, 7). It occurs in 48% of GC that have identifiable elements (Fig. 1). The number of conchs varies from 1 to exceptionally 6; 82% of hyolithid-bearing GC have only 1 or 2 conchs; 62% of the conchs are ca. 3–6 mm long and 0.6–3 mm wide (Table 1). Hyolithids in GC are 3D-preserved and show no visible trace of physical breakdown or chemical dissolution, the conch and the operculum being sometimes connected (Fig. 6D). The very rare presence of helens within GC, either attached or detached from the conch, suggests that the majority of hyolithids became partly disarticulated as they entered the digestive tract of the worm (e.g. by the muscular contractions of pharynx). Helens may have been weakly attached in life, which may account for the low percentage (ca. 7%; [52]) of fully articulated hyolithids in the fossil assemblages. Hyolithid conchs show a remarkably consistent orientation with 77% of them pointing towards the mouth of Ottoia. This indicates that hyolithids were preferentially grasped and drawn into the gut by their anterior side, where they probably offered a stronger grip point to the pharyngeal teeth of Ottoia. (b) Brachiopods. Articulate brachiopods (Table 2) are represented in GC by Micromitra burgessensis [1,53,54] characterized by a very distinctive lozenge-like reticulated pattern (Figs. 6I–K, 8A–F) and, possibly Diraphora [1,53,54], although much more rarely. The best-preserved specimens of Micromitra burgessensis (not in GC) are fringed with long and delicate setae which indicates that the animal did not live buried in the sediment [1] but more likely at the water sediment interface. (c) Arthropods. Arthropod skeletal elements (Table 3) are frequent, represented mainly by agnostids, small trilobites and bradoriids (Figs. 6L–P; 8G–J). The agnostids Ptychagnostus praecurrens [1,55] and possibly Pagetia bootes [1,55] occur as mainly disarticulated exoskeletal elements (anterior and posterior shields, thoracic segments), except for one complete specimen found within the anterior-most section of the gut just behind the pharynx majority of specimens were studied in the ROM and the USNM. A small number of them were obtained on loan and returned. Results Gut Content Analysis As with the majority of non-biomineralizing fossils from the Burgess Shale, Ottoia prolifica is preserved as compressed aluminosilicate and carbonaceous films [46,47] (Fig. 2). Ottoia resembles Recent priapulids [48,49] (Fig. 3) in having a retractile introvert armed with hooks and an invaginable pharynx lined with small teeth, two features of key-importance in locomotion and feeding [50]. The gut of Ottoia appears as a colored or reflective strip of constant width (1.4 to 2.3 mm in specimens 60–100 mm long [21]) running axially from the pharynx to the anus. It is either straight, sinuous or looped. EDX elemental mapping reveals anatomical partitioning of the gut with elevated C, Fe and P that probably reflects its organic-rich original composition and early diagenetic mineralizations in pyrite, apatite or calcite (Fig. 4). More than 50% of the studied specimens possess empty guts (Fig. 1) and about 20% display three-dimensionally preserved gut contents (GC) that preferentially concentrate in the posterior half of their digestive tract. GC typically occur as compacted ribbon-like features or fragmented blobs containing skeletal elements (e.g. hyolithid conchs, brachiopod valves), smaller debris of uncertain origin, and sediment. Thin section, SEM and EDX analyses do not show any significant compositional difference between GC and the aluminosilicate host rock, except from being enriched in organic matter (Fig. 5). Furthermore, acritarchs and sponge spicules found in comparable quantities in GC and the host rock [21] confirm that Ottoia ingested sediment. (a) Hyolithids. The most frequent animal in Ottoia’s GC (Fig. 1, Tables 1, 2, 3, 4 and Table S1) is the hyolithid Haplophrentis carinatus [1,51] characterized by a mineralized exoskeleton with a PLOS ONE \| www.plosone.org 7 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Table 3. Arthropod elements in the gut contents of Ottoia prolifica from the Middle Cambrian Burgess Shale: countings and measurements. 3-BRADORIIDS RQ+RT % WQ+WT % USNM % ALL % Ottoia prolifica with agnostids 6 100 9 100 1 100 17 100 with Pagetia bootes 5 62.5 2 40 1 25 9 47 with Ptychagnostus praecurrens 2 25 2 40 0 0 3 23.5 with undet. agnostids 1 12.5 1 20 3 75 5 29.5 number of agnostid elements 8 100 6 100 6 100 20 100 position 1 (anterior) 2 25 1 16.5 0 0 3 15 position 2 (mid-anterior) 1 12.5 0 0 1 16.5 3 15 position 3 (mid-posterior) 1 12.5 3 50 1 16.5 10 50 position 4 (posterior) 4 50 2 33.5 4 67 4 20 number of measured agnostid elements 8 100 5 100 2 100 15 100 width: 0–0.99 mm 1 12.5 0 0 0 0 1 6.5 width: 1–1.99 mm 4 50 1 20 0 0 5 33.5 width: 2–2.99 mm 3 37.5 1 20 0 0 4 26.5 width: 3–3.99 mm 0 0 1 20 2 100 3 20 width: 4–4.99 mm 0 0 2 40 0 0 2 13.5 width: 5–5.99 mm 0 0 0 0 0 0 0 0 width: 6–6.99 mm 0 0 0 0 0 0 0 0 2- TRILOBITES RQ+RT % WQ+WT % USNM % ALL % Ottoia prolifica with trilobites 1 100 5 100 2 100 8 100 with Ehmaniella waptensis 0 0 2 40 0 0 2 25 with undet. trilobites 1 100 3 60 2 100 6 75 number of trilobite elements 1 100 6 100 2 100 9 100 position 1 (anterior) 0 0 1 16.5 0 0 0 0 position 2 (mid-anterior) 0 0 0 0 1 50 3 33.5 position 3 (mid-posterior) 1 100 1 16.5 0 0 2 22 position 4 (posterior) 0 0 4 67 1 50 4 44.5 number of measured trilobite elements 1 100 5 100 0 0 6 100 width: 0–0.99 mm 0 0 0 0 0 0 0 0 width: 1–1.99 mm 1 100 1 20 0 0 2 33.3 width: 2–2.99 mm 0 0 0 0 0 0 0 0 width: 3–3.99 mm 0 0 2 40 0 0 2 33.3 width: 4–4.99 mm 0 0 2 40 0 0 2 33.3 width: 5–5.99 mm 0 0 0 0 0 0 0 0 width: 6–6.99 mm 0 0 0 0 0 0 0 0 3- BRADORIIDS RQ+RT % WQ+WT % USNM % ALL % Ottoia prolifica with bradoriids 6 100 9 100 1 100 17 100 number of bradoriid elements 6 100 11 100 1 100 22 100 position 1 (anterior) 1 17 2 18 0 0 4 18 position 2 (mid-anterior) 2 33 2 18 1 100 4 18 position 3 (mid-posterior) 1 17 4 36.5 0 0 6 27.5 position 4 (posterior) 2 33 3 27.5 0 0 8 36.5 number of measured valves/carapaces 6 100 11 100 1 100 20 100 valve/carapace length: 0–0.99 mm 0 0 1 9 0 0 1 5 valve/carapace length: 1–1.99 mm 5 83.5 7 64 1 100 16 80 valve/carapace length: 2–2.99 mm 1 16.5 3 27 0 0 3 15 valve/carapace length: 3–3.99 mm 0 0 0 0 0 0 0 0 valve/carapace length: 4–4.99 mm 0 0 0 0 0 0 0 0 PLOS ONE \| www.plosone.org 8 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Table 3. Cont. 3-BRADORIIDS RQ+RT % WQ+WT % USNM % ALL % valve/carapace length: 5–5.99 mm 0 0 0 0 0 0 0 0 valve/carapace length: 6–6.99 mm 0 0 0 0 0 0 0 0 RQ, RT, WQ, WT: collections of the Royal Ontario Museum, Toronto, Raymond Quarry and talus, Walcott Quarry and talus, respectively. USNM, collections of the Smithsonian National Museum of Natural History, Washington D.C. Raw data in Table S1. doi:10.1371/journal.pone.0052200.t003 found in SLE. That SLE are arthropod setae is unlikely because of the lack of tergites, shields or appendages associated with them. SLE are interpreted as the chaetae of the polychaete worm Burgessochaeta setigera [1,59] (Figs. 10G–J, 11) that effectively cooccurs with Ottoia (Table 5). Supporting evidence comes from the high number of chaetae in Burgessochaeta (.1000 attached to more than 20 pairs of biramous parapodia), their size range (diameter 30–90 mm) and frequent groupings in bundles (Fig. 10B–E). The size of SLE is consistent with Ottoia feeding on juveniles of Burgessochaeta (Fig. 11). Polychaete chaetae are frequent in the feces of Recent priapulid worms such as Priapulus (Fig. 3I). (e) Almond-shape elements (ASE). ASE (Table 4) have a consistent almond shape, are slightly convex, and bear at least 6 ribs parallel to their margins (Fig. 12). They typically occur in GC as aligned elements (N = 1 to 12; 34% over 9; Table 1) often overlapping each other. Their length varies from 1.5 to 6 mm (63% between 2 and 3.5 mm). More than 88% of ASE point towards the anus of Ottoia - i.e. - the opposite direction of hyolithid shells in GC (compare with Figs. 6A–H, 7). The only skeletal elements comparable in size, shape and ornament with ASE are the scale-like sclerites of wiwaxiids, especially those of Wiwaxia (Fig. 6L, M). The trilobite Ehmaniella [1,55] is represented by isolated cephalons, pygidia and disarticulated thoracic segments (Figs. 6N–P, 9A–E). The bradoriid Liangshanella burgessensis [56] is a tiny arthropod capped by a dorsally folded shield. Although extremely abundant in the Burgess Shale biota [2], L. burgessensis is a rare element in GC (Fig. 9F, G). Indeterminate bivalved arthropods different from bradoriids also occur as shield-like folded features (Fig. 9I, J). In addition to these readily identifiable undigested remains are setae-like (SLE) and almond-shape (ASE) skeletal elements. (d) Setae-like elements (SLE). SLE generally occur as large concentrations of straight or slightly curved 3D-preserved cylindrical elements (Fig. 10). Their size (length and diameter 50–950 and 17–55 mm, respectively; Fig. 11) is not consistent with a sponge origin (Figs. 11, Fig. S1). Most sponges occurring in the same horizon or associated with Ottoia on the same bedding plane [57,58] have monaxial needle-like elements (diameter between 10 and 20 mm) usually tightly clustered to form tracts or tufts. Pirania has strong radial spicules (length .7 mm and diameter .100 mm). No cross-shaped or rayed structure typical of hexactinellid (e.g. Protospongia) or stem-group calcareous (e.g. Eiffelia) sponges was Table 4. Almond-shape elements (ASE; see Fig. 12) in the gut contents of Ottoia prolifica from the Middle Cambrian Burgess Shale: countings and measurements. ASE RQ+RT % WQ+WT % USNM % ALL % Ottoia prolifica with ASE 5 100 13 100 4 100 22 100 number of ASE 15 100 66 100 26 100 107 100 position 1 (anterior) 0 0 0 0 0 0 0 0 position 2 (mid-anterior) 0 0 0 0 1 4 1 1 position 3 (mid-posterior) 6 40 16 24 9 34.5 31 29 position 4 (posterior) 9 60 50 76 16 61.5 75 70 number of measured ASE 13 100 45 100 16 100 74 100 length: 1–1.49 mm 0 0 0 0 0 0 0 0 length: 1.5–1.99 mm 0 0 6 13 0 0 6 8 length: 2–2.49 mm 2 15 7 15.5 2 12.5 11 15 length: 2.5–2.99 mm 3 23 9 20 4 25 16 22 length: 3–3.49 mm 5 38 10 22 5 31 20 27 length: 3.5–3.99 mm 1 8 4 9 2 12.5 7 9.5 length: 4–4.99 mm 1 8 2 4.5 2 12.5 5 7 length: 5–5.49 mm 0 0 3 7 0 0 3 4 length: 5.–5.49 mm 0 0 2 4.5 0 0 2 2.5 length: 5.5–5.99 mm 1 8 2 4.5 1 6.5 4 5 length: 6. 6.49 mm 0 0 0 0 0 0 0 0 RQ, RT, WQ, WT: collections of the Royal Ontario Museum, Toronto, Raymond Quarry and talus, Walcott Quarry and talus, respectively. USNM, collections of the Smithsonian National Museum of Natural History, Washington D.C. Raw data in Table S1. doi:10.1371/journal.pone.0052200.t004 PLOS ONE \| www.plosone.org 9 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 6. Hyolithids, brachiopods and arthropods within the gut of Ottoia prolifica from the middle Cambrian Burgess Shale. A–C, ROM 61747, with 4 hyolithid shells (Haplophrentis carinatus), their apex pointing anteriorly. D, ROM 61767, hyolithid with operculum and conch in connexion. E, F, ROM 61749, hyolithid conch and a pair of disarticulated helens. G, ROM 61774, hyolithid conch and disarticulated operculum. H, USNM 202777, hyolithid conch within the posteriormost part of the gut (bursa everted). I–K, ROM 61779 with two brachiopods (Micromitra burgessensis) in posterior gut. L, M, ROM 61775 with complete agnostid arthropod (Ptychagnostus praecurrens) within the anterior gut. N, O, ROM 61777 with a trilobite pygidium (Ehmaniella burgessensis) inside the gut. P, ROM 61785 with a trilobite cephalon (E. burgessensis). Abbreviations: a, anus; ag, agnostid; an, trunk annulation, ANT, anterior; ase, almond-shape element; br, brachiopod; br1, br2, from anterior, brachiopod 1 and 2; bu, bursa; ce, cephalon; co, hyolithid conch; gc, gut content; gu, gut; gw, gut wall; h1–h4, from anterior, hyolithid 1 to 4; he, helen; in, introvert; m, mouth; op, hyolithid operculum; ph, posterior hook; POST, posterior; pt, pharyngeal teeth; py, pygidium; tr, trunk. Scale bar: 1 cm for A, I, L; 5 mm for B, C, E, H, M; 2 mm for K; 1 mm for D, F, G, J, N–P. doi:10.1371/journal.pone.0052200.g006 [35] is not confirmed here although this behavior clearly remains plausible (see recent priapulid worms such as Priapulus; [61]). I reexamined this specimen (USNM 198922). The spinules and proboscis hooks that are assumed to be present within its gut are most probably preservational artefacts or due to the chance juxtaposition of two ill-preserved Ottoia specimens as suggested by L. Wilison [21]. Gut contents from the Raymond Quarry are largely dominated by hyolithids, whereas SLE (assumed polychaetes), hyolithids and ASE (assumed wiwaxiids) prevail in GC from the Walcott Quarry (Fig. 1). This suggests that Ottoia was not corrugata [1,60] (Fig. 12J) that co-occurs with Ottoia (Table 5). The relatively low number of ASE in GC, the absence of typical spiny and crescentic elements, and the average size of Wiwaxia (.20 mm vs. gut diameter of Ottoia ,3 mm) is not consistent with wiwaxiids being ingested whole by Ottoia. More likely it suggests that Ottoia fed on decaying wiwaxiids by ingesting lumps of soft tissues where small sclerites were still attached. The consistent orientation of ASE in GC may be explained by both the unidirectional imbricated pattern of the Wiwaxia scleritome [60] and also by capture constraints (see hyolithids). The cannibalistic behavior of Ottoia based on a single poorly preserved specimen PLOS ONE \| www.plosone.org 10 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 7. Hyolithids in the gut of Ottoia prolifica from the middle Cambrian Burgess Shale. A, ROM 61753a, showing 3 hyolithids preserved with their opercule. B–E, ROM 61782, with 5 hyolithid shells (Haplophrentis carinatus) within the gut, their apex pointing anteriorly; general view and details. F–H, USNM 196381, with 6 hyolithid shells within the gut. I, USNM 188604, with 3 hyolithids (h3 close to the anus). Abbreviations: ANT, anterior; an, trunk annulation; co1–co3, hyolithid conch 1 to 3; gc, gut content; gu, gut; he, helen; h1–h6, from anterior, hyolithid 1 to 6; op1–op3, hyolithid operculum 1 to 3; lo, loop; ph, posterior hook; POST, posterior; tr, trunk. Scale bar: 1 cm for B, F, H, I; 5 mm for A, C–E; 2 mm for G. doi:10.1371/journal.pone.0052200.g007 PLOS ONE \| www.plosone.org 11 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 8. Other skeletal elements in the gut of Ottoia prolifica from the Middle Cambrian Burgess Shale. A–D, USNM 196204, with articulate brachiopod, possibly Diraphora bellicostata [1,50,51]. E, ROM 61756, with the inarticulate brachiopod Micromitra burgessensis [1,50,51] and undetermined gut contents. F, ROM 61750 with Micromitra burgessensis and a hyolithid. G, H, ROM 61770 with agnostid (possibly Pagetia bootes [1,52]), general view and close-up. I, J, ROM 61783 with complete agnostid and hyolithid. Abbreviations: ag, agnostid; an, trunk annulation; ANT, anterior; br, brachiopod; ce, cephalon; gc, gut contents; gu, gut; h, hyolithid; op, operculum of hyolithid; ph, posterior hook; POST, posterior; py, pygidium. All light photographs. Scale bar: 1 cm for A, G; 2 mm for B–F, H–J. doi:10.1371/journal.pone.0052200.g008 and storage process. Nutrients were probably chemically extracted from food via digestive fluids produced in the midgut lumen as in Recent priapulids [48]. The assumed low nutritional value of some of the food items such as hyolithids, brachiopods that probably contained less protein-rich tissues than arthropods and worms; [62]) may have been offset by the richer intake of dead tissues from carcasses (Fig. 13). Ottoia lacked visual and complex sensory organs, in contrast with the arthropods from the same horizons that had potential features (e.g. compound eyes, antennae) for visual [10] and chemo-tactile recognition. Attraction to food was probably triggered by chemical cues released from living and dead tissues (Fig. 14A). Chemoreceptors were possibly located in the well-developed circumoral scalids (Fig. 2G), as is the case in modern priapulids worms ([63] and Fig. 3E, G). dependent on one particular food source but could adapt its diet with local food availability. Fossil Associations Two fossil associations with several specimens of Ottoia forming a wreath around the carcass of the arthropod Sidneyia [31,32] indicate that Ottoia had possible scavenging habits (Fig. 13). Decaying carcasses of relatively large epibenthic animals such as Sidneyia (length up to ca 140 mm [31]) may have represented a substantial food source for Ottoia, easily accessible from its supposed shallow subhorizontal burrows [50]. The tiny pharyngeal teeth of Ottoia (Fig. 2E, F) are interpreted as a possible adaptation for scraping soft material such as decaying tissues. Discussion Trophic Complexity of the Cambrian Ecosystem Feeding Process Ottoia obtained food from diverse animal sources (nine species in GC) and by using different techniques: 1) predation on small invertebrates that lived at or near the water-sediment interface (e.g., hyolithids, brachiopods, and polychaetes); and 2) scavenging The feeding mechanism of Ottoia was remarkably simple, being limited to the transit of food via a tubular gut with no physical breakdown (except the disarticulation of composite exoskeletons) PLOS ONE \| www.plosone.org 12 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 9. Other skeletal elements in the gut of Ottoia prolifica from the middle Cambrian Burgess Shale. A, B, ROM 61785, showing gut contents with a trilobite cephalon (probably Ehmaniella burgessensis [1,52]). C, D, ROM 61761, with contents containing trilobite remains (e.g. thoracic segments). E, USNM 196425, with gut contents containing possible trilobite remains. F, G, ROM 61776, with bradoriid arthropod [53] in anterior part of gut. H, I, ROM 61778, with possible shields of bivalved arthropods in posterior gut, general view and detail. J, ROM 61771, with possible shields of bivalved arthropods. Abbreviations: ANT, anterior; ba, bivalved arthropod (shield); bd, bradoriid; ce, cephalon; gc, gut contents; gu, gut; ph, posterior hook; POST, posterior; px(e), everted pharynx; tr, trilobite; ts, thoracic segment. All light photographs (J, whitened with ammonium chloride). Scale bar: 1 cm for A–C; 5 mm for F; 2 mm for D, E, G; 1 mm for I, J. doi:10.1371/journal.pone.0052200.g009 Figure 10. Setae-like elements (SLE) within the gut of Ottoia prolifica from the middle Cambrian Burgess Shale, compared with the chaetae of Burgessochaeta. A–D, ROM 61755b, general view, accumulations and details of SLE in gut. E, ROM 61772b, SLE in gut. F, ROM 61746a, SLE in cross section, preserved in aluminosilicate. G–I, Burgessochaeta setigera (Polychaeta; [1,54]); G, ROM 56967 complete specimen with numerous chaetae on parapodia; H, ROM 56968a(1), ROM 56968a(1), decayed specimen; I, ROM 56968a(2), chaetae on parapodia. J, ROM 56969a, bundle of chaetae (compare with C). Abbreviations: ANT, anterior; bd, possible bundle of SLE; cf, carbonaceous film; ch, chaetae; gc, gut content; gu, gut; pa, parapodium; POST, posterior. Scale bar: 1 cm for A, G; 1 mm for E, H–J; 500 mm for B; 100 mm for D; 20 mm for F. doi:10.1371/journal.pone.0052200.g010 PLOS ONE \| www.plosone.org 13 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 11. Comparative measurements between the setae-like elements (SLE) within the gut of Ottoia prolifica, the chaetae of Burgessochaeta setigera [1,59] and sponge spicules. The diameter of most spicules of sponges occurring in the same horizons as Ottoia ranges between 10 and 20 mm and is lower than that of most SLE. Interpolation (up right diagram) suggests that SLE are undigested chaetae of small individuals of Burgessochaeta, possibly between 5 and 10 mm long. Size distribution of chaetae (blue) and SLE (red) lengths are given for three wellpreserved Burgessochaeta specimens (A–C) and one Ottoia gut content (number 4). Diagonella (bottom left) is a typical sponge in the Burgess Shale biota. Abbreviations: ANT, anterior; BL, body length; bo, body; ch, chaetae; CL, chaeta length; pa, parapodium; POST, posterior; sp, spicule. 1, ROM 61786b; 2, ROM 61787; 3, ROM 61772b; 4, ROM 61755; 5, ROM 61788; 6, ROM 61789; 7, ROM 61746. A, ROM 56968a; B, ROM 56968b; C, ROM 56967; D, ROM 56968b; E, ROM 56969a. doi:10.1371/journal.pone.0052200.g011 ‘‘hunted’’ [39] may not reflect the exact reality of feeding relationships. More likely these small invertebrates that often lived in large populations were taken off randomly by Ottoia which may have lived in sub-horizontal burrows just below the water sediment interface [50]. The presence of disarticulated elements in GC, typically trilobites, cannot be interpreted as unambiguous evidence of predation, because it may result from chance ingestion during scavenging. Similarly, fine sediment was inevitably ingested along with consumable food. The high percentage of empty guts indicates that Ottoia was neither a sediment eater sensu stricto nor on carcasses and detritus. The brachiopods and hyolithids from the Burgess Shale biota were most probably slow moving animals that could have been equally ingested alive or scavenged after death by Ottoia. No fossil evidence indicates that Ottoia favored predation over scavenging or the reverse. In contrast, polychaetes such as Burgessochaeta were probably far more active errant and burrowing animals with capabilities to escape predators such as Ottoia. Again, Ottoia may have fed indiscriminately upon dead and living polychaetes in various proportion depending on its hunting abilities and the rapidity of the prey. The idea that hyolithids were PLOS ONE \| www.plosone.org 14 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Table 5. Numerical abundance of Ottoia prolifica and the animal taxa that constituted its diet (evidence from gut contents and feeding assemblages, present paper) through successive bed assemblages in the Great Phyllopod Bed (Walcott Quarry Member, Burgess Shale Formation, Middle Cambrian). BA nb. ind. nb. taxa A B(1) C(2) D(3) E(4) F(5) G(5) H(5) I(6) J(7) K +120 516 43 11 8 0 1 23 21 10 4 1 0 2 0 1423 55 6 2 14 1 53 11 24 0 0 0 3 240 229 26 1 1 9 3 10 7 0 0 0 0 0 2110 585 53 6 4 3 1 134 3 38 194 0 1 10 2120 3312 92 272 8 54 1 165 4 106 164 16 16 12 2130 3267 79 27 4 5 1 107 2 156 7 5 6 44 2150 2930 85 3 27 0 1 326 35 757 106 9 44 54 2170 1488 73 10 8 0 28 47 10 393 4 11 5 9 2210 4609 105 35 29 0 31 274 18 1011 65 54 12 139 2220 93 28 1 0 0 5 6 1 2 0 1 0 1 2235 2247 84 63 12 0 51 284 4 175 33 6 5 38 2245 4614 62 3 13 0 44 1400 14 238 248 1 13 54 2250 2478 74 12 8 0 7 833 7 91 98 2 8 18 2260 3844 79 46 10 25 25 1079 19 203 51 2 19 76 2265 1842 70 22 2 2 2 414 9 140 11 4 3 25 2270 216 33 3 0 0 1 28 2 4 2 0 0 0 2310 915 63 4 3 0 15 49 16 115 1 1 1 21 2315 189 22 0 0 0 1 3 23 30 0 0 2 11 2320 1561 66 16 5 1 14 11 12 64 1 2 1 10 2350 4258 40 44 0 2 100 13 14 22 0 1 0 1 2355 233 27 0 1 2 38 6 6 57 1 0 1 6 2360 2392 43 2 1 2 4 29 21 74 1 4 2 1 2370 582 48 0 3 0 3 19 26 76 21 0 3 14 2380 455 38 9 0 9 5 69 4 20 2 5 0 1 2400 2548 92 56 7 32 29 65 23 41 2 12 1 62 2410 172 32 1 1 0 1 3 1 6 0 0 0 3 2418 115 18 1 0 0 1 29 1 1 1 0 0 0 2420 1570 40 12 8 9 2 475 3 10 0 1 1 14 2430 430 31 1 1 0 7 40 2 15 2 1 0 6 2445 1563 41 0 1 0 5 98 41 72 1 1 2 13 2455 404 29 1 0 11 31 13 5 0 0 1 1 2 2465 686 23 0 7 0 1 13 5 19 2 0 6 9 2480 381 59 8 4 2 1 25 13 8 0 1 6 1 2495 101 24 1 1 6 1 9 5 0 0 0 0 0 2500 192 27 0 7 0 0 33 3 7 0 1 1 4 2502 180 25 0 0 1 2 15 1 0 0 1 0 0 Faunal data courtesy J.-B. Caron and [2,42,52]. A, Ottoia prolifica (Priapulida); B, Haplophrentis carinatus (hyolithid); C, Burgessochaeta setigera (Polychaeta); D, Wiwaxia corrugata (wiwaxiid); E, Liangshanella burgessensis (bradoriid arthropod); F, Ehmaniella ssp. (Trilobita); G, Ptychagnostus praecurrens (agnostid arthropod); H, Pagetia bootes (agnostid arthropod); I, Sidneyia inexpectans (Arthropoda); J, Mitromitra burgessensis (Brachiopoda); K, Diraphora bellicostata (Brachiopoda). (1) including individual shell operculum or shell whichever is greater; (2) all collected specimens; (3) excluding isolated sclerites. Count of one specimen when presence of isolated remains only (levels: 120, 2110, 2130, 2150, 2270, 2315, 2418, 2465, 2495); (4) number of specimens without soft tissues divided by two to compensate for the presence of dissociated valves;(5) including number of cephala or pygidia whichever is greater; (6) excluding isolated thoracic tergites. Count of one specimen when presence of isolated remains only (levels: 120, 2350, 2430, 2445, 2455, 2500, 2500); (7) excluding fragments of shells (exception 2500 with a single occurrence). doi:10.1371/journal.pone.0052200.t005 PLOS ONE \| www.plosone.org 15 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 12. Almond-shape elements (ASE) within the gut of Ottoia prolifica from the middle Cambrian Burgess Shale, compared to the sclerites of Wiwaxia [60]. A–C, G, H, ROM 61768, general views, and details of ASE (bulbous feature in G is an artefact due to mineralization). D, ROM 61763b with aligned ASE. E, ROM 61773a, with ASE and other skeletal elements. F, ROM 61781a, three aligned ribbed ASE. I, ROM 61745b, isolated ASE within gut. J, ROM 61747, Wiwaxia corrugata [1,60] with sclerites in situ. K, L, ROM 56965, W. corrugata, ribbed sclerite and general view of decayed specimen. Abbreviations: a, anus; ANT, anterior; ase, almond-shape element; gc, gut content; m, mouth; ma, mouth apparatus; POST, posterior; scs, scale-like sclerite; se, skeletal element; sps, spine-like sclerite. Scale bar: 5 mm for A–C, J, L; 2 mm for D, E, K; 1 mm for F–I. doi:10.1371/journal.pone.0052200.g012 a continuous feeder. Its straight cylindrical gut is also poorly consistent with continuous deposit feeding exemplified by modern sipunculans [64]. The gut of Recent and Cambrian [65] sipunculans is typically U-shaped and highly coiled. Although we cannot exclude that Ottoia collected and ingested undifferentiated particles and detritus (as possibly indicated by the organic enrichment of GC), this worm had none of the characteristics of a surface deposit feeder (e.g., introvert with small tentacles). Moreover, the ratio of its body to gut volume (0.8–1.5%) [21] is much lower than in typical deposit feeders. Ottoia was more likely an intermittent omnivorous feeder with low maintenance requirements. Possible modern analogues are macrobenthic priapulids such as Priapulus and Halicryptus [66,67], in which guts are frequently empty and contain detritus mixed with identifiable animal food items (Table 6; [66–68]). Our study undermines the status of Ottoia as an iconic predator and selective hunter [39] and gives this taxon the more realistic status of being a generalist and possibly facultative feeder [69] – i.e., an animal with the capacity to vary its diet with local availability. In recent marine ecosystems, facultative feeders play an important role in conferring resilience in the benthic communities to environmental disturbances and habitat changes [69]. Ottoia may have played a comparable and important role at a critical time when the first modern-style ecosystems started to build up. The recognition of genuinely generalist feeding strategies, as seen here in Ottoia, reveals a high level of trophic complexity and flexibility that has no equivalent in preceding eras (e.g., Ediacaran ecosystem; [70,71]) and foreshadows modern-style ecosystems. PLOS ONE \| www.plosone.org Direct documentation of this behavior in the immediate aftermath of the Cambrian explosion indicates that the marine ecosystem had already become too complex to be understood in terms of simple linear dynamics. More likely, the ecosystem already functioned as an interactive web, with multiple interactions between animal species and the exploitation of diverse food sources. This mode of functioning, which probably set up in the Early Cambrian, is likely to have generated important feedback and accelerating effects on diversity, ecosystem stability and macroevolutionary dynamics. Early Onset of Parallel Trophic Pathways Predation was undoubtedly one of the driving forces in the early diversification of metazoans and the build-up of complex animal interactions and trophic web [12,16,19,29,35,72]. Grazing [11,20] and suspensivory [73] were also major feeding techniques used by numerous Cambrian animals. The case of Ottoia highlights the role of scavenging as another key-consumption mode. We think that the rise of epibenthic communities [2] resulting from the Cambrian radiation fuelled the food web with a new pool of detrital material that became a major and abundant food source for numerous scavengers and detritivores thus promoting and boosting the detrital pathway. The input of animal-derived organic matter into the ecosystem probably deeply modified the food supply in terms of quantity, energy, chemical quality and digestibility with probable feedback effects on the evolution of digestive systems [21] and feeding modes. In common with Ottoia, 16 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 13. Three fossil associations from the Burgess Shale Formation, middle Cambrian, showing Ottoia prolifica around and below the carcass of the arthropod Sidneyia inexpectans and suggesting scavenging behaviour in Ottoia prolifica. A, B, USNM 196241, showing at least 5 worms around the decaying carcass of Sidneyia. This specimen was interpreted [32] as a death assemblage with the worms feeding around the collapsed and decaying carcass of Sidneyia inexpectans. I follow this interpretation here, although the number of worms is more likely to be five than nine [32]. C, D, ROM 61748a, showing an assemblage very similar to USNM 196241; four worms form a wreath-like arrangement around the remains of Sidneyia. E, F, USNM 250218, showing a curved specimen of Ottoia closely associated with Sidneyia. All light photographs (A, courtesy Jean-Bernard Caron, ROM). Scale bar: 1 cm. Abbreviations: an, annulation; dg, digestive glands; gc, gut content; gu, gut; hy, hyolithid conch; ph, posterior hook; px, pharynx; w1–5, worm (Ottoia) 1–5. doi:10.1371/journal.pone.0052200.g013 PLOS ONE \| www.plosone.org 17 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Figure 14. Major components of the diet of Ottoia prolifica from the middle Cambrian Burgess Shale. 1, hyolithids (Haplophrentis); 2, brachiopods (Micromitra); 3, polychaete worms (Burgessochaeta); 4, bradoriids (Liangshanella); 5, trilobites (Ehmaniella); 6, agnostids (Ptychagnostus); 7, 8, carcasses of Sidneyia and Wiwaxia. (A–C), feeding behavior of Ottoia: detection of food via possible chemical cues and ingestion. gu, gut; in, introvert with spiny scalids; px(e), everted pharynx; px(i), inverted pharynx; sc, scalid; tr, trunk. doi:10.1371/journal.pone.0052200.g014 Table 6. Diet of Recent macrobenthic priapulid worms exemplified by Priapulus caudatus and Halicryptus spinulosus (see morphology in Fig. 3). Diet of Priapulus caudatus higher taxa 1 Aphrodite Annelida Polychaeta feces [61] Amphiura chiaji Echinodermata Ophiurida feces; feeding exp. [61] Terrebellides strömi Annelida Polychaeta feeding exp. [61] Mellina costata Annelida Polychaeta feeding exp. [61] Amphicteis gunneri Annelida Polychaeta feeding exp. [61] Priapulus caudatus (cannibalism) Priapulida – live observations [61] Priapulus caudatus (cannibalism) Priapulida – live observations [68] Saccoglossus kowalewskyi (fragment) Hemichordata Enteropneusta feeding exp. [68] Cerebratulus marginatus (fragment) Nemertea – feeding exp. [68] algal remains – – gut contents [61] mud and unrecognizable debris – – feces [68] mud – – gut contents [61] Diet of Halicryptus spinulosus higher taxa 1 higher taxa 2 source of data refs Halicryptus spinulosus Priapulida – gut contents [67] Harmothoe sarsi Annelida Polychaeta gut contents [67] higher taxa 2 source of data refs Pygospio elegans Annelida Polychaeta gut contents [67] Naididae undet. Annelida Oligochaeta gut contents [67] Oligochaeta undet. Annelida Oligochaeta gut contents [67] Monoporeia affinis Arthropoda (Cru.) Amphipoda gut contents [67] Pontoporeia femorata Arthropoda (Cru.) Amphipoda gut contents [67] Crustacea undet. Arthropoda – gut contents [67] Tanypodinae undet. Arthropoda (Ins.) Chironomidae gut contents [67] Chironominae undet. Arthropoda (Ins.) Chironomidae gut contents [67] Arthropoda undet. – – gut contents [67] animal remains undet. – – gut contents [67] eggs – – gut contents [67] algal remains undet. – – gut contents [67] detritus – – gut contents [67] Abbreviations: Cru, Crustacea; exp, experiments; Ins, Insecta, doi:10.1371/journal.pone.0052200.t006 PLOS ONE \| www.plosone.org 18 December 2012 \| Volume 7 \| Issue 12 \| e52200 Gut Contents of Cambrian Worm Ottoia Table S1 Studied material (Ottoia prolifica from the middle Cambrian Burgess Shale, British Columbia, Canada). The specimens are housed in the Royal Ontario Museum (ROM), Toronto, Canada and the Smithsonian National Museum of Natural History (originally US National Museum; USNM), Washington D.C. All the specimens have preserved digestive tracts with or without gut contents. (XLS) arthropods may have acquired adaptations to exploiting this detrital food store with relatively low cost of energy expenditure. This requires testing from detailed studies on the digestive systems and appendage functionalities of Cambrian arthropods and their possible modern analogues. Parallel circuits such as the ‘‘green pathway’’ (through primary producers, herbivore/grazers to carnivores) and the detrital pathway that is essential in the energy flow of modern marine ecosystems [41] may have been already operating in the Cambrian adding to the trophic complexity. Acknowledgments Supporting Information I thank Jean-Bernard Caron, Peter Fenton, Douglas Erwin and Mark Florence for access to the fossil material deposited in the Royal Ontario Museum (ROM), Toronto and the National Museum of Natural History (USNM), Washington D.C. I am thankful to Fredrik Pleijel, Sylve Robertsson, Stefan Agrenius (Sven Lovén Center for Marine Sciences, Sweden) and Alexander Tzetlin (White Sea Biological Station Kartesh, Russia) for collecting Recent priapulids, Tara Macdonald and Brenda Burd for information on recent marine ecosystems, and Nick Butterfield, Jean-Bernard Caron Mark Williams and Stephen Mojzsis for critical reading of the early versions of the MS. I am thankful to Tom Harvey and two anonymous reviewers for their constructive comments. Lucy Wilson is thanked for images used in Figure 5A–D. This is Royal Ontario Museum Burgess Shale project number 40. Figure S1 Sponge species that co-occur with Ottoia prolifica in level -120 [2,42,52] of the Walcott Quarry (Burgess Shale Formation, middle Cambrian). A, B, Hazelia nodulifera Walcott, ROM 40317B(1), general view and details. C, D, Hazelia palmata Walcott, ROM 53585, general view in polarized light and details of closely packed spicules. E, F, Falospongia falata Rigby, ROM 40317B(2), general view and details of skeletal tracts. G, H, Pirania muricata Walcott, ROM 53309, general view and details of radiating spicules. I–K, Diagonella hindei Walcott, ROM 61766, general view and details of the spicule network of a small and larger specimen on the same slab. L, Eiffelia globosa Walcott, ROM 53567, details of six-rayed spicules. msp, monaxial spicule; rsp, radial thick spicule; rtr, radial tract; tr, tract composed of numerous spicules. (Scale bar, 2 mm for A, C, E, G, I; 1 mm F, H, J-L; 500 mm for B, D. (PDF) Author Contributions Conceived and designed the experiments: JV. Performed the experiments: JV. Analyzed the data: JV. Contributed reagents/materials/analysis tools: JV. Wrote the paper: JV. References 18. Waloszek D, Maas A, Chen J-Y, Stein M (2007) Evolution of cephalic feeding structures and the phylogeny of Arthropoda. Palaeogeography, Palaeoclimatology, Palaeoecology 254: 273–287. 19. 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https://openalex.org/W4224924112	https://www.frontiersin.org/articles/10.3389/fpubh.2022.743053/pdf	English	null	Depression and Psychological Stress Among Health Workers in Remote Areas in Indonesia	Frontiers in public health	2,022	cc-by	5,642	Depression and Psychological Stress Among Health Workers in Remote Areas in Indonesia Sri Idaiani 1* and Lukman Waris 2 1 National Research and Innovation Agency, Jakarta, Indonesia, 2 Department of Public Health, Universitas Falatehan, Banten, Indonesia Background: The Indonesian government launched the Nusantara Sehat program in 2015, under which teams of health workers were assigned to community health care centers in remote, border, and island areas for 2 years. The deployment to remote areas is likely to affect their psychological condition if they are not equipped with facilities and strong motivation. This study aimed to describe the psychological condition of health workers in remote areas in Indonesia, focusing on the proportion of the prevalence of depression and psychological stress. Materials and Methods: This cross-sectional study was conducted between April and December of 2018. Participants were 140 health workers placed in 26 community healthcare centers in 13 provinces. Interviews were conducted by enumerators using a questionnaire that included questions from the Mini International Neuropsychiatric Interview (MINI) and Self-Reporting Questionnaire-20 (SRQ-20). Edited by: Andrew T. Olagunju, McMaster University, Canada Reviewed by: Katalin Papp, University of Debrecen, Hungary Adegboyega Ogunwale, Neuropsychiatric Hospital, Aro, Abeokuta, Nigeria Correspondence: Sri Idaiani sriidaiani@gmail.com; sri.idaiani@brin.go.id orcid.org/0000-0001-9010-9103 Edited by: Andrew T. Olagunju, McMaster University, Canada Reviewed by: Katalin Papp, University of Debrecen, Hungary Adegboyega Ogunwale, Neuropsychiatric Hospital, Aro, Abeokuta, Nigeria Results: Of the participants, 7.1% experienced depression and 10.0% experienced psychological stress. Motivation was related to psychological stress in participants with an odds ratio of 0,218 (95% conﬁdence interval = 0.065–0.729, p = 0.013). Health workers with high motivation tend not to experience psychological stress compared to individuals with lower motivation. Correspondence: Sri Idaiani sriidaiani@gmail.com; sri.idaiani@brin.go.id orcid.org/0000-0001-9010-9103 Conclusion: Health workers with high motivation experience relatively low levels of psychological stress. To overcome stress, high motivation is needed to control psychological risk factors before and during placement. Specialty section: This article was submitted to Public Mental Health, a section of the journal Frontiers in Public Health Keywords: health worker, remote area, depression, psychological stress, motivation INTRODUCTION Received: 06 October 2021 Accepted: 18 March 2022 Published: 27 April 2022 Living in a remote area, far from family and friends, with a lack of facilities is a stressor for health workers (1). The most common psychological disorder among workers is burnout. Burnout generally occurs in workers in humanitarian and social ﬁelds. There are three aspects of burnout: emotional exhaustion characterized by anxiety and depression, depersonalization, and personal achievement (2). Emotional exhaustion has been found to have the highest correlation with workers’ mental health (3). ORIGINAL RESEARCH published: 27 April 2022 doi: 10.3389/fpubh.2022.743053 ORIGINAL RESEARCH published: 27 April 2022 doi: 10.3389/fpubh.2022.743053 Study Population The study population was comprised with teams of health workers placed at 26 CHCs in remote areas across 13 provinces between 2015 and 2017 (waves 6 and 8). The CHCs were selected by random sampling from among 39 CHCs in 28 provinces. The health workers were selected by purposive sampling. We did not include health workers from previous waves as they had already completed the program. “Batch” or “wave” is the period when the team is deployed to the location. When this study was conducted, there were two waves or batches in the location. Wave 6 had been on location longer than wave 8. Deployment is considered a proxy for work experience. y g j Stress and depression, or other mental disorders, are known to decrease productivity; although many factors, such as motivation, aﬀect them. Motivation is inﬂuenced by rewards, ﬁnances, and reinforcements, which is also related to other problems, such as workload and family factors (6). Decreased productivity causes losses for providers and, in the case of health workers, the government loses providers should healthcare workers decrease productivity. The government spends a considerable amount of money to place health workers and have to ensure that they work eﬃciently. As a subject criterion, all TBNS members who were in the selected location were eligible as participants. Members who were not present at the time the research team visited the site or were unwilling to participate in the study were excluded. y In 2015, the government of Indonesia launched the Team Based Nusantara Sehat (TBNS) program to meet health service needs. According to the Minister of Health Regulation Number 16 of 2017, each team consists of at least ﬁve types of health professionals, namely, general practitioners, dentists, public health workers, sanitarians, nutritionists, laboratory analysts, pharmacists, midwives, and nurses. The teams are placed for two years in community health care centers (CHCs) in remote areas far from the district capital that lack basic infrastructure. Remote areas, in this context, are determined by the Ministry of Village, Development of Disadvantaged Regions, and Transmigration by considering the economic factors, human resources, infrastructure accessibility, and other regional characteristics. Citation: Idaiani S and Waris L (2022) Depression and Psychological Stress Among Health Workers in Remote Areas in Indonesia. Front. Public Health 10:743053. doi: 10.3389/fpubh.2022.743053 Idaiani S and Waris L (2022) Depression and Psychological Stress Among Health Workers in Remote Areas in Indonesia. Front. Public Health 10:743053. doi: 10.3389/fpubh.2022.743053 Various factors, both internal and external, inﬂuence the psychosocial condition of health workers. Internal factors include workers’ health, social and family networks, health-related April 2022 \| Volume 10 \| Article 743053 Frontiers in Public Health \| www.frontiersin.org Health Worker in Remote Area Idaiani and Waris behavior, and motivation, while work environment, residence, workload, income, supervision, guidance, national politics, national policies, income inequality, and employment conditions constitute external factors (4). never been assessed. Therefore, it is necessary to assess the mental status of health workers in remote areas. With this mind, the Ministry of Health, in 2018, approved a study to assess the psychological condition of health workers in remote areas in Indonesia. The purpose of this study was to describe the psychological conditions (depression and psychological stress) of health workers placed at CHCs in remote areas between 2015 and 2017. From the preceding description, it is clear that emotional fatigue often occurs in workers in the form of anxiety and depression, and also, in a milder form, general stress or psychological stress. Anxiety is more commonly characterized by autonomic symptoms, and depression by low mood. Unresolved psychological stress is likely to develop into more speciﬁc disorders, such as anxiety, depression, and other psychological conditions. In Indonesia, according to the National Health Survey (NHS), the prevalence of depression in the general population aged 15 years is 6.1%, while in the general population who have a job, it varies from 2.4 to 6.9%. Additionally, the prevalence of psychological stress in people over the age of 15 years is 9.8%, while among those who have a job is 3.9–10.8% (5). Frontiers in Public Health \| www.frontiersin.org Study Population Minimum sample size was estimated using the formula n = N ∗X/(X + N – 1) where X = Z2 α/2 ∗p ∗(1 – p) / MOE2 (8), where p = 0.108 (from p which produces the most n, namely, p of psychological stress), N = 210 (prediction of total number of TNBS personnel with assumption 1 team consisted of 6 person from 35 CHCs), conﬁdence level of 95%, α is 0.05 and Zα/2 = 1.96 thus Z2 α/2 = 3.8416), and MOE is the margin of error = 2%. Based on this formula, the minimum required sample size was 88. Measurements Community health center District Province 0 Jakarta (the capital) DKI Jakarta 1 North Rupat Bengkalis Riau 2 Sungai Guntung Indragiri Hilir Riau 3 Tambelan Bintan Riau Islands 4 East Serasan Natuna Riau Islands 5 Enggano North Bengkulu West Kalimantan 6 Great Sajingan Sambas West Kalimantan 7 Badau Kapuas Hulu West Kalimantan 8 Balai Karangan Sanggau West Kalimantan 9 Tiong Ohang Mahakam Ulu East Kalimantan 10 Long Nawang Malinau North Kalimantan 11 Long Ampung Malinau North Kalimantan 12 Sei Menggaris Nunukan North Kalimantan 13 Ogodeide Toli Toli Central Sulawesi 14 Gemeh Talaud North Sulawesi 15 Kandahe Sangihe North Sulawesi 16 Ndao Rote Ndao East Nusa Tenggara 17 Silawan Belu East Nusa Tenggara 18 Wedomu Belu East Nusa Tenggara 19 Maritaing Alor East Nusa Tenggara 20 Bere Bere Morotai Island North Maluku 21 Ilwaki Southwest Maluku Maluku 22 Lelang Southwest Maluku Maluku 23 Adaut Western Southeast Maluku Maluku 24 Dorekar Raja Ampat West Papua 25 Sabar Miokre Supiori Papua 26 Bupul Merauke Papua FIGURE 1 \| Location of selected community health centers. and depression. The cut-oﬀpoint of 6 is applied to all characteristics of the participants, both men and women, scale. Based on the assessment method, motivation was divided into three categories: low (score < 25), medium (score = No. Community health center District Province 0 Jakarta (the capital) DKI Jakarta 1 North Rupat Bengkalis Riau 2 Sungai Guntung Indragiri Hilir Riau 3 Tambelan Bintan Riau Islands 4 East Serasan Natuna Riau Islands 5 Enggano North Bengkulu West Kalimantan 6 Great Sajingan Sambas West Kalimantan 7 Badau Kapuas Hulu West Kalimantan 8 Balai Karangan Sanggau West Kalimantan 9 Tiong Ohang Mahakam Ulu East Kalimantan 10 Long Nawang Malinau North Kalimantan 11 Long Ampung Malinau North Kalimantan 12 Sei Menggaris Nunukan North Kalimantan 13 Ogodeide Toli Toli Central Sulawesi 14 Gemeh Talaud North Sulawesi 15 Kandahe Sangihe North Sulawesi 16 Ndao Rote Ndao East Nusa Tenggara 17 Silawan Belu East Nusa Tenggara 18 Wedomu Belu East Nusa Tenggara 19 Maritaing Alor East Nusa Tenggara 20 Bere Bere Morotai Island North Maluku 21 Ilwaki Southwest Maluku Maluku 22 Lelang Southwest Maluku Maluku 23 Adaut Western Southeast Maluku Maluku 24 Dorekar Raja Ampat West Papua 25 Sabar Miokre Supiori Papua 26 Bupul Merauke Papua FIGURE 1 \| Location of selected community health centers. No. Frontiers in Public Health \| www.frontiersin.org Measurements The interview questionnaire collected demographic data and included items on motivation, depression, and psychological stress. Depression was measured using the depression questionnaire (9–11) from version 6 of the Mini International Neuropsychiatric Interview (MINI). The MINI is an interview- based diagnostic tool which assesses depression in the past 2 weeks or over the lifetime. It consists of three screening questions and seven main questions. The data from the tool are inputted into an algorithm which produces an output assessing whether a person is depressed or not. In Indonesia, the validity and reliability of the tool have been measured with good results (12, 13). Psychological stress was assessed using 20 questions from the Self-Reporting Questionnaire (SRQ) (14). Respondents were categorized as depressed if they met the requirements according to the MINI questionnaire, whereas those who answered “yes” to at least 6 of the 20 SRQ questions suﬀered from psychological stress, a condition characterized by symptoms of anxiety In general, the health workers deployed to remote areas are fresh graduates with a health education background. The maximum age limit is 30 years for general practitioners and 25 years for other health workers. The health workers are expected to smoothly and successfully pass the two-year deployment period. Those who fail to carry out their duties receive sanctions, making it diﬃcult for them to participate in other health programs. The TBNS personnel are placed in remote areas. Therefore, the government has to ensure that the deployed team is working optimally and productively. Considering this, the evaluation of both the physical and mental conditions of the team placed in remote areas is necessary. This paper will focus on the mental health of the team because poor psychological conditions lead to burnout and low productivity (3, 7). In addition, until recently, the mental condition of health workers in remote areas has April 2022 \| Volume 10 \| Article 743053 2 Idaiani and Waris Health Worker in Remote Area No. Measurements Community health center District Province 0 Jakarta (the capital) DKI Jakarta 1 North Rupat Bengkalis Riau 2 Sungai Guntung Indragiri Hilir Riau 3 Tambelan Bintan Riau Islands 4 East Serasan Natuna Riau Islands 5 Enggano North Bengkulu West Kalimantan 6 Great Sajingan Sambas West Kalimantan 7 Badau Kapuas Hulu West Kalimantan 8 Balai Karangan Sanggau West Kalimantan 9 Tiong Ohang Mahakam Ulu East Kalimantan 10 Long Nawang Malinau North Kalimantan 11 Long Ampung Malinau North Kalimantan 12 Sei Menggaris Nunukan North Kalimantan 13 Ogodeide Toli Toli Central Sulawesi 14 Gemeh Talaud North Sulawesi 15 Kandahe Sangihe North Sulawesi 16 Ndao Rote Ndao East Nusa Tenggara 17 Silawan Belu East Nusa Tenggara 18 Wedomu Belu East Nusa Tenggara 19 Maritaing Alor East Nusa Tenggara 20 Bere Bere Morotai Island North Maluku 21 Ilwaki Southwest Maluku Maluku 22 Lelang Southwest Maluku Maluku 23 Adaut Western Southeast Maluku Maluku 24 Dorekar Raja Ampat West Papua 25 Sabar Miokre Supiori Papua 26 Bupul Merauke Papua FIGURE 1 \| Location of selected community health centers. FIGURE 1 \| Location of selected community health centers. scale. Based on the assessment method, motivation was divided into three categories: low (score < 25), medium (score = 25–74), and high (score ≥75). There is no information regarding validation of this questionnaire in Indonesia; however, it has been adapted and translated into Indonesian and has been used in the 2017 National Health Worker Research (17). The data collectors (enumerators) read all questions to the respondents. The enumerators received training on data collection procedures and interviewing techniques related to the questionnaire material. and depression. The cut-oﬀpoint of 6 is applied to all characteristics of the participants, both men and women, according to the results of validity tests that have been previously carried out and have been used in routine surveys in Indonesia (15). Motivation was assessed using a motivation questionnaire that was originally developed for workers in rural areas in Zambia (16). It consists of 23 questions about general motivation, job satisfaction, intrinsic job satisfaction, burnout, organization commitment, contentiousness, and timeliness using a Likert-type April 2022 \| Volume 10 \| Article 743053 Frontiers in Public Health \| www.frontiersin.org 3 Health Worker in Remote Area Idaiani and Waris TABLE 1 \| Number of community health centers and respondents by province. RESULTS Figure 1 shows that most community health centers (CHCs) are located in the outer border areas and islands. There were no selected CHCs in Java and Bali. Table 1 details the distribution of community health centers (CHCs) and respondents by province. There were 140 respondents from 26 CHCs in 13 provinces. psychological stress (3.9–10.8%) in workers is lower than the national rate. Comparing the results of this study with those of other studies is diﬃcult because previous studies assessed depression in health workers in disaster areas (18, 19) and not in remote areas. The proportion of health workers who experienced depression was 7.1%, while 10.0% experienced psychological stress (Table 2). There were no respondents with low motivation. Therefore, motivation is classiﬁed only as medium or high. Wave of participation and motivation had a signiﬁcant relationship with depression (p < 0.25). Psychological stress, wave and motivation, and gender, had a signiﬁcant relationship (p < 0.25). Health workers in wave 6 were placed at CHCs ∼6 months before those in wave 8. The variables of interest were subjected to multivariate analysis and the results are displayed in Tables 3, 4. Although gender was not associated with depression and psychological stress, the prevalence of depression was higher in women than in men. This is consistent with the results of studies on mental health in general and NHS 2018. As for stress in the workplace, men tend to ignore the symptoms of depression (20). Another study states that depression and anxiety at the workplace are associated with musculoskeletal disorders (21). Furthermore, victims of workplace violence in health care settings are generally women, widows, and youngsters who are at high risk of psychiatric morbidity (19). Table 3 shows that more recent work periods (wave 8) have almost no eﬀect on depression (conﬁdence interval is 0.016– 1.078). Motivation is also not related to depression (p > 0.05). Table 4 shows a signiﬁcant relationship between motivation and psychological stress. That is, people with high motivation have lower risk of psychological stress compared to those with medium motivation (p = 0.013). Both gender and wave of participation did not show any relationship with psychological stress (conﬁdence interval for gender is 0.045–1.234 and wave is 0.087–1.302, p > 0.05). Health workers who were often absent from duty appeared to have more depression and stress, although there were no statistically signiﬁcant diﬀerences. RESULTS This is similar to the results of previous studies that absence at work is related to stress, fatigue, and burnout (3). In a study of ethnic Latin workers, most cases of depression, though moderate in degree, occurred during the ﬁrst year of work. The questionnaire used in this study did not categorize the degree of depression (22). DATA ANALYSIS STATA® version 14 used spmap command to display the location of the selected CHCs. IBM®SPSS® version 24 was used to undertake a chi square test of independence to establish initial bivariate relationships between variables (gender, wave, type of health worker, motivation). We included variables in subsequent multivariate analyses if they were signiﬁcant at p < 0.25. Included variables were simultaneously entered into two logistic regression equations – one using the outcome variable of depression and the other psychological distress. An association between predictor and outcome variables were assumed at p < 0.05. Measurements Province n CHC n Respondents % Respondents Riau 2 10 7.1 Bengkulu 1 6 4.3 Riau Islands 2 11 7.9 West Kalimantan 3 17 2.1 East Kalimantan 1 5 3.6 North Kalimantan 3 18 2.9 North Sulawesi 1 6 4.3 Central Sulawesi 1 6 4.3 East Nusa Tenggara 4 22 15.7 Maluku 4 17 12.1 North Maluku 1 5 3.6 West Papua 1 6 4.3 Papua 2 11 7.9 Total 26 140 100 TABLE 1 \| Number of community health centers and respondents by province. Frontiers in Public Health \| www.frontiersin.org DISCUSSION In this study, the type of profession did not have an association with depression or psychological stress. Because of the small number of respondents, some professionals were placed into two groups. In general, health workers have a similar risk of experiencing stress as other professionals. Moreover, as doctors and dentists are more experienced than other health workers, they are better able to cope with stress. Doctors and dentists have the possibility of starting an independent practice after the completion of the health program, whereas other health workers are dependent on employment opportunities at institutions or healthcare facilities. The proportion of remote health workers who experienced depression was 7.1%. This number was higher than the national rate of 6.1% for population aged ≥15 years based on the NHS conducted in 2018 (14). The NHS assessed depression using the same tool as this study, that is, the depression questionnaire from MINI version 6. The prevalence of psychological stress among remote health workers was 10%, which was also similar to NHS 2018 (9.8% in population aged ≥15 years). The prevalence of depression and psychological stress varies depending on the type of work. According to NHS 2018, in general, the prevalence of depression (2.4–6.9%) and Furthermore, work motivation only related to psychological stress in this study. Work motivation is inﬂuenced by many April 2022 \| Volume 10 \| Article 743053 Frontiers in Public Health \| www.frontiersin.org 4 Health Worker in Remote Area Idaiani and Waris TABLE 2 \| Factors related to depression and psychological stress. Depression p* Psychological stress p* Yes No Yes No n % n % n % n % Gender Male 2 20.0 47 36.2 0.492 2 14.3 47 37.3 0.138 Female 8 80.0 83 63.8 12 85.7 79 62.7 Wave 6 1 10.0 62 47.7 0.023 3 21.4 60 47.6 0.089 8 9 90.0 68 52.3 11 78.6 66 52.4 Type of health worker Doctor (general practitioner), dentist,midwife, nurse 2 20.0 51 39.2 0.319 3 21.4 50 38.7 0.296 Public health worker, sanitarian, nutritionist, laboratory analyst, pharmacist 8 80.0 79 60.8 11 78.6 76 60.3 Motivation Medium 9 90.0 127 97.7 0.259 12 85.7 124 98.4 0.050 High 1 10.0 3 2.3 2 14.3 2 1.6 Total 10 7.1 130 92.9 14 10.0 126 90.0 *Fisher’s exact test. TABLE 3 \| Results of multivariate analysis of factors related to depression. ETHICS STATEMENT This study has several limitations. First, a number of important determinants of depression and psychological stress were not examined, such as job satisfaction, career pathway, income, social support, turnover, availability of medicines, skills, safe and supportive environment, and access to health facilities (28, 36). Second, the cross-sectional design limits the inference and direction of causality. The strength of this study is that it was conducted on a hard-to-reach population, given the geography of Indonesia. It focuses on a population that had not been previously studied in the country. Second, it utilized standardized instruments for the diagnosis of depression and the measurement of psychological stress. The study protocol was reviewed and approved by the Scientiﬁc Committee and Health Research Ethics Commission of the National Institute of Health Research and Development in Indonesia (number: LB.02.01/2/KE.116/2018). All procedures were performed in accordance with the Helsinki Declaration. Written informed consent was obtained from all the participants prior to the interviews. ACKNOWLEDGMENTS The authors would like to acknowledge the researchers of National Institute of Health Research and Development who are in charge of monitoring the Nusantara Sehat program for writing and data analysis collaboration. Special thanks for Aliza Hunt for the suggestion and correction on data analysis writing. These results should be further interogated with studies speciﬁcally assessing the psychological conditions of health workers in remote areas using a larger sample size and varying working periods or wave placements. In terms of work place policy, the Health Oﬃce should conduct periodic supervision DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. It is thus important to deal with stress and depression at work so as not to aﬀect the quality of service. The ways of dealing with stress vary depending on age and other factors. Providing clear work instructions and conducting risk assessments is one way to prevent stress in the workforce (33–35). AUTHOR CONTRIBUTIONS SI participated in manuscript writing and data analysis. LW led the project and made revisions to the manuscript. All authors read and approved the ﬁnal manuscript. DISCUSSION Depression OR SE Z 95% CI p Wave 6 Ref 8 0.132 0.141 −1.89 0.016–1.078 0.059 Motivation Medium Ref High 0.342 0.234 −1.56 0.089–1.312 0.118 OR, odds ratio; CI, conﬁdence interval. TABLE 4 \| Results of multivariate analysis of factors related to psychological stress. Psychological stress OR SE Z 95% CI p Gender Male Ref Female 4.484 3.675 1.83 0.045–1.234 0.067 Wave 6 Ref 8 0.287 0.202 −1.77 0.087–1.302 0.076 Motivation Medium Ref High 0.218 0.134 −2.47 0.065–0.729 0.013 OR, odds ratio; CI, conﬁdence interval. factors such as leadership job satisfaction income social Other factors that aﬀect health workers in remote areas TABLE 2 \| Factors related to depression and psychological stress. OR, odds ratio; CI, conﬁdence interval. Other factors that aﬀect health workers in remote areas include health system, ﬁnancial and socio-economic conditions, job satisfaction, retention, and turnover (27–29). Studies on health workers in remote areas have discussed turnover intention factors such as leadership, job satisfaction, income, social support, and work skills (23–26). Therefore, living in remote areas is not the only factor inﬂuencing the retention of health workers. April 2022 \| Volume 10 \| Article 743053 Frontiers in Public Health \| www.frontiersin.org 5 Idaiani and Waris Health Worker in Remote Area to improve motivation and skills and ensure the welfare of remote health workers throughout Indonesia. Stress risk control is required both before and during placement, such as stress management, work training, and supervision. as a cause of burnout and stress (28, 30). As for stress at work, studies have generally focused on informal or part-time workers. Part-time male workers tend not to complain signiﬁcantly (31). Stress can have both long- and short-term eﬀects. Long-term stress aﬀects the retirement period, while short-term stress causes a lack of productivity, which ultimately aﬀects the quality of health services (20, 32). FUNDING The prevalence of depression and psychological stress among health workers in remote areas of Indonesia is slightly higher than in the general adult population of Indonesia. This number is also higher compared with other types of workers in Indonesia. Motivation is one of the factors associated with psychological stress. In this study, there was no association between respondents’ working experience and gender with both depression and psychological stress. This study was supported by the Indonesian Ministry of Health. The fund was directed to cover the travel and accommodation of researchers from Jakarta to study locations. 8. Select-statistics.co.uk. Population Proportion - Sample Size - Select Statistical Consultants. Available at: https://select-statistics.co.uk/calculators/sample- size-calculator-population-proportion/ (accessed March 7, 2022). 7. Canavan ME, Sipsma HL, Adhvaryu A, Ofori-Atta A, Jack H, Udry C, et al. Psychological distress in Ghana: associations with employment and lost productivity. Int J Ment Health Syst. 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(2019) 17:2. doi: 10.1186/s12960-018-0340-6 24. Musinguzi C, Namale L, Rutebemberwa E, Dahal A, Nahirya-Ntege P, Kekitiinwa A. The relationship between leadership style and health worker motivation, job satisfaction and teamwork in Uganda. J Healthc Leadersh. (2018) 10:21–32. doi: 10.2147/JHL.S147885 Copyright © 2022 Idaiani and Waris. 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. 25. Alhassan RK, Nketiah-Amponsah E, Spieker N, Arhinful DK, Rinke de, Wit TF. Assessing the impact of community engagement interventions on health worker motivation and experiences with clients in primary April 2022 \| Volume 10 \| Article 743053 Frontiers in Public Health \| www.frontiersin.org
https://openalex.org/W2808615798	https://cancerandmetabolism.biomedcentral.com/track/pdf/10.1186/s40170-018-0180-9.pdf	English	null	Beta-hydroxybutyrate (3-OHB) can influence the energetic phenotype of breast cancer cells, but does not impact their proliferation and the response to chemotherapy or radiation	Cancer & metabolism	2,018	cc-by	15,731	Beta-hydroxybutyrate (3-OHB) can influence the energetic phenotype of breast cancer cells, but does not impact their proliferation and the response to chemotherapy or radiation Beta-hydroxybutyrate (3-OHB) can influence the energetic phenotype of breast cancer cells, but does not impact their proliferation and the response to chemotherapy or radiation Catharina Bartmann1, Sudha R. Janaki Raman2, Jessica Flöter2, Almut Schulze2, Katrin Bahlke1, Jana Willingstorfer1, Maria Strunz1, Achim Wöckel1, Rainer J. Klement3, Michaela Kapp1, Cholpon S. Djuzenova4, Christoph Otto5 and Ulrike Kämmerer1* RESEARCH Open Access Beta-hydroxybutyrate (3-OHB) can influence the energetic phenotype of breast cancer cells, but does not impact their proliferation and the response to chemotherapy or radiation Catharina Bartmann1, Sudha R. Janaki Raman2, Jessica Flöter2, Almut Schulze2, Katrin Bahlke1, Jana Willingstorfer1, Maria Strunz1, Achim Wöckel1, Rainer J. Klement3, Michaela Kapp1, Cholpon S. Djuzenova4, Christoph Otto5 and Ulrike Kämmerer1* © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Ketogenic diets (KDs) or short-term fasting are popular trends amongst supportive approaches for cancer patients. Beta-hydroxybutyrate (3-OHB) is the main physiological ketone body, whose concentration can reach plasma levels of 2–6 mM during KDs or fasting. The impact of 3-OHB on the biology of tumor cells described so far is contradictory. Therefore, we investigated the effect of a physiological concentration of 3 mM 3-OHB on metabolism, proliferation, and viability of breast cancer (BC) cells in vitro. Methods: Seven different human BC cell lines (BT20, BT474, HBL100, MCF-7, MDA-MB 231, MDA-MB 468, and T47D) were cultured in medium with 5 mM glucose in the presence of 3 mM 3-OHB at mild hypoxia (5% oxygen) or normoxia (21% oxygen). Metabolic profiling was performed by quantification of the turnover of glucose, lactate, and 3-OHB and by Seahorse metabolic flux analysis. Expression of key enzymes of ketolysis as well as the main monocarboxylic acid transporter MCT2 and the glucose-transporter GLUT1 was analyzed by RT-qPCR and Western blotting. The effect of 3-OHB on short- and long-term cell proliferation as well as chemo- and radiosensitivity were also analyzed. Results: 3-OHB significantly changed the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in BT20 cells resulting in a more oxidative energetic phenotype. MCF-7 and MDA-MB 468 cells had increased ECAR only in response to 3-OHB, while the other three cell types remained uninfluenced. All cells expressed MCT2 and GLUT1, thus being able to uptake the metabolites. The consumption of 3-OHB was not strongly linked to mRNA overexpression of key enzymes of ketolysis and did not correlate with lactate production and glucose consumption. Neither 3-OHB nor acetoacetate did interfere with proliferation. Further, 3-OHB incubation did not modify the response of the tested BC cell lines to chemotherapy or radiation. * Correspondence: frak057@mail.uni-wuerzburg.de 1Department of Obstetrics and Gynaecology, University Hospital of Würzburg, Josef-Schneider-Str. 4, 97080 Würzburg, Germany Full list of author information is available at the end of the article Bartmann et al. Cancer & Metabolism (2018) 6:8 https://doi.org/10.1186/s40170-018-0180-9 Bartmann et al. Cancer & Metabolism (2018) 6:8 https://doi.org/10.1186/s40170-018-0180-9 Background higher affinity for lactate [27, 28]. In mitochondria, 3-OHB is degraded via ketolysis into acetyl-CoA, which then is metabolized within the Krebs cycle and the re- spiratory chain to generate energy [29–32]. Since the oxidation of 3-OHB generates more energy per mol oxy- gen used compared to glucose, it is sometimes labeled a “superfuel” [33]. However, cells need functioning mito- chondria as well as sufficient oxygen supply to generate energy from 3-OHB. The latter is hampered in the hyp- oxic microenvironment of larger tumors, which has already been shown in vivo for breast cancer tissue in patients [34]. Breast cancer (BC) is one of the most common cancers and affects about one in eight women during their life- time [1]. In general, modern BC therapy includes differ- ent therapeutic approaches, such as surgical removal of the tumor, chemotherapy, radiation, and hormone ther- apy [2]. In addition to these conventional therapies, a large number of patients seek supportive therapies like specific diets to improve their outcome. The correlation between different types of diet and the incidence and progression of cancer is increasingly becoming the focus of research [3–7]. In this respect, avoiding carbohydrates to specifically “starve cancer cells” is the most popular trend amongst “cancer diets.” The rationale for this diet- ary regime is often based on the “Warburg effect,” which describes the preferential fermentation of glucose to lac- tate even under availability of sufficient oxygen [8, 9]. Therefore, reducing carbohydrate intake and thus lower- ing blood glucose seems to be a promising strategy for cutting cancer off from glucose supply [10–12]. There are somewhat contradicting results regarding the effect of 3-OHB on growth and biology of tumor cells cultured in vitro and in experimental tumors in mice. In some studies, ketone bodies seem to be associ- ated with cancer progression, metastasis, and poor clin- ical outcome [35, 36]. In contrast, it was shown that a ketogenic diet significantly reduces tumor growth in mice [37, 38]. Further, an antiproliferative effect of 3-OHB was already shown for different cancer cells, such as glioblastoma and tumor stem cells [37], melan- oma, cervical carcinoma, or neuroblastoma [39–41]. Several studies also described a significant delay of tumor growth in mice and humans in a systemic ketosis [16, 18, 37, 38, 40, 42–49]. In this respect, Rodrigues and coworkers reported evidence for a “β-hydroxybutyrate paradox” [50]. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 19 Bartmann et al. Cancer & Metabolism (2018) 6:8 (Continued from previous page) (Continued from previous page) Conclusions: We found that a physiological level of 3-OHB can change the energetic profile of some BC cell lines. However, 3-OHB failed to influence different biologic processes in these cells, e.g., cell proliferation and the response to common breast cancer chemotherapy and radiotherapy. Thus, we have no evidence that 3-OHB generally influences the biology of breast cancer cells in vitro. Keywords: Ketogenic diet, β-Hydroxybutyrate, Ketone bodies, Breast cancer, Seahorse, Metabolic profile, Chemotherapy, Ionizing radiation Background They postulated that the effect of 3-OHB on cancer growth would depend on the tumor’s energetic phenotype. Thus, “oxidative cells” would use 3-OHB as an additional energy source so that tumors with predomin- antly “oxidative cells” increase their growth when this me- tabolite is available. Other cells with a more “glycolytic, Warburg-like phenotype” would be unable to metabolize 3-OHB in which case it could accumulate intracellularly and inhibit tumor growth via signaling and epigenetic mechanisms [50]. Besides fasting, the strictest form of such a “very low carb” diet is called the ketogenic diet (KD). The KD is characterized by consuming the predominant proportion of calories from fat, balancing those derived from protein and thus consuming very few calories from glucose or other carbohydrates. Different KD regimens were shown to be safe and well tolerated in a variety of malignancies [13–19] and lead to the metabolic state of a physiological ketosis [20]. During ketosis, the “ketone bodies” acetoace- tate (AcAc) and D-β-hydroxybutyrate (R-3-hydroxybuty- rate: 3-OHB) are predominantly produced in the liver and can be detected in the peripheral blood and urine above normal levels [21]. 3-OHB is found at similar or higher concentrations than AcAc and therefore, considered the principal “ketone body.” In humans, the median concen- tration of 3-OHB in plasma reaches approximately 3 mM under short-term fasting conditions [22], up to 6 mM dur- ing long-term starvation [23] and regularly at least 2 mM under a ketogenic diet [24]. In view of this preclinical pro- and contra evidence and the fact that increasing numbers of patients are adopting a ketogenic diet or short-term fasting during oncological therapy, we studied the impact of 3-OHB on seven different BC cell lines in vitro. Here, we initially analyzed the energetic profile of these cells and corre- lated this to the effect of 3-OHB on cell proliferation. 3-OHB is transported into cells via monocarboxylic acid transporters (MCT). The isoforms MCT1, MCT2, and MCT4 can transport lactate and ketone bodies across the cell membrane [25, 26]. Here, MCT2 has the highest affinity for 3-OHB, whereas MCT1 and 4 have a Page 3 of 19 Bartmann et al. Cancer & Metabolism (2018) 6:8 Further, we investigated the possibility of synergism be- tween ketosis and radio- or chemotherapy [51]. Turnover of metabolites For quantification of glucose, lactate, and 3-OHB metab- olism, cells were seeded and cultured at conditions de- scribed in the cell proliferation assay. After 5 days, supernatants were collected and the levels of 3-OHB were analyzed by the PrecisionXceed® instrument with the corresponding test strips FreeStyle Precision® β-Ketone (Abbott, Wiesbaden, Germany). The concen- trations of glucose and lactate were measured with the Cobas 8000 modular analyzer series (Roche Diagnostics; Mannheim, Germany) at the central laboratory of the University Hospital of Würzburg. Concentrations of me- tabolites were expressed in millimolar per optical density (OD) of crystal violet dye extracts in each well at day 3 or 5 of culture. The amount of solubilized dye in OD is directly proportional to the cell number. Therefore, after removing the supernatant carefully for metabolite quan- tification, adherent cells were fixed with 100 μl methanol Background To mimic a physiological state of metabolites found in the circulation of patients performing a KD, we performed experiments with 3 mM 3-OHB, representing pro- nounced ketosis, and 5 mM glucose, typical for the blood glucose level found in persons on a KD (range 4.9–5.2 mM) [19, 52–57]. Furthermore, we investigated cells at both an oxygen supply of 5% oxygen (= 5 kPA), a typical mean concentration between well-vascularized benign breast tissue (6.5 kPA) and non-hypoxic tumor regions (3.2–4.7 kPA) in vivo [58] and 21% oxygen (21 kPA), as a common condition used in cell culture. 75-cm2 cell culture flasks (TPP, Trasadingen, Switzerland) in Dulbecco’s modified Eagle’s medium (DMEM)/Hams F12 (1:1) medium (Gibco, ThermoFisher Scientific, Darm- stadt, Germany) supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany) and 50 ng/ml Gentamy- cin (Sigma-Aldrich, Munich, Germany) in the presence of 5% CO2 and 21% oxygen, respectively. The subtype of the breast cancer cell lines used was classified before by gene expression profile and the expression of the estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (Her2) receptor. The abbreviations of receptors and breast cancer subtype classification shown here are published [59–61]. Semiquantified receptor status: results with “−”, “(−)”, “0”, and “0–1+” are classified as negative and results with “+”, “(+)”, “+/−”, 6, and 8 are classified as positive for receptor expression (also summarized by [62]) np not published Mutations shown here are described by [63–65] Breast cancer cell lines and culture Breast cancer cell lines and culture The BC cell lines BT20, BT474, HBL100, MCF-7, MDA-MB 231, MDA-MB 468, and T47D were obtained from Cell Lines Service GmbH (CLS; Eppelheim, Germany), and their receptor status, subtype, and mutation status are summarized in Table 1 [59–65]. The BT474 and the MDA-MB 231 cell lines were purchased directly from CLS for the experiments and used at low passage. All other cell lines were authenticated via genetic profiling of SRT loci by CLS before running the experiments. Aliquots of the cell lines were freshly cultured from frozen samples in Table 1 Subtype of the breast cancer cell lines used in the experiments Cell line Receptor Subtype Ref. Mutations ER PR Her2 BT20 − (−) np Basal A [60] ATM; BRCA2; CBLB; CDKN2A; COL1A1; RAP1GDS1; RB1; PIK3CA TP53 0 0 0–1 Basal [61] BT474 + (+) + Luminal [60] EPS15; HIST1H3B; NSD1; PIK3CA; PPP2R1A; RHOA; TP53 0 8 3+ Luminal B [61] + +/− + Luminal B [59] HBL100 − (−) np Basal B [60] np MCF-7 + (+) np Luminal [60] ATP2B3; CDKN2A; EP300; ERBB4; MAP3K13; PIK3CA 6 6 0–1 Luminal A [61] + +/− − Luminal A [59] MDA-MB 231 − (−) np Basal B [60] BRAF; CD79A; KRAS; CNKN2A; NF2; PBRM1; PDGFRA; TP53 0 0 0–1+ Basal [61] MDA-MB 468 (−) (−) np Basal A [60] CACNA1D; INPP4B; PTEN; RB1; TP53 0 0 0 Basal [61] − − − Basal [59] T47D + (+) np Luminal [60] ACVR1; ARID1A; PIK3CA; TP53 + + − Luminal A [59] The subtype of the breast cancer cell lines used was classified before by gene expression profile and the expression of the estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (Her2) receptor. The abbreviations of receptors and breast cancer subtype classification shown here are published [59–61]. Semiquantified receptor status: results with “−”, “(−)”, “0”, and “0–1+” are classified as negative and results with “+”, “(+)”, “+/−”, 6, and 8 are classified as positive for receptor expression (also summarized by [62]) np not published Mutations shown here are described by [63–65] Page 4 of 19 Page 4 of 19 Bartmann et al. Cancer & Metabolism (2018) 6:8 cell line to reach semiconfluency after 3 days under the respective oxygen and low glucose conditions and to reach confluency after 5 days via preliminary testing. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Western blotting For RT-qPCR and Western blot analyses, aliquots of cells were cultured under the same medium (DMEM/10% FCS/Gentamycin/5 mM glucose; with or without 3 mM 3-OHB) and oxygen (21 or 5%) conditions to reach sub- confluency after 5 days in 75-cm2 cell culture flasks. RNA extraction, cDNA synthesis (iScript, Bio-Rad), and qPCR (Mesa Green containing Meteor Taq hotstart polymerase, Eurogentic) as well as Western blotting were performed as described previously [66]. The primers and conditions of the qPCR are described in Table 2. In brief, qPCR reactions were performed on a Colony formation assay Colony formation assay For the colony formation assay, cells were cultured at 100 cells/well in 48 well plates (TTP) with 500 μl medium (DMEM/10% FCS/Gentamycin/5 mM glucose) with or without 3 mM 3-OHB at oxygen concentrations of 5 or 21% for 14 to 18 days, depending on the cell line. Half of the medium was replenished every 4 days. Crystal violet staining was performed for evaluation of the adherent colonies. Therefore, supernatant was care- fully removed; adherent cells were fixed with 250 μl methanol (Sigma-Aldrich) for 10 min at RT and then dried. Colonies were stained by incubation in crystal vio- let solution (same as above) for 10 min and then washed several times with distilled water. Stained colonies were documented by using the ImmunoCapture 6.2 in the ImmunoSpot Analyzer® (Cellular Technology, Shaker Heights, OH, USA). Two independent experiments were performed with 8 replicate wells per condition (5 and 21% oxygen with and without 3-OHB) for each cell line. Breast cancer cell lines and culture Thus, 250–350 cells per well, depending on the cell line, were seeded in 200 μl DMEM/10%FCS/Gentamycin/ 5 mM glucose. Cell plates were cultured for 5 days at 5% CO2 and 37 °C in humidified chambers at oxygen concentrations of 21 or 5% in hypoxia-incubators (Coy Laboratories Products Inc., Grass Lake, MI, USA) respectively. At least 3 independent wells per cell line were tested either with or without 3 mM 3-OHB or 1.5 mM acetoacetate (lithium salt) and LiCl (both Sigma-Aldrich) as control in parallel. Four independent experiments were performed with fresh cultured cell aliquots. At day 5, supernatants were removed for metabolite testing, 100 μl of fresh medium containing 5-bromo-2′-deoxyuridine (BrdU) were added for an- other 24 h, and cell proliferation rate was then analyzed by the BrdU test (Roche; Cell Proliferation ELISA) ac- cording to the manufacturer’s instructions. (Sigma-Aldrich) for 10 min at room temperature (RT) and then dried. Cells were stained by incubation in 100 μl crystal violet solution per well (0.4% crystal violet [Merck, Darmstadt, Germany] in 1:3 methanol: phosphate-buffered saline) for 10 min at RT and then washed several times with distilled water. Crystal vio- let was extracted from cells with 100 μl of 10% acetic acid per well on a plate shaker for 30 min, and OD was determined at 570 nm by using a standard ELISA-Plate reader. (Sigma-Aldrich) for 10 min at room temperature (RT) and then dried. Cells were stained by incubation in 100 μl crystal violet solution per well (0.4% crystal violet [Merck, Darmstadt, Germany] in 1:3 methanol: phosphate-buffered saline) for 10 min at RT and then washed several times with distilled water. Crystal vio- let was extracted from cells with 100 μl of 10% acetic acid per well on a plate shaker for 30 min, and OD was determined at 570 nm by using a standard ELISA-Plate reader. Energetic profiling by Seahorse technique g p g y q The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed with the Seahorse XF Cell Mito Stress Test (Part #103015-100; Agilent Technologies, Santa Clara, CA, USA) in a Seahorse XFe96 Analyzer (Agilent Technologies). The day before the experiment, 40,000 cells per well were plated in a 96-well Seahorse plate in 100 μl DMEM/10% FCS/Gentamy- cin/5 mM glucose medium with or without 3 mM 3-OHB (sodium-hydroxybutyrate, Sigma-Aldrich). The Agilent Sea- horse XFe96 Sensor Cartridge was hydrated with 200 μl/well of XF calibrant solution overnight in a non-CO2 incubator at 37 °C. On the day of the experiment, 100 ml of Seahorse assay medium containing 1 mM pyruvate, 2 mM glutamine, and 5 mM glucose was prepared. The pH of the pre-warmed (37 °C) medium was adjusted to 7.4 with 0.1 N NaOH. Twenty milliliters of the assay medium was used to prepare 3 mM 3-OHB, and the pH was readjusted to 7.4 with 0.1 N HCl. Cells were washed twice with 200 μl of the corresponding Seahorse medium and incubated in 175 μl of the respective Seahorse medium per well in a non-CO2 incubator at 37 °C for 1 h. Meanwhile, the Seahorse sensor cartridge ports were loaded with 25 μl of inhibitors to have a final concentration of 2 μM oligomycin (port A, Calbiochem), 1 μM FCCP (port B, Sigma-Aldrich), and 0.5 μM rotenone/antimycin A (port C, Sigma-Aldrich). The experimental design was setup using the WAVE soft- ware program, and measurements were performed in the Seahorse XFe96 Analyzer. After the measurement, super- natant from the cells was removed and the cells were fixed by addition of 100 μl methanol (Sigma-Aldrich) for 10 min at RT and air dried. Subsequently, the cells were stained using crystal violet solution as described for the colony formation assay (see below). For quantification, stained plates were incubated with 200 μl of 10% acetic acid per well with shaking for 15 min and the resulting solution was analyzed in a plate reader (Tecan GENios plus, Tecan Deutschland GmbH, Crailsheim, Germany) at 630 nm. Chemosensitivity and radiosensitivity For both sensitivity tests, the cells were seeded as in the prior tests in 96-well plates in standard medium with 5 mM glucose and incubated for 48 h at oxygen concen- trations of 5 or 21% respectively. For chemosensitivity testing, the test drug concentration (TDC) of epirubicin was defined as 0.5 μg/ml, of paclitaxel as 13.6 μg/ml, and of carboplatin as 15.8 μg/ml, as de- scribed in the literature [69–72]. The cell culture medium (see above) with or without 3 mM 3-OHB and eight different dilutions of the chemotherapeutic drugs (epirubicin: highest concentration: 25% TDC and two- fold dilution series; paclitaxel highest concentration: Cell proliferation assay The cycler protocol was 5 min at 95 °C (initial denaturation), 40 cycles of 15 s at 95 °C, 60 s at 60 °C (two-step protocol), and 5 min at 72 °C (final extension) for all primer pairs used. Fold expression of genes of interest (Table 2) expression relative to refer- ence genes PPIA and β-actin was calculated with the ΔΔCq method [67]. Post-amplification melting curves were con- trolled to exclude primer-dimer artifacts and contamina- tions (not shown). Data from two independent cell culture experiments in triplicate reactions for each primer pair were summarized (Fig. 3b). CFX96 real-time PCR system (Bio-Rad) operated by CFX Manager Software (version 3.0). The cycler protocol was 5 min at 95 °C (initial denaturation), 40 cycles of 15 s at 95 °C, 60 s at 60 °C (two-step protocol), and 5 min at 72 °C (final extension) for all primer pairs used. Fold expression of genes of interest (Table 2) expression relative to refer- ence genes PPIA and β-actin was calculated with the ΔΔCq method [67]. Post-amplification melting curves were con- trolled to exclude primer-dimer artifacts and contamina- tions (not shown). Data from two independent cell culture experiments in triplicate reactions for each primer pair were summarized (Fig. 3b). For radiosensitivity testing, subconfluent monolayers of cells were irradiated in the culture plates filled with 200 μl/well with the corresponding growth medium with graded single doses (0–8 Gy) and then cultivated for 10 days at either 5 or 21% oxygen con- centration. Irradiation was performed using a 6 MV Siemens linear accelerator (Siemens, Concord, CA, USA) at a dose rate of 2 Gy/min. Half of the medium was changed every 4 days. At the end of the incuba- tion period, BrdU was added and cell proliferation was measured by the BrdU test 24 h thereafter. For Western blotting, pellets of 1 × 106 tumor cells were lysed and protein concentrations were determined using the Bradford method [68] and Coomassie Brilliant Blue (Roti-Quant; Roth, Karlsruhe, Germany) reagent. Afterwards, samples were mixed with 5× loading buffer (Fermentas GmbH, St. Leon-Roth, Germany), denatured at 95 °C for 5 min, chilled on ice, and stored at −20 °C for further analysis. Equal amounts of proteins (20 μg) were separated on a 10% polyacrylamide gel. The anti- bodies used are listed in Table 3. Statistics Data are presented as means (± standard error of mean [SEM]). p values lower than 0.05 in the non-parametric Mann-Whitney U test were considered significant. The software GraphPad Prism 6 (La Jolla, CA 92037 USA) was used to create the figures and to perform statistical analysis. IC50 was determined via the nonlinear regres- sion dose-response curve (inhibition) function of the Prism software. Cell proliferation assay Adherent growing cells were seeded in 96-well flat bot- tom plates (TPP) at cell numbers determined for each Bartmann et al. Cancer & Metabolism (2018) 6:8 Page 5 of 19 Table 2 Details of the primer pairs used in this study Gene Forward primer/reverse primer cDNA size (bp) PubMed access no. β-Actin 5′-CCT TGC CAT CCT AAA AGC C-3′ 5′-CAC GAA AGC AAT GCT ATC AC-3′ 96 NM_001101 PPIA 5′-TGT CCA TGG CAA ATG CTG GAC CC-3′ 5′-GCG CTC CAT GGC CTC CAC AA-3′ 140 NM_021130.3 BDH1 5′-CGC CGG GTG AAG GCG-3′ 5′-GAA TGG CCC AGT TCC TCC C-3′ 92 NM_004051.4 SCOT 5′-GCC ATT GCC AGT AAG CCA AG-3′ 5′-CCA GGC TTT CAC CAA AGC AA-3′ 99 NM_000436.3 ACAT1 5′-CGG CAG ATG CAG CGA AGA GG-3′ 5′-AGG TTC TAC AGC AGC GTC AGC A-3′ 77 NM_000019.3 DNA sequences and PubMed accession numbers for each gene are indicated. qPCR reactions were performed on a CFX96 real-time PCR system (Bio-Rad) operated by CFX Manager Software (version 3.0). The two-step cycler protocol was 5 min at 95 °C, 40 cycles of 15 s at 95 °C, 60 s at 60 °C, followed by 5 min at 72 °C and used for all primer pairs PPIA peptidylprolyl isomerase A, BDH 3-hydroxybutyrate dehydrogenase, SCOT succinyl-CoA transferase (3-oxoacid CoA transferase), ACAT acetyl-CoA-acetyltransferase DNA sequences and PubMed accession numbers for each gene are indicated. qPCR reactions were performed on a CFX96 real-time PCR system (Bio-Rad) operated by CFX Manager Software (version 3.0). The two-step cycler protocol was 5 min at 95 °C, 40 cycles of 15 s at 95 °C, 60 s at 60 °C, followed by 5 min at 72 °C and used for all primer pairs PPIA peptidylprolyl isomerase A, BDH 3-hydroxybutyrate dehydrogenase, SCOT succinyl-CoA transferase (3-oxoacid CoA transferase), ACAT acetyl-CoA-acetyltransferase 50% TDC and tenfold dilution series or carboplatin highest concentration: 400% TDC and twofold dilu- tion series) were added. TDC concentrations for the experiments were selected by cell viability assays per- formed in preliminary tests with all cell lines (not shown). After 3 days of cell culture, BrdU was added for the final 24 h of culture and the BrdU test per- formed as described above. CFX96 real-time PCR system (Bio-Rad) operated by CFX Manager Software (version 3.0). 3-OHB changes oxygen consumption rate and extracellular acidification rate in BT20 cells in response to 3-OHB and reduced oxygen conditions varied between the tested BC cell lines. We observed an overall decrease in expression of all three enzymes in MCF7 and MDA-MB 231 cells with further decrease (MCF7) or a moderate increase of BDH1 and ACAT in the presence of 3-OHB. All enzymes were downregu- lated in HBL100 and MCF7 cells, but and only BDH1 and ACAT in MDA-MB 231 cells in response to low oxygen (5% oxygen). This regulation was not observed in BT20, BT474, MDA-MB 468, and T47D. In summary, the data show no strong correlation between mRNA overexpression and consumption of 3-OHB or oxygen concentration. Seahorse analysis confirmed the metabolic phenotype found in the glucose consumption/lactate production rate analysis. All cell lines except T47D exhibited a more aerobic/energetic cell type, corresponding to cells that divide but generate their energy predominantly from oxidative phosphorylation (OXPHOS) (Fig. 2a). This basal metabolic phenotype did not correlate with the reaction of these cells to 3-OHB, since 3-OHB significantly influenced the oxygen consump- tion rate (OCR) in BT20 cells only. Here, OCR in- creased from 77.8 ± 5.8 to 124.0 ± 10.0 pmol/min (mean ± SEM; p < 0.001), when cells were cultured in the presence of 3-OHB (Fig. 2b, c). For all other cell lines, we found no evidence that 3-OHB influenced oxygen consumption (Fig. 2c, left graph). In addition, the effect of 3-OHB on extracellular acidification rate (ECAR) was analyzed. In the case of BT20 cells, ECAR significantly increased from 61.2 ± 4.3 mpH (milli pH)/min to 80.3 ± 7.7 mpH/min (p < 0.05) with 3-OHB. An increase from 83.6 ± 3.1 mpH/min to 99.5 ± 5.2 mpH/min (p < 0.05) and from 54.2 ± 1.8 mpH/min to 62.5 ± 2.3 mpH/min (p < 0.01) was ob- served in MCF7 and MDA-MB 468 cells under the influence of 3-OHB, respectively (Fig. 2c, right side). Moreover, the extent of 3-OHB uptake was not linked with the extent of the Warburg effect in each cell line as shown in Fig. 2a. There was also no significant difference in the concentration of 3-OHB remaining in the medium after incubation with cells at 5 or 21% oxygen (Fig. 3a). By Western blot analysis (Fig. 3c), we showed that MCT2, the key transporter for 3-OHB into cells [27], was expressed highly by all seven cell lines. 3-OHB changes oxygen consumption rate and extracellular acidification rate in BT20 cells In par- ticular, MCT2 expression was higher at mild hypoxia in the majority of cell lines. Therefore, the transport of 3-OHB across the plasma membrane seems not to be a limiting factor for 3-OHB consumption by BC cells. GLUT1 as a key transporter for glucose into tumor cells was detected in all cell lines, and its expression was not influenced by 3-OHB (Fig. 3c). Together, our results demonstrate that 3-OHB does not significantly change the expression of ketolytic enzymes or of the transporter molecules MCT2 and GLUT1. Nevertheless, BC cell lines show marked differences in their ability to deplete 3-OHB from the medium indicating that mRNA expres- sion patterns of ketolytic enzymes were not associated with consumption rate of 3-OHB. Thus in general, no direct correlation between the metabolic reaction to 3-OHB and the basal energetic phenotype of the cell lines was seen. Glucose consumption and lactate production is not influenced by 3-OHB Glucose consumption and production of lactate were normalized to the optical density (OD) of crystal violet dye extracts, which is directly proportional to the cell Page 6 of 19 Bartmann et al. Cancer & Metabolism (2018) 6:8 Table 3 Antibodies for Western blot Antibody Gen/name Company Clone Species Dilution MW of antigen Primary Beta-actin Abcam mAbcam 8226 Mouse 1:10000 42 kDa Monocarboxylic acid transporter 2 (MCT-2) Abcam Polyclonal Rabbit 1:2000 52 kDa Glucose transporter 1 (GLUT1) USBiological Polyclonal Rabbit 1:1000 55 kDa Secondary HRP-labeled goat-anti-mouse KPL Polyclonal Goat 1:10000 HRP-labeled goat-anti-rabbit Abcam Polyclonal Goat 1:20000 MW molecular weight Table 3 Antibodies for Western blot MW molecular weight 468, and T47D consume relatively low amounts of glucose (< 3.5 mM/OD) and show low lactate production (< 10 mM/OD), while BT474 show very low levels of glucose consumption (< 1 mM/OD) and lactate production (< 5 mM/OD). Interestingly, independent of the respective metabolic characteristics, 3 mM 3-OHB failed to signifi- cantly influence the consumption of glucose or produc- tion of lactate in the presence of either 5 or 21% oxygen (Fig. 1). number, as readout for the rate of aerobic glycolysis (Warburg effect) or respiration, respectively. With these measurements, we were able to categorize the metabolic characteristics of the seven tested cell lines as follows: BT20 and MDA-MB 231 exhibit high glucose consump- tion (with an average of more than 5.0 mM) normalized to cell number as reflected by OD readout of the crystal violet assay) and high lactate production (with an average of more than 15 mM/OD). HBL100, MCF-7, MDA-MB Fig. 1 The graphs represent the rate of lactate production (mmol × l−1 × optical density (OD)−1; upper panel) and glucose consumption (mmol × l−1 × OD−1; lower panel) normalized to total cell content (OD) after 5 days of culture in 5 or 21% oxygen (gray column = 3 mM 3-OHB; black column = control). Each column represents mean ± SEM of four independent experiments Fig. 1 The graphs represent the rate of lactate production (mmol × l−1 × optical density (OD)−1; upper panel) and glucose consumption (mmol × l−1 × OD−1; lower panel) normalized to total cell content (OD) after 5 days of culture in 5 or 21% oxygen (gray column = 3 mM 3-OHB; black column = control). Each column represents mean ± SEM of four independent experiments Page 7 of 19 Page 7 of 19 Bartmann et al. Glucose consumption and lactate production is not influenced by 3-OHB Cancer & Metabolism (2018) 6:8 Consumption of 3-OHB is not strongly linked to overexpression of ketolytic enzymes and does not correlate with the observed effects on metabolic phenotype p yp To investigate whether BC cells were able to use 3-OHB as a substrate for intermediate metabolism, we next de- termined uptake rates thereof in the panel of breast can- cer cell lines. All cell lines depleted 3-OHB from the culture medium but the magnitude of depletion differed substantially between them (Fig. 3a). Interestingly, we detected variable levels of mRNA expression for key en- zymes of ketolysis, namely 3-hydroxybutyrate dehydro- genase 1 (BDH1), succinyl-CoA transferase (SCOT), and acetyl-CoA-acetyltransferase (ACAT), in the seven human breast cancer cell lines (Fig. 3b). In BT474 cells, which showed a relevant consumption of 3-OHB (Fig. 3a), we detected high levels of mRNA for all three key enzymes. SCOT and ACAT, but not BDH1, were overexpressed in HBL100 cells, which showed reduced 3-OHB consumption compared to BT474 cells. All other cell lines revealed low levels of mRNA expression for ketolytic enzymes with a moderate ACAT mRNA ex- pression in MDA-MB 231 and T47D cells. However, changes in mRNA expression levels of ketolytic enzymes Short-term and long-term cell proliferation of breast cancer cells is not affected by incubation with 3-OHB cancer cells is not affected by incubation with 3-OHB To analyze whether 3-OHB can affect cancer cell prolif- eration independent of their metabolic phenotype, we incubated the BC cell lines with 5 mM glucose, with and without addition of 3 mM 3-OHB in the presence of 5 or 21% oxygen, respectively. Here, we found a slight re- duction (< 10%) in short-term (5 days) proliferation fol- lowing 3-OHB treatment in BT20, MCF-7, MDA-MB 231, MDA-MB 468, and T47D cells at either oxygen concentration, while BT474 and HBL100 cells were not affected (Fig. 4a). Since AcAc, the second ketone body which rises in circulation upon a ketogenic diet, was described to increase proliferation in BRAF V600E melanoma cells [73], we performed cell proliferation as- says in the presence of this metabolite over 5 days in parallel to the 3-OHB experiments. As shown in the Additional file 1, there was no significant effect of AcAc Bartmann et al. Cancer & Metabolism (2018) 6:8 Page 8 of 19 A B C Fig. 2 (See legend on next page.) A B B C C Bartmann et al. Cancer & Metabolism (2018) 6:8 Page 9 of 19 (See figure on previous page.) Fig. 2 a Energetic phenotype as revealed by Seahorse flux analysis in cultures without 3-OHB (black symbols) and with 3 mM 3-OHB (gray symbols). Arrow indicates the significant (p < 0.05) shift in energetic phenotype observed with the BT20 cell line. Graph summarizes the results of four independent seahorse experiments with four replicate wells for each cell line. b The curves of OCR and ECAR for the BC cell lines with the most prominent changes (BT20) and without any changes (HBL100) depending on the addition of 3-OHB are shown here. The graph represents the three measuring points of basal levels of respiration/acidification, and changes after addition of oligomycin, FCCP, and antimycin A/rotenone (black line and dots = control, gray line and boxes = 3-OHB). c Column statistics of the baseline OCR and ECAR of BC cell lines with 3-OHB (gray column) compared to control (black column) (p < 0.001; p < 0.01; p < 0.05). Each column summarizes mean ± SEM of four independent seahorse experiments with four replicate wells per experiment for each cell line Further, we found that 3-OHB did not significantly sensitize BC cells to radiation at either 21 or 5% oxygen. Short-term and long-term cell proliferation of breast cancer cells is not affected by incubation with 3-OHB Hypoxia per se, however, showed a tendency to confer a higher radio-resistance to the tumor cells (Fig. 6a + b) consistent with the known action of oxygen as a radio- sensitizer. Two representative examples of dose-response curves are shown for the highest responders to radiation at both oxygen conditions in Fig. 6c–f. In analyzing the cell lines individually, however, a tendency to confer a higher radiosensitivity is seen for those tumor cells grown in 3-OHB at 5% oxygen in all but the MDA-MB 321 cell lines (Additional file 3). on the proliferation rate of the BC cell lines tested. A slight increase in proliferation of BT20 cells at 5% oxy- gen concentration did not reach statistical significance. Since AcAc was used as lithium salt, control experi- ments with LiCl at corresponding Li concentration were performed, but did not differ from the proliferation rates seen with Li-free cell culture medium (not shown). on the proliferation rate of the BC cell lines tested. A slight increase in proliferation of BT20 cells at 5% oxy- gen concentration did not reach statistical significance. Since AcAc was used as lithium salt, control experi- ments with LiCl at corresponding Li concentration were performed, but did not differ from the proliferation rates seen with Li-free cell culture medium (not shown). To test the effect of 3 mM 3-OHB on long-term pro- liferation, we performed a colony formation assay for at least 14 days of culture. Similar to the results of the short-term proliferation assay, we found no significant differences in number and size of cell colonies between cultures treated with 3-OHB and control cultures. Of note, oxygen concentration influenced size and number of cell colonies of BT474, HBL100, and MDA-MB 231 cells (Fig. 4b). Discussion In this study, we have shown that beta-hydroxybutyrate (3-OHB), the main ketone body found in the circulation after fasting or ketogenic diets (KDs), was able to change the energetic phenotype of BT20 breast cancer cells when applied at physiological concentrations of 3 mM. However, this effect of 3-OHB on energy metabolism was not observed in any of the other BC cell lines inves- tigated herein. Moreover, 3-OHB did not interfere with turnover of glucose and lactate and neither significantly affected short- and long-term cancer cell proliferation, or their sensitivity to chemotherapy or ionizing radiation in any of the cell lines tested. This implies that 3-OHB at a physiological concentration of 3 mM seems to be inert in affecting energetic processes essential for prolif- eration or cell survival in the tested BC cell lines in vitro. In addition, we found that AcAc, the second ketone body elevated under a KD, also did not significantly in- fluence proliferation of any BC cell line measured over 5 days. While preclinical data have shown that AcAc promotes proliferation of BRAF-V600-positive melan- oma cells [73], it is interesting that in a pilot study [13] it was a patient with BRAF V600E-positive/BRAF-inhibi- tor-resistant melanoma who responded favorably to a KD; this highlights the limitations of translating preclin- ical study results to humans. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation py g We next addressed the question whether exposure to 3-OHB may affect the sensitivity of BC cells to different treatment modalities. We therefore exposed the BC cell lines to epirubicin, paclitaxel, or carboplatin, three che- motherapeutic agents commonly used in breast cancer treatment. Here, we did not find any significant influence of 3-OHB on the effect of these drugs on can- cer cell viability at either 5 or 21% oxygen (Fig. 5a and Additional file 2). The cumulative IC50 of paclitaxel was 4.0 ± 1.1 ng/ml (mean ± SEM) (control) versus 2.7 ± 0.8 ng/ml (3 mM 3-OHB) at 5% oxygen and 3.7 ± 1.3 ng/ml (control) versus 3.8 ± 1.2 ng/ml (3 mM 3-OHB) at 21% oxygen. The mean cumulative IC50 of epirubicin was 28.3 ± 6.1 ng/ml in control cells versus 28.5 ± 7.8 ng/ml (3 mM 3-OHB) at 5% oxygen concen- tration and 18.9 ± 3.6 ng/ml (control) versus 18.0 ± 2.7 ng/ml (3 mM 3-OHB) at 21% oxygen concentration. In the case of carboplatin, the mean cumulative IC50 was 6.6 ± 1.4 μg/ml (control) versus 6.0 ± 1.4 μg/ml (3-OHB) at 5% oxygen and 5.0 ± 2.4 μg/ml (control) ver- sus 4.8 ± 2.3 μg/ml (3-OHB) at 21% oxygen concentra- tion. Representative results of the sensitivity tests are shown in Fig. 5b, c. This research was performed, since a possible influ- ence of 3-OHB on cancer growth has gained substantial interest over the past years. In particular, different regimes for KDs are currently investigated in several Page 10 of 19 Bartmann et al. Cancer & Metabolism (2018) 6:8 A B C Fig. 3 (See legend on next page.) A B C B C g. 3 (See legend on next page.) C Page 11 of 19 Bartmann et al. Cancer & Metabolism (2018) 6:8 (See figure on previous page.) Fig. 3 a The columns show the amount of 3-OHB (in mM) consumed by the cells normalized to their cell number as given by optical density (OD) measured with the crystal violet assay. Columns represent mean ± SEM of two independent experiments with three replicate wells per experiment. There was no significant difference in the consumption of 3-OHB between cultivation at 21 and 5% oxygen. A tendency to reduced 3-OHB consumption was observed at 5% oxygen. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation b Relative expression of mRNA for the ketolytic enzymes BDH1 (β-hydroxybutyrate dehydrogenase), SCOT (succinyl-CoA:3-ketoacid coenzyme A transferase), and ACAT (acetyl-CoA acetyltransferases) in the tested BC cell lines. Each column represents mean ± SEM of data from two independent cell culture experiments in triplicate reactions for each primer pair. c All cell lines express the most important transporter for 3-OHB, the monocarboxylate transporter 2 (MCT2), and the glucose transporter 1 (GLUT1) on protein level. Beta-actin served as loading control. Representative Western blot images of the four test conditions (21 and 5% oxygen with and without 3-OHB) for each cell line are shown clinical trials to improve the outcome for cancer patients [74]. Several reports indicate a significant benefit of ketosis and especially 3-OHB on slowing cancer progres- sion in different preclinical cancer models and patients [13, 37, 42, 44, 49, 75–77], amongst them one recent case study successfully applying a KD as part of a multi- modal pro-oxidative therapy in a stage IV triple-negative breast cancer patient [78]. On the other hand, there are other publications describing a negative impact of keto- sis/3-OHB on tumor cell growth in vitro and in mouse models [35, 36, 79, 80]. Therefore, we initially analyzed the energetic profile of seven different BC cell lines using substrate turnover quanti- fication and energetic flux analysis. The basic energetic A B Fig. 4 a The graphs show the proliferation rate (BrdU; in % of control cells) of the different BC cell lines cultured in medium containing 3 mM 3-OHB (gray column) compared to control without 3-OHB (black column) at 5 or 21% oxygen concentration after 5 days of culture (differences are not statistically significant). The columns summarize mean ± SEM of data of four independent experiments with three replicate wells per experiment for each cell line. b The figure shows representative results (one out of eight replicates for each cell line) of the colony formation assay for the tested BC cell lines after 14 days of culture. The cell lines show no significant alteration in number and size of colonies upon addition of 3-OHB. BT474, HBL-100, and MDA-MB 231 showed an overall reduced colony size at 5% oxygen concentration compared to 21% oxygen concentration A A A B B Fig. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation 4 a The graphs show the proliferation rate (BrdU; in % of control cells) of the different BC cell lines cultured in medium containing 3 mM 3-OHB (gray column) compared to control without 3-OHB (black column) at 5 or 21% oxygen concentration after 5 days of culture (differences are not statistically significant). The columns summarize mean ± SEM of data of four independent experiments with three replicate wells per experiment for each cell line. b The figure shows representative results (one out of eight replicates for each cell line) of the colony formation assay for the tested BC cell lines after 14 days of culture. The cell lines show no significant alteration in number and size of colonies upon addition of 3-OHB. BT474, HBL-100, and MDA-MB 231 showed an overall reduced colony size at 5% oxygen concentration compared to 21% oxygen concentration Fig. 4 a The graphs show the proliferation rate (BrdU; in % of control cells) of the different BC cell lines cultured in medium containing 3 mM 3-OHB (gray column) compared to control without 3-OHB (black column) at 5 or 21% oxygen concentration after 5 days of culture (differences are not statistically significant). The columns summarize mean ± SEM of data of four independent experiments with three replicate wells per experiment for each cell line. b The figure shows representative results (one out of eight replicates for each cell line) of the colony formation assay for the tested BC cell lines after 14 days of culture. The cell lines show no significant alteration in number and size of colonies upon addition of 3-OHB. BT474, HBL-100, and MDA-MB 231 showed an overall reduced colony size at 5% oxygen concentration compared to 21% oxygen concentration Bartmann et al. Cancer & Metabolism (2018) 6:8 Page 12 of 19 A B C Fig. 5 a The column-graphs show the cumulative IC50 of epirubicin, paclitaxel, and carboplatin in control cells (dark gray box) and cells cultured with 3 mM 3-OHB (light gray box). Per cell line, three to four each independent dose-response experiments with six replicate wells per experiment were calculated. b Representative dose-response curves obtained for BT-20 cells at 5% oxygen in chemotherapy sensitivity testing with the chemotherapeutic drugs (epirubicin, paclitaxel, carboplatin) which was used for the calculation of the IC50 (dashed line) (black box = control, white box = 3-OHB). c Same as b but for 21% oxygen. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation Curves summarize four independent experiments with six replicate wells per experiment A C B C B Fig. 5 a The column-graphs show the cumulative IC50 of epirubicin, paclitaxel, and carboplatin in control cells (dark gray box) and cells cultured with 3 mM 3-OHB (light gray box). Per cell line, three to four each independent dose-response experiments with six replicate wells per experiment were calculated. b Representative dose-response curves obtained for BT-20 cells at 5% oxygen in chemotherapy sensitivity testing with the chemotherapeutic drugs (epirubicin, paclitaxel, carboplatin) which was used for the calculation of the IC50 (dashed line) (black box = control, white box = 3-OHB). c Same as b but for 21% oxygen. Curves summarize four independent experiments with six replicate wells per experiment colleagues. However, in the study of Pelicano and colleagues, both MDA-MB cell lines were very similar to the energetic phenotype of BT20 cells. In contrast, in our study, the BT20 cells showed a relatively low basal OCR compared to other TNBC cell lines in our experiments. This more oxidative basal phenotype of the TNBC cell line MDA-MB 468 was recently also shown by Lanning and colleagues, even at high glucose conditions of 10 mM [82]. However, this study also described a low oxidative phenotype for the MDA-MB 231 cell line which was remarkably lower than that of MDA-MB 468, while in our investigation, both cell lines showed phenotypes differed somewhat from the results reported by Pelicano and coworkers [81], who found that triple-negative breast cancer (TNBC) cell lines were in general characterized by a higher extracellular acidification rate (ECAR) and lower oxygen consumption rate (OCR) compared to hormone re- ceptor positive cell lines [81]. For the TNBC cell line BT20, we found an energetic profile that was low in OCR and ECAR and was comparable to the hormone receptor positive T47D cell line. Furthermore, the TNBC cell lines MDA-MB 231 and MDA-MB 468 showed very similar energetic pro- files in our experiments and those of Pelicano and Page 13 of 19 Bartmann et al. Cancer & Metabolism (2018) 6:8 A B C D E F Fig. 6 Cell proliferation after irradiation measured by BrdU, summarized for all BC cell lines at 21% (a) and 5% (b) oxygen concentration (black column = control; gray column = 3 mM 3-OHB). No significant influence of 3-OHB was seen. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation This indicates that 3-OHB alters mitochondrial capacity, for example, by increasing the expression/activity of respiratory complexes or by indu- cing mitochondrial biogenesis. In addition, glycolysis was also increased after 3-OHB exposure, indicating an overall increase in metabolic activity of these cells (as displayed in Fig. 2a). However, this phenotype was not observed in the other breast cancer cell lines used in our study. One reason for this difference could be that BT20 is the only TNBC cell line that is mutant for BRCA2 (Table 1). Indeed, a large-scale metabolomics study re- cently found that BT20 cells differ in their metabolic profile compared to other TNBC cells, based on un- supervised hierarchical clustering [85]. While further ex- perimentation is required to identify the underlying mechanisms for the overall increase in metabolic activity caused by 3-OHB exposure in these cells, our data clearly show that 3-OHB does not affect the viability of BT20 cells or their response to chemotherapy or radi- ation, despite this metabolic effect. Notably, ECAR is not only dependent on medium acidification via lactic acid but can also be affected by the production of car- bonic acid (H2CO3) as an end product of the oxidative degradation of metabolites in the tricarboxylic acid (TCA) cycle [86, 87]. Here, a very important aspect is the observation that breast cancer cell lines, and espe- cially the TNBC cell lines, often use glutamine as a rele- vant nutrient to support their metabolic demands [88, 89]. Moreover, glutamine can also be converted to pyru- vate and lactate by malic enzyme [90]. Since glutamine is not a limiting factor in our cell culture medium, we cannot exclude that the observed increase in ECAR is Here, we found no evidence that 3-OHB fuels the me- tabolism of BC cells in vitro. This is in accordance to an in vivo study, which showed that 3-OHB did not influ- ence growth of melanoma cells either when injected in- traperitoneally or when elevated by a high-fat diet [73]. However, at the same time, it is in contrast to a previous study that has shown an increased growth of breast can- cer xenografts derived from MDA-MB 231 cells when mice were injected with 3-OHB intraperitoneally [36]. In our in vitro experiments, the MDA-MB 231 cell line showed no increased proliferation in the presence of 3-OHB. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation Columns represent mean ± SEM of three independent experiments with six replicate wells per experiment. Two representative dose-response curves for MCF7 and MDA-MB 468 are shown (c–f). MDA-MB 468 cells were sensitive to radiation (c, d), while MCF7 cells were relatively insensitive even to high doses (e, f). Open and filled symbols represent mean (± SD) of S-phase cell counts in 3-OHB-untreated control and 3-OHB-treated cells, respectively. The data were normalized to the corresponding values of non-irradiated cells at 21 or 5% oxygen, respectively A B A B D C D E F F Fig. 6 Cell proliferation after irradiation measured by BrdU, summarized for all BC cell lines at 21% (a) and 5% (b) oxygen concentration (black column = control; gray column = 3 mM 3-OHB). No significant influence of 3-OHB was seen. Columns represent mean ± SEM of three independent experiments with six replicate wells per experiment. Two representative dose-response curves for MCF7 and MDA-MB 468 are shown (c–f). MDA-MB 468 cells were sensitive to radiation (c, d), while MCF7 cells were relatively insensitive even to high doses (e, f). Open and filled symbols represent mean (± SD) of S-phase cell counts in 3-OHB-untreated control and 3-OHB-treated cells, respectively. The data were normalized to the corresponding values of non-irradiated cells at 21 or 5% oxygen, respectively Fig. 6 Cell proliferation after irradiation measured by BrdU, summarized for all BC cell lines at 21% (a) and 5% (b) oxygen concentration (black column = control; gray column = 3 mM 3-OHB). No significant influence of 3-OHB was seen. Columns represent mean ± SEM of three independent experiments with six replicate wells per experiment. Two representative dose-response curves for MCF7 and MDA-MB 468 are shown (c–f). MDA-MB 468 cells were sensitive to radiation (c, d), while MCF7 cells were relatively insensitive even to high doses (e, f). Open and filled symbols represent mean (± SD) of S-phase cell counts in 3-OHB-untreated control and 3-OHB-treated cells, respectively. The data were normalized to the corresponding values of non-irradiated cells at 21 or 5% oxygen, respectively Page 14 of 19 Bartmann et al. Cancer & Metabolism (2018) 6:8 Page 14 of 19 due to glutamine metabolism in the cell lines investi- gated. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation Therefore, the increase in ECAR in parallel to elevated OCR in BT20 cells is likely to correspond to a generally more “energetic phenotype” of enhanced respiration and CO2 or lactate-induced acidification [86, 87]. Another possible interpretation would be mitochon- drial uncoupling via overexpression of uncoupling pro- tein 2 (UCP2) that, although not evaluated in our study, has been shown to occur in BC cell lines [91]. UCP2 overexpression has a metabolic action by supporting glu- cose and glutamine fermentation at the expense of mito- chondrial oxidation [92]. If mitochondrial uncoupling increased, then oxygen consumption would not strictly be linked to respiratory capacity. comparable values of OCR. It is likely that these dis- crepancies between metabolic profiling data can arise from small differences in the culture conditions, for example different metabolite concentrations in fetal calf serum. Nevertheless, we can conclude that under the experimental conditions used here, only BT20 cells shifted their metabolic profile upon exposure to reduced oxygen concentration. Those discrepancies between our and other metabolic profiling data could also be due to different expression profiles of the cell lines in different laboratories based on different sub- clones [83, 84]. Of note, our quotient of OCR/ECAR for BT20 had a range from 0.9–2.4 (not shown), which is closer to the original findings of Warburg, who found that the quo- tient of respiration to aerobic glycolysis was 0.3–0.9 in tumor tissue sections [8, 9]. Apart from BT20, the other BC cell lines remained relatively stable with respect to the OCR/ECAR ratio in the presence of 3-OHB. Anyway, the shift to a more energetic phenotype seems not to correlate with the metabolism of 3-OHB as an energy source. As proven by RT-qPCR, the BT20 cells express very low levels of mRNA for BDH1, the key entry enzyme of ketolysis, and in line with this, consume only small amounts of 3-OHB. Of note, BT20 cells also displayed the highest basal rate of glucose consumption and lactic acid production of all cell lines tested and this rate was not influenced by 3-OHB at physiological con- centrations. Further, the proliferation rate of BT20 cells was unaffected by 3-OHB, similar to the other six cell lines tested. p We found that 3-OHB increases both basic OCR and maximal OCR after inhibition of ATP synthesis by oligo- mycin and in the presence of the uncoupling agent FCCP in BT20 cells. Additional file 1: The graphs show the proliferation rate (BrdU; in % of control cells) for the seven different breast cancer cell lines cultured in medium containing 1.5 mM AcAc (white column) compared to control without AcAc (black column) at 5% or 21% oxygen concentration after 5 days of culture (differences are not statistically significant). The columns represent mean ± SEM of data of 4 independent experiments with 3 replicate wells per experiment for each cell line. (PPTX 104 kb) Conclusions The intent of the study was to investigate the effect of 3-OHB on seven BC cell lines in vitro under conditions likely to be found in patients on a KD or short-term starvation. We have found strong evidence that a physiological concen- tration of 3 mM 3-OHB and AcAc did not impact cell prolif- eration and the response to standard BC chemotherapy and ionizing radiation is not changed by 3-OHB. These findings were independent from the diverse genetic background of the cell lines and differences in mRNA expression of ketoly- tic enzymes and 3-OHB uptake. Taken together, we found that 3-OHB at physiological concentrations has no major impact on BC cell proliferative behavior and the metabolic activity in vitro and especially does not fuel tumor cell growth. These results support clinical observations that physiologically increased 3-OHB serum concentrations in- duced either by a ketogenic diet or by short-term starvation do neither support nor inhibit breast cancer cell proliferation. Thus, a ketogenic diet should be safe for breast cancer pa- tients as already described for patients with diverse cancer types (for review, see [114]). p j g y y Cells take up ketone bodies by monocarboxylate trans- porters (MCTs), a family of proton-linked plasma mem- brane transporters that carry ketone bodies across biological membranes. The most important transporter for 3-OHB into cancer cells is MCT2 [25, 99–101], and previous studies describe an overexpression of MCT2 in BC cells [102]. Here, we found a strong expression of MCT2 in all seven BC cell lines, so that the absence of 3-OHB effects in the cells cannot be explained by defect- ive MCT expression. Since the expression of GLUT1 was found to be related to poor prognosis in breast can- cer [103, 104], we analyzed if its expression could be re- duced by 3-OHB. However, as for MCT2, we could not detect any modulation of the GLUT1 receptor expres- sion in dependency of 3-OHB, an observation also de- scribed in cardiomyocytes [105]. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation Altogether, the ex- pression of ketolytic enzymes on mRNA level seems not to be associated with the rate of 3-OHB consumption and unrelated to levels of glucose consumption and lac- tate production; however, we are aware, that mRNA ex- pression did not allow to judge about enzymatic activity. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation In accordance to the results of Antalis and colleagues [98], we found very low mRNA transcripts for ACAT1 for BDH1 in MCF-7 cells, which was linked to their inability to consume 3-OHB. Altogether, the ex- pression of ketolytic enzymes on mRNA level seems not to be associated with the rate of 3-OHB consumption and unrelated to levels of glucose consumption and lac- tate production; however, we are aware, that mRNA ex- pression did not allow to judge about enzymatic activity. Cells take up ketone bodies by monocarboxylate trans- porters (MCTs), a family of proton-linked plasma mem- brane transporters that carry ketone bodies across biological membranes. The most important transporter for 3-OHB into cancer cells is MCT2 [25, 99–101], and previous studies describe an overexpression of MCT2 in BC cells [102]. Here, we found a strong expression of MCT2 in all seven BC cell lines, so that the absence of 3-OHB effects in the cells cannot be explained by defect- ive MCT expression. Since the expression of GLUT1 was found to be related to poor prognosis in breast can- cer [103, 104], we analyzed if its expression could be re- duced by 3-OHB. However, as for MCT2, we could not detect any modulation of the GLUT1 receptor expres- sion in dependency of 3-OHB, an observation also de- scribed in cardiomyocytes [105]. Previous studies have shown variable gene expression levels of key enzymes involved in ketolytic metabolism in cancer cell lines of different entities [41, 93–97]. In line with these data, we found different mRNA expres- sion patterns of ketolytic enzymes in BC cell lines. In BT474 and HBL100, mRNA transcripts for all ketolytic enzymes were detectable. This was associated with the highest relative consumption rate of 3-OHB by these cells. In contrast, the other BC cell lines failed to express increased levels of at least one of these key enzymes. This mRNA expression pattern was independent from the subtype of BC [59–61] (Table 1) and not influenced by 3-OHB. In accordance to the results of Antalis and colleagues [98], we found very low mRNA transcripts for ACAT1 for BDH1 in MCF-7 cells, which was linked to their inability to consume 3-OHB. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation Our intent was to investi- gate the effect of 3-OHB in vitro under conditions more likely to be found in cancer patients on KDs. That is why we used physiological concentration of 3-OHB (3 mM) and glucose (5 mM) [22–24]. Using cell culture conditions comparable to our experi- ments, Martuscelli described an antiproliferative effect of 3-OHB in glioblastoma cell lines and tumor stem cells with half maximal inhibitory concentration (IC50) of 2 mM 3-OHB in the presence of low (4 mM) and physiological (5–6 mM) glucose concen- trations [37]. These contrary results described for the effect of 3-OHB on BC cells and glioma cells may re- flect differences in their ability to consume 3-OHB. physiological concentration of 5 mM 3-OHB in brain tumor cells, but at a very high glucose concentration of 25 mM that represents a more pathophysiological ketoacidotic situation [40]. Our intent was to investi- gate the effect of 3-OHB in vitro under conditions more likely to be found in cancer patients on KDs. That is why we used physiological concentration of 3-OHB (3 mM) and glucose (5 mM) [22–24]. Using cell culture conditions comparable to our experi- ments, Martuscelli described an antiproliferative effect of 3-OHB in glioblastoma cell lines and tumor stem cells with half maximal inhibitory concentration (IC50) of 2 mM 3-OHB in the presence of low (4 mM) and physiological (5–6 mM) glucose concen- trations [37]. These contrary results described for the effect of 3-OHB on BC cells and glioma cells may re- flect differences in their ability to consume 3-OHB. [ ] y effect of 3-OHB on BC cells and glioma cells may re- flect differences in their ability to consume 3-OHB. Previous studies have shown variable gene expression levels of key enzymes involved in ketolytic metabolism in cancer cell lines of different entities [41, 93–97]. In line with these data, we found different mRNA expres- sion patterns of ketolytic enzymes in BC cell lines. In BT474 and HBL100, mRNA transcripts for all ketolytic enzymes were detectable. This was associated with the highest relative consumption rate of 3-OHB by these cells. In contrast, the other BC cell lines failed to express increased levels of at least one of these key enzymes. This mRNA expression pattern was independent from the subtype of BC [59–61] (Table 1) and not influenced by 3-OHB. 3-OHB incubation does not influence the response of BC cells to chemotherapy or ionizing radiation Moreover, no notable inhibition of proliferation by 3-OHB could be seen in our short-term (5 days) and long-term (at least 14 days) experiments, as described for other cell lines [39–41]. In this context, it should be noted that the growth inhibitory effects for 3-OHB pre- viously reported were predominantly seen with very high and non-physiological concentrations of 3-OHB (5– 40 mM), an observation we have seen in our cell lines as well (not shown). In detail, the first description of an an- tiproliferative effect of 3-OHB on different cancer cell lines was published by Magee et al. in 1979. The authors tested concentrations of 3-OHB between 10 and 40 mM [39]. In 2009, Skinner et al. described an effect of 3-OHB on the viability of human neuroblastoma cells. Again, the authors used very high concentrations of 3-OHB between 24 and 43 mM [41]. Interestingly, an antiproliferative effect of 3-OHB was also described for a Bartmann et al. Cancer & Metabolism (2018) 6:8 Page 15 of 19 The KD is increasing in popularity, and an increasing number of cancer patients are trying the KD simultan- eously with chemotherapy and radiation therapy. Recent clinical trials, e.g., NCT01419483, investigate safety and tolerance of a KD during combined chemotherapy and ra- diation. We analyzed the response of 3-OHB-treated BC cells to chemotherapy and radiation in vitro. No signifi- cant changes in the dose-response to three chemothera- peutical drugs most commonly used in BC treatment [106–110] were observed. Thus, the sensitizing effect of a ketogenic diet on radiochemotherapy in vivo [111] might be mediated by effects other than direct influences on cancer cells. In this context, the clinical study published by Klement and Sweeney [112] is of interest. The authors described an adequate tumor regression for a small cohort of cancer patients undergoing a KD and radiation therapy. Further, two mouse studies with glioma and lung cancer confirm the radio-sensitizing effect of a ketogenic diet [111, 113]. To date, no information is available on a pos- sible radio-sensitizing effect of 3-OHB. Our in vitro results with 3 mM 3-OHB indicate a non-significant tendency of this ketone body to sensitize most BC cells to ionizing radiation. physiological concentration of 5 mM 3-OHB in brain tumor cells, but at a very high glucose concentration of 25 mM that represents a more pathophysiological ketoacidotic situation [40]. Funding 11. Ryu TY, Park J, Scherer PE. Hyperglycemia as a risk factor for cancer progression. Diabetes Metab J. 2014;38:330–6. Part of this work was supported by a grant of the Interdisciplinary Centre for Clinical Research (IZKF) University Hospital of Würzburg (Z-2/66 to C.B.). Part of this work was supported by a grant of the Interdisciplinary Centre for Clinical Research (IZKF) University Hospital of Würzburg (Z-2/66 to C.B.). 12. Monzavi-Karbassi B, Gentry R, Kaur V, Siegel ER, Jousheghany F, Medarametla S, Fuhrman BJ, Safar AM, Hutchins LF, Kieber-Emmons T. Pre- diagnosis blood glucose and prognosis in women with breast cancer. Cancer Metab. 2016;4:7. Availability of data and materials The datasets used and/or analyzed during the current study are included in this published article or available from the corresponding author on reasonable request. 13. Tan-Shalaby JL, Carrick J, Edinger K, Genovese D, Liman AD, Passero VA, Shah RB. Modified Atkins diet in advanced malignancies—final results of a safety and feasibility trial within the Veterans Affairs Pittsburgh Healthcare System. Nutr Metab (Lond). 2016;13:52. 13. Tan-Shalaby JL, Carrick J, Edinger K, Genovese D, Liman AD, Passero VA, Shah RB. Modified Atkins diet in advanced malignancies—final results of a safety and feasibility trial within the Veterans Affairs Pittsburgh Healthcare System. Nutr Metab (Lond). 2016;13:52. Publisher’s Note 18. Fine EJ, Segal-Isaacson CJ, Feinman RD, Herszkopf S, Romano MC, Tomuta N, Bontempo AF, Negassa A, Sparano JA. Targeting insulin inhibition as a metabolic therapy in advanced cancer: a pilot safety and feasibility dietary trial in 10 patients. Nutrition. 2012;28:1028–35. 18. Fine EJ, Segal-Isaacson CJ, Feinman RD, Herszkopf S, Romano MC, Tomuta N, Bontempo AF, Negassa A, Sparano JA. Targeting insulin inhibition as a metabolic therapy in advanced cancer: a pilot safety and feasibility dietary trial in 10 patients. Nutrition. 2012;28:1028–35. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Abbreviations 3-OHB: Beta-hydroxybutyrate; AcAc: Acetoacetate; ACAT: Homo sapiens acetyl-CoA-acetyltransferase; BDH: 3-Hydroxybutyrate dehydrogenase; BrdU: 5-Bromo-2′-deoxyuridine; CLS: Cell Lines Service GmbH; DMEM: Dulbecco’s modified Eagle’s medium; ECAR: Extracellular acidification rate; FCS: Fetal calf serum; GLUT1: Glucose transporter 1; KD: Ketogenic diet; MCT: Monocarboxylic acid transporter; mpH: Milli pH; MW: Molecular weight; OCR: Oxygen consumption rate; OD: Optical density; OXPHOS: Oxidative phosphorylation; PPIA: Peptidylprolyl isomerase A; SCOT: Succinyl-CoA transferase; SEM: Standard error of mean; TDC: Test drug concentration; TNBC: Triple-negative breast cancer 6. Palacios C, Daniel CR, Tirado-Gomez M, Gonzalez-Mercado V, Vallejo L, Lozada J, Ortiz A, Hughes DC, Basen-Engquist K. Dietary patterns in Puerto Rican and Mexican-American breast cancer survivors: a pilot study. J Immigr Minor Health. 2017;19:341–8. 6. Palacios C, Daniel CR, Tirado-Gomez M, Gonzalez-Mercado V, Vallejo L, Lozada J, Ortiz A, Hughes DC, Basen-Engquist K. Dietary patterns in Puerto Rican and Mexican-American breast cancer survivors: a pilot study. J Immigr Minor Health. 2017;19:341–8. 7. Augustin LS, Libra M, Crispo A, Grimaldi M, De Laurentiis M, Rinaldo M, D'Aiuto M, Catalano F, Banna G, Ferrau F, et al. Low glycemic index diet, exercise and vitamin D to reduce breast cancer recurrence (DEDiCa): design of a clinical trial. BMC Cancer. 2017;17:69. 7. Augustin LS, Libra M, Crispo A, Grimaldi M, De Laurentiis M, Rinaldo M, D'Aiuto M, Catalano F, Banna G, Ferrau F, et al. Low glycemic index diet, exercise and vitamin D to reduce breast cancer recurrence (DEDiCa): design of a clinical trial. BMC Cancer. 2017;17:69. 8. Warburg O. On respiratory impairment in cancer cells. Science. 1956;124: 269–70. Received: 31 August 2017 Accepted: 17 May 2018 Additional file 3: Columns represent mean ± SEM of cell proliferation after irradiation shown for the seven cell lines at 21% and 5% oxygen concentration (gray column = with 3-OHB; black column without 3-OHB) (summarized in Fig. 6). The BT20, BT474 and T47D cell lines cultured in the presence of 3-OHB showed a trend towards increased radio- resistance at 21% oxygen (with some significant results at single doses). In contrast, in MCF-7 and MDA-MB 468, 3-OHB cultured cells showed a trend towards impaired cell proliferation following radiation at the same oxy- gen concentration. At 5% oxygen concentration, 3-OHB seemed to have a sensitizing effect to radiation in some cell lines. Columns represent mean ± SEM of 3 independent experiments with 6 replicate wells per experiment. < 0.05, p < 0.01, *p < 0.001. (PPTX 152 kb) Acknowledgements h k k 9. Warburg OP, Karl, Negelein E. Über den Stoffwechsel der Carcinomzelle. In: Biochemische Zeitschrift, vol 152; 1924. p. 115–47. We thank Monika Koospal and Susanne Kolb for the support of the RT-qPCR experiments and Astrid Katzer for the assistance with the radiation. We thank Monika Koospal and Susanne Kolb for the support of the RT-qPCR experiments and Astrid Katzer for the assistance with the radiation. 10. Klement RJ, Kammerer U. Is there a role for carbohydrate restriction in the treatment and prevention of cancer? Nutr Metab (Lond). 2011;8:75. References 1 D S i 1. DeSantis CE, Fedewa SA, Goding Sauer A, Kramer JL, Smith RA, Jemal A. Breast cancer statistics, 2015: convergence of incidence rates between black and white women. CA Cancer J Clin. 2016;66:31–42. 2. Gnant M, Thomssen C, Harbeck N. St. Gallen/Vienna 2015: a brief summary of the consensus discussion. Breast Care (Basel). 2015;10:124–30. 2. Gnant M, Thomssen C, Harbeck N. St. Gallen/Vienna 2015: a brief summary of the consensus discussion. Breast Care (Basel). 2015;10:124–30. 3. 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Champ CE, Palmer JD, Volek JS, Werner-Wasik M, Andrews DW, Evans JJ, Glass J, Kim L, Shi W. Targeting metabolism with a ketogenic diet during the treatment of glioblastoma multiforme. J Neuro-Oncol. 2014;117:125–31. 17. Champ CE, Palmer JD, Volek JS, Werner-Wasik M, Andrews DW, Evans JJ, Glass J, Kim L, Shi W. Targeting metabolism with a ketogenic diet during the treatment of glioblastoma multiforme. J Neuro-Oncol. 2014;117:125–31. p g The authors declare that they have no competing interests. Ethics approval and consent to participate Not applicable Ethics approval and consent to participate Not applicable 16. Rieger J, Bahr O, Maurer GD, Hattingen E, Franz K, Brucker D, Walenta S, Kammerer U, Coy JF, Weller M, Steinbach JP. ERGO: a pilot study of ketogenic diet in recurrent glioblastoma. Int J Oncol. 2014;44:1843–52. 16. Rieger J, Bahr O, Maurer GD, Hattingen E, Franz K, Brucker D, Walenta S, Kammerer U, Coy JF, Weller M, Steinbach JP. ERGO: a pilot study of ketogenic diet in recurrent glioblastoma. Int J Oncol. 2014;44:1843–52. Additional files Page 16 of 19 Page 16 of 19 Bartmann et al. Cancer & Metabolism (2018) 6:8 University of Würzburg, 97070 Würzburg, Germany. 3Department of Radiotherapy and Radiation Oncology, Leopoldina Hospital, 97422 Schweinfurt, Germany. 4Department of Radiotherapy, University Hospital of Würzburg, 97080 Würzburg, Germany. 5Experimental Surgery, Department of General, Visceral, Vascular, and Pediatric Surgery, University Hospital of Würzburg, 97080 Würzburg, Germany. Additional file 2: Graphs present the IC50 with the 95% confidence intervals for the seven tested cell lines obtained for the three cytostatic drugs epirubicin, paclitaxel and carboplatin comparing the IC50 obtained for cells cultured with 3 mM 3-OHB (gray blots) with the control cells grown in medium free of 3-OHB (black boxes). Each blot represents 3–4 independent dose-response experiments with 6 replicate wells per ex- periment. None of the differences are statistically significant; however a strong tendency to a reduction in IC50 of paclitaxel is seen for T47D grown in 3-OHB medium compared to the control. (PPTX 104 kb) Received: 31 August 2017 Accepted: 17 May 2018 Authors’ contributions 14. Klement RF, Richard D, Gross EC, Champ CE, D'Agostino DP, Fine EJ, Kämmerer U, Poff A, Rho JM, Seyfried TN, Scheck AC. Need for new review of article on ketogenic dietary regimes for cancer patients. Med Oncol. 2017;34:1–4. 14. Klement RF, Richard D, Gross EC, Champ CE, D'Agostino DP, Fine EJ, Kämmerer U, Poff A, Rho JM, Seyfried TN, Scheck AC. Need for new review of article on ketogenic dietary regimes for cancer patients. Med Oncol. 2017;34:1–4. CB and UK originated the study and were responsible for the data collection, analysis, interpretation, and drafting the manuscript. SRJR, JF, AS, CSD, and CO performed the experiments, analyzed the data, and participated in writing the manuscript. RJK and AW helped with editing the manuscript and discussing the data. KB, JW, MS, and MK performed the experiments. All authors read and approved the final manuscript. 15. Schwartz K, Chang HT, Nikolai M, Pernicone J, Rhee S, Olson K, Kurniali PC, Hord NG, Noel M. Treatment of glioma patients with ketogenic diets: report of two cases treated with an IRB-approved energy-restricted ketogenic diet protocol and review of the literature. Cancer Metab. 2015;3:3. 15. Schwartz K, Chang HT, Nikolai M, Pernicone J, Rhee S, Olson K, Kurniali PC, Hord NG, Noel M. Treatment of glioma patients with ketogenic diets: report of two cases treated with an IRB-approved energy-restricted ketogenic diet protocol and review of the literature. Cancer Metab. 2015;3:3. 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https://openalex.org/W3004987506	https://flore.unifi.it/bitstream/2158/1190172/1/ijerph-17-01036-v2.pdf	English	null	A Scoping Review on How to Make Hospitals Health Literate Healthcare Organizations	International journal of environmental research and public health/International journal of environmental research and public health	2,020	cc-by	10,549	Received: 3 January 2020; Accepted: 4 February 2020; Published: 6 February 2020 Abstract: The concept of health literacy is increasingly being recognised as not just an individual trait, but also as a characteristic related to families, communities, and organisations providing health and social services. The aim of this study is to identify and describe, through a scoping review approach, the characteristics and the interventions that make a hospital a health literate health care organisation (HLHO), in order to develop an integrated conceptual model. We followed Arksey and O’Malley’s ﬁve-stage scoping review framework, reﬁned with the Joanna Briggs Institute methodology, to identify the research questions, identify relevant studies, select studies, chart the data, and collate and summarize the data. Of the 1532 titles and abstracts screened, 106 were included. Few studies have explored the eﬀect of environmental support on health professionals, and few outcomes related to staﬀsatisfaction/perception of helpfulness have been reported. The most common types of interventions and outcomes were related to the patients. The logical framework developed can be an eﬀective tool to deﬁne and understand priorities and related consequences, thereby helping researchers and policymakers to have a wider vision and a more homogeneous approach to health literacy and its use and promotion in healthcare organizations. Keywords: health literacy; health literate healthcare organizations; hospital; health equity; logical framework International Journal of Environmental Research and Public Health A Scoping Review on How to Make Hospitals Health Literate Healthcare Organizations Patrizio Zanobini 1,, Chiara Lorini 1 , Alberto Baldasseroni 2 , Claudia Dellisanti 3 and Guglielmo Bonaccorsi 1 1 Department of Health Sciences, University of Florence, Viale GB Morgagni 48, 50134 Florence, Italy chiara.lorini@uniﬁ.it (C.L.); guglielmo.bonaccorsi@uniﬁ.it (G.B.) chiara.lorini@uniﬁ.it (C.L.); guglielmo.bonaccorsi@uniﬁ.it (G.B.) 2 Tuscany Regional Centre for Occupational Injuries and Diseases (CeRIMP), Central Tuscany LHU, Via di San Salvi, 12, 50135 Florence, Italy; baldasse1955@gmail.com 3 Department of Epidemiology, Regional Health Agency of Tuscany, Via Pietro Dazzi, 1, 50141 Florence, Italy; claudiadellisanti70@libero.it C d t i i bi i@ il T l +39 3663435179 chiara.lorini@uniﬁ.it (C.L.); guglielmo.bonaccorsi@uniﬁ.it (G.B.) 2 Tuscany Regional Centre for Occupational Injuries and Diseases (CeRIMP), Central Tuscany LHU, Via di San Salvi, 12, 50135 Florence, Italy; baldasse1955@gmail.com 2 Tuscany Regional Centre for Occupational Injuries and Diseases (CeRIMP), Central Tuscany LHU, Via di San Salvi, 12, 50135 Florence, Italy; baldasse1955@gmail.com 3 Department of Epidemiology, Regional Health Agency of Tuscany, Via Pietro Dazzi, 1, 50141 Florence, Italy; claudiadellisanti70@libero.it , , , y; g 3 Department of Epidemiology, Regional Health Agency of Tuscany, Via Pietro Dazzi, 1, 50141 Florence, Italy claudiadellisanti70@libero.it * Correspondence: patriziozanobini@gmail.com; Tel.: +39-3663435179 2.1. Identifying the Research Questions The ﬁrst step in the process of conducting a scoping literature review is to determine the research questions to be addressed in the study. The research question addressed in this review was based on the PCC (population–concept–context) model of the Joanna Briggs Institute [15]: “What interventions and what outcomes are pursued in health literate hospitals?” where HLHO is deﬁned as described in the introduction [8]. An HLHO 1. Has leadership that makes health literacy integral to its mission, structure, and operations. 2. Integrates health literacy into planning, evaluation measures, patient safety, and quality improvement. 3. Prepares the workforce to be health literate and monitors progress. 4. Includes populations served in the design, implementation, and evaluation of health information and services. 5. Meets the needs of populations with a range of health literacy skills while avoiding stigmatisation. 6. Uses health literacy strategies in interpersonal communications and conﬁrms understanding at all points of contact. 7. Provides easy access to health information and services, as well as navigation assistance. 8. Designs and distributes print, audiovisual, and social media content that is easy to understand and act on. 9. Addresses health literacy in high-risk situations, including care transitions and communications about medicines. 10. Communicates clearly what health plans cover and what individuals will have to pay for services. 1. Has leadership that makes health literacy integral to its mission, structure, and operations. p p g y g g 6. Uses health literacy strategies in interpersonal communications and conﬁrms understanding at all poin of contact. 7. Provides easy access to health information and services, as well as navigation assistance. 8. Designs and distributes print, audiovisual, and social media content that is easy to understand and act 9. Addresses health literacy in high-risk situations, including care transitions and communications about medicines. 10. Communicates clearly what health plans cover and what individuals will have to pay for service Facing such attributes, many authors have proposed health literacy intervention toolkits for health care organizations [9,10], as well as correlated measurement tools [11–13]; however, to date a systematisation of the conceptual model based on the experiences described in the literature is still lacking. The aim of this study is to identify and describe, through a scoping review approach, the characteristics and the interventions that make a hospital an HLHO, according to the deﬁnition of Brach et al. [8], in order to develop an integrated conceptual model capturing the most comprehensive framework of HLHOs. The research has been restricted to hospitals instead of examining all health care settings, due to the low variability of this type of setting between diﬀerent countries. 2. Materials and Methods We followed Arksey and O’Malley’s ﬁve-stage scoping review framework [14], reﬁned with the Joanna Briggs Institute methodology [15], in order to (1) identify the research questions, (2) identify relevant studies, (3) select studies, (4) chart the data, and (5) collate and summarize the data. 1. Introduction Health literacy (HL) is a multidimensional concept that has been developed since the 1970s [1], moving from an individual to a public health perspective [2]. In one of its deﬁnitions, HL is described as the degree to which individuals can obtain, process, and understand the basic health information and services they need to make appropriate health decisions [3]. Thus, given that health has biological, psychological, and social determinants, HL is increasingly being recognised as not just an individual trait, but also as a characteristic related to families, communities, and organisations providing health and social services [4]. For this reason, attention has also shifted to the speciﬁc context in which care is provided: patients’ abilities to understand medical information and navigate care-seeking processes are in fact related to the demands that the health delivery systems place on them, and the speciﬁc organizational context in which care is provided may contribute to compensating for patients’ limited HL [5–7]. www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2020, 17, 1036; doi:10.3390/ijerph17031036 www.mdpi.com/journal/ijerph 2 of 16 Int. J. Environ. Res. Public Health 2020, 17, 1036 The concept of health literate health care organisations (HLHOs) is proposed to asses health care organization performance with patients’ HL issues. This kind of organization will make it easier for people to navigate, understand. and use information and services to take care of their health. p p g Brach et al. propose ten speciﬁc attributes (see Table 1) of HLHOs [8]. Speciﬁcally, the ten attributes of an HLHO have been described, as reported in Table 1. Table 1. Ten attributes to the health literate health care organizations (HLHOs), according to the Institute of Medicine (IOM) [8]. Table 1. Ten attributes to the health literate health care organizations (HLHOs), according to the Institute of Medicine (IOM) [8]. Strings Strings Database ((“health literacy” AND implementation) OR (“Health Literacy/nursing”[Mesh] OR “Health Literacy/organization and administration”[Mesh] OR “Health Literacy/utilization”[Mesh]) OR “health literate” OR (“health literacy” AND (organizat * OR organisat )) AND (“hospitals”[MeSH Terms] OR “hospitals”[All Fields] OR “hospital”[All Fields] OR hospital OR “health facility ” OR “Health Facilities”[Mesh]) Pubmed ((“health literacy” AND implementation) OR (“Health Literacy” AND nursing) OR ((“Health Literacy” OR “health literate”) AND (organizat OR administrat * OR utilizat ))) AND (hospital OR “health facility ”) WoS, Cinahl, Scopus, Psycinfo “health literacy” OR “health literate” Sociological Abstract “health literacy” OR “health literate” Database searches were also conducted in other four adjunctive databases: Cinahl, Scopus, Psycinfo, and Sociological Abstract. The last search was completed on 1 July 2019. No date limits were applied. 2.2. Identifying Relevant Studies The search strategy on Pubmed and Web of Science was built by selecting groups of keywords for each part of the PCC. Each group was combined to others through the Boolean operator AND (Table 2). 3 of 16 Int. J. Environ. Res. Public Health 2020, 17, 1036 Table 2. Search strings. Table 2. Search strings. 2.3. Selecting Articles Public Health 2020, 17, x 4 of 16 4 of 17 • Attribute 9 (high-risk situations): every study where the intervention or outcomes were related to high-risk situations, such as informed consent for surgery, administration of medicines, advanced directives, and transitions in care, such as discharge from the hospital. • Attribute 9 (high-risk situations): every study where the intervention or outcomes were related to high-risk situations, such as informed consent for surgery, administration of medicines, advanced directives, and transitions in care, such as discharge from the hospital. Att ib t 10 ( t t ) t di h i t l if i ti b t • Attribute 9 (high-risk situations): every study where the intervention or outcomes were related to high-risk situations, such as informed consent for surgery, administration of medicines, advanced directives, and transitions in care, such as discharge from the hospital. • Attribute 9 (high-risk situations): every study where the intervention or outcomes were related to high-risk situations, such as informed consent for surgery, administration of medicines, advanced directives, and transitions in care, such as discharge from the hospital. Att ib t 10 ( t t ) t di h i t l if i ti b t • Attribute 10 (payment transparency): studies whose aim was to clarify communications about health service costs to patients or evaluate the impact of interventions that make communications about insurance coverage and costs more transparent. • Attribute 10 (payment transparency): studies whose aim was to clarify communications about health service costs to patients or evaluate the impact of interventions that make communications about insurance coverage and costs more transparent. • Attribute 10 (payment transparency): studies whose aim was to clarify communications about health service costs to patients or evaluate the impact of interventions that make communications about insurance coverage and costs more transparent. • Attribute 10 (payment transparency): studies whose aim was to clarify communications about health service costs to patients or evaluate the impact of interventions that make communications about insurance coverage and costs more transparent. All electronic database search results were combined in Endnote, and duplicate records were removed. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) ﬂow diagram guidance was used to display studies that were identiﬁed by the database search and met the inclusion and exclusion criteria (Figure 1). All electronic database search results were combined in Endnote, and duplicate records were removed. 2.3. Selecting Articles Two reviewers performed the data extraction and appraisal independently, with an a priori study protocol. The study protocol included the following requirements: Two reviewers performed the data extraction and appraisal independently, with an a priori study protocol. The study protocol included the following requirements: Only primary studies, systematic reviews, and meta-analyses were considered. Studies should be focused on hospitals. Only primary studies, systematic reviews, and meta-analyses were considered. Studies should be focused on hospitals. They must describe any intervention and outcome that concerns one or more of the ten attributes of HLHOs, as follows: They must describe any intervention and outcome that concerns one or more of the ten attributes of HLHOs, as follows: • Attribute 1 (leadership): studies whose aim was to involve leadership or to assess the eﬀect of leadership involvement; • Attribute 2 (planning): interventions whose aim was to introduce or test the eﬀect of planning activities related to health literacy. This also includes every intervention aimed at developing or using tools/instruments for assessing organizational health literacy; Attribute 3 (workforce): every intervention could evaluate the impact of health literacy training on healthcare workers (HCWs), or whose aim was to develop, change, or adopt health literacy training • Attribute 4 (population): studies whose aim was to include the population in the design, implementation, and evaluation of health information and services, or every study assessing the eﬀects of population engagement; • Attribute 5 (meets the needs of the population): studies whose purpose was to assess the eﬀect of interventions that meet “the needs of populations with a range of health literacy skills while avoiding stigmatization”; • Attribute 6 (communication): studies whose aim was to implement a communication technique or that assess the eﬀect of implementing communication techniques; • Attribute 7 (navigation): studies whose aim was to implement or evaluate the impact of interventions to provide easy access to health information, both in the physical and electronic environment; • Attribute 8 (contents easy to understand): every study whose aim was to assess the suitability of materials for their target audience or the impact of the development/use of suitable material; Int. J. Environ. Res. Public Health 2020, 17, 1036 Int. J. Environ. Res. 2.3. Selecting Articles The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram guidance was used to display studies that were identified by the database search and met the inclusion and exclusion criteria (Figure 1). Figure 1. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow Figure 1. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) ﬂow diagram. Figure 1. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow Figure 1. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) ﬂow diagram. diagram. In accordance with the standard approach to conducting scoping reviews, a quality appraisa In accordance with the standard approach to conducting scoping reviews, a quality appraisal was not performed. agram. accordance with the standard approach to conducting scoping reviews, a quality appraisal accordance with the standard approach to conducting scoping reviews, a quality appraisal was formed. was not performed. 2.4. Charting the Data 2.4. Charting the Data To answer the research questions, we created a data charting form in Excel with the following elements: authors, year of publication, country of the study, study design, sample characteristics, aim To answer the research questions, we created a data charting form in Excel with the following elements: authors, year of publication, country of the study, study design, sample characteristics, aim of the study, conclusions, related HLHO attributes, interventions, and outcome measures (Appendix). of the study, conclusions, related HLHO attrib (A di ) 2.5. Collating, Summarising, and Reporting the Results • Support for staﬀ: interventions aimed at facilitating health professionals in helping patients. This can be achieved by health literacy training, or by tools/technologies/environments that improve the healthcare worker–patient relationship. • Support for governance: interventions designed to better evaluate and manage system eﬀorts in becoming an HLHO. This includes all the interventions aimed at developing tools/instruments for assessing organizational health literacy, as well as quality improvement actions related to health literacy: establishing measures, setting aims, speciﬁc assessment analysis, forming teams, communicating awareness, developing health literacy improvement plan, testing changes, and tracking progress. Outcomes were also grouped into categories and subcategories: Outcomes were also grouped into categories and subcategories: Patient outcomes: divided into changes in knowledge/skills/behaviors, perception of intervention satisfaction, and patient health outcomes. Staﬀoutcomes: including perception of intervention satisfaction and changes in knowledge skills/behaviors. • System outcomes: including changes in the scores for tools that assess organizational health literacy, the quality improvements perceived/obtained, measures of validation for the tools developed, and costs. • System outcomes: including changes in the scores for tools that assess organizational health literacy, the quality improvements perceived/obtained, measures of validation for the tools developed, and costs. The research group, with the contribution of a sociologist, developed a theoretical logical framework for a generic healthcare organization and combined it with interventions and outcomes to better synthetize the data. A logical framework is a diagram mapping out a chain of hypothesized causal relationships and providing a structure to describe the interventions that are available to reach speciﬁed public health goals [16]. The purpose of a logical framework is organizing, grouping, and selecting the interventions for the health issues under consideration, and for choosing the outcomes used to deﬁne the success of each intervention [17]. of the study, conclusions, related HLHO attrib (A di ) 2.5. Collating, Summarising, and Reporting the Results of the study, conclusions, related HLHO attrib (A di ) 2.5. Collating, Summarising, and Reporting the Results of the study, conclusions, related HLHO attrib (Appendix) 2.5. Collating, Summarising, and Reporting the Results Appendix). 2 5 Collating, Summarising, and Reporting the Results We used information from the data charting form to summarize the overall number of stud years of publication, countries where studies were conducted, and the focus and purpose of the stud ( pp ) 2.5. Collating, Summarising, and Reporting the Results We used information from the data charting form to summarize the overall number of studies, years of publication countries here studies ere conducted and the focus and purpose of the We used information from the data charting form to summarize the overall number of studies, years of publication, countries where studies were conducted, and the focus and purpose of the studies. The charted interventions were grouped into three categories, and each category was divided into two subcategories: 2.5. Collating, Summarising, and Reporting the Results We used information from the data charting form to summarize the overall number of studies, yea of publi atio ou t ie he e tudie e e o du ted a d the fo u a d pu po e of the y p p p The charted interventions were grouped into three categories, and each category was divided into two subcategories: years of publication, countries where studies were conducted, and the focus and purpose of the studies. The charted interventions were grouped into three categories, and each category was divided b • Support for patients: every intervention was designed to help patients access and use health information better. Patients may receive support directly by health professionals (staﬀsupport) years of publication, countries where studies were conducted, and the focus and purpose of the studies. The charted interventions were grouped into three categories, and each category was divided • Support for patients: every intervention was designed to help patients access and use health information better. Patients may receive support directly by health professionals (staﬀsupport) Int. J. Environ. Res. Public Health 2020, 17, 1036 5 of 16 or by material, electronic tools, conditions, and objects belonging to the hospital’s structure (environmental support); • Support for staﬀ: interventions aimed at facilitating health professionals in helping patients. This can be achieved by health literacy training, or by tools/technologies/environments that improve the healthcare worker–patient relationship. Figure 1 shows the article selection. Figure 1 shows the article selection. Of the 1532 titles and abstracts screened, 106 were included, of which 97 were primary studies and 9 were systematic reviews. Among the primary studies, 24 were randomized controlled trials, 42 were quasi-experimental studies, 19 were descriptive studies, and 12 were validation studies. The majority (70%) of the selected primary studies were performed in the United States, followed by Europe (13%), Australia (6%), Canada (3%), and others (6%). In Tables 3 and 4, respectively, the interventions and outcomes divided into subcategories are summarized. Int. J. Environ. Res. Public Health 2020, 17, 1036 6 of 16 Table 3. Interventions (see supplementary ﬁle for more detail). Intervention Subcategories Code Ref. No. of Studies Support for patient Environmental 1a [18–83] 66 Staﬀ 1b [19,21,24,28–30,34, 40,41,49,51,61,63, 66–68,75,80,82–99] 35 Support for staﬀ Training 2a [67,80,100–109] 12 Environmental 2b [50,64,67,68,76,110] 6 Support for governance Developing/usig tools/instruments for assessing organizational health literacy 3a [11,108,111–118] 10 Actions for quality improvements 3b [50,111,116,119– 122] 7 Table 4. Outcomes (see supplementary ﬁle for more detail). Target Subcategories Code Ref. No. of Studies Patient Knowledge/skills/behaviour 1a [18,22–27,29–33,35–38,40– 44,46,47,49–55,57,59–61,63, 65,66,68,70,75–83,87,88,93, 95,96,121,122] 57 Satisfaction/acceptability/ helpfulness/ 1b [20–22,28,30,31,47,48,54,56, 59,63,67,73,79,81,84,85,94, 106,107,117,120] 23 Patient health outcomes 1c [19,27,34,37,41,45,63,66,71, 76,80,86,87,89–93,97–99] 22 Staﬀ Knowledge/skills/behaviour 2a [38,64,67,68,101–103,105, 108–110,122] 12 Staﬀperception of satisfaction/helpfulness 2b [23,100–104] 6 System Assessment tools scores (organisation) 3a [11,84,94,116,117,121] 11 Quality improvements perceived/gained 3b [108,111,116,118,119,122] 6 Validation/feasibility/usability/ 3c [39,58,69,72,114,117] 8 Costs 3d [40,80,98] 3 Table 3. Interventions (see supplementary ﬁle for more detail). 3.3. Attributes (See Table 5 for References) The most common attribute investigated was the eighth (with 67 studies, followed by attribute 9 with 53 studies and 7 with 36 studies]. No study was found to be related to attribute 10. Attribute 5 is very generic, so it can be included in any of the remaining nine attributes. This will be further explained in the discussion section. Only 18 studies (see Supplementary File for references) analysed a single attribute. Table 5. Attributes (see Supplementary File for more detail). N Description No. of Studies Ref. 1 Leadership 7 [65,105,106,111,114–116] 2 Planning 19 [11,69,72,73,88,94,106,107,110–117, 119,121,122] 3 Workforce 13 [67,80,94,100–107,109,110] 4 Population 10 [11,28,38,42,50,56,58,59,67,118] 5 Meets the needs of the population 0–106 6 Communication 25 [19,36,38,41,49,51,61,63,67,78–80,82, 89,90,93,94,96,99–102,104,107,109] 7 Navigation 36 [19–21,24,28,30–32,34,36,38,40,46,48, 53,54,56–60,63,68,72,82,84,86,87,89,90, 97–99,112,121,122] 8 Contents easy to understand 67 [18–20,22–33,35–45,47,49–58,60–67,69, 70,72–83,90,93,110,112,118,120–122] 9 High-risk situations 53 [18–21,23,25–28,32,34,35,37–41,43–45, 49,52,54,59,61–68,74–76,80,82–88,90– 93,95,96,98,99,113,119] 10 Payment transparency 0 / * further explained in the Discussion section. Table 5. Attributes (see Supplementary File for more detail). N Description No. of Studies Ref. 1 Leadership 7 [65,105,106,111,114–116] 2 Planning 19 [11,69,72,73,88,94,106,107,110–117, 119,121,122] 3 Workforce 13 [67,80,94,100–107,109,110] 4 Population 10 [11,28,38,42,50,56,58,59,67,118] 5 Meets the needs of the population 0–106 * 6 Communication 25 [19,36,38,41,49,51,61,63,67,78–80,82, 89,90,93,94,96,99–102,104,107,109] 7 Navigation 36 [19–21,24,28,30–32,34,36,38,40,46,48, 53,54,56–60,63,68,72,82,84,86,87,89,90, 97–99,112,121,122] 8 Contents easy to understand 67 [18–20,22–33,35–45,47,49–58,60–67,69, 70,72–83,90,93,110,112,118,120–122] 9 High-risk situations 53 [18–21,23,25–28,32,34,35,37–41,43–45, 49,52,54,59,61–68,74–76,80,82–88,90– 93,95,96,98,99,113,119] 10 Payment transparency 0 / * further explained in the Discussion section. Table 5. Attributes (see Supplementary File for more detail). * further explained in the Discussion section. 3.2. Outputs/Outcomes (See Table 4 for References) The most common outcome was a change in knowledge/skill/behaviour of patients (57 studies); twenty-three studies used subjective outcomes, such as perceptions of satisfactions and helpfulness, and 22 studies reported patient health outcomes. Only 15 studies used staﬀoutcomes: 12 for changes in knowledge/skill/behaviour, and 6 for subjective perceptions. Fifteen studies have analysed system outcomes: 11 of these have utilized scores of tools for conducting organizational assessments, 6 have measured quality improvement changes, 8 have measured the validity of particular tools developed by the organization, and 3 have evaluated the costs related to the interventions. 3.1. Interventions (See Table 3 for References) The majority of studies investigate the eﬀects of interventions that support patients. A total of 66 [18–83] have investigated environmental support. This includes both material support (informative brochures, ﬂyers, or pamphlets) and digital technology (software/apps) for patient education or to help patients to better access or manage their health information. Thirty-ﬁve studies investigated the eﬀects of staﬀinterventions in helping patients by describing interventions aimed at educating or helping patients in their healthcare pathways, such as during medical reconciliations and follow-ups. Only 15 studies examined the eﬀect of interventions targeting hospital staﬀ. Most of them (12) were related to health literacy training programs to improve staﬀcommunication skills, and six examined interventions to help doctors create easily understandable material, such as templates for discharge instructions. Fifteen studies examined interventions to support hospital governance: 10 illustrate the development and use of instruments or tools for assessing organizational health literacy, while 7 sought to evaluate quality improvements such as health literacy interventions and activities. Int. J. Environ. Res. Public Health 2020, 17, 1036 7 of 16 7 of 16 4. Discussion 4. Discussion In the years following its publication, the “Ten Attributes of Health Literate Health Care Organizations” has been used as both an assessment tool and a guidebook for building health literate organisations [10,123]. However, the 10 attributes have been criticized for being developed inductively and for lacking theoretical backing [124]. With our work, we have tried to overcome this issue by searching and analyzing the literature and building a logical framework to support the 10 In the years following its publication, the “Ten Attributes of Health Literate Health Care Organizations” has been used as both an assessment tool and a guidebook for building health literate organisations [10,123]. However, the 10 attributes have been criticized for being developed inductively and for lacking theoretical backing [124]. With our work, we have tried to overcome this issue by searching and analyzing the literature and building a logical framework to support the 10 attributes in deﬁning an HLHO. y g y g g g pp attributes in defining an HLHO. Most literature has investigated attributes 8 and 9, which combined together are related to the use of educational material and are strictly related to the environmental phase of our logical framework. No study was found to be related to the 10th attribute. This could be because patients do not have to pay for services in every health care system. However, in our review, the majority of studies were set in the United States. Clear communication of costs for patients is not considered to be an issue related to health literacy. In addition, our work evidenced that the majority of interventions belonged to two or more attributes. The cause of this is to be found in some redundancies related to the 10 attributes. The fifth attribute is a perfect example of how difficult it is to define the limits of the domains for each attribute. The description says that an HLHO “meets the needs of populations with a range of health literacy skills while avoiding stigmatization”. If the focus is meeting the needs of a population, then every intervention found in our review could belong to this attribute; on the contrary, when interventions focus on just avoiding stigmatisations, then no study could be related to this attribute. While this redundancy can be useful for broadly describing every aspect of an HLHO, it can generate confusion at a decision-making level. 4. Discussion Thanks to our logical framework, it should be easier to identify and to determine to which area every type of intervention belongs. For instance, as described above, most literature reports interventions and outcomes related to attributes 8 and 9, but this does not give enough information about the context they refer to. Analyzing our results using our logical framework, we can clearly see that the vast majority of Most literature has investigated attributes 8 and 9, which combined together are related to the use of educational material and are strictly related to the environmental phase of our logical framework. No study was found to be related to the 10th attribute. This could be because patients do not have to pay for services in every health care system. However, in our review, the majority of studies were set in the United States. Clear communication of costs for patients is not considered to be an issue related to health literacy. In addition, our work evidenced that the majority of interventions belonged to two or more attributes. The cause of this is to be found in some redundancies related to the 10 attributes. The ﬁfth attribute is a perfect example of how diﬃcult it is to deﬁne the limits of the domains for each attribute. The description says that an HLHO “meets the needs of populations with a range of health literacy skills while avoiding stigmatization”. If the focus is meeting the needs of a population, then every intervention found in our review could belong to this attribute; on the contrary, when interventions focus on just avoiding stigmatisations, then no study could be related to this attribute. While this redundancy can be useful for broadly describing every aspect of an HLHO, it can generate confusion at a decision-making level. Thanks to our logical framework, it should be easier to identify and to determine to which area every type of intervention belongs. For instance, as described above, most literature reports interventions and outcomes related to attributes 8 and 9, but this does not give enough information about the context they refer to. Analyzing our results using our logical framework, we can clearly see that the vast majority of research has investigated the role of interventions to support patients, while focusing on environmental content that is easy to understand and act on. 3.4. Logical Framework (Figure 2) The logical framework developed depicts the relationship between the HLHO and its intended eﬀects on patient health outcomes. It is composed of the determinants of an HLHO (health literacy deﬁnition, reference population, and features of the organization) and phases (governance, staﬀ, and environment) that identify the ways in which HLHOs interact with patients. At the end of the logical framework, patient health outcomes are shown. However, this was not meant to be a deﬁnitive guide to the relationship between these components, because many of these relationships have not been explicitly tested. Each phase has its own interventions and proximal outcomes (outputs) that deﬁne success for each intervention. Interventions and outputs, obtained by analysing the results of our literature search, were organized in a matrix linked to the phases. Governance interventions were applied only to the governance phase, while interventions of support staﬀand patients were applied to both the staﬀand environmental phases. 8 of 16 s were pplied Int. J. Environ. Res. Public Health 2020, 17, 1036 literature search, were organized in a applied only to the governance phase w Figure 2. Logical framework. Figure 2. Logical framework. Figure 2. Logical framework Figure 2. Logical framework. 4. Discussion For this reason, the most common types of outcomes reported were related to the patients—in particular, changes in knowledge, skill, and behaviour. However, this process includes, for the most part, materials related to patient education, and no study was found on navigation issues in the physical environment of a Int. J. Environ. Res. Public Health 2020, 17, 1036 9 of 16 hospital, even though navigation was ﬁrst raised as a health literacy problem out of concern for the complexity of health care facilities and their poor signage. There are many interventions, like using color coded pathways, standardizing plain language directions, having volunteer escorts, and posting directions in commonly used languages and navigation apps [125], but we did not ﬁnd any studies that evaluated their eﬀects. In addition, a reasonable number of patient health outcomes were investigated, but they were all related to interventions to support patients. No study related to staﬀor governance intervention reported any kind of patient health outcomes. Very few studies [50,64,67,68,76,110] have explored the eﬀect of environmental support on health professionals, and few outcomes related to staﬀsatisfaction/perception of helpfulness have been reported [23,100–104]. At the same time, studies examining interventions to support the governance of the organization, despite receiving more attention, often had methodological limitations, due to their weak study designs. As such, the generalizability of the ﬁndings from these studies was limited. For example, some of them were on using organizational health literacy assessment tools. While these tools were all pilot-tested for overall usability, none of them were clearly demonstrated to be reliable to measure improvement. In the literature, it is clear that limited health literacy is a signiﬁcant factor associated with increased healthcare utilization and costs [126,127], and that meeting the needs of people with limited health literacy could produce savings of approximately 8% of the total costs for this population [127]. However, only three studies [40,80,98] reported “costs” as an outcome. Health organizations need resources and strategies to save staﬀtime and costs [128]. It would be desirable to justify health literate interventions by linking them to saving staﬀtime or reducing costs to convince more health organizations (including business-driven health organizations) to transform themselves to meet health literacy goals. Our work has some limitations. First, we only take hospitals into account, and due to the characteristics of a scoping review, we did not evaluate the quality of studies. 4. Discussion Consequently, this review cannot report the best intervention for a health organization wishing to become health literate. Our logical framework shows every group of interventions and related consequences reported by the literature so far. Hopefully, this would help researchers and policymakers to move beyond the single-intervention-based improvement mindset, and to implement groups of interventions for each area of the organization, making health literacy integral to all operations. When an organization sets a goal of becoming health literate, it replaces fragmented quality improvement activities with a systematic and comprehensive approach [129]. Author Contributions: Conceptualization, P.Z., C.D., A.B., C.L. and G.B.; methodology, P.Z., C.D. and A.B.; data search and analysis, P.Z., C.L. and G.B.; writing—original draft, P.Z.; writing—review and editing, P.Z., C.L. and G.B.; supervision, C.D., A.B., C.L. and G.B.; project administration, G.B. All authors have read and agreed to the published version of the manuscript. Supplementary Materials: The following are available online at https://www.mdpi.com/1660-4601/17/3/103 Table S1: Primary Studies; Table S2: Systematic Reviews. References 1. Simonds, S.K. Health education as social policy. Health Educ. Monogr. 1974, 2, 1–25. [CrossRef] 2. Sørensen, K.; Van den Broucke, S.; Fullam, J.; Doyle, G.; Pelikan, J.; Slonska, Z.; Brand, H.; (HLS-EU) Consortium Health Literacy Project European. Health literacy and public health: A systematic review and integration of deﬁnitions and models. BMC Public Health 2012, 12, 80. [CrossRef] [PubMed] 3. Institute of Medicine. Health Literacy: A Prescription to End Confusion; National Academies Press: Washington, DC, USA, 2004. 4. Batterham, R.W.; Hawkins, M.; Collins, P.A.; Buchbinder, R.; Osborne, R.H. Health literacy: Applying current concepts to improve health services and reduce health inequalities. Public Health 2016, 132, 3–12. 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Batterham, R.W.; Buchbinder, R.; Beauchamp, A.; Dodson, S.; Elsworth, G.R.; Osborne, R.H. The OPtimising HEalth LIterAcy (Ophelia) process: Study protocol for using health literacy proﬁling and community engagement to create and implement health reform. BMC Public Health 2014, 14, 694. [CrossRef] 10. Abrams, M.A.; Kurtz-Rossi, S.; Riﬀenburgh, A.; Savage, B.A. Building Health Literate Organizations: A Guidebook to Achieving Organizational Change; UnityPoint Health: West Des Moines, IA, USA, 2014. 11. Kowalski, C.; Lee, S.Y.; Schmidt, A.; Wesselmann, S.; Wirtz, M.A.; Pfaﬀ, H.; Ernstmann, N. The health literate health care organization 10 item questionnaire (HLHO-10): Development and validation. BMC Health Serv. Res. 2015, 15, 47. [CrossRef] 12. Altin, S.V.; Lorrek, K.; Stock, S. Development and validation of a brief screener to measure the Health Literacy Responsiveness of Primary Care Practices (HLPC). BMC Fam. Pract. 2015, 16, 122. [CrossRef] 13. Kripalani, S.; Wallston, K.A.; Cavanaugh, K.L.; Osborn, C.Y.; Mulvaney, S.; McDougald Scott, A.; Rothman, R.L. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions This scoping review identiﬁes and describes the characteristics and the interventions that make a hospital an HLHO. So far, in the literature, little attention has been given to the eﬀect of environmental support on health professionals, and few outcomes related to staﬀsatisfaction/perception of helpfulness have been reported; the most common types of interventions and outcomes reported have been related to the patients. We also build a logical framework here to support the 10 attributes in deﬁning an HLHO, which, despite some limitations, can be an eﬀective tool to better deﬁne and more speciﬁcally understand priorities and related consequences, thereby helping researchers and policymakers to have a wider vision and a more homogeneous approach to health literacy and its development in healthcare organizations. Supplementary Materials: The following are available online at https://www.mdpi.com/1660-4601/17/3/1036/s1, Table S1: Primary Studies; Table S2: Systematic Reviews. Author Contributions: Conceptualization, P.Z., C.D., A.B., C.L. and G.B.; methodology, P.Z., C.D. and A.B.; data search and analysis, P.Z., C.L. and G.B.; writing—original draft, P.Z.; writing—review and editing, P.Z., C.L. and G.B.; supervision, C.D., A.B., C.L. and G.B.; project administration, G.B. All authors have read and agreed to the published version of the manuscript. 10 of 16 Int. J. Environ. Res. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2647756991	https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1006858&type=printable	English	null	Lack of a peroxiredoxin suppresses the lethality of cells devoid of electron donors by channelling electrons to oxidized ribonucleotide reductase	PLOS genetics	2,017	cc-by	13,793	RESEARCH ARTICLE Susanna Boronat1☯, Alba Domènech1☯, Mercè Carmona1, Sarela Garcı´a-Santamarina1¤, M. Carmen Baño´ 2, Jose´ Ayte´1, Elena Hidalgo1 1 Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, Barcelona, Spain, 2 Departamento de Bioquı´mica y Biologı´a Molecular, Universitat de València, Valencia, Spain 1 Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, Barcelona, Spain, 2 Departamento d Bioquı´mica y Biologı´a Molecular, Universitat de València, Valencia, Spain 1 Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, Barcelona, Spain, 2 Departamento de Bioquı´mica y Biologı´a Molecular, Universitat de València, Valencia, Spain a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. ¤ Current address: Department of Pharmacology and Cancer Biology, Duke University Medical School, Durham, United States of America * elena.hidalgo@upf.edu (EH); jose.ayte@upf.edu (JA) OPEN ACCESS The thioredoxin and glutaredoxin pathways are responsible of recycling several enzymes which undergo intramolecular disulfide bond formation as part of their catalytic cycles such as the peroxide scavengers peroxiredoxins or the enzyme ribonucleotide reductase (RNR). RNR, the rate-limiting enzyme of deoxyribonucleotide synthesis, is an essential enzyme relying on these electron flow cascades for recycling. RNR is tightly regulated in a cell cycle- dependent manner at different levels, but little is known about the participation of electron donors in such regulation. Here, we show that cytosolic thioredoxins Trx1 and Trx3 are the primary electron donors for RNR in fission yeast. Unexpectedly, trx1 transcript and Trx1 pro- tein levels are up-regulated in a G1-to-S phase-dependent manner, indicating that the sup- ply of electron donors is also cell cycle-regulated. Indeed, genetic depletion of thioredoxins triggers a DNA replication checkpoint ruled by Rad3 and Cds1, with the final goal of up-regu- lating transcription of S phase genes and constitutive RNR synthesis. Regarding the thiore- doxin and glutaredoxin cascades, one combination of gene deletions is synthetic lethal in fission yeast: cells lacking both thioredoxin reductase and cytosolic dithiol glutaredoxin. We have isolated a suppressor of this lethal phenotype: a mutation at the Tpx1-coding gene, leading to a frame shift and a loss-of-function of Tpx1, the main client of electron donors. We propose that in a mutant strain compromised in reducing equivalents, the absence of an abundant and competitive substrate such as the peroxiredoxin Tpx1 has been selected as a lethality suppressor to favor RNR function at the expense of the non-essential peroxide scavenging function, to allow DNA synthesis and cell growth. Citation: Boronat S, Domènech A, Carmona M, Garcı´a-Santamarina S, Baño´ M.C, Ayte´ J, et al. (2017) Lack of a peroxiredoxin suppresses the lethality of cells devoid of electron donors by channelling electrons to oxidized ribonucleotide reductase. PLoS Genet 13(6): e1006858. https:// doi.org/10.1371/journal.pgen.1006858 Editor: Julian E. Sale, MRC Laboratory of Molecular Biology, UNITED KINGDOM Received: February 24, 2017 Accepted: June 8, 2017 Published: June 22, 2017 Copyright: © 2017 Boronat 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 24, 2017 Accepted: June 8, 2017 Published: June 22, 2017 Copyright: © 2017 Boronat et al. Lack of a peroxiredoxin suppresses the lethality of cells devoid of electron donors by channelling electrons to oxidized ribonucleotide reductase Susanna Boronat1☯, Alba Domènech1☯, Mercè Carmona1, Sarela Garcı´a-Santamarina1¤, M. Carmen Baño´ 2, Jose´ Ayte´1, Elena Hidalgo1 Introduction Cysteine residues are not very abundant in proteins, but they are over-represented in functional regions of proteins, such as surfaces and catalytic centers [1]. The thiol group of cysteines is sub- ject of post-translational modifications altering its redox state; several of these oxidation states are reversible, such as sulfenic acid and disulfides. In particular, reversible thiol to disulfide switches happen as a consequence of cellular responses to oxidative stress, and several proteins with reactive cysteine residues undergo oxidations as part of their catalytic cycles (for a review, see [2]). Cells are provided with two major systems meant to control the thiol-disulfide status, the thioredoxin (Trx) and the glutaredoxin/glutathione (Grx/GSH) systems. Trxs and Grxs catalyze thiol-disulfide exchange reactions, and share a motif known as the Trx fold [3]. Thermodynamically, both types of reductants use as the ultimate electron donor NADPH [4]. Electrons are therefore transferred from NADPH to final substrates through gra- dients in redox potentials. In the case of Trxs, Trx reductase is the intermediate between NADPH and Trx, while GSH reduces oxidized Grxs, GSH reductase being the link between NADPH and oxidized GSH. Trx was first identified in 1964 as an electron donor for Escherichia coli ribonucleotide reductase (RNR), an enzyme required for DNA synthesis [5]. Grx was later discovered as an alternative electron donor for the same enzyme in E. coli mutants lacking Trx [6]. Many reports indicate that there is cross-talk between both branches of these electron transfer sys- tems and certain redundancy, but it is also clear that there is substrate specificity. From then onwards, it became clear that these oxido-reductases regulate a wide number of processes in eukaryotic and prokaryotic organisms, apart from DNA synthesis and repair, including antioxidant defense and redox regulation, sulfur metabolism or apoptosis; the sub- strates of Trxs and Grxs mediating these effects are peroxiredoxins (Prxs), GSH peroxidases, methionine sulfoxide reductases, phosphoadenylyl sulfate (PAPS) reductase or RNRs (for reviews on these and other functions of the electron donor cascades, see [2,7–12]). In most cell types, the only substrate of electron donors which is essential for survival (and not only for spe- cific cellular processes such as cysteine biosynthesis or oxidative stress tolerance) is RNR. RNR catalyzes the reduction of ribonucleosides into deoxyribonucleosides, and is therefore essential to provide the building blocks, deoxyribonucleotides (dNTPs), during DNA replica- tion and repair. Trx deficiency triggers DNA replication stress Catalunya (Spain). EH is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. OPEN ACCESS This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Ministerio de Economı´a y Competitividad (Spain), PLAN E and FEDER (BFU2015-68350-P to EH, BFU2015-66347-P to JA, BFU2014-58429-P to MCB), and by 2014-SGR-154 from Generalitat de Catalunya (Spain) to EH and JA. AD is recipient of a pre-doctoral fellowship from Generalitat de 1 / 21 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 Author summary The essential enzyme ribonucleotide reductase (RNR), the rate-limiting enzyme of deoxy- ribonucleotide synthesis, relies on the thioredoxin and glutaredoxin electron flow cas- cades for recycling. RNR is tightly regulated in a cell cycle-dependent manner at different levels. Here, we show that cytosolic thioredoxin Trx1 is the primary electron donor for RNR in fission yeast, and that trx1 transcript and protein levels are up-regulated at G1-to- S phase transition. Genetic depletion of thioredoxins triggers the DNA replication check- point up-regulating RNR synthesis. Furthermore, deletion of the genes coding for thiore- doxin reductase and dithiol glutaredoxin is synthetic lethal, and we show that a loss-of- function mutation at the peroxiredoxin Tpx1-coding gene acts as a genetic suppressor. We propose that in a mutant strain compromised in reducing equivalents, the absence of an abundant and competitive substrate of redoxins, the peroxiredoxin Tpx1, has been selected as a lethality suppressor to favor channeling of electrons to the essential RNR. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 Trx deficiency triggers DNA replication stress reduced (specificity site) as well as the rate of reduction (activity site) [13,14], and a small sub- unit, β, containing a stable diferric-tyrosyl radical cofactor (oxygen is required for the assembly of the diferric-tyrosyl radical cofactor in RNRs), which initiates nucleotide reduction through the transient oxidation of a cysteine to a thiyl radical in the catalytic site of the α subunit. Dur- ing this process, two local cysteines in the large subunit provide reducing equivalents, and the disulfide bond generated between them, after isomerizing within the same α monomer towards a solvent-exposed position, is reduced by Trx or Grx to yield active RNR. Balanced and sufficient pools of dNTPs have to be present during S phase of the cell cycle, and also to assist in DNA repair. In fact, several studies suggest that a correct supply of dNTPs during DNA replication is important for genome stability and for the prevention of cancer [15,16]. Inhibition of RNR activity by the radical scavenger hydroxyurea (HU) and other com- pounds has been used as a chemotherapeutic strategy of numerous cancer types [16,17]. RNRs are tightly regulated through many different mechanisms, which include allosteric and oligo- meric regulation, transcription of the α and/or β-coding genes to modulate protein levels, inhibi- tion of RNR catalytic activity and regulation of the subcellular localization of the RNR subunits (for reviews on RNR regulation, see [18,19]). In Schizosaccharomyces pombe, Cdc22 and Suc22 are the large and small subunits of RNR, respectively [20]. Most studies concerning regulation of fission yeast RNR activity have centered on the RNR inhibitor Spd1, which affects activity and subunit localization of α and β [21–23], and on the up-regulation of cdc22 transcription during the S phase and after DNA damage (for a review, see [19]). While the suc22 transcript does not fluctuate with the cell cycle, transcription of cdc22 is up-regulated by the MBF transcription fac- tor, which triggers expression of genes required for the S phase [20,24,25]. Regarding regulation of cdc22 expression by checkpoint kinases under stress conditions, treatment with HU, which inhibits RNR, decreases the available pool of dNTPs and causes the formation of stalled replica- tion forks and the activation of the DNA replication checkpoint driven by the Rad3 and Cds1 kinases; activated Cds1 phosphorylates and inactivates the Yox1 transcriptional repressor, pro- moting MBF-dependent cdc22 expression [26]. Regarding the regulation of RNR by cofactors and post-transcriptional modifications, changes in RNR subunit localization in response to iron bioavailability have been recently demonstrated in budding yeast [27]. Nevertheless, very little is known about the redox-dependent cell cycle regulation of RNR activity [28]. In fact, the identity of the S. pombe electron transfer components required for RNR recycling is unknown. Here, we identify Trx1 and Trx3 as the main electron donors of fission yeast RNR, we demonstrate that Trx1 expression is actually up-regulated at S phase at the transcript and protein levels, and that in the absence of Trx1 and Trx3 the DNA replication checkpoint is activated. With the expectation that a complete block of electrons flow should drive to cell lethality by blocking RNR at its oxidized form unless a continuous synthesis of RNR is triggered, we have managed to generate a synthetic lethal combination by deleting the Trx reductase and the Grx1-coding genes. A spontaneous suppressor of this synthetically lethal phenotype is a frame-shift mutation at the beginning of tpx1, the gene coding for the most abundant consumer of electrons in the cell, the Prx Tpx1. Our experiments suggest that in the triple knockout strain Δtrr1 Δgrx1 Δtpx1, the elimination of the main sink of electron favors the reduction of the essential substrate RNR. Introduction In eukaryotes, class Ia RNRs are composed of a large subunit, α, containing the catalytic site and two allosteric effector binding sites, that control which substrate is PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 2 / 21 Trxs 1 to 3, and Grxs 1 to 5 are indicated, with their proposed subcellular l li ti ( t li t it h d i l it ) T d t (T 1) d GSH d t (P 1) l Fig 1. The large subunit of RNR, Cdc22, is substrate of cytosolic Trxs. (A) Schematic representation of the electron flow cascades in fission yeast. Trxs 1 to 3, and Grxs 1 to 5 are indicated, with their proposed subcellular localization (cytosolic, cyt; mitochondrial, mito). Trx reductase (Trr1) and GSH reductase (Pgr1) are also indicated, as well as some substrates of these electron donors cascades, such as the large subunit of RNR Cdc22, the main Prx Tpx1, methionine sulfoxide reductase (MetSO red.) Mxr1 or phosphoadenylyl sulfate reductase (PAPS red.) Met16. (B) Trxs 1 and 3 are involved in the recycling of Cdc22. TCA extracts from YE cultures of strains SB104 (WT), SB105 (Δtrr1), SB106 (Δtrx1), SB134 (Δtrx2), SB133 (Δtrx1 Δtrx2), SB115 (Δtrx3) and SB121 (Δtrx1 Δtrx3), all of them carrying an HA-tagged version of cdc22 at their endogenous loci, were obtained and processed by SDS-PAGE in the presence or absence of DTT and analyzed by Western blot using antibody against HA. Reduced/active (red. Cdc22-HA) and oxidized/inactive Cdc22 (ox. Cdc22-HA) are indicated. (C) The Grx system is not required to reduce Cdc22. TCA extracts from strains SB104 (WT), SB140 (Δgrx1), SB131 (Δgrx2), AD178 (Δgrx3), SB111 (Δgrx4), AD181 (Δgrx5), AD104 (Δpgr1), SB141 (Δtrx1 Δgrx1), SB121 (Δtrx1 Δtrx3) and SB308 (Δtrx1 Δtrx3 Δgrx1), all of them carrying HA-tagged version of Cdc22, were processed by non-reducing SDS-PAGE as described in B. (D) Percentage of oxidized Cdc22-HA in the indicated strains. Quantification was performed as described in Materials and Methods. SEM from three independent experiments are shown. (E) dGTP levels in wild-type and Trx mutants. Strains SB117 (WT), SB137 (Δtrx1) and SB138 (Δtrx1 Δtrx3) were grown in YE and dGTP levels were determined by the DNA polymerase-based enzymatic assay. The values are represented relative to those of the wild-type strain. Error bars (SEM) from three independent experiments are shown. (F) DNA content analysis of isolated nuclei. Samples taken from asynchronous cultures of strains 972 (WT), MJ15 (Δtrx1) and SG248 (Δtrx1 Δtrx3) were ethanol fixed. Nuclei were isolated and DNA content was determined as described in Materials and Methods. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 Trxs are the preferred electron donors of S. pombe RNR large subunit Cdc22 S. pombe contains three genes coding for Trxs [29] and one for Trx reductase, trr1 (Fig 1A). Trx1 is the main cytoplasmic Trx [30]; Trx2 is localized to the mitochondria [31]; and Trx3/ 3 / 21 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 Trx deficiency triggers DNA replication stress Fig 1. The large subunit of RNR, Cdc22, is substrate of cytosolic Trxs. (A) Schematic representation of the electron flow cascades in fission yeast. Trxs 1 to 3, and Grxs 1 to 5 are indicated, with their proposed subcellular localization (cytosolic, cyt; mitochondrial, mito). Trx reductase (Trr1) and GSH reductase (Pgr1) are also indicated, as well as some substrates of these electron donors cascades, such as the large subunit of RNR Cdc22, the main Prx Tpx1, methionine sulfoxide reductase (MetSO red.) Mxr1 or phosphoadenylyl sulfate reductase (PAPS red.) Met16. (B) Trxs 1 and 3 are involved in the recycling of Cdc22. TCA extracts from YE cultures of strains SB104 (WT), SB105 (Δtrr1), SB106 (Δtrx1), SB134 (Δtrx2), SB133 (Δtrx1 Δtrx2), SB115 (Δtrx3) and SB121 (Δtrx1 Δtrx3), all of them carrying an HA-tagged version of cdc22 at their endogenous loci, were obtained and processed by SDS-PAGE in the presence or absence of DTT and analyzed by Western blot using antibody against HA. Reduced/active (red. Cdc22-HA) and oxidized/inactive Cdc22 (ox. Cdc22-HA) are indicated. (C) The Grx system is not required to reduce Cdc22. TCA extracts from strains SB104 (WT), SB140 (Δgrx1), SB131 (Δgrx2), AD178 (Δgrx3), SB111 (Δgrx4), AD181 (Δgrx5), AD104 (Δpgr1), SB141 (Δtrx1 Δgrx1), SB121 (Δtrx1 Δtrx3) and SB308 (Δtrx1 Δtrx3 Δgrx1), all of them carrying HA-tagged version of Cdc22, were processed by non-reducing SDS-PAGE as described in B. (D) Percentage of oxidized Cdc22-HA in the indicated strains. Quantification was performed as described in Materials and Methods. SEM from three independent experiments are shown. (E) dGTP levels in wild-type and Trx mutants. Strains SB117 (WT), SB137 (Δtrx1) and SB138 (Δtrx1 Δtrx3) were grown in YE and dGTP levels were determined by the DNA polymerase-based enzymatic assay. The values are represented relative to those of the wild-type strain. Error bars (SEM) from three independent experiments are shown. (F) DNA content analysis of isolated nuclei. Samples taken from asynchronous cultures of strains 972 (WT), MJ15 (Δtrx1) and SG248 (Δtrx1 Δtrx3) were ethanol fixed. Nuclei were isolated and DNA content was determined as described in Materials and Methods. Histograms represent the number of cells (Counts) with different FL1-A (DNA content), 200 corresponding to 1C Fig 1. The large subunit of RNR, Cdc22, is substrate of cytosolic Trxs. (A) Schematic representation of the electron flow cascades in fission yeast. Trx deficiency triggers DNA replication stress as a gray rectangle in each of the histograms. Gates for G1, S and G2/M are indicated with dashed lines. The percentage of cells in G1, S and G2 has been calculated from three independent experiments and is shown below each histogram. SEM from three independent experiments are shown. https://doi.org/10.1371/journal.pgen.1006858.g001 Txl1 has cytoplasm localization, although it is also associated with the proteasome [32–34]. Trx1 and, to a minor extent, Trx3 are the electron donors of the Prx Tpx1, essential for aerobic scavenging of peroxides and for signal transduction [35–37]. Thus, while cells lacking Trx1 are extremely sensitive to hydrogen peroxide (H2O2), Trx2 and Trx3 appear to be dispensable for the defense against oxidative stress [35]. Regarding the other branch of the disulfide reduc- tases, fission yeast expresses only two dithiol Grxs, cytosolic Grx1 and mitochondrial Grx2, several monothiol Grxs such as Grx4 (involved in the iron starvation response) [38–40], endo- plasmic reticulum-located Grx3 [41] and mitochondrial Grx5 (involved in mitochondrial iron-sulfur cluster assembly) [42], and one GSH reductase, Pgr1 (Fig 1A). Pgr1 has been reported to be essential at least during aerobic growth [43], but cells lacking the reductase can grow under semi-anaerobic conditions. These two cascades have to recycle enzymes which suffer disulfide formation as part of their catalytic functions, such as the essential Cdc22 and the non-essential Tpx1, Mxr1 or Met16 (Fig 1A). To test the role of these cascades in the turnover of the large RNR subunit, Cdc22, we com- bined single or multiple deletion mutations with the expression of a tagged version of the pro- tein, Cdc22-HA. This modification, performed at the chromosomal locus, did not affect cell fitness or cell tolerance to the RNR inhibitor HU (S1 Fig). As shown in Fig 1B, a DTT sensitive, slow migrating band was detected using anti-HA immune-blotting of extracts from asynchro- nous cultures of wild-type cells expressing Cdc22-HA, corresponding to 10.2 ± 1.6% of total Cdc22; the sensitivity to the dithiol DTT indicates that the slower migrating band corresponds to a disulfide-containing RNR. In extracts from cells lacking Trx reductase, a band between oxidized and reduced Cdc22 was detected, probably a transient intermediate between RNR and its electron donor. Importantly, cells lacking cytosolic Trx1, but not the mitochondrial Trx2, displayed 35 ± 2.7% of Cdc22 in its oxidized form. The lack of Trx3 did not significantly affect the amount of Cdc22 disulfide form, but it enhanced the ratio of oxidized-to-reduced form (50 ± 3.5%) in a Δtrx1 background. On the contrary, disruption of the Grx branch by deletion of the genes coding for Grx1-to-Grx5, or GSH reductase, Pgr1, did not exacerbate disulfide accumulation, unless added to the Δtrx1 Δtrx3 background (Fig 1C). The percentages of Cdc22 oxidation in these and other mutants of the Trx and Grx branches are indicated in Fig 1D. To confirm that Trx deficiencies have an effect on Cdc22 activity, we measured dNTP levels of asynchronous wild-type, Δtrx1 and Δtrx1 Δtrx3 cultures (Fig 1E for dGTP, and S2A Fig for dATP), and detected small but significant decreases in the absence of Trxs. We also measured the percentage of cells at G1, S and G2 phases in asynchronous cultures from these cells. As shown in Fig 1F, cells lacking Trx1 and, to a larger extent, Trx1 and Trx3 displayed an enlarged population of cells at S phase, indicating that these strains completed DNA replication slower than wild-type cells. Histograms represent the number of cells (Counts) with different FL1-A (DNA content), 200 corresponding to 1C and 400 corresponding to 2C. A representative histogram of each strain is shown. Cells in S phase are indicated Fig 1. The large subunit of RNR, Cdc22, is substrate of cytosolic Trxs. (A) Schematic representation o PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 4 / 21 Trx deficiency triggers DNA replication stress non-permissive temperature, cdc25-22 cells are arrested at the G2/M transition and after drop- ping the temperature, cells are synchronically released from the arrest. As shown in Fig 2A, Cdc22-HA oxidation cycles in wild-type cells and in cells lacking Trx3, with a transient peak from 60 to 120 minutes after the release. This peak overlaps with that of the septation index (Fig 2B), which in fission yeast is concomitant with S phase. The peaks of Cdc22-HA oxidation and septation index are delayed and more sustained in cells lacking Trx1 (or Trx1 and Grx1). Strikingly, in cells devoid of cytosolic Trxs, Δtrx1 Δtrx3, Cdc22-HA oxidation does not cycle Fig 2. Oxidation of Cdc22 occurs during S phase in synchronous cultures and its cycling is dependent on Trx1 and Trx3. (A) Analysis of TCA protein extracts from YE cultures synchronized using the cdc25-22 mutation. Strains SB117 (WT), SB142 (Δtrx3), SB137 (Δtrx1), SB141 (Δtrx1 Δgrx1) and SB138 (Δtrx1 Δtrx3), carrying the temperature sensitive cdc25-22 allele and expressing Cdc22-HA, were arrested in late G2 by a temperature shift to 36˚C for 4 h, and then synchronously released from the arrest at 25˚C. TCA protein extracts of asynchronous (AS) cell cultures, from arrested cells (time 0) and after release from the block (20 to 180 min) were analyzed as in Fig 1B. The solid black line at the top of each panel indicates the time points where the majority of the cells were in S phase, according to D. (B) Septation index was measured from samples taken at the indicated time points. (C) dGTP levels of cell cultures as in A at the indicated times points were determined and represented as in Fig 1E. (D) DNA content analysis of isolated nuclei. Samples taken from synchronized cultures of A were ethanol fixed, nuclei were isolated and DNA content analyzed by flow cytometry as described in Fig 1F. https://doi.org/10.1371/journal.pgen.1006858.g002 Fig 2. Oxidation of Cdc22 occurs during S phase in synchronous cultures and its cycling is dependent on Trx1 and Trx3. (A) Analysis of TCA protein extracts from YE cultures synchronized using the cdc25-22 mutation. Strains SB117 (WT), SB142 (Δtrx3), SB137 (Δtrx1), SB141 (Δtrx1 Δgrx1) and SB138 (Δtrx1 Δtrx3), carrying the temperature sensitive cdc25-22 allele and expressing Cdc22-HA, were arrested in late G2 by a temperature shift to 36˚C for 4 h, and then synchronously released from the arrest at 25˚C. Transient disulfide accumulation of Cdc22 during S phase is reversed by Trx1 and Trx3 We synchronized cultures of cells expressing Cdc22-HA and carrying mutations in several components of the reducing cascades by means of the cdc25-22 allele. Cdc25 is the G2-to-M activating phosphatase of the cyclin-dependent kinase of S. pombe, Cdc2. Upon shift to the PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 5 / 21 https://doi.org/10.1371/journal.pgen.1006858.g002 The trx1 gene is up-regulated at the G1-to-S transition of the cell cycle Once confirmed that Cdc22 oxidation is exacerbated during catalysis, we tested whether the expression of its main electron donor was also up-regulated during S phase. As shown in Fig 3A, Trx1 protein levels were enhanced at S phase as determined in extracts from block and release experiments. To test whether this protein up-regulation was dependent on a transcrip- tional event, we used Cyclebase, a repository of published cell cycle experiments [44], to inter- rogate genome-wide studies on block and release experiments performed in fission yeast. As shown in Fig 3B, there is a small but consistent cell cycle regulation of trx1 mRNA. The com- bined peaktime of all published datasets occurs at the beginning of G1 (Fig 3C), a bit later than other S phase transcripts such as cdc22, cdc18, yox1 or nrm1 (S3 Fig). All these genes are up- regulated by the MBF transcription factor. To test whether the increase of trx1 mRNA is dependent on MBF, we analyzed its transcript levels upon HU treatment or in cells lacking the MBF repressor Yox1. As shown in Fig 3D, trx1 transcription does not seem to depend on the MBF complex, contrary to cdc22. Future work will help us elucidating who triggers the accu- mulation of trx1 mRNA at G1-to-S transition. The absence of Trx1 and Trx3 triggers the DNA replication checkpoint and induces transcription of cdc22 Inhibition of RNR activity by HU treatment triggers a DNA replication stress, probably through the decrease in dNTP concentrations and stalling of DNA polymerase at replication forks. In S. pombe, the DNA replication checkpoint is driven by the Rad3 and Cds1 kinases. One target of this cascade is the transcriptional repressor Yox1, which after phosphorylation by Cds1 is released from the MBF complex and its S phase promoters [26,45] (Fig 4A). To test whether RNR inhibition by Trx deficiency can trigger replication stress, we first tested whether cells lacking Trx1 and Trx3 are sensitive to the presence of the RNR inhibitor HU. As shown in Fig 4B, Δtrx1 and Δtrx1 Δtrx3 cells are moderately and severely sensitive to HU, respectively, highlighting their defects in DNA synthesis. In wild-type cells, HU treatment exacerbates the accumulation of total and oxidized Cdc22-HA, as well as of the inhibitory phosphorylation of Yox1 (Fig 4C). Both Trx mutant strains, but specially Δtrx1 Δtrx3, display constitutive activation of the Rad3-Cds1 checkpoint cascade, as demonstrated by the presence of phosphorylated Yox1 even in the absence of HU stress in this strain background (Fig 4C), and by the enhanced levels of cdc22 mRNA under basal conditions (Fig 4D). We generated a Δtrx1 Δcds1 strain to demonstrate that the weak phosphorylation of Yox1 in Δtrx1 cells is dependent on Cds1 (Fig 4E). Δtrx1 Δtrx3 deletions are synthetic lethal with deletion of cds1, while a triple Δtrx1 Δtrx3 Δchk1 mutant is viable (S4 Fig); Chk1 is the effector kinase of the Inhibition of RNR activity by HU treatment triggers a DNA replication stress, probably through the decrease in dNTP concentrations and stalling of DNA polymerase at replication forks. In S. pombe, the DNA replication checkpoint is driven by the Rad3 and Cds1 kinases. One target of this cascade is the transcriptional repressor Yox1, which after phosphorylation by Cds1 is released from the MBF complex and its S phase promoters [26,45] (Fig 4A). To test whether RNR inhibition by Trx deficiency can trigger replication stress, we first tested whether cells lacking Trx1 and Trx3 are sensitive to the presence of the RNR inhibitor HU. As shown in Fig 4B, Δtrx1 and Δtrx1 Δtrx3 cells are moderately and severely sensitive to HU, respectively, highlighting their defects in DNA synthesis. Trx deficiency triggers DNA replication stress and the protein is maintained at its oxidized form at high levels, around 50–60%. In fact, these cells do not have a clear septation peak. Next, and to confirm that the activity of Cdc22 was compromised in the mutants lacking Trxs, we measured dNTP levels in the previous synchronous cultures. As shown in Fig 2C (dGTP) and S2B Fig (dATP), while the levels of dNTPs increased in wild-type cells during cell cycle progression (60 and 100 min after release), cells lacking Trx1 or Trx1 and Trx3 displayed a reduction of dNTPs levels, pointing that these strains could have compromised DNA synthe- sis. In fact, when we analyzed the DNA content from the synchronous cultures, we indeed observed a delayed and extended S phase in Δtrx1 cells (Fig 2D). This is even more noticeable in cells in which all the cytosolic Trxs were absent: in Δtrx1 Δtrx3 S phase was not detected by FACS until 120 min after the release, which represents 60 minutes of delay when compared to wild-type cells. TCA protein extracts of asynchronous (AS) cell cultures, from arrested cells (time 0) and after release from the block (20 to 180 min) were analyzed as in Fig 1B. The solid black line at the top of each panel indicates the time points where the majority of the cells were in S phase, according to D. (B) Septation index was measured from samples taken at the indicated time points. (C) dGTP levels of cell cultures as in A at the indicated times points were determined and represented as in Fig 1E. (D) DNA content analysis of isolated nuclei. Samples taken from synchronized cultures of A were ethanol fixed, nuclei were isolated and DNA content analyzed by flow cytometry as described in Fig 1F. https://doi.org/10.1371/journal.pgen.1006858.g002 6 / 21 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 The absence of Trx1 and Trx3 triggers the DNA replication checkpoint and induces transcription of cdc22 We propose that the survival of cells lacking both Trxs depends at least partially on the Cds1-dependent transcriptional up-regulation of the cdc22 gene (Fig 4A). DNA damage checkpoint. We propose that the survival of cells lacking both Trxs depends at least partially on the Cds1-dependent transcriptional up-regulation of the cdc22 gene (Fig 4A). The absence of Trx1 and Trx3 triggers the DNA replication checkpoint and induces transcription of cdc22 (A) Strain SB117 (WT cdc25-22 cdc22-HA) was processed as in Fig 2A and analyzed with antibodies against HA or Trx1, as indicated. Tubulin was used as a loading control. The graph below shows the Trx1 fold change relative to the arrested cells (0 min). Error bars (SEM) from three independent experiments are shown. (B) Normalized expression of cdc22 and trx1 mRNA through the cell cycle according to the database Cyclebase 3.0 (http:// cyclebase.org/). The expression values of cdc22 and trx1 through the cell cycle from six independent experiments were first transformed to log2, then the mean value was substracted over all time points, and finally, the expression values were all normalized. (C) Combined peaktime of the trx1 transcript in a regular fission yeast cell cycle. Data obtained from the database Cyclebase 2.0. (D) Up-regulation of trx1 is not dependent on the MBF complex. Total RNA prepared from untreated (-) or HU-treated (+) (180 min, 10 mM HU) cultures of strains 972 (WT), JA804 (Δyox1) and MJ15 (Δtrx1) was analyzed by Northern blot with probes for trx1 and cdc22, a MBF-dependent gene; act1 was used as a loading control. Fig 3. The Trx1 protein and trx1 mRNA levels are up-regulated during G1/S. (A) Strain SB117 (WT cdc25-22 cdc22-HA) was processed as in Fig 2A and analyzed with antibodies against HA or Trx1, as indicated. Tubulin was used as a loading control. The graph below shows the Trx1 fold change relative to the arrested cells (0 min). Error bars (SEM) from three independent experiments are shown. (B) Normalized expression of cdc22 and trx1 mRNA through the cell cycle according to the database Cyclebase 3.0 (http:// cyclebase.org/). The expression values of cdc22 and trx1 through the cell cycle from six independent experiments were first transformed to log2, then the mean value was substracted over all time points, and finally, the expression values were all normalized. (C) Combined peaktime of the trx1 transcript in a regular fission yeast cell cycle. Data obtained from the database Cyclebase 2.0. (D) Up-regulation of trx1 is not dependent on the MBF complex. Total RNA prepared from untreated (-) or HU-treated (+) (180 min, 10 mM HU) cultures of strains 972 (WT), JA804 (Δyox1) and MJ15 (Δtrx1) was analyzed by Northern blot with probes for trx1 and cdc22, a MBF-dependent gene; act1 was used as a loading control. DNA damage checkpoint. The absence of Trx1 and Trx3 triggers the DNA replication checkpoint and induces transcription of cdc22 In wild-type cells, HU treatment exacerbates the accumulation of total and oxidized Cdc22-HA, as well as of the inhibitory phosphorylation of Yox1 (Fig 4C). Both Trx mutant strains, but specially Δtrx1 Δtrx3, display constitutive activation of the Rad3-Cds1 checkpoint cascade, as demonstrated by the presence of phosphorylated Yox1 even in the absence of HU stress in this strain background (Fig 4C), and by the enhanced levels of cdc22 mRNA under basal conditions (Fig 4D). We generated a Δtrx1 Δcds1 strain to demonstrate that the weak phosphorylation of Yox1 in Δtrx1 cells is dependent on Cds1 (Fig 4E). Δtrx1 Δtrx3 deletions are synthetic lethal with deletion of cds1, while a triple Δtrx1 Δtrx3 Δchk1 mutant is viable (S4 Fig); Chk1 is the effector kinase of the PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 7 / 21 Trx deficiency triggers DNA replication stress Fig 3. The Trx1 protein and trx1 mRNA levels are up-regulated during G1/S. (A) Strain SB117 (WT cdc25-22 cdc22-HA) was processed as in Fig 2A and analyzed with antibodies against HA or Trx1, as indicated. Tubulin was used as a loading control. The graph below shows the Trx1 fold change relative to the arrested cells (0 min). Error bars (SEM) from three independent experiments are shown. (B) Normalized expression of cdc22 and trx1 mRNA through the cell cycle according to the database Cyclebase 3.0 (http:// cyclebase.org/). The expression values of cdc22 and trx1 through the cell cycle from six independent experiments were first transformed to log2, then the mean value was substracted over all time points, and finally, the expression values were all normalized. (C) Combined peaktime of the trx1 transcript in a regular fission yeast cell cycle. Data obtained from the database Cyclebase 2.0. (D) Up-regulation of trx1 is not dependent on the MBF complex. Total RNA prepared from untreated (-) or HU-treated (+) (180 min, 10 mM HU) cultures of strains 972 (WT), JA804 (Δyox1) and MJ15 (Δtrx1) was analyzed by Northern blot with probes for trx1 and cdc22, a MBF-dependent gene; act1 was used as a loading control. htt //d i /10 1371/j l 1006858 003 Fig 3. The Trx1 protein and trx1 mRNA levels are up-regulated during G1/S. (A) Strain SB117 (WT Fig 3. The Trx1 protein and trx1 mRNA levels are up-regulated during G1/S. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 Δtrr1 Δgrx1, inactivating both the Trx and the Grx electron donor branches, is synthetic lethal One of the substrates of activated Cds1 is Yox1, a transcriptional repressor of the MBF complex, an activator of G1/S genes. Among other genes, transcription of cdc22 is triggered to compensate the decline in dNTP concentrations. (B) Serial dilutions of YE cultures of strains 972 (WT), JA1158 (Δcds1), AV18 (Δsty1), MJ15 (Δtrx1) and IC76 (Δtrx1 Δtrx3) were spotted on YE agar plates without or with 4 mM HU, and grown for 3 days at 30˚C. (C) TCA extracts prepared from untreated (-) or HU-treated (+) (180 min, 10 mM HU) YE cultures of strains SB149 (WT), SB150 (Δtrx1) and SB153 (Δtrx1 Δtrx3), all expressing Cdc22-HA and Yox1-13Myc, were analyzed as in Fig 1B with antibodies against HA or Myc, as indicated. (D) Total RNA prepared from untreated (-) or HU-treated (+) (180 min, 10 mM HU) YE cultures of strains 972 (WT), JA943 (Δcds1), MJ15 (Δtrx1) and SG248 (Δtrx1 Δtrx3) was analyzed as in Fig 3D with probes for cdc22 and act1, used as a loading control. (E) TCA extracts from untreated YE cultures of SB149 (WT), SB150 (Δtrx1), SB153 (Δtrx1 Δtrx3), SB200 (Δcds1) and SB199 (Δcds1 Δtrx1) were analyzed as in Fig 1B. Sty1 levels were used as loading control. https://doi.org/10.1371/journal.pgen.1006858.g004 1A). Many of the mutants displayed severe growth defects, which could often be rescued by growing the cells in semi-anaerobiosis or in the presence of exogenous GSH (S1 Table, Fig 5A). Indeed, exogenous addition of GSH was sufficient to decrease the ratio of oxidized-to- reduced Cdc22 in mutants lacking Trxs (Fig 5B and 5C) and to alleviate some of their growth defects in liquid media (Fig 5D). This suggests that the Grx-GSH branch is a back-up mecha- nism of reduction of the essential RNR. 1A). Many of the mutants displayed severe growth defects, which could often be rescued by growing the cells in semi-anaerobiosis or in the presence of exogenous GSH (S1 Table, Fig 5A). Indeed, exogenous addition of GSH was sufficient to decrease the ratio of oxidized-to- reduced Cdc22 in mutants lacking Trxs (Fig 5B and 5C) and to alleviate some of their growth defects in liquid media (Fig 5D). This suggests that the Grx-GSH branch is a back-up mecha- nism of reduction of the essential RNR. Δtrr1 Δgrx1, inactivating both the Trx and the Grx electron donor branches, is synthetic lethal After exhaustive combination of gene deletions, only two crosses lead to lethality in fission yeast: double deletions of the trr1 (coding for Trx reductase) and gcs1 (codes for glutamate-cys- teine ligase, the rate-limiting enzyme on the GSH biosynthetic pathway) genes, or the double knock-out trr1 and grx1 (coding for the only dithiol cytosolic Grx) (Fig 5E). We propose that in this Δtrr1 Δgrx1 strain background RNR would remain oxidized. Δtrr1 Δgrx1, inactivating both the Trx and the Grx electron donor branches, is synthetic lethal So far, we have demonstrated that Trxs are the main electron donors of Cdc22, and that cells lacking Trx1 and Trx3 have important defects and activate the replication checkpoint. Taking into account that RNR is an essential protein, we attempted to induce lethality by combining a number of deletions of genes coding for components of the electron pathway cascades (see Fig PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 8 / 21 Trx deficiency triggers DNA replication stress Fig 4. Trx deficiency triggers the DNA replication checkpoint. (A) Scheme showing the activation by HU, an RNR inhibitor, of the Rad3-Cds1 checkpoint cascade. The decrease in the total concentration of dNTPs induces stalling of the replication fork and the activation of the checkpoint kinases Rad3 and Cds1. One of the substrates of activated Cds1 is Yox1, a transcriptional repressor of the MBF complex, an activator of G1/S genes. Among other genes, transcription of cdc22 is triggered to compensate the decline in dNTP concentrations. (B) Serial dilutions of YE cultures of strains 972 (WT), JA1158 (Δcds1), AV18 (Δsty1), MJ15 (Δtrx1) and IC76 (Δtrx1 Δtrx3) were spotted on YE agar plates without or with 4 mM HU, and grown for 3 days at 30˚C. (C) TCA extracts prepared from untreated (-) or HU-treated (+) (180 min, 10 mM HU) YE cultures of strains SB149 (WT), SB150 (Δtrx1) and SB153 (Δtrx1 Δtrx3), all expressing Cdc22-HA and Yox1-13Myc, were analyzed as in Fig 1B with antibodies against HA or Myc, as indicated. (D) Total RNA prepared from untreated (-) or HU-treated (+) (180 min, 10 mM HU) YE cultures of strains 972 (WT), JA943 (Δcds1), MJ15 (Δtrx1) and SG248 (Δtrx1 Δtrx3) was analyzed as in Fig 3D with probes for cdc22 and act1, used as a loading control. (E) TCA extracts from untreated YE cultures of SB149 (WT), SB150 (Δtrx1), SB153 (Δtrx1 Δtrx3), SB200 (Δcds1) and SB199 (Δcds1 Δtrx1) were analyzed as in Fig 1B. Sty1 levels were used as loading control. https://doi org/10 1371/journal pgen 1006858 g004 Fig 4. Trx deficiency triggers the DNA replication checkpoint. (A) Scheme showing the activation by HU, Fig 4. Trx deficiency triggers the DNA replication checkpoint. (A) Scheme showing the activation by HU, an RNR inhibitor, of the Rad3-Cds1 checkpoint cascade. The decrease in the total concentration of dNTPs induces stalling of the replication fork and the activation of the checkpoint kinases Rad3 and Cds1. Trx deficiency triggers DNA replication stress Fig 5. Deletion of trr1 and grx1 is synthetic lethal. (A) Survival of several multiple deletion mutants in the Trx and Grx cascades, and growth recovery with GSH or semi-anaerobic conditions. Serial dilutions of YE cultures of strains 972 (WT), AV18 (Δsty1), SG70 (Δtrx1), SG248 (Δtrx1 Δtrx3), IC146 (Δtrx1 Δtrx2 Δtrx3), SB160 (Δtrx1 Δtrx3 Δgrx1) and SB226 (Δtrx1 Δtrx2 Δtrx3 Δgrx1) were spotted on YE plates in the presence or not of 2 mM GSH, and plates were grown at 30˚C under aerobic (+O2) or semi-anaerobic (-O2) conditions. (B) TCA extracts prepared from untreated (-) or 2 mM GSH-treated (+) YE cultures of strains SB150 (Δtrx1) and SB153 (Δtrx1 Δtrx3), all expressing Cdc22-HA and Yox1-13Myc, were obtained and analyzed as in Fig 4C. Sty1 levels were used as loading control. (C) Percentage of oxidized Cdc22-HA in SB104 (WT), SB106 (Δtrx1), SB121 (Δtrx1 Δtrx3) and SB308 (Δtrx1 Δtrx3 Δgrx1) all expressing Cdc22-HA grown in the presence or absence of 2 mM GSH. Error bars (SEM) from three independent experiments are shown. (D) GSH partially suppresses the growth defects of liquid cultures of cells lacking electron donors. Growth of strains as in C was monitored by recording OD600 for a period of 40 hours of cultures containing (dashed lines) or not (solid lines) 2 mM GSH. (E) Double deletion of the trr1 and grx1 genes is synthetic lethal. Schematic representation of tetrad dissection. Strains SG166 (Δtrr1; trr1::nat) and SB34 (Δgrx1; grx1::hph) were crossed and the spores resulting from four tetrads were separated using a Singer tetrad micromanipulator and germinated on YE plates under semi-anaerobic conditions. The markers of the deletions were determined by replica-plating on antibiotic-containing plates (nourseothricin or hygromycin), and the final genotypes of each colony are indicated in the panel. No growth was detected for the double deletion (Δ1 Δ2: Δtrr1 Δgrx1 †), which was inferred after subtracting all the markers accumulated in the remaining spores of each tetrad. Fig 5. Deletion of trr1 and grx1 is synthetic lethal. (A) Survival of several multiple deletion mutants in the Trx and Grx cascades, and growth recovery with GSH or semi-anaerobic conditions. Lack of Tpx1 restores the growth of cells lacking Trr1 and Grx1 In spite of the results shown above, and using random spore selection, we unexpectedly obtained a single colony lacking Trr1 and Grx1 and therefore containing a suppressor muta- tion. To our surprise, we determined by sequence analysis that this mutation laid on the gene encoding the Prx Tpx1, introducing a one-base deletion at the 26th codon of the open reading frame and subsequently a frame shift and a stop codon at position 71 (Fig 6A). To confirm PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 9 / 21 https://doi.org/10.1371/journal.pgen.1006858.g005 Trx deficiency triggers DNA replication stress severe growth defects even under semi-anaerobic conditions, which can be partially overcome by GSH addition. Tpx1 is probably the most demanding substrate of electron donors: there are more than 400,000 copies of the protein per cell [46], and Tpx1 is continuously catalyzing H2O2 detoxifi- cation during aerobic growth with the participation of Trx1, Trx3 and, probably, Grx1 [35,36]. The fact that strains such as Δtrx1 Δtrx3 Δgrx1 grow better under semi-anaerobic condition (Fig 5A) is an indication that Tpx1 may be competing with Cdc22 for reducing equivalents in cells devoid of the main cytosolic electron donors: the levels of peroxides during semi-anaero- bic metabolism are lower than in the presence of oxygen, and therefore Tpx1 is not cycling and demanding electrons to the same extent. To demonstrate that the absence of Tpx1 could positively impinge on the reduced-to-oxi- dized ratio of RNR, we measured the amount of oxidized and reduced Cdc22-HA in different strains expressing or not Tpx1. As shown in Fig 6C and 6D, deletion of tpx1 always reduced the percentage of oxidized Cdc22 in three different Trx-deficient strains. We conclude that tpx1 deletion allows the channeling of electrons into the disulfide-bonded RNR, and this is particularly relevant in redoxin mutants. To test whether the competition between Tpx1 and Cdc22 for reducing equivalents could occur in a wild-type background, we forced temporal depletion of reduced Trx1 by Tpx1 dur- ing S phase. Exhaustion of reduced Trx1 by Tpx1 can only be accomplished when the Prx is actively scavenging peroxides, but an excess of H2O2 triggers Tpx1 over-oxidation and avoids Trx1 depletion [35]. Therefore, we applied mild oxidative stress in a continuous manner to S phase cultures, by synchronizing wild-type cells expressing Cdc22-HA using the cdc25-22 allele as shown in Fig 2, and adding or not 100 μM H2O2 at the onset of S phase, with subsequent additions of 25 μM every five minutes, to force Tpx1 oxidation and Trx1-dependent recycling (Fig 7). Trx1 oxidation was followed in extracts prepared in the presence of 4-acetamido-40- maleimidylstilbene-2,20-disulfonic acid (AMS) as described before [35]. AMS is a bulky thiol alkylating agent: while three moieties of AMS are incorporated in Trx1 when it is in the reduced form, only one AMS is incorporated when Trx1 is oxidized and two of its cysteine res- idues form a disulfide; slower migrating bands, corresponding to the transient mixed disulfides between Trx1 and its substrates, can also be detected by Western blot upon Trx1 oxidation. As shown in Fig 7A, the S phase-dependent oxidation of Cdc22 does not cause an apparent con- sumption of reducing equivalents, since the majority of Trx1 remains in the reduced form dur- ing the whole cycle. When a continuous addition of mild H2O2 is applied starting at 60 min (at the onset of S phase), a sustained oxidation of Trx1 is accomplished (Fig 7B), which is fully dependent on peroxide scavenging by Tpx1 [35]. Importantly, this Tpx1-dependent depletion of reduced Trx1 enhances the amount of oxidized Cdc22 (from 12% to 25%; Fig 7C), and the disulfide form is maintained for a longer period that in the absence of peroxides (Fig 7A, 7B and 7C). A small but significant cell cycle delay can be observed as a consequence of an elon- gated S phase, as demonstrated with the septation index (Fig 7D). In conclusion, if oxidative stress emerges during S phase, Tpx1 enzymatic activity jeopardizes the RNR-dependent syn- thesis of dNTPs through depletion of reduced Trx1. Serial dilutions of YE cultures of strains 972 (WT), AV18 (Δsty1), SG70 (Δtrx1), SG248 (Δtrx1 Δtrx3), IC146 (Δtrx1 Δtrx2 Δtrx3), SB160 (Δtrx1 Δtrx3 Δgrx1) and SB226 (Δtrx1 Δtrx2 Δtrx3 Δgrx1) were spotted on YE plates in the presence or not of 2 mM GSH, and plates were grown at 30˚C under aerobic (+O2) or semi-anaerobic (-O2) conditions. (B) TCA extracts prepared from untreated (-) or 2 mM GSH-treated (+) YE cultures of strains SB150 (Δtrx1) and SB153 (Δtrx1 Δtrx3), all expressing Cdc22-HA and Yox1-13Myc, were obtained and analyzed as in Fig 4C. Sty1 levels were used as loading control. (C) Percentage of oxidized Cdc22-HA in SB104 (WT), SB106 (Δtrx1), SB121 (Δtrx1 Δtrx3) and SB308 (Δtrx1 Δtrx3 Δgrx1) all expressing Cdc22-HA grown in the presence or absence of 2 mM GSH. Error bars (SEM) from three independent experiments are shown. (D) GSH partially suppresses the growth defects of liquid cultures of cells lacking electron donors. Growth of strains as in C was monitored by recording OD600 for a period of 40 hours of cultures containing (dashed lines) or not (solid lines) 2 mM GSH. (E) Double deletion of the trr1 and grx1 genes is synthetic lethal. Schematic representation of tetrad dissection. Strains SG166 (Δtrr1; trr1::nat) and SB34 (Δgrx1; grx1::hph) were crossed and the spores resulting from four tetrads were separated using a Singer tetrad micromanipulator and germinated on YE plates under semi-anaerobic conditions. The markers of the deletions were determined by replica-plating on antibiotic-containing plates (nourseothricin or hygromycin), and the final genotypes of each colony are indicated in the panel. No growth was detected for the double deletion (Δ1 Δ2: Δtrr1 Δgrx1 †), which was inferred after subtracting all the markers accumulated in the remaining spores of each tetrad. https://doi.org/10.1371/journal.pgen.1006858.g005 https://doi.org/10.1371/journal.pgen.1006858.g005 that the suppressor mutation was linked to loss-of-function of Tpx1, we performed tetrad anal- ysis to select a triple Δtrr1 Δgrx1 Δtpx1 knock-out strain. As shown in Fig 6B by tetrad dissec- tion, the double Δtrr1 Δgrx1 is synthetic lethal, while tiny colonies of the Δtrr1 Δgrx1 Δtpx1 strain were isolated under semi-anaerobic conditions and could be recovered on plates con- taining GSH. As shown in S5 Fig, the growth of this triple delete, Δtrr1 Δgrx1 Δtpx1, displays PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 10 / 21 Discussion Balanced pools of dNTPs have to be accumulated during S phase and after DNA damage and replication stress, and these DNA building blocks are synthesized on demand. In these two scenarios, replication and checkpoint activation, RNR activity is up-regulated through several different mechanisms. We have shown here that the catalytic disulfide formed at the large sub- unit of RNR, Cdc22, is reduced by the main cytosolic Trx, Trx1. Three important conclusions PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 11 / 21 Trx deficiency triggers DNA replication stress Fig 6. Loss-of-function of Tpx1 suppresses the lethal phenotype of a Δtrr1 Δgrx1 double deletion. (A) Sequence comparison of the wild-type and mutant tpx1 loci. The suppressor mutation is a nucleotide deletion at codon 26 (indicated in red in both loci) causing a frame shift leading to a truncated polypeptide of 70 amino acids (stop codon indicated in blue in the mutant allele). (B) Deletion of tpx1 is a suppressor of the synthetic lethality of Δtrr1 Δgrx1. Strains SG169 (trr1::kan) and MC138 (grx1::hph tpx1::nat) were crossed and analyzed as in Fig 5E. Four very small colonies were isolated from four tetrads and recovered in GSH-containing plates grown under semi-anaerobic conditions. Again, no growth was detected for the double deletion (Δ1 Δ2: Δtrr1 Δgrx1 †), which was inferred after subtracting all the markers accumulated in the remaining spores of the second tetrad. (C) TCA extracts from YE cultures of strains SB104 (WT), SB112 (Δtpx1), SB106 (Δtrx1), AD175 (Δtrx1 Δtpx1), SB121 (Δtrx1 Δtrx3), AD176 (Δtrx1 Δtrx3 Δtpx1), SB308 (Δtrx1 Δtrx3 Δgrx1) and AD172 (Δtrx1 Δtrx3 Δgrx1 Δtpx1), all of them carrying an HA-tagged version of cdc22 at the endogenous locus, were analyzed as in Fig 1B. Reduced/active (red. Cdc22-HA) and oxidized/inactive Cdc22 (ox. Cdc22-HA) are indicated. (D) Graph shows the average percentage of oxidized Cdc22-HA from three independent experiments as in C. Error bars (SEM) are shown. https://doi.org/10.1371/journal.pgen.1006858.g006 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 Trx deficiency triggers DNA replication stress Fig 7. Transient depletion of reduced Trx1 in S phase by oxidized Tpx1 impairs timely recycling of Cdc22. (A, B) YE cultures of strain SB149 (WT cdc25-22 cdc22-HA yox1-13myc) were blocked and released as in Fig 2A. TCA extracts were prepared at the indicated timepoints and separated into two fractions. One fraction was used to visualize Cdc22-HA and was alkylated with iodoacetamide. The other fraction was used to visualize Trx1 and was alkylated with AMS. Alkylated extracts were analyzed by non-reducing gel electrophoresis and Western blotting with an HA antibody and with a specific Trx1 antibody, respectively. Reduced and oxidized Cdc22 are indicated. Reduced [Trx1-3AMS (red.)] and oxidized [Trx1-1AMS (ox.)] Trx1 are also indicated, as well as bands corresponding to transient mixed disulfides of Trx1 with its substrates, mainly Tpx1 (Trx1 mixed disulfides). (B) Same experiment performed as in A, but 0.1 mM H2O2 was added 60 minutes after release and then 0.025 mM H2O2 was added every 5 minutes thereafter. (C) Graph shows the percentage of oxidized Cdc22-HA at the indicated timepoints both in untreated (panel A) and H2O2 treated (panel B) cells. (D) Septation index from cells blocked and released in the presence (panel B) or absence (panel A) of H2O2. https://doi.org/10.1371/journal.pgen.1006858.g007 Fig 7. Transient depletion of reduced Trx1 in S phase by oxidized Tpx1 impairs timely recycling of Cdc22. (A, B) YE cultures of strain SB149 (WT cdc25-22 cdc22-HA yox1-13myc) were blocked and released as in Fig 2A. TCA extracts were prepared at the indicated timepoints and separated into two fractions. One fraction was used to visualize Cdc22-HA and was alkylated with iodoacetamide. The other fraction was used to visualize Trx1 and was alkylated with AMS. Alkylated extracts were analyzed by non-reducing gel electrophoresis and Western blotting with an HA antibody and with a specific Trx1 antibody, respectively. Reduced and oxidized Cdc22 are indicated. Reduced [Trx1-3AMS (red.)] and oxidized [Trx1-1AMS (ox.)] Trx1 are also indicated, as well as bands corresponding to transient mixed disulfides of Trx1 with its substrates, mainly Tpx1 (Trx1 mixed disulfides). (B) Same experiment performed as in A, but 0.1 mM H2O2 was added 60 minutes after release and then 0.025 mM H2O2 was added every 5 minutes thereafter. (C) Graph shows the percentage of oxidized Cdc22-HA at the indicated timepoints both in untreated (panel A) and H2O2 treated (panel B) cells. Fig 6. Loss-of-function of Tpx1 suppresses the lethal phenotype of a Δtrr1 Δgrx1 double deletion. (A) Sequence comparison of the wild-type and mutant tpx1 loci. The suppressor mutation is a nucleotide deletion at codon 26 (indicated in red in both loci) causing a frame shift leading to a truncated polypeptide of 70 amino acids (stop codon indicated in blue in the mutant allele). (B) Deletion of tpx1 is a suppressor of the synthetic lethality of Δtrr1 Δgrx1. Strains SG169 (trr1::kan) and MC138 (grx1::hph tpx1::nat) were crossed and analyzed as in Fig 5E. Four very small colonies were isolated from four tetrads and recovered in GSH-containing plates grown under semi-anaerobic conditions. Again, no growth was detected for the double deletion (Δ1 Δ2: Δtrr1 Δgrx1 †), which was inferred after subtracting all the markers accumulated in the remaining spores of the second tetrad. (C) TCA extracts from YE cultures of strains SB104 (WT), SB112 (Δtpx1), SB106 (Δtrx1), AD175 (Δtrx1 Δtpx1), SB121 (Δtrx1 Δtrx3), AD176 (Δtrx1 Δtrx3 Δtpx1), SB308 (Δtrx1 Δtrx3 Δgrx1) and AD172 (Δtrx1 Δtrx3 Δgrx1 Δtpx1), all of them carrying an HA-tagged version of cdc22 at the endogenous locus, were analyzed as in Fig 1B. Reduced/active (red. Cdc22-HA) and oxidized/inactive Cdc22 (ox. Cdc22-HA) are indicated. (D) Graph shows the average percentage of oxidized Cdc22-HA from three independent experiments as in C. Error bars (SEM) are shown. https://doi.org/10.1371/journal.pgen.1006858.g006 https://doi.org/10.1371/journal.pgen.1006858.g006 can be extracted from our work: first, the mRNA and protein levels of Trx1 are up-regulated during S phase, what demonstrates a new layer of regulation of RNR. Second, the other cyto- solic Trx, Trx3, may support Trx1 in RNR recycling, so that cells lacking both electron donors suffer from severe replication stress which is partially overcome by the activation of the Rad3-Cds1 checkpoint. Third, the fitness phenotypes of mutants defective in electron donor capacity can be partially alleviated by depletion of another substrate of Trx1, the Prx Tpx1; elimination of an abundant competitor funnels electrons towards the essential RNR. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 12 / 21 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 Trx deficiency triggers DNA replication stress lacking Trx1 and Trx2 display a longer S phase [49], that the total pool of dNTPs from asyn- chronous cultures is unaffected thanks to the novo synthesized RNR [50], but that dNTP levels of cells synchronized in S phase are significantly lower than those of wild-type cells [51]. In view of our results, this is probably due to a reduced pool of dNTPs to assist on DNA synthesis [28]. We show here that the checkpoint kinases Rad3 and Cds1 are constitutively active in Δtrx1 Δtrx3 cells, and that this activation is required for the survival of this strain, since the tri- ple Δtrx1 Δtrx3 Δcds1 combination is lethal. We propose that in Δtrx1 Δtrx3 cells the main source of active/reduced Cdc22 is de novo synthesized protein, which arises from the constitu- tive up-regulation of cdc22 transcription in a Rad3-Cds1-Yox1 dependent manner. The complete lack of Cdc22 recycling should drive cells to lethality. With this idea in mind, we have generated an extensive combination of deletion mutants in most components of the Trx and Grx branches, and one of these combinations resulted in synthetic lethality: Δtrr1 Δgrx1. The reason why other mutants, such as the quadruple Δtrx1 Δtrx2 Δtrx3 Δgrx1 and Δtrx1 Δtrx3 Δgrx1 Δgrx2 strains, could be isolated under semi-anaerobic conditions but not the aforementioned Δtrr1 Δgrx1 strain is still intriguing to us. It can be speculated that the lack of Trx reductase is more pervasive that the elimination of its substrates due to the accumula- tion of oxidized Trxs, which may invert their role towards thiol oxidases [52,53], or which may bind to Trx substrates with the same affinity as reduced Trx [54] and block their reduction by other electron donors. A similar result has been reported in other organisms such as E. coli, where lethality or severe sickness can only be accomplished by deletion of the Trx reductase coding gene in combination with a defect in the Grx branch [55]. In S. cerevisiae, deletion of the trx1, trx2, grx1 and grx2 genes has been reported to be lethal [56]. Prxs are probably the most demanding cellular substrates of electron donors: they are con- tinuously catalyzing peroxide scavenging during aerobic metabolism, and they are among the most abundant proteins in most cell types. We reported before that Trx1 and, secondarily, Trx3 are the main electron donors of Tpx1, and cells lacking both cytoplasmic Trxs (Trx1 and Trx3) display constitutively oxidized Tpx1 [35]. Cells lacking Tpx1 cannot grow aerobically on plates; however, strain Δtrx1 Δtrx3 is still viable aerobically, suggesting a secondary role for the Grx/GSH system in Tpx1 reduction. Therefore, Tpx1 and Cdc22 compete for Trx1, Trx3 and, probably, another component(s) of the Grx/GSH cascade. This is not a problem in a wild-type background under most conditions: reduction of Tpx1 by Trx1 is hardly saturated, unless mild oxidative stress is applied, and only for a limited amount of time unless a continuous sup- ply of peroxides is provided (Fig 7A and 7B); indeed, upon severe H2O2 stress, Tpx1 becomes hyper-oxidized to sulfinic acid and temporarily inactivated [35,37,57], which may be beneficial to avoid inhibition of RNR recycling. However, when electron donors become limiting by genetic interventions it is probably advantageous to promote reduction of an essential sub- strate, RNR, by eliminating a non essential one, a Prx. In fact, other processes improving the fitness of redoxin mutants as well as the oxidized-to-reduced ratio of RNR are semi-anaerobic growth (by minimizing the activity and electron consumption of Tpx1 in peroxide scavenging; Fig 5A) and GSH addition (by providing unlimited reducing power; Fig 5A and S5 Fig). Prxs are probably the most demanding cellular substrates of electron donors: they are con- tinuously catalyzing peroxide scavenging during aerobic metabolism, and they are among the most abundant proteins in most cell types. We reported before that Trx1 and, secondarily, In our study, we present evidence for the existence of a novel electron donor for RNR, as the synthetic lethal phenotype of Δtrr1 Δgrx1 mutant can be rescued by eliminating Tpx1, the major competitor substrate for electrons. It was similarly proposed by Grant and colleagues that PAPS reductase could have an alternative hydrogen donor to Trx1 and Trx2 in budding yeast, since a Δtrx1 Δtrx2 strain grew on minimal media without sulphate under low-aeration growth conditions reducing the generation of reactive oxygen species [56], and probably mini- mizing the function of Prxs or GSH peroxidases. We have not identified yet the alternative electron donor of RNR in the Δtrr1 Δgrx1 Δtpx1 background. (D) Septation index from cells blocked and released in the presence (panel B) or absence (panel A) of H2O2. https://doi.org/10.1371/journal.pgen.1006858.g007 https://doi.org/10.1371/journal.pgen.1006858.g007 Regarding the cell cycle-dependent regulation of Trx1, all the experiments performed so far with synchronized S. pombe cultures highlight the smooth but consistent waves of trx1 tran- scripts, with a G1 peaktime (http://cyclebase2.jensenlab.org/) (Fig 3B). We have discarded the participation of the main transcriptional activator of G1-S phase genes, the MBF complex, in trx1 cycling (Fig 3D). Further work will be required to characterize this cell cycle-regulated event. Interestingly, it has recently been reported that colorectal cancer tissues display enhanced protein levels of both RNR and Trx1, and that inhibition of both proteins simulta- neously produced a synergistic anti-proliferation effect in this model [47]. To the best of our knowledge, this is the first report demonstrating that eukaryotic cells car- rying Trx deficiencies suffer from replication stress and constitutively trigger the DNA replica- tion checkpoint. In E. coli, an interesting connection between electron donor supplies, activation of DNA replication by DnaA and transcription up-regulation of RNR was proposed To the best of our knowledge, this is the first report demonstrating that eukaryotic cells car- rying Trx deficiencies suffer from replication stress and constitutively trigger the DNA replica- tion checkpoint. In E. coli, an interesting connection between electron donor supplies, activation of DNA replication by DnaA and transcription up-regulation of RNR was proposed by the group of Beckwith [48]. In Saccharomyces cerevisiae, it has been published that mutants activation of DNA replication by DnaA and transcription up-regulation of RNR was proposed by the group of Beckwith [48]. In Saccharomyces cerevisiae, it has been published that mutants PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 13 / 21 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 TCA extracts and immuno blot analysis Modified trichloroacetic acid (TCA) extracts were prepared blocking thiols with either iodoa- cetamide or AMS and separated in non-reducing denaturing electrophoresis as previously described [37]. Only when indicated, the reducing agent dithiothreitol (DTT) was added to the sample buffer prior to electrophoresis. Cdc22-HA and Yox1-Myc were immuno-detected with monoclonal house-made anti-HA or anti-Myc antibodies, respectively. Trx1 was immuno- detected with anti-Trx1 polyclonal antibody [60]. Anti-Sty1 polyclonal antibody [36] was used as loading control. Relative quantification of protein levels in Western blots was performed by scanning membranes with a Licor 3600 CDigit Blot Scanner (Licor Inc., USA) and using the Image Studio 4.0 software. dGTP and dATP measurements Growth conditions, genetic manipulations and strains Growth conditions, genetic manipulations and strains Cells were grown in rich medium (YE) at 30˚C as described previously [59]. When cells were crossed, we chose tetrad dissection or random spore analysis as indicated in the text. For tetrad analysis, asci were dissected by micromanipulation with a Singer Micromanipulator MSM 400 (Singer Instruments, UK). After growth of the dissected spores on YE agar plates under semi- anaerobic conditions, genetic markers were scored by replica-plating on YE-agar plates con- taining or not the indicated antibiotics, and placing the plates at 30˚C under semi-anaerobic conditions in the presence or not of 2 mM GSH, as indicated. Anaerobic liquid cultures were grown in flasks filled to the top with medium at 30˚C without shaking. Origins and genotypes of strains used in this study are outlined in Appendix S2 Table, and most of them were con- structed by standard genetic methods. A strain with tagged Cdc22-HA, SB104, was con- structed by replacing the cdc22-YFP::kanMX6 cassette of strain AWS16 (h+ cdc22-YFP:: kanMX6 ade6-704 leu1-32 ura4-D18, kindly provided by A. Carr), with a cdc22-HA::natMX6 cassette and by cleaning the auxotrophies. The natMX6 cassette in SB104 was replaced by the hphMX6 cassette, resulting in strain SB110. Derived strains containing additional deletions were obtained by crossing SB104 or SB110 with the corresponding strains, and plating spores in appropriate media, with the exception of strain AD104 that was obtained by deletion of the pgr1 gene in SB110. Strains with tagged Yox1-13Myc were obtained by crossing appropriate strains with JA778 (h- yox1-13myc::kanMX6 ura4-D18) or JA779 (h+ yox1-13myc::kanMX6 ura4-D18). Trx deficiency triggers DNA replication stress proposed to participate in processes other than disulfide reduction. At least in mammalian RNR, a GSH-mixed disulfide mechanism for Grx-mediated reduction of RNR has been described [58]. Whether S. pombe monothiol Grxs are mediating the channeling of electrons to RNR in a GSH-dependent manner, or whether GSH itself can reduce the disulfide in Cdc22 will have to be elucidated. There are still two or three genes in fission yeast coding for monothiol glutaredoxins (Grx3, Grx4, Grx5), which have been PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 14 / 21 RNA analysis Total RNA from S. pombe YE cultures was obtained, processed and transferred to membranes as described previously [63]. Membranes were hybridized with the [α-32P]dCTP-labelled cdc22, trx1 and act1 probes, containing the complete open reading frames. Trx deficiency triggers DNA replication stress Cell cycle block and release experiments Temperature-sensitive strains carrying the allele cdc25-22 were cultured in YE at the permis- sive temperature (25˚C) in a shaker water bath until reaching OD600 of 0.3, shifted to the non- permissive temperature (36˚C) for 4 hours and then allowed to resume the cell cycle by grow- ing them at 25˚C during 3 hours as described [62]. Full arrest at G2/M was checked by micros- copy. 5 ml aliquots were taken from non-arrested cells and at different times after release to prepare TCA extracts. Cell cycle progression was monitored with fluorescence microscopy by measuring the septation index of calcofluor-stained cells and by flow cytometry. Flow cytometry analysis of DNA content in isolated nuclei We followed a previously published protocol for determining DNA content on isolated nuclei [61]. Briefly, 1x107 cells were fixed in 70% ethanol and nuclei were prepared. Isolated nuclei were treated with RNase A (37˚C overnight) and DNA was stained in PBS solution containing 1 μM Sytox green. Growth curves Yeast cells were grown in YE from an initial OD600 of 0.2, with or without the addition of 2 mM GSH, using an assay based on automatic measurements of optical densities, as previously described [64]. HU and oxygen sensitivity assay on solid plates For survival on solid plates, S. pombe strains were grown, diluted and spotted on YE plates con- taining or not HU at the indicated concentrations and plates were incubated at 30˚C for 2–3 days as previously described [26]. To study the survival of strains on solid plates under aerobic or semi-anaerobic conditions, S. pombe strains were grown, diluted and spotted in YE, and plates were incubated at 30˚C under aerobic or semi-anaerobic conditions. To grow cells in solid media in an semi-anaerobic environment, we incubated the plates at 30˚C in a tightly sealed plastic bag containing a water-activated Anaerocult A sachet (Merck, Darmstadt, Ger- many) [36], or alternatively in a nitrogen-filled anaerobic chamber (Forma Anaerobic System, Thermo Electron Corp.). When indicated 2 mM GSH was added to YE agar plates. dGTP and dATP measurements The dATP and dGTP levels were determined by the DNA polymerase-based enzymatic assay as described before [27]. In brief, the incorporation of dATP and dGTP into specific oligonu- cleotides, containing poly(AAAT) and poly(AAAC) sequences respectively, by the Klenow DNA polymerase was determined in the presence of excess [3H]-labeled dTTP. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 15 / 21 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 Acknowledgments We are thankful to Oscar Fornàs, from the UPF-CRG Flow Cytometry Facility (Barcelona, S i ) f hi d i i th bl k d l i t W th k T C f idi We are thankful to Oscar Fornàs, from the UPF-CRG Flow Cytometry Facility (Barcelona, Spain) for his advice in the block and release experiments. We thank Tony Carr for providing strain expressing Cdc22-YFP. We are thankful to Oscar Fornàs, from the UPF-CRG Flow Cytometry Facility (Barcelona, Spain) for his advice in the block and release experiments. We thank Tony Carr for providing strain expressing Cdc22-YFP. We are thankful to Oscar Fornàs, from the UPF CRG Flow Cytometry Facility (Barcelona, Spain) for his advice in the block and release experiments. We thank Tony Carr for providing strain expressing Cdc22-YFP. Author Contributions Conceptualization: SB AD SGS JA EH. Supporting information S1 Fig. C-terminal tagging of Cdc22 does not affect cell growth under basal or HU condi- tions. Serial dilutions of strains 972 (WT), JA1158 (Δcds1), SB62 (cdc22-YFP), SB104 (cdc22-HA) and SB212 (cdc22-myc) were spotted on agar plates without (YE) or with 2 and 4 mM HU, and grown for 3 days at 30˚C. (TIF) S2 Fig. Determination of dATP levels in wild-type and mutant strains. (A) dATP levels in wild-type and Trx mutants. Strains SB117 (WT), SB137 (Δtrx1) and SB138 (Δtrx1 Δtrx3) were grown in YE and dATP levels were determined by the DNA polymerase-based enzymatic assay. The values are represented relative to those of the wild-type strain. Error bars (SEM) PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1006858 June 22, 2017 16 / 21 Trx deficiency triggers DNA replication stress from three independent experiments are shown. (B) dATP levels of cell cultures as in Fig 2A at the indicated times points were determined and represented as in Fig 1E. (TIF) S3 Fig. The MBF-dependent mRNAs peak at the end of M phase or beginning of G1. Com- bined peaktime of the S phase transcripts cdc22, yox1, cdc18, cdt1, cig2 and nrm1. Data obtained from the database Cyclebase 2.0. (TIF) S4 Fig. Deletion of the Trx-coding genes does not interact genetically with the DNA dam- age checkpoint. (A) Scheme depicting the activation of Chk1 upon DNA damage. DNA dam- age activates the kinases Rad3 and Chk1. Cdc10, a member of the MBF complex, is then phosphorylated by Chk1, resulting in its release from chromatin and leading to repression of MBF dependent genes. (B) Δchk1 does not display genetic interaction with Δtrx1 Δtrx3. Growth of 972 (WT), JA804 (Δrad3), SG248 (Δtrx1 Δtrx3) and AD151 (Δtrx1 Δtrx3 Δchk1) cells was monitored by recording the OD600 for a period of 30 hours. (C) Serial dilutions of strains 972 (WT), JA804 (Δrad3), JA795 (Δyox1), SG248 (Δtrx1 Δtrx3) and AD151 (Δtrx1 Δtrx3 Δchk1) were spotted on agar plates without (YE) or with 1 mM H2O2 or with 5 mM HU, and grown for 3 days at 30˚C. (TIF) S5 Fig. The triple delete Δtrr1 Δgrx1 Δtpx1 displays severe growth defects. Serial dilutions of strains 972 (WT), SG4 (Δtpx1), SG248 (Δtrx1 Δtrx3), SB304 (Δtrx1 Δtrx3 Δgrx1) and MC144 (Δtrr1 Δgrx1 Δtpx1) were spotted on YE agar plates without or with 2 mM GSH under semi- anaerobic (-O2) or aerobic (+O2) conditions and grown for 3 days at 30˚C. (TIF) S5 Fig. The triple delete Δtrr1 Δgrx1 Δtpx1 displays severe growth defects. Serial dilutions of strains 972 (WT), SG4 (Δtpx1), SG248 (Δtrx1 Δtrx3), SB304 (Δtrx1 Δtrx3 Δgrx1) and MC144 (Δtrr1 Δgrx1 Δtpx1) were spotted on YE agar plates without or with 2 mM GSH under semi- anaerobic (-O2) or aerobic (+O2) conditions and grown for 3 days at 30˚C. (TIF) Conceptualization: SB AD SGS JA EH. 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https://openalex.org/W2461549381	https://bora.uib.no/bora-xmlui/bitstream/1956/15898/3/acs%25252Eest%25252E6b01723.pdf	English	null	Effects and Location of Coplanar and Noncoplanar PCB in a Lipid Bilayer: A Solid-State NMR Study	Environmental science & technology	2,016	cc-by	5,984	■INTRODUCTION of noncoplanar PCBs may be far-reaching compared to coplanar PCBs. The use of polychlorinated biphenyls (PCBs) has been highly restricted or banned in several countries since the 1970s and 1980s, although a worldwide ban was not in place until 2001.1 However, the PCBs low biodegradability and high lipophilicity cause these compounds to accumulate in the adipose tissue of mammals as well as other creatures such as birds and ﬁsh.2,3 Despite slow degradation, PCBs are still found in the atmosphere, even in remote locations such as Antarctica.4 Due to the high environmental persistence and high resistance to decomposition in living organisms,5,6 PCBs accumulate up the food chain, causing human exposure through both aquatic and terrestrial food sources. The possible eﬀects of prolonged human exposure have therefore received considerable atten- tion.7 Most previous literature focuses on eﬀects of PCBs on cellular membranes, which illuminates eﬀects of PCBs but makes it diﬃcult to investigate the mechanisms of action. In order to access the core mechanisms it is helpful to utilize a model lipid membrane. PCB 77 and PCB 52 have been used in several earlier studies,14,16,17 representing coplanar and non- coplanar PCB, respectively. Tan et al. showed that PCB 52 causes rapid cell death in thymocytes and cerebellar granule cell neurons, altering multiple membrane components, while PCB 77 showed none of these eﬀects.14,16 It was further shown from ﬂuorescent polarization measurements that PCB 52 increases membrane ﬂuidity, which was linked to the stereochemistry of the more bulky PCB 52 compared to PCB 77, causing a greater impact on membrane properties when intercalated in the bilayer structure.16 Campbell et al. investigated interaction of these PCBs with a model 1,2-dimyristoyl-sn-glycero-3-phos- phocholine (DMPC) lipid membrane using atomic force microscopy (ATM) and diﬀerential scanning calorimetry (DSC).17 It was observed that bilayers with PCB 52 had a lower gel-to-liquid phase transition temperature. * S Supporting Information * S Supporting Information ABSTRACT: Coplanar and noncoplanar polychlorinated biphen- yls (PCBs) are known to have diﬀerent routes and degree of toxicity. Here, the eﬀects of noncoplanar PCB 52 and coplanar PCB 77 present at 2 mol % in a model system consisting of POPC liposomes (50% hydrated) are investigated by solid-state 13C and 31P NMR at 298 K. Both PCBs intercalate horizontally in the outer part of the bilayer, near the segments of the acyl chain close to the glycerol group. Despite similar membrane locations, the coplanar PCB 77 shows little eﬀect on the bilayer properties overall, except for the four nearest neighboring lipids, while the eﬀect of PCB 52 is more dramatic. The ﬁrst ∼2 layers of lipids around each PCB 52 in the bilayer form a high ﬂuidity lamellar phase, whereas lipids beyond these layers form a lamellar phase with a slight increase in ﬂuidity compared to a bilayer without PCB 52. Further, a third high mobility domain is observed. The explanation for this is the interference of several high ﬂuidity lamellar phases caused by interactions of PCB 52 molecules in diﬀerent leaﬂets of the model bilayer. This causes formation of high curvature toroidal region in the bilayer and might induce formation of channels. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. pubs.acs.org/est © 2016 American Chemical Society DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295 Eﬀects and Location of Coplanar and Noncoplanar PCB in a Lipid Bilayer: A Solid-State NMR Study Christian Totland,,† Willy Nerdal,† and Signe Steinkopf‡ †Department of Chemistry, University of Bergen, Norway, Allégaten 41, N-5007 Bergen, Norway ‡Department of Biomedical Laboratory Sciences and Chemical Engineering, Bergen University College, 5020 Bergen, Norway ■MATERIALS AND METHODS Although not visible in the static spectrum, the corresponding MAS spectrum of the POPC + PCB 77 sample (Figure 1 C) shows the presence of a small additional lamellar phase, corresponding to 8.5% of the sample lipids. This lamellar phase is not observed in the static spectrum due to low abundance. If this lamellar phase is related to lipids interacting with PCB 77, it corresponds to about four aﬀected lipids per PCB 77 molecule. NMR. The NMR data was obtained on a Bruker AV III HD 500 instrument. The instrument is equipped with magic angle spinning (MAS) hardware for 4 mm rotors (ZrO2). Experi- ments were carried out at a sample temperature of 298 K. 13C spectra were obtained at a sample spinning rate of 6 kHz with high-power proton decoupling during acquisition. The 13C experiments were acquired with a relaxation delay of 5 s and 15000 scans. For the static 31P spectra 5000 scans were collected with a relaxation delay of 3 s between transients. MAS 31P experiments were carried out with a rotor spinning speed of 2.5 kHz. Simulations of static 31P NMR spectra were carried out with the Topspin software (version 3.5). NMR. The NMR data was obtained on a Bruker AV III HD 500 instrument. The instrument is equipped with magic angle spinning (MAS) hardware for 4 mm rotors (ZrO2). Experi- ments were carried out at a sample temperature of 298 K. 13C spectra were obtained at a sample spinning rate of 6 kHz with high-power proton decoupling during acquisition. The 13C experiments were acquired with a relaxation delay of 5 s and 15000 scans. For the static 31P spectra 5000 scans were collected with a relaxation delay of 3 s between transients. MAS 31P experiments were carried out with a rotor spinning speed of 2.5 kHz. Simulations of static 31P NMR spectra were carried out with the Topspin software (version 3.5). However, changes in POPC morphology in the presence of PCB 52 are clearly seen in the 31P spectra (Figure 1), where a high-mobility phase is indicated by a sharp peak in the static spectrum (Figure 1 A and B), which is typical for a micellar-like phase. This phase corresponds to 18% of sample lipids. In addition, there are two lamellar phases with greater ﬂuidity than that observed for the POPC and POPC+PCB 77 samples. ■MATERIALS AND METHODS Materials. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocho- line (POPC) was obtained from Avanti Polar Lipids (Birmingham, AL). PCB 52 and PCB 77 were obtained from Sigma-Aldrich. Figure 1. Recorded (A) and simulated (B) static 31P NMR spectra of POPC samples (50 wt % hydration) with and without PCB 52 or 77. The central peak of corresponding MAS spectra with 2 kHz spinning are shown in C: An asterisk marks an isotropic high mobility phase (does not give spinning side bands), while the remaining peaks represent lamellar phases. The amount of PCB in the samples is 2 mol % relative to the lipid. Liposome Preparation. The amount of PCB in the samples is 2.0 mol %, which is above the limit where nonspeciﬁc toxicity occurs for noncoplanar PCB.24 The lipid samples were dissolved in chloroform, which was then evaporated, and the samples were lyophilized overnight. The dry samples were suspended in degassed, argon bubbled water and left on a water bath for 1 h at 40 °C. In order to obtain mostly unilamellar liposomes the sample was freeze−thawed seven times using liquid N2.22 The samples were pH-adjusted to 7.4 by adding a small amount of 0.05 M NaOH with freezing and thawing between each adjustment. The electrolyte concentration as a result of the pH adjustment was in the order of 10−4 M. Samples were subjected to 24 h of lyophilization giving partially hydrated liposomes with a hydration level of ∼12 water molecules per lipid molecule.21,22 Thus, samples with about 50 wt % water (fully hydrated lipids) could be obtained by adding degassed argon-bubbled water.25,26 The samples were then equilibrated at 40 °C for 48 h and packed in NMR rotors in an argon-atmosphere and stored in the freezer. POPC, POPC + PCB 77, or POPC + PCB 52 with 50 wt % hydration. The spectrum of pure POPC shows as expected a single lamellar phase with chemical shift anisotropy (CSA) at 43 ppm. The static spectrum of the POPC+PCB 77 sample (Figure 1 A) shows the same lamellar phase but with a small fraction of a higher mobility isotropic phase. As seen from the simulated spectrum (Figure 1 B), this isotropic phase consists of a negligible fraction of POPC in the sample. It is not clear whether this isotropic phase is related to interaction with PCB 77, as a small fraction of isotropic POPC may occur during sample preparation regardless of PCB. ■RESULTS AND DISCUSSION ■RESULTS AND DISCUSSION that PCB 52 diﬀuses more slowly than PCB 77 in the bilayer.17 It was further concluded that, contrary to PCB 52, PCB 77 does not intercalate into the lipid bilayers. This latter conclusion was later questioned by Jonker and van der Heijden.18 31P NMR. Figure 1 shows the static (A), simulated (B), and MAS (C) 31P NMR spectra of the three samples containing Figure 1. Recorded (A) and simulated (B) static 31P NMR spectra of POPC samples (50 wt % hydration) with and without PCB 52 or 77. The central peak of corresponding MAS spectra with 2 kHz spinning are shown in C: An asterisk marks an isotropic high mobility phase (does not give spinning side bands), while the remaining peaks represent lamellar phases. The amount of PCB in the samples is 2 mol % relative to the lipid. In order to fully evaluate the eﬀect and location of diﬀerent PCBs in a lipid bilayer, atomic-level investigation of the bilayer is required. A technique capable of this is nuclear magnetic resonance (NMR) spectroscopy.19−22 The amount of PCB interacting with a membrane relevant to human exposure is relatively small and remains below the detection limit of 13C NMR. Still, high-resolution magic angle spinning (MAS) 13C NMR can give detailed insight into the lipid-PCB interaction by monitoring changes in lipid 13C spectra. Additionally, 31P NMR gives direct and precise information on bilayer morphology and ﬂuidity.23 These techniques are applied here on a system comprising of unilamellar liposomes of POPC −a biologically abundant lipid −with and without PCB 52 or PCB 77. The purpose of this work is to establish how noncoplanar and coplanar PCBs interact with a lipid bilayer and what possible consequences such interactions have to the bilayer properties. DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295 Environmental Science & Technology Article ■INTRODUCTION Additionally, ﬂuorescence correlation spectroscopy of PCB in 1,2-dilinoleoyl- sn-glycero-3-phosphocholine (DLPC) ﬂuid bilayers showed There are as much as 209 possible conﬁgurations of PCB, where the degree and route of toxicity of various congeners varies signiﬁcantly.7 From studies of structure−activity relation- ships it is suggested that coplanar PCBs without chlorine substitution in the ortho position have biological properties similar to dioxin (2,3,7,8-tetrachlorobibenzo-p-dioxin), showing carcinogenic eﬀects by acting on the aryl hydrocarbon receptor.7,8 The noncoplanar PCBs, on the other hand, show diﬀerent and more complex routes of toxicity, with for example estrogenic and neurotoxic activity.9−11 Additionally, non- coplanar PCBs have shown to increase the ﬂuidity of cellular membranes,12−14 which may aﬀect the activity of multiple membrane proteins.15 Consequently, the physiological eﬀects Received: April 7, 2016 Revised: June 28, 2016 Accepted: July 5, 2016 Published: July 5, 2016 0 DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295 Received: April 7, 2016 Revised: June 28, 2016 Accepted: July 5, 2016 Published: July 5, 2016 © 2016 American Chemical Society 8290 8290 DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295 Environmental Science & Technology ■MATERIALS AND METHODS It has previously been proposed that PCB 77 does not intercalate between the lipid molecules and only interacts with the headgroup,17 something that was later argued as unlikely due to the lipophilic nature of PCB.18 In this section the locations of PCB 52 and 77 are determined by 13C NMR. y Figures 2 and 3 show the eﬀect of PCB intercalation in the POPC bilayer. Compared to the reference sample with only Figure 3. 13C MAS NMR spectra of the carbonyl (A) and CC (B) regions of the POPC (top), POPC + PCB 77 (middle), and POPC + PCB 52 (bottom) samples at 298 K with 50 wt % hydration. Peak labels are in accordance with the molecular structure given in Figure 2. The amount of PCB in the sample is 2 mol % relative to the lipid. The peak labeled with an asterisk is interpreted as POPC in a diﬀerent phase. Assignments are based on ref 27. Figure 2. A: Molecular structure of POPC. B: Diﬀerences in 13C chemical shifts (Δδ) of the POPC lamellar phase in the presence of PCB 77 (◆) and PCB 52 (□), compared to a sample of POPC only. A positive value of Δδ means that the peak in question shifts to higher ppm values in the presence of PCB (Δδ = δw/PCB −δPOPC). The molecular structure of POPC is shown with labeling of the carbons according to the assignment in the plot and in Figure 3 and Figure S1 and S2 of the Supporting Information. change where the angle between the acyl chain and headgroup increases upon intercalation of PCB. The increased angle reduces stearic interactions between the carbonyl carbon and surrounding nuclei which in turn reduces the chemical shift. An accuracy of <0.02 ppm can be expected when internal shift referencing is used. The more pronounced shift diﬀerences (∼0.5 ppm) observed for some resonances in the PCB 52 sample, for example acyl chain carbons 4 and 7 (Figure 2 B), is most likely due to the higher angle between the phenyl planes and consequent more bulky stereochemistry of PCB 52 compared to PCB 77. As discussed in the next section, the 31P NMR results suggest that this diﬀerence causes PCB 52 to aﬀect lipids in a greater radius compared to PCB 77. Figure 2. A: Molecular structure of POPC. ■MATERIALS AND METHODS B: Diﬀerences in 13C chemical shifts (Δδ) of the POPC lamellar phase in the presence of PCB 77 (◆) and PCB 52 (□), compared to a sample of POPC only. A positive value of Δδ means that the peak in question shifts to higher ppm values in the presence of PCB (Δδ = δw/PCB −δPOPC). The molecular structure of POPC is shown with labeling of the carbons according to the assignment in the plot and in Figure 3 and Figure S1 and S2 of the Supporting Information. g For the model POPC bilayer, the location of PCB below the carbonyl segments and above carbon number 9 from the carbonyl of the acyl chains is further illustrated by the CC resonances shown in Figure 3 B. Here, carbon 9, which is closest to the headgroup, is clearly aﬀected by the presence of both PCB 52 and 77, while carbon 10 remains unaﬀected. These results suggest that the PCB molecules are oriented with the long molecular axis horizontally to the bilayer plane, as a vertical orientation would be expected to aﬀect 13C resonances further down the acyl chains. The small dipole moments present when the phenyl plane conﬁgurations are as in Figure 4 A can then be oriented with the positive part toward the negative charge of the POPC headgroup. The resulting PCB orientations are shown schematically in Figure 4 B for this scenario. However, dynamics such as rotational averaging of the PCB and lipid molecules are not considered in the ﬁgure. POPC, the 13C chemical shifts of lamellar POPC are altered in samples containing PCB 77 or PCB 52 (Figure 2 B). From the plot of chemical shift diﬀerences in Figure 2, two main observations are made. First, the pattern of chemical shift diﬀerences is similar for both the PCB 52 and PCB 77 sample. However, the changes in chemical shift are slightly larger for the PCB 52 sample. Second, the changes in chemical shift are more pronounced for the carbonyl carbons (1′, 1) and subsequent acyl chain carbons. This indicates both that PCB 52 and 77 intercalate at the same position in the POPC bilayer and that this position is below the POPC carbonyl where POPC carbons in general experience a more crowded chemical environment, as evident from the decrease in chemical shift. ■MATERIALS AND METHODS The CSAs of the two lamellar phases with 2 mol % PCB 52 present are 31 and 17 ppm, representing 50% and 32% of sample lipids, respectively. The CSA value is inversely proportional to both the axial rotation of the lipids and the degree of lipid oscillation. Hence, higher lipid mobility or larger angle of the oscillation 8291 DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295 Figure 3. 13C MAS NMR spectra of the carbonyl (A) and CC (B) regions of the POPC (top), POPC + PCB 77 (middle), and POPC + PCB 52 (bottom) samples at 298 K with 50 wt % hydration. Peak labels are in accordance with the molecular structure given in Figure 2. The amount of PCB in the sample is 2 mol % relative to the lipid. The peak labeled with an asterisk is interpreted as POPC in a diﬀerent phase. Assignments are based on ref 27. Article Environmental Science & Technology Article Article will induce higher bilayer ﬂuidity and cause lower CSA values. This indicates increased ﬂuidity compared to the lower mobility lamellar phase observed in the POPC and POPC+PCB 77 samples (CSA = 43 ppm). will induce higher bilayer ﬂuidity and cause lower CSA values. This indicates increased ﬂuidity compared to the lower mobility lamellar phase observed in the POPC and POPC+PCB 77 samples (CSA = 43 ppm). will induce higher bilayer ﬂuidity and cause lower CSA values. This indicates increased ﬂuidity compared to the lower mobility lamellar phase observed in the POPC and POPC+PCB 77 samples (CSA = 43 ppm). p ( pp ) The 31P NMR data conﬁrm results from earlier studies on both cellular and model lipid bilayer samples where the presence of PCB 52 is found to increase bilayer ﬂuidity.14,17,26 Similar to previous studies, PCB 77 do not signiﬁcantly aﬀect the membrane ﬂuidity; however, 4 lipids per PCB 77 adopt a lamellar phase with slightly diﬀerent properties. The nature of the lipid morphologies is discussed in a separate section. 13 g 13C NMR. There have been controversies regarding the localizations of PCB 52 and 77 with respect to the bilayer. DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295 ■MATERIALS AND METHODS The displacement of the carbonyl resonance to higher Another notable feature in the 13C spectra is the appearance of additional POPC peaks, particularly in the PCB 52 sample. This is shown in Figure 3 A for the carbonyl spectral region. The origins of these peaks are various POPC morphologies in the PCB 52 sample that are not present to a signiﬁcant extent in the absence of PCB 52. The small additional peaks seen in The displacement of the carbonyl resonance to higher chemical shift values (Figure 3 A) reﬂects a conformational 8292 DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295 Environmental Science & Technology Article Figure 4. A: PCB77 (left) and PCB52 (right). The stereochemistry is calculated by the use of density functional theory (Spartan ’14). The torsion angles between phenyl planes for the shown conﬁgurations are ∼76° for PCB52 and ∼37° for PCB77. These conﬁgurations correspond to dipole moments of 0.28 and 0.86 D, respectively (directions indicated). Note however that dipole moments will vary as the phenyl rings twist.18 B: Schematic side view of PCB 77 and 52 intercalated in a POPC layer, showing the more bulky stereochemistry of PCB 52. particularly those of the headgroup region. For example, 13C resonances from carbons near the headgroup region of tetradecyltrimethylammonium bromide increase as much as 1.5 ppm upon transition from bilayer to micelle.28 Therefore, the additional carbonyl peak at higher ppm values in the PCB 52 sample (Figure 3) most likely correlates to the high mobility micellar-like phase seen in the 31P spectrum. Additional peaks also appear around other resonances from carbons near the headgroup region, including the glycerol (A, B, and C) and cholin (γ, α, and β) carbons, as well as the ﬁrst three carbons of the acyl chains (1, 1′−3, 3′) (see the Supporting Information). y pp g Lipid Morphologies in the PCB 52 and PCB 77 Samples. The changes in bilayer morphology and ﬂuidity in the presence of PCB 52 are related to stearic interactions between the POPC acyl chain and PCB 52. With 2 mol % PCB 52, the most notable changes in bilayer morphology are a high ﬂuidity lamellar phase constituting 32% of sample lipids and a high mobility micellar-like phase constituting 18% of sample lipids. ■MATERIALS AND METHODS As mentioned, the remaining lipids form a less mobile lamellar phase, which still has higher ﬂuidity than the single lamellar phase seen in the POPC+PCB 77 and POPC samples (Figure 1). g The sample contains 2 mol % PCB 52 relative to the amount of lipid. A reasonable assumption is that PCB 52 has highest impact on nearest and next nearest neighboring lipids and that the most mobile of the lamellar phases relates to these regions. Further, if PCB 52 molecules localized in separate leaﬂets of the bilayer become close in space, the mobile lamellar phases of both leaﬂets will interact and destabilize the bilayer. The result of this interaction is formation of a curved toroidal region which will resemble a micellar-like phase in the NMR spectra and facilitate formation of channels in the bilayer. The location of PCB 52 close to the choline headgroup will increase the headgroup cross-sectional area relative to the acyl chain. This gives a packing parameter that facilitates increased curvature, similar to a micellar phase, when several PCB 52s are near each other at both sides of the bilayer. Conversely, a PCB location closer to the core of the bilayer could reduce the curvature and cause inverse hexagonal phases when multiple PCB 52s interact in the bilayer. Such inverse hexagonal phases result in powder pattern 31P resonances which are not seen here. Hence, the presence of a micellar-like phase supports that PCB locates in the outer part of the bilayer hydrophobic core. A schematic presentation of the bilayer is illustrated in Figure 5 where the Figure 4. A: PCB77 (left) and PCB52 (right). The stereochemistry is calculated by the use of density functional theory (Spartan ’14). The torsion angles between phenyl planes for the shown conﬁgurations are ∼76° for PCB52 and ∼37° for PCB77. These conﬁgurations correspond to dipole moments of 0.28 and 0.86 D, respectively (directions indicated). Note however that dipole moments will vary as the phenyl rings twist.18 B: Schematic side view of PCB 77 and 52 intercalated in a POPC layer, showing the more bulky stereochemistry of PCB 52. the carbonyl and headgroup regions of the POPC and POPC + PCB 77 13C spectra are likely related to the sample packing, where bilayer regions with extended curvature can occur in the turns of ﬂattened liposomes and at the sample-rotor interface. ■REFERENCES The results show that PCB 77 does not aﬀect membrane properties signiﬁcantly, despite a similar location to PCB 52. However, the 31P MAS NMR spectra suggests that PCB 77 in fact does have an eﬀect on neighboring lipids which form a higher mobility lamellar phase similar to the one observed in the PCB 52 sample, due to the similar isotropic 31P chemical shifts. The diﬀerence is the extent of the eﬀect, where one PCB 77 aﬀects about 4 lipids and one PCB 52 aﬀect 16 lipids, likely due to the diﬀerent stereochemistry of the two PCBs. The small eﬀect of PCB 77 will not be detectable by techniques used in previous studies and was e.g. misinterpreted in an earlier study by Campbell et al. to suggest that PCB 77 does not intercalate in the bilayer.17 (1) Porta, M.; Zumeta, E. Implementing the Stockholm Treaty on Persistent Organic Pollutants. Occup. Environ. Med. 2002, 59 (10), 651−652. (1) Porta, M.; Zumeta, E. Implementing the Stockholm Treaty on Persistent Organic Pollutants. Occup. Environ. Med. 2002, 59 (10), 651−652. (2) McFarland, V. A.; Clarke, J. U. Environmental occurrence, abundance, and potential toxicity of polychlorinated biphenyl congeners: considerations for a congener-specific analysis. Environ. Health Perspect. 1989, 81, 225−239. (3) Dorneles, P. R.; Lailson-Brito, J.; Secchi, E. R.; Dirtu, A. C.; Weijs, L.; Dalla Rosa, L.; Bassoi, M.; Cunha, H. A.; Azevedo, A. F.; Covaci, A. Levels and profiles of chlorinated and brominated contaminants in Southern Hemisphere humpback whales, Megaptera novaeangliae. Environ. Res. 2015, 138, 49−57. (3) Dorneles, P. R.; Lailson-Brito, J.; Secchi, E. R.; Dirtu, A. C.; Weijs, L.; Dalla Rosa, L.; Bassoi, M.; Cunha, H. A.; Azevedo, A. F.; Covaci, A. Levels and profiles of chlorinated and brominated contaminants in Southern Hemisphere humpback whales, Megaptera novaeangliae. Environ. Res. 2015, 138, 49−57. (4) Kallenborn, R.; Breivik, K.; Eckhardt, S.; Lunder, C. R.; Manø, S.; Schlabach, M.; Stohl, A. Long-term monitoring of persistent organic pollutants (POPs) at the Norwegian Troll station in Dronning Maud Land, Antarctica. Atmos. Chem. Phys. 2013, 13 (14), 6983−6992. y When utilizing a model lipid membrane, the biological relevance of the model system is of importance in order to translate the eﬀects to biological systems. Here, the biological abundant POPC is used, which means that general properties of the bilayer, such as thickness and chain saturation, resemble a cellular membrane. ■ABBREVIATIONS POPC 1-palmitoyl-2-oeloylphosphatidylcholine DMPC dimyristoylphosphatidylcholine DPPC dipalmitoylphosphatidylcholine PCB polychlorinated biphenyls NMR nuclear magnetic resonance MAS magic angle spinning POPC 1-palmitoyl-2-oeloylphosphatidylcholine p y y p p y DMPC dimyristoylphosphatidylcholine DPPC dipalmitoylphosphatidylcholine NMR nuclear magnetic resonance Notes The authors declare no competing ﬁnancial interest. In the schematic bilayer representations shown in Figure 5 it is indicated that where a large enough number of PCB 52 molecules can be found in a limited region, the POPC bilayer will collapse and form a micellar-like phase. The POPC lipids in such a phase can be expected to return to bilayer when the PCB molecules that cause the micellar phase diﬀuse apart and that these processes will move around in the POPC bilayer depending on the number of PCB molecules found in a limited region. It should be noted that this study is conducted at a single PCB concentration using a model POPC bilayer. In a biological system other toxicological mechanisms may come into play at various concentrations; however, at the current PCB bilayer concentration the high ﬂuidity phase occurring about each PCB 52 likely has highly adverse eﬀects, also in a biological system. ■ACKNOWLEDGMENTS We would like to acknowledge Dr.scient. Sonnich Meier, Institute of Marine Research, Bergen, Norway and Prof. Knut Teigen, The Department of Biomedicine, University of Bergen, Norway for providing some of the sample material used. ■REFERENCES In particular, the formation of curved toroidal regions may depend on such bilayer properties. Still, the results presented here display many of the same trends as previous studies using more typical saturated model lipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphoch (DPPC),14 DMPC, and DLPC,17 where increased ﬂuidity in the presence of PCB 52 is observed. PCB 52 is found to be among the more abundant PCB congeners in both animals and in the atmosphere,3,4 making knowledge about its toxicological routes important. (5) von Waldow, H.; MacLeod, M.; Jones, K.; Scheringer, M.; Hungerbühler, K. Remoteness from emission sources explains the fractionation pattern of polychlorinated biphenyls in the northern hemisphere. Environ. Sci. Technol. 2010, 44 (16), 6183−6188. (6) Beyer, A.; Biziuk, M. Environmental fate and global distribution of polychlorinated biphenyls. Rev. Environ. Contam. Toxicol. 2009, 201, 137−158. (7) Carpenter David, D. O. Polychlorinated Biphenyls (PCBs): Routes of exposure and effects on human health. Rev. Environ. Health 2006, 21, 1. (8) Safe, S. H. Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment. Crit. Rev. Toxicol. 1994, 24 (2), 87−149. (9) Arnold, D. L.; Mes, J.; Bryce, F.; Karpinski, K.; Bickis, M. G.; Zawidzka, Z. Z.; Stapley, R. A pilot study on the effects of Aroclor 1254 ingestion by rhesus and cynomolgus monkeys as a model for human ingestion of PCBs. Food Chem. Toxicol. 1990, 28 (12), 847− 857. Environmental Science & Technology Article Article ■AUTHOR INFORMATION ■AUTHOR INFORMATION micellar phase is represented as a curved toroidal region which forms a channel in the bilayer. micellar phase is represented as a curved toroidal region which forms a channel in the bilayer. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the ﬁnal version of the manuscript. ■MATERIALS AND METHODS The 31P NMR spectra (Figure 1) conﬁrm the presence of diﬀerent morphological states of POPC in the PCB 52 sample. The morphology of aggregated amphiphilic molecules has potentially a large impact on the molecules’ 13C chemical shifts, the carbonyl and headgroup regions of the POPC and POPC + PCB 77 13C spectra are likely related to the sample packing, where bilayer regions with extended curvature can occur in the turns of ﬂattened liposomes and at the sample-rotor interface. The 31P NMR spectra (Figure 1) conﬁrm the presence of diﬀerent morphological states of POPC in the PCB 52 sample. The morphology of aggregated amphiphilic molecules has potentially a large impact on the molecules’ 13C chemical shifts, Figure 5. Schematic view of the POPC bilayer with intercalated PCB 52, illustrating the three POPC phases observed from the 31P spectra with 2 mol % PCB 52. The simulated 31P spectra of the various phases are shown (from Figure 1 B). White orbs represent the less mobile lamellar phase (50%), gray orbs represent the mobile lamellar phase (32%), and blue orbs represent the curved toroidal/micellar phase (18%). The intercalated PCB 52 molecules (2%) are represented as red orbs. The number of orbs representing each phase approximately equals the fraction percentage found by 31P NMR and thus gives a realistic distribution of the phases and PCB 52 in the bilayer. Figure 5. Schematic view of the POPC bilayer with intercalated PCB 52, illustrating the three POPC phases observed from the 31P spectra with 2 mol % PCB 52. The simulated 31P spectra of the various phases are shown (from Figure 1 B). White orbs represent the less mobile lamellar phase (50%), gray orbs represent the mobile lamellar phase (32%), and blue orbs represent the curved toroidal/micellar phase (18%). The intercalated PCB 52 molecules (2%) are represented as red orbs. The number of orbs representing each phase approximately equals the fraction percentage found by 31P NMR and thus gives a realistic distribution of the phases and PCB 52 in the bilayer. DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295 8293 Environmental Science & Technology Corresponding Author The data presented here gives molecular-level details about the interaction of coplanar and noncoplanar PCBs with a lipid bilayer, beyond what has been shown earlier. Particularly, the ability of noncoplanar PCB 52 to potentially disrupt the bilayer upon formation of curved toroidal regions may explain the cytotoxic eﬀects of these chemicals.14 Moreover, the interaction of the high ﬂuidity lamellar phase occurring around each PCB 52 with membrane proteins will signiﬁcantly aﬀect protein function, as observed earlier.12−15 Phone: +47 55588233. E-mail: christian.totland@uib.no. Phone: +47 55588233. E-mail: christian.totland@uib.no. Environmental Science & Technology (11) Fischer, L. J.; Seegal, R. F.; Ganey, P. E.; Pessah, I. N.; Kodavanti, P. R. Symposium overview: toxicity of non-coplanar PCBs. Toxicol. Sci. 1998, 41 (1), 49−61. (12) Bonora, S.; Torreggiani, A.; Fini, G. DSC and Raman study on the interaction between polychlorinated biphenyls (PCB) and phospholipid liposomes. Thermochim. Acta 2003, 408 (1−2), 55−65. (13) Gonzalez, A.; Odjélé, A.; Weber, J.-M. PCB-153 and temperature cause restructuring of goldfish membranes: Homeo- viscous response to a chemical fluidiser. Aquat. Toxicol. 2013, 144− 145, 11−18. (14) Tan, Y.; Chen, C. H.; Lawrence, D.; Carpenter, D. O. Ortho- substituted PCBs kill cells by altering membrane structure. Toxicol. Sci. 2004, 80 (1), 54−59. ̌ (15) Šimečková, P.; Vondráček, J.; Procházková, J.; Kozubík, A.; Krčmář, P.; Machala, M. 2,2′,4,4′,5,5′-Hexachlorobiphenyl (PCB 153) induces degradation of adherens junction proteins and inhibits β- catenin-dependent transcription in liver epithelial cells. Toxicology 2009, 260 (1−3), 104−111. (16) Tan, Y.; Li, D.; Song, R.; Lawrence, D.; Carpenter, D. O. Ortho- substituted PCBs kill thymocytes. Toxicol. Sci. 2003, 76 (2), 328−337. ( ) (17) Campbell, A. S.; Yu, Y.; Granick, S.; Gewirth, A. A. PCB association with model phospholipid bilayers. Environ. Sci. Technol. 2008, 42 (19), 7496−7501. (18) Jonker, M. T. O.; van der Heijden, S. A. Comment on “PCB association with model phospholipid bilayers. Environ. Sci. Technol. 2009, 43 (13), 5155−5156. (19) Grélard, A.; Loudet, C.; Diller, A.; Dufourc, E. J. NMR spectroscopy of lipid bilayers. In Membrane Protein Structure Determination: Methods and Protocols; Lacapère, J.-J., Ed.; Humana Press: Totowa, NJ, 2010; pp 341−359. , J, ; pp (20) Bonev, B. B. Chapter Eleven - High-Resolution Solid-State NMR of Lipid Membranes. In Advances in Planar Lipid Bilayers and Liposomes; Aleš, I., Julia, G., Eds.; Academic Press: 2013; Vol. Vol. 17, pp 299−329. (21) Song, C.; Holmsen, H.; Nerdal, W. Existence of lipid microdomains in bilayer of dipalmitoyl phosphatidylcholine (DPPC) and 1-stearoyl-2-docosahexenoyl phosphatidylserine (SDPS) and their perturbation by chlorpromazine: a 13C and 31P solid-state NMR study. Biophys. Chem. 2006, 120 (3), 178−187. (22) Nerdal, W.; Gundersen, S. A.; Thorsen, V.; Høiland, H.; Holmsen, H. Chlorpromazine interaction with glycerophospholipid liposomes studied by magic angle spinning solid state 13C-NMR and differential scanning calorimetry. Biochim. Biophys. Acta, Biomembr. 2000, 1464 (1), 165−175. (23) Picard, F.; Paquet, M.-J.; Levesque, J.; Bélanger, A.; Auger, M. 31P NMR first spectral moment study of the partial magnetic orientation of phospholipid membranes. Biophys. J. 1999, 77 (2), 888−902. DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295 S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b01723. (10) Saint-Amour, D.; Roy, M.-S.; Bastien, C.; Ayotte, P.; Dewailly, É.; Després, C.; Gingras, S.; Muckle, G. Alterations of visual evoked potentials in preschool Inuit children exposed to methylmercury and polychlorinated biphenyls from a marine diet. NeuroToxicology 2006, 27 (4), 567−578. (10) Saint-Amour, D.; Roy, M.-S.; Bastien, C.; Ayotte, P.; Dewailly, É.; Després, C.; Gingras, S.; Muckle, G. Alterations of visual evoked potentials in preschool Inuit children exposed to methylmercury and polychlorinated biphenyls from a marine diet. NeuroToxicology 2006, 27 (4), 567−578. 13C spectra (all spectral regions); 31P MAS spectra; Figures S1−S5; reference S1 (PDF) 8294 DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295 Article Environmental Science & Technology Environmental Science & Technology (24) van Wezel, A. P.; Opperhuizen, A. Narcosis due to environmental pollutants in aquatic organisms: Residue based toxicity, mechanisms and membrane burdens. Crit. Rev. Toxicol. 1995, 25 (3), 255−279. (25) Janiak, M. J.; Small, D. M.; Shipley, G. G. Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin. J. Biol. Chem. 1979, 254 (13), 6068−6078. lecithin. J. Biol. Chem. 1979, 254 (13), 6068−6078. (26) Small, D. M. Phase equilibria and structure of d J ( ) (26) Small, D. M. Phase equilibria and structure of dry and hydrated egg lecithin. J. Lipid Res. 1967, 8 (6), 551−557. egg lecithin. J. Lipid Res. 1967, 8 (6), 551−557. (27) Ferreira, T. M.; Coreta-Gomes, F.; Ollila, O. H.; Moreno, M. J.; Vaz, W. L.; Topgaard, D. Cholesterol and POPC segmental order parameters in lipid membranes: solid state 1H-13C NMR and MD simulation studies. Phys. Chem. Chem. Phys. 2013, 15 (6), 1976−1989. (27) Ferreira, T. M.; Coreta-Gomes, F.; Ollila, O. H.; Moreno, M. J.; Vaz, W. L.; Topgaard, D. Cholesterol and POPC segmental order y y ( ) (28) Totland, C.; Nerdal, W. Thermotropic behavior of a cationic surfactant in the adsorbed and micellar state: An NMR study. Langmuir 2012, 28 (16), 6569−6576. 8295 DOI: 10.1021/acs.est.6b01723 Environ. Sci. Technol. 2016, 50, 8290−8295
W4286900568.txt	https://zenodo.org/records/5578147/files/Heimatland%20Brochure.pdf	fr		Bienvenue à Heimatland! Saurez-vous trouver votre place?	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	11,307	Edité par Laure Sandoz, Dina Bader et Nicolas Yazgi Dans le cadre du Théâtre de la Connaissance 2021 urez-vous trouver votre place ? BIENVENUE À HE ! Sa TLAND A IM Neuchâtel, 2021 Edité par Laure Sandoz, Dina Bader et Nicolas Yazgi urez-vous trouver votre place ? BIENVENUE À HE ! Sa TLAND A IM Chère lectrice, cher lecteur, Malgré les restrictions liées à la pandémie, le nccr - on the move, un Pôle national de recherche financé par la Confédération et coordonné par l’Université de Neuchâtel, a poursuivi ses recherches et s’est montré très intéressé à discuter de ses résultats et de ses réflexions avec la Cité. Dans le cadre de « Bienvenue à Heimatland ! », nous sommes heureux d’engager ces discussions lors d’un événement aussi prestigieux que NeuchàToi 2021. Ce rendez-vous récurrent revêt une grande importance pour les échanges interculturels du canton de Neuchâtel qui rayonnent bien au-delà de ses frontières. C’est donc pour nous un grand privilège de participer à un tel événement. « Bienvenue à Heimatland ! » est un jeu théâtral dans lequel vous jouez le rôle principal. Dans cette expérience immersive, vous êtes mis.e au défi, en tant que migrant.e, de trouver votre place dans la société. Dans la tradition du Théâtre de la Connaissance, vous êtes confronté.e aux réalités et contraintes de la migration de manière ludique. Dans un dialogue entre l’art et la science, une des questions centrales de la Suisse – la migration et ses acteur.rice.s – est négociée avec votre aide d’une manière riche en émotions. Qu’un tel échange ait lieu à Neuchâtel n’est pas un hasard. L’Université de Neuchâtel est un centre d’excellence européen sur les questions de migration. Le SFM (Forum suisse pour l’étude des migrations et de la population), la MAPS (Maison d’analyse des processus sociaux), le CDM (Centre de droit des migrations) et enfin notre nccr - on the move en témoignent. Cette concentration de spécialistes permet de développer une perception différente de la société et de penser sous d’autres angles. Elle offre une condition unique pour le développement de ce Théâtre de la Connaissance. Nous avons eu l’occasion de tester le jeu de rôle dont « Bienvenue à Heimatland ! » s’inspire pour la première fois lors d’un événement interne du nccr – on the move. Concept développé par ISA Berne, un service spécialisé en migrations, ce jeu a ensuite été affiné pour NeuchàToi 2021, le rendant plus pointu tant sur le plan artistique que scientifique. Une telle réalisation ne serait pas possible sans le soutien énergique de professionnel.le.s du théâtre et de chercheur.euse.s engagé.e.s. Je tiens à remercier pour la coordination, la conception et l’organisation, Nicolas Yazgi, anthropologue, dramaturge et metteur en scène, ainsi que Laure Sandoz qui a supervisé le projet. Nous remercions également Dina Bader et Annique Lombard du nccr – on the move, les collègues de la MAPS, de la FLSH et du Bureau presse et promotion de l’Université de Neuchâtel pour leur engagement, ainsi que nos sponsors. Un remerciement tout particulier à Yves Maumary pour le développement graphique des idées mises en scène et l’affiche, et à Gina Morelli pour le design de cette publication. J’espère que ce Théâtre de la Connaissance aura été pour vous inspirant et passionnant. Cette brochure prolonge l’expérience en explicitant certains des éléments qui ont inspiré la conception de l’événement et en proposant des ressources pour poursuivre la réflexion. Gianni D’Amato Directeur nccr – on the move Bienvenue à HEIMATLAND ! Saurez-vous trouver votre place ? Jeu de rôle théâtral suivi d’une discussion participative sur le thème de la gestion migratoire Université de Neuchâtel, Rue A.-L. Breguet 2, du 17 au 19 septembre 2021 Direction & conception : Laure Sandoz & Nicolas Yazgi Inspiré librement d’une idée originale développée par ISA Berne Entrée libre - sur inscription ● Informations : www.unine.ch/theatre-connaissance Pourquoi cet événement ? Depuis le début du XXe siècle, la Suisse est devenue un pays d’immigration. La proportion de la population étrangère résidant sur le territoire helvétique est aujourd’hui de 25 %, ce qui est bien plus que dans la plupart des autres pays européens. En outre, plus de la moitié des personnes récemment arrivées en Suisse possède une formation tertiaire, ce qui constitue également un record en comparaison internationale. Ces chiffres s’expliquent par le fait que la Suisse dispose d’un système de libre circulation des personnes avec l’Union Européenne et d’une économie dynamique, avec notamment 28’622 entreprises faisant partie d’un groupe multinational et employant environ 1,4 million de personnes, soit un peu plus du quart de l’emploi total en Suisse. Néanmoins, la Suisse se caractérise également par une politique d’admission particulièrement restrictive pour les ressortissant.e.s non-européen.ne.s qui dépend dans une large mesure du pouvoir discrétionnaire des autorités. Cette politique s’inscrit depuis le milieu des années 1960 dans une longue série de mesures visant à limiter l’immigration. Ainsi, les conditions d’entrée en Suisse sont très variables et contribuent à structurer un système à plusieurs vitesses dans lequel certaines personnes étrangères disposent d’un large éventail de possibilités pour s’adapter à leur nouvel environnement tandis que d’autres sont limitées dans leurs options et se retrouvent dans des situations de vulnérabilité et de marginalité. Dans un contexte politique qui place bien souvent l’immigration au cœur des débats, il nous a semblé fécond d’engager ce dialogue entre recherche, création artistique et public de la cité afin de promouvoir une réflexion critique autour de l’inclusion des personnes migrantes et du rôle de chacun.e dans le processus de construction d’une société commune. Laure Sandoz Docteure en anthropologie, chercheuse postdoctorante à l’Institut de géographie et au nccr - on the move Nicolas Yazgi Dramaturge et anthropologue Table des matières Dans les coulisses de « Bienvenue à Heimatland ! » 10 – 11 12 – 15 16 – 19 L’ esprit de la mise en scène Nicolas Yazgi Les différents livrets pour étranger.e.s Laure Sandoz Le livret dans le jeu Nicolas Yazgi Et en Suisse, comment ça se passe ? 20 – 23 24 – 25 Le jeu de l’échelle de l’intégration juridique en Suisse Stefanie Kurt « Dis-moi ton permis… » Anne-Laure Bertrand Dans les coulisses de « Bienvenue à Heimatland ! » 26 – 27 28 – 29 30 – 33 Le ruban de Möbius Nicolas Yazgi L’administration étatique Laure Sandoz Préparer et développer les rôles Nicolas Yazgi Et en Suisse, comment ça se passe ? 34 – 37 38 – 41 42 – 47 Bureaucrates anonymes ou êtres humains agissant en « leur âme et conscience » ? Christin Achermann Distinguer les réfugié.e.s des migrant.e.s : pratique arbitraire ou simple application de la loi ? Marion Fresia L’asile en marges : les requérant.e.s d’asile face à la gestion migratoire Raphaël Rey Dans les coulisses de « Bienvenue à Heimatland ! » 48 – 49 Critères d’intégration et règles implicites Laure Sandoz Et en Suisse, comment ça se passe ? 50 – 53 54 – 57 Une intégration « réussie » ? La fabrique de l’intégration Tania Zittoun, Pascal Mahon, Anne Lavanchy, Flora Di Donato La discrimination en Suisse : résultats d’une étude expérimentale sur l’embauche des jeunes descendant.e.s d’immigré.e.s Robin Stünzi 59 Pour en savoir plus sur la migration en Suisse 60 – 61 Partenaires impliqués Dans les coulisses de « Bienvenue à Heimatland ! » L’ esprit de la mise en scène Heimatland ne représente pas littéralement la Suisse, mais en propose plutôt un miroir fictionnel déformant. Ce qui s’y passe n’est donc pas censé être un reflet exact de la réalité : celle-ci est à la fois grandement simplifiée et légèrement décalée. Les décalages viennent rappeler qu’il s’agit, justement, d’un environnement fictif et amener parfois un zeste d’humour. En revanche, les situations que rencontrent les participant.e.s durant le jeu sont basées sur les recherches et l’expérience de terrain et sont donc réalistes. Pour mettre l’administration du pays en scène et imaginer le système de jeu, il a fallu s’éloigner des réflexes de construction d’un « bon spectacle ». En effet, l’objectif principal n’était pas de mettre sur pied un spectacle, mais bien, au travers d’un jeu de rôle théâtral, d’induire un processus participatif expérimental permettant de réfléchir ensemble différemment sur le thème de la gestion migratoire (différemment que, par exemple, au travers d’une classique « conférence sur... »). Ce processus commence pour le public par le jeu de rôle théâtral, donc par une immersion sensible qui mobilise le corps et les émotions, en plus de l’intellect. Dans cette optique, les files d’attente, une forme de répétitivité, les frustrations, les simplifications ainsi que les limites du dispositif que nous avons mis en place sont assumées : elles sont potentiellement fécondes pour les parties réflexives de l’événement qui suivent le jeu de rôle théâtral. Pour ce qui est du développement de l’univers de Heimatland, de son administration et des situations que le public allait rencontrer, il a été décidé assez tôt, lors d’une séance avec une équipe scientifique étendue, de rester terre à terre, c’est-àdire de ne pas chercher à rendre l’expérience trop « théâtrale » ni à trop pousser les choses vers la satire ou la parodie. L’idée était que, pour ce thème et ce dispositif, une forme de réalisme était préférable pour induire le processus de réflexion souhaité. Il a donc ensuite beaucoup été question de trouver le bon degré de décalage tout en restant suffisamment réaliste. 11 Prenons le drapeau de Heimatland comme première illustration d’élément décalé. Il conserve les couleurs du drapeau suisse et l’idée d’un motif archétypal central. En lieu et place de la croix, se trouve « un cercle impossible ». Le cercle, de manière générale, est symboliquement associé à des notions comme l’unité, l’harmonie, la complétude, la perfection, l’infini, etc. Un objet impossible est la représentation d’un objet fictif contraire aux lois physiques connues de la nature : l’objet tridimensionnel qui devrait correspondre au dessin théorique est impossible à matérialiser. C’est bien sûr cette tension entre une représentation idéale (une administration « parfaite » : impartiale, juste et équitable pour tout.e.s) et sa matérialisation impossible dans la réalité qui a été la source d’inspiration (et qui fait écho aux recherches). La devise du pays, « tous comme les autres / chacun pour soi », est un des rares éléments un peu plus satiriques et exprime de manière concise la nécessité de se conformer – de faire comme les autres – tout en n’ayant, in fine, d’autre choix que d’être livré à soi-même. Pour présenter d’autres éléments de la mise en scène, j’ai préparé quelques compléments qui sont placés en regard des textes qui suivent pour enrichir l’effet de miroir, déformant mais espérons fécond, entre l’univers fictionnel de Heimatland et la réalité de la gestion migratoire en Suisse. Nicolas Yazgi Dramaturge et anthropologue Dans les coulisses de « Bienvenue à Heimatland ! » Les différents livrets pour étranger.e.s A Heimatland, chaque étranger.e reçoit dès son arrivée un livret qui détermine son statut légal et ses possibilités d’évolution dans son nouveau pays de résidence. Lequel avez-vous reçu ? Le texte ci-dessous vous éclairera sur sa signification. Livret X Il est attribué aux personnes admises à titre provisoire. Leur renvoi est impossible pour des raisons humanitaires ou pratiques, mais au moindre changement de situation, les autorités peuvent leur ordonner de quitter Heimatland. Pour espérer rester et obtenir un permis plus stable, les admis.e.s provisoires doivent faire preuve d’efforts importants pour s’intégrer car la vie ne leur est pas rendue facile. Vous avez peut-être remarqué, si vous avez reçu un tel statut, que vous étiez limité.e à des dés 1 à 4 pour votre progression et que les autorités se montraient particulièrement sévères lors de l’évaluation de votre niveau d’intégration. 13 Photo© Guillaume Perret Dans les coulisses de « Bienvenue à Heimatland ! » Livret V Il est attribué aux personnes qui correspondent à la définition officielle de réfugié.e. Celles-ci bénéficient d’une protection bien plus stable à Heimatland que les admis.e.s provisoires. Elles ne peuvent pas être renvoyées et, après quelques années, il leur est possible d’obtenir une autorisation d’établissement leur octroyant quasiment les mêmes droits qu’à la population locale. Pour cela, il est important qu’elles démontrent leurs efforts d’intégration et leur capacité à subvenir à leurs propres besoins sans dépendre d’aides étatiques. Tout comme les admis.e.s provisoires, elles se trouvent dans une situation de dette vis-à-vis de l’Etat qui leur a octroyé sa protection, ce qui peut compliquer leur progression vers un statut meilleur. Livrets Y1 et Y2 Ils correspondent à une autorisation de séjour qui doit être régulièrement renouvelée et qui peut être révoquée en cas de mauvaise intégration. Une distinction est établie entre les ressortissant.e.s d’état dits « alliés » (livret Y1) et les ressortissant.e.s d’état dits « tiers » (livret Y2). Les états alliés étant généralement considérés comme culturellement plus proches que les états tiers, la pression pour s’intégrer exercée par les autorités est moins forte pour les premier.e.s que pour les second.e.s, ce qui peut faciliter l’accès à un statut plus durable. De plus, le risque de perdre son permis de séjour en raison d’une mauvaise intégration est moins important pour les ressortissant.e.s d’états alliés. 15 Livrets Z1 et Z2 Ils correspondent à une autorisation d’établissement permanente qui peut donner accès à la procédure de naturalisation. Ils sont attribués à quelques rares catégories d’étranger.e.s dès leur arrivée à Heimatland lorsque des motifs de politique générale ou des intérêts scientifiques ou économiques à haut niveau le justifient. Là aussi, une distinction est établie entre états alliés (livret Z1) et états tiers (livret Z2), et les exigences sont plus sévères pour les ressortissant.e.s d’états tiers que pour les ressortissant.e.s d’états alliés. Les autorités attendent néanmoins un effort d’intégration de la part de tou.te.s les détenteur.rice.s d’autorisations d’établissement et elles peuvent faire régresser celles et ceux qui ne répondent pas à leurs exigences vers un statut moins stable. « Bienvenue à Heimatland ! » s’inspire librement et volontairement de la politique migratoire suisse, avec son régime de protection pour réfugié.e.s et son système dual en matière d’admission, qui distingue entre citoyen.ne.s européen.ne.s et le reste du monde. Le concept d’intégration y joue un rôle important en tant qu’outil pour sélectionner les personnes qui obtiendront le droit de rester à long terme dans le pays et pour exclure celles considérées comme pouvant nuire à la cohésion sociale, l’ordre public ou la stabilité économique du pays. Laure Sandoz Docteure en anthropologie, chercheuse postdoctorante à l’Institut de géographie et au nccr - on the move Dans les coulisses de « Bienvenue à Heimatland ! » Le livret dans le jeu Le livret pour étranger.e.s a été imaginé comme un croisement entre son équivalent dans la réalité et le carnet scolaire, exprimant ainsi l’infantilisation que représente le rapport à une administration toute puissante pour un individu et sa trajectoire de vie (infantilisation souvent ressentie, comme le montrent les recherches). La partie dédiée à « l’espace pour le développement de votre potentiel intégratoire » au dos du livret, continue de jouer autour de ce croisement : une surface à décorer et colorier, surtout sans sortir du cadre, pour montrer que l’on est « bon élève » du processus d’intégration. L’intérieur du livret a été conçu pour garder une trace de la « carrière administrative » de chacun.e des participant.e.s. Le terme de carrière permet d’englober l’ensemble des étapes où les migrant.e.s ont été amené.e.s à interagir avec l’administration. Elle représente un processus cumulatif qui se traduit comme la résultante toujours (ré)actualisée d’une suite d’interactions entre des individus singuliers et une administration qui les gère par réduction. Dans ce sens, si chaque nouvelle situation est unique, elle sera aussi influencée par toutes celles qui ont précédé. Ainsi, toutes les situations rencontrées durant le jeu sont agrafées dans les pages centrales du livret par ordre d’occurrence et peuvent être parcourues à tout moment par les membres de l’administration dans leurs différents rôles, venant ainsi influencer leurs réactions. 17 H E IMATL A N D H E I MATL A ND Livret pour étranger Livret pour étranger Permis Z1 Permis Y2 Administration Centrale et Omnisciente Administration Centrale et Omnisciente Form.2369985764.34 bis Form.2369985764.34 bis H E IMATL A N D H E I MATL A ND Livret pour étranger Livret pour étranger Permis Z2 Permis V Administration Centrale et Omnisciente Form.2369985764.34 bis Administration Centrale et Omnisciente Form.2369985764.34 bis Livrets de jeu. Dans les coulisses de « Bienvenue à Heimatland ! » Test d’un prototype du livret de jeu en développement. Photo© Guillaume Perret 19 Ensuite, le livret intègre différents supports pour garder les traces du parcours dans le jeu : progression temporelle, dettes, bons ou mauvais points selon les critères d’évaluation officiels, etc. Le système de notation est conçu pour être cryptique : les symboles dénotant les différents critères ne sont pas littéraux et la notation même des bons points ou mauvais points se fait à l’envers de ce que le bon sens voudrait (et il n’est jamais dit durant le jeu qu’il s’agit de tels points). Ce qui est inscrit dans le livret représente donc une « boîte noire » pour les participant.e.s. L’idée est de faire à la fois référence à l’opacité du fonctionnement de l’administration pour les usager.e.s et d’offrir un support pour le processus d’interprétation lors du débrief en petits groupes de participant.e.s qui prend place après le jeu et avant la table ronde avec les spécialistes. Finalement, l’ensemble des traces qui s’inscrivent sur le livret durant le jeu sert de support pour le processus d’évaluation lors du ou des passages devant les grand.e.s integrateur.rice.s, déterminant ainsi grandement la suite du parcours pour les participant.e.s et leur destin à Heimatland. Nicolas Yazgi Dramaturge et anthropologue Et en Suisse, comment ça se passe ? Le jeu de l’échelle de l’intégration juridique en Suisse « Plus d'intégration, cela signifie concrètement renforcer notre cohésion sociale. » Robert Cramer, conseiller d’Etat du canton de Genève, 11 décembre 2013. Cinq ans après la déclaration de Robert Cramer, la Suisse introduit un modèle d’intégration graduel plus restrictif avec l’entrée en vigueur de la loi fédérale sur la nationalité (LN) en 2018 et la loi fédérale sur les étranger.e.s et l’intégration (LEI) en 2019. Ce modèle repose sur le principe selon lequel plus il y a de droits rattachés à un statut juridique, plus les exigences en matière d’intégration doivent être élevées. La nouvelle structure de la LEI est basée sur les deux mots clés de la politique d’intégration en Suisse : « encourager et exiger ». L’Etat contribue à l’encouragement de l’intégration notamment dans les domaines de la formation, du travail, de la sécurité sociale, de la santé, de l’aménagement du territoire et des espaces urbains, dans le sport, les médias et la culture. Des mesures d’encouragement spécifiques existent également pour les personnes étrangères ayant des besoins d’intégration particuliers, notamment dans le cadre des programmes d’intégration cantonaux (PIC). L’exigence de l’intégration est quant à elle régulée par plusieurs critères et instruments. Le respect de la sécurité et de l’ordre public, le respect des valeurs de la Constitution, les compétences linguistiques et la participation à la vie économique ou l’acquisition d’une formation sont ainsi pris en compte pour évaluer le niveau d’intégration d’une personne étrangère. Les autorités compétentes peuvent exiger qu’une convention d’intégration soit conclue afin d'indiquer clairement à la personne étrangère ce qui est attendu d’elle. Elles peuvent également formuler des recommandations pour les personnes de l’UE/AELE. 21 Des conséquences juridiques sont rattachées au non-respect des conventions d’intégration : les autorités peuvent retirer ou refuser de prolonger une autorisation de séjour tant qu’une convention d’intégration n’a pas été conclue ou respectée. La convention d’intégration est également prévue pour les personnes admises à titre provisoire (livret F pour des personnes qui n’ont pas obtenu de permis de séjour ou d’établissement mais dont le renvoi est illicite, inexigible ou matériellement impossible). De plus, les autorités compétentes peuvent révoquer un permis d’établissement (livret C valable de manière indéterminée) et le remplacer par un permis de séjour (livret B à renouveler tous les 5 ans de manière générale) si la personne étrangère ne remplit pas les critères d’intégration requis. L’encouragement et les exigences en matière d’intégration sont encadrés par un échange régulier entre les diverses autorités compétentes. Les autorités chargées de l’exécution de la LEI s’assistent mutuellement dans l’accomplissement de leurs tâches, communiquent entre elles des renseignements sur les personnes étrangères et s’accordent, sur demande, le droit de consulter leurs dossiers. L’ouverture d’enquêtes pénales, les jugements de droit civil ou pénal, les changements d’état civil, le versement de prestations de l’aide sociale ou de prestations complémentaires, ainsi que d’autres décisions indiquant l’existence de besoins d’intégration particuliers, sont autant d’informations auxquelles les autorités ont accès pour évaluer le niveau d’intégration d’une personne. Et en Suisse, comment ça se passe ? En résumé, la LEI prévoit un modèle complexe entre exigences et encouragements qui s’apparente à une sorte de jeu de l’échelle. Au cours de leur parcours d’intégration, les personnes étrangères devront se laisser « examiner » à plusieurs reprises sur la base des critères d’intégration établis, ce qui pourra déboucher sur un renouvellement ou une prolongation de leur permis de séjour, mais également donner lieu à sa révocation. Bien que ce modèle d’intégration graduel impose aux autorités de mettre à disposition des structures et des instruments qui soutiennent et encouragent les personnes étrangères dans leur intégration économique et socioculturelle, les exigences s’en trouvent renforcées et légitimées. De manière analogue, « Bienvenue à Heimatland ! » reproduit un système d’autorisations de séjour, d’étapes, d’examens et d’instruments d’intégration qui permet de réfléchir autrement aux différents enjeux, obstacles et contradictions du processus d’intégration tel qu’il est défini par la loi. L’événement met en lumière le jeu de l’échelle auquel sont soumises les personnes étrangères, même nées en Suisse, pour avancer dans leur parcours d’intégration. Stefanie Kurt Professeure à la HES-SO Valais-Wallis, Haute Ecole de Travail Social, Sierre 23 Poursuivre la réflexion • Kurt, Stefanie, Christin Achermann, Lisa Marie Borelli et Luca Pfirter. 2020. The Making of Social Cohesion : A Critique of Migration Law and Practices in Switzerland. nccr on the move, blog. 3 mars. https://nccr-onthemove.ch/blog/ the-making-of-social-cohesion-acritique-of-migration-law-andpractices-in-switzerland/ • Kurt, Stefanie. 2017. Nouvelles exigences en matière d’intégration des étrangers. Plaidoyer 04/2017 du 3 juillet 2017, p. 20-24. https://www.plaidoyer.ch/article/ artikeldetail/nouvelles-exigencesen-matiere-dintegration-desetrangers/ • Loi fédérale sur les étrangers et l’intégration (LEI) du 16 décembre 2005 (Etat le 1er avril 2020), RS 142.20. https://www.fedlex.admin.ch/eli/ cc/2007/758/fr Extrait du livret de jeu. Et en Suisse, comment ça se passe ? « Dis-moi ton permis… » En Suisse, comme dans l’ensemble des pays européens, les parcours de vie des personnes étrangères sont fortement influencés par les différents statuts et permis de séjour qui leur sont attribués. C’est un système administratif complexe qui dicte les possibilités et les obstacles auxquels elles sont confrontées. En effet, nombreux sont les domaines où le permis de séjour joue un rôle essentiel : « Dis-moi ton permis, et je te dirai si tu peux travailler, vivre avec ta famille, choisir ton lieu de vie, te déplacer librement… » Cette hiérarchisation administrative des individus via leurs permis de séjour s’observe dans tous les domaines de la migration (migration de travail, familiale, etc.). Il est possible de l’illustrer à partir de l’exemple des migrations d’asile. Schématiquement, la législation helvétique classe les permis de la façon suivante : au bas de l’échelle se situent les requérant.e.s d’asile (permis N) dont les droits concernant l’accès au marché du travail, au regroupement familial, à la mobilité et à l’aide sociale sont les plus limités – lorsqu’ils ne sont pas tout simplement inexistants. Viennent ensuite les titulaires d’admissions provisoires (permis F et permis « F réfugié.e »), puis les réfugié.e.s statutaires ayant obtenu l’asile (permis « B réfugié.e »). Ces dernier.e.s bénéficient d’une plus grande liberté, qui n’égale cependant pas celle des détenteur.rice.s d’une autorisation d’établissement (permis C), ni celle de la population de nationalité suisse, notamment en ce qui concerne le droit au regroupement familial et la mobilité. Les requérant.e.s d’asile dont la demande a été rejetée n’ont pour leur part pas de statut légal. Ils vivent en attente de leur renvoi, dans des conditions particulièrement difficiles. Au fil du temps passé en Suisse, on observe un « cumul des désavantages », avec des conséquences négatives pour les personnes qui conservent les permis les plus précaires (N et F) durant de longues années. Sans oublier que d’autres facteurs, tels que le sexe, la nationalité ou le canton 25 Poursuivre la réflexion • Berthoud, Carole. 2012. Déqualifiés ! Le potentiel inexploité des migrantes et des migrants en Suisse. Berne : Croix-Rouge suisse. https://edudoc.ch/ record/106417?ln=fr de résidence (qui est attribué par l’administration selon une clé de répartition définie par voie légale au début de la procédure d’asile), viennent parfois creuser encore les inégalités. Comme le souligne Bolzman (2001 : 135), « le processus d’intégration dépend en grande partie de ceux qui définissent les règles du jeu, à savoir les Etats récepteurs ». La volonté des autorités suisses d’encourager la participation sociale et économique des réfugié.e.s tout en restreignant leurs possibilités d’accès aux permis de séjour les plus stables démontre ainsi le paradoxe des politiques menées en matière d’asile et d’intégration. Anne-Laure Bertrand Docteure en démographie, maître-assistante en méthodes quantitatives, MAPS, Université de Neuchâtel • Bertrand, Anne-Laure. 2020. Dans la jungle des permis de séjour : Parcours administratifs et intégration professionnelle des réfugiés en Suisse. Zurich et Genève : Seismo. https://seismoverlag.ch/de/daten/ dans-la-jungle-des-permis-desejour/ • Bolzman, Claudio. 2001. Politiques d’asile et trajectoires sociales des réfugiés : une exclusion programmée. Les cas de la Suisse. Sociologie et Sociétés XXXIII(2), 133-158. https://www.erudit.org/en/journals/ socsoc/2001-v33-n2socsoc730/008315ar.pdf • Fresia, Marion, David Bozzini, et Alice Sala. 2013. Les rouages de l’asile en Suisse : Regards ethnographiques sur une procédure administrative. Neuchâtel : Forum suisse pour l’étude des migrations et de la population. http://doc.rero.ch/record/258654 • ODAE romand. 2015. Permis F : Admission provisoire ou exclusion durable ? Genève : Observatoire romand du droit d’asile et des étrangers. https://odae-romand.ch/projet/ permis-f-admission-provisoire/ Dans les coulisses de « Bienvenue à Heimatland ! » Le ruban de Möbius Le motif que l’on trouve au centre de l’affiche et sur la couverture du livret pour étranger.e.s de Heimatland est ce qu’on appelle un ruban de Möbius, sur lequel un système de quadrillage est inscrit. Lorsque matérialisé en trois dimensions, un tel ruban devient une surface qui n’a qu’une face. Un modèle simple se réalise en opérant une torsion d'un demi-tour à une longue bande de papier puis en collant les deux extrémités, créant ainsi un ruban sans fin. L’image symbolise librement différentes dimensions de l’univers que nous avons développé : l’aspect relativement unidimensionnel de la gestion administrative des migrant.e.s, un système qui tourne sur luimême et qui ne s’arrête jamais (même si des individus peuvent en sortir par la naturalisation ou le renvoi), un système labyrinthique dont on n’a jamais la vue d’ensemble et dans lequel on peut se perdre, les cases dans lesquelles il faut rentrer, la tension entre possible progression et le risque de tourner en rond, etc. Nicolas Yazgi Dramaturge et anthropologue 27 Le ruban de Möbius. Dans les coulisses de « Bienvenue à Heimatland ! » L’administration étatique « Bienvenue à Heimatland ! » reproduit une administration étatique qui régule le séjour des personnes étrangères et contrôle que leur comportement s’adapte aux règles du pays. Qui sont les fonctionnaires que vous avez rencontrés durant votre parcours ? Les fonctionnaires du destin : Ils. elles vous permettent d’avancer dans votre parcours administratif en distribuant les événements auxquels vous devrez faire face. En lançant les dés, ils.elles marquent le temps passé à Heimatland qui sera pris en compte pour évaluer votre niveau d’intégration. Cependant, vous avez peut-être remarqué que leurs dés sont pipés. En fonction du type de permis, le temps s’écoule plus ou moins vite ! Les conseiller.e.s en orientation intégrative : Ils.elles accompagnent le parcours des personnes étrangères en les conseillant et en les informant de ce qui est attendu d’elles. Bien que leur rôle consiste avant tout à encourager l’intégration, ces fonctionnaires sont en contact direct avec le reste de l’administration étatique et communiquent les informations nécessaires pour permettre une évaluation rigoureuse du comportement des migrant.e.s. Les agent.e.s d’intégration : Ils.elles ont pour mission de faire comprendre aux personnes étrangères que l’intégration n’est pas une option mais une exigence. Les autorités de Heimatland estiment mettre beaucoup de moyens à disposition pour favoriser la cohésion sociale, aussi est-il attendu des migrant.e.s qu’ils.elles fassent des efforts pour s’adapter aux normes de leur nouveau pays. Lorsque ces efforts sont jugés insuffisants, les agent.e.s d’intégration sont là pour rappeler à l’ordre les personnes concernées. 29 Les grand.e.s intégrateurs.rices : Sur la base des évaluations transmises par les conseiller.e.s en orientation intégrative et les agent.e.s d’intégration, ces fonctionnaires ont le pouvoir de faire progresser les migrant.e.s vers un statut légal meilleur, ou au contraire de les faire régresser, voire de les renvoyer du pays. Alors que certaines personnes, dont la présence à Heimatland est jugée peu problématique, voire bénéfique, ne rencontreront les grand.e.s intégrateur.rice.s que rarement, voire jamais, durant leur parcours, d’autres, dont la présence est perçue comme moins désirable, seront régulièrement appelées à les voir. Les agent.e.s de sécurité : Ils.elles assurent l’ordre et se chargent de placer les personnes à renvoyer en détention provisoire. Bien que la discrimination soit sanctionnée à Heimatland, il leur arrive d’être influencé.e.s par certaines caractéristiques physiques lors des contrôles d’identité... Laure Sandoz Docteure en anthropologie, chercheuse postdoctorante à l’Institut de géographie et au nccr - on the move Dans les coulisses de « Bienvenue à Heimatland ! » Préparer et développer les rôles Notre équipe était formée d’acteur.rice.s qui n’étaient pas familier.e.s avec le thème de la gestion migratoire. Elles et ils ont donc fourni un travail conséquent pour se l’approprier afin de pouvoir incarner leurs rôles et improviser autour des situations du jeu. L’équipe était constituée à deux tiers d’étudiant.e.s intéressé.e.s par le théâtre (et/ou l’improvisation) et à un tiers de comédien.ne.s professionnel.le.s qui avaient comme mission, en plus de leur rôles, de coacher les étudiant.e.s individuellement afin d’enrichir leur expérience. Contrairement à un processus théâtral classique, il n’était pas possible de seulement répéter entre nous : le dispositif ne peut fonctionner pleinement qu’avec un public extérieur. Il a été dès lors essentiel pour nous de déployer des « crash tests » grandeur nature, suivis de débriefs collectifs : d’abord pour poser le squelette du système, tester les flux et repérer les problèmes, puis pour enrichir et affiner l’expérience finalement proposée au public. Ces tests furent riches, non seulement pour nourrir le travail en cours et pratiquer les improvisations avec un public varié, mais aussi pour les participant.e.s et ce de manière très différente pour les un.e.s et les autres, selon si, par exemple, il s’agissait de spécialistes du thème, parfois renvoyé.e.s à leurs « raccourcis interprétatifs » automatisés, ou d’un public externe qui prenait conscience de certaines choses ou revisitait des expériences personnelles. 31 Afin de libérer l’esprit des actrices et acteurs pour les improvisations, nous avons mis sur pied un système de « partition performative » pour chaque rôle, soit une séquence d’actions servant de colonne vertébrale aux interactions durant le jeu. Par exemple, pour un.e agent.e d’intégration : accueillir le.la migrant.e à son bureau, lui demander ce qui l’amène, prendre son livret, repérer la dernière situation agrafée, regarder dans le classeur les indications relatives à celle-ci comme si c’était la chose la plus naturelle du monde, etc. Ensuite, nous avons créé le classeur qui rassemble le matériel de référence, notamment toutes les situations et les éléments relatifs à inclure lors des improvisation (p.ex : mettre en garde à propos de x, sanctionner y, mettre un mauvais point selon critère z, etc.). Comme les fonctionnaires eux-mêmes le disent à qui les questionne sur le classeur durant le jeu : « il permet d’éviter l’erreur humaine et de traiter tout le monde équitablement ». Le classeur contient aussi un mémo général, le rappel de la partition performative pour chaque rôle, des « trucs » pour gérer certaines situations délicates, etc. Il est utilisé comme un appui de jeu central durant les interactions. Les acteur.rice.s ont en parallèle travaillé la caractérisation de leur personnage (ce qui l’individualise et le fait sortir de sa fonction professionnelle générique) et leur « vie de bureau ». Cette vie de bureau prend parfois le premier plan pour venir rappeler aux participant.e.s-migrant.e.s qu’ils.elles ne sont pas nécessairement la priorité pour leurs interlocuteur. rice.s, voire qu’ils.elles peuvent déranger. Dans les coulisses de « Bienvenue à Heimatland ! » Il a fallu travailler des éléments permettant d’établir le « feeling » entre les membres de l’administration et le public, important car susceptible d’influencer le déroulement des interactions. Pour chaque situation, l’établissement du feeling, plus ou moins positif ou négatif, se base sur la « carrière administrative » des participant.e.s (voir encart livret dans le jeu), sur la catégorie de leur permis de séjour, sur la situation elle-même, sur le comportement des participant.e.s (poli ? coopératif ? défiant ? provocateur ? etc.), mais aussi sur des critères de « racisme invisible » que nous avons choisi de baser sur des critères tout aussi arbitraires que ceux du racisme réel, mais non essentialisables (par exemple longueur des cheveux, type de chaussures portées, etc.). Ce travail de préparation a été collectivement très enrichissant (y compris pour le duo de conception grâce aux questions et suggestions de l’équipe), mais n’enlève pas une certaine vulnérabilité au processus, que nous assumons entièrement : les situations restent vécues et improvisées en temps réel avec un public qui réagit en direct – et de manière très diversifiée. Si parfois la bulle fictionnelle se fragilise voire se dissout en cours d’exercice, ce n’est pas un problème : cela rappelle simplement que derrière l’immersion, il y a un dispositif expérimental au travers duquel nous avons choisi, acteur.rice.s et public, de « jouer ensemble à » pour un temps, afin d’ensuite réfléchir collectivement d’une manière renouvelée. Nicolas Yazgi Dramaturge et anthropologue 33 H E I M A T L A N D bienvenue Affiche de bienvenue placée à l’entrée des services de l’administration durant le jeu. Et en Suisse, comment ça se passe ? Bureaucrates anonymes ou êtres humains agissant en « leur âme et conscience » ? Au quotidien, une multitude de collaborateur.rice.s agissent au nom d’institutions telles que le Secrétariat d’État aux migrations (SEM) ou « le service des migrations » des cantons. Même s’ils.elles restent souvent dans l’ombre de leurs offices, les membres des administrations de la migration sont des acteur.rice.s particulièrement important.e.s, car leurs actions traduisent des normes abstraites en des décisions avec des effets concrets. La somme de tous ces actes définit la réalité pratique du droit et de la politique migratoire. Si les tâches des personnes administrant la migration consistent aussi à organiser le recrutement de la main-d’œuvre étrangère ou à promouvoir l’émigration, l’administration de la migration consiste en bonne partie à décider qui peut légalement entrer et séjourner sur le territoire national avec quels droits et quelles obligations. Dans l’administration de la migration, les autorités disposent d’un pouvoir discrétionnaire important. D’un côté, le pouvoir discrétionnaire permet de tenir compte des spécificités individuelles. De l’autre, il implique le risque d’aboutir à des décisions inégales si, par exemple, une décision d’accorder une prestation est influencée par des stéréotypes racistes ou sexistes. De manière plus générale, le pouvoir discrétionnaire démontre que les actions des personnes chargées d’appliquer une loi ne sont pas le reflet direct de celle-ci. Les règles juridiques ne déterminent donc pas directement l’action, mais établissent un cadre qui oriente, qui offre des possibilités et qui définit des limites aux actions des agent.e.s de terrain. 35 Contrairement à l’image dépersonnalisée des bureaucraties et des personnes y travaillant, les fonctionnaires sont des sujets individuels avec des caractéristiques, des émotions et des histoires. Ce constat banal d’un point de vue sociologique a pourtant des implications importantes. En effet, ces caractéristiques jouent un rôle dans leur travail quotidien, notamment à travers leur pouvoir d’appréciation. En raison des marges de manœuvre prévues par la loi, les bureaucrates peuvent, et souvent doivent, faire recours à des interprétations subjectives et à leurs expériences. Cela influe sur leur manière de poser des questions et de pondérer des éléments, sur leur posture visà-vis de quelqu’un, et donc sur des décisions et des actes qui in fine constituent ce qu’est la politique migratoire en pratique. Du côté des personnes migrantes, il y a également une grande diversité de situations, de parcours, d’intérêts et de manières de faire. Même si leurs demandes peuvent être classées dans des catégories légales (asile, séjour, nationalité), chacune est liée à un individu particulier. Le travail des fonctionnaires des migrations consiste à traiter administrativement des cas spécifiques afin de les classer en des termes qui entrent dans le cadre donné par la loi – des motifs crédibles ou pas, des personnes intégrées ou non, des connaissances suffisantes ou non, des intérêts privés suffisamment importants ou pas. Si des normes, des directives et la jurisprudence orientent ce travail, les personnes administrant la migration doivent prendre leurs décisions sur la base d’informations forcément limitées et réductrices. Ces fonctionnaires sont confrontés à des fragments d’histoires de vie présentés dans des conditions particulières (telles qu’une audition d’asile ou un entretien avec une commission de naturalisation), avec des formulaires et des dossiers qui ne reflètent que la partie d’une vie qui est visible et gérée administrativement. Et en Suisse, comment ça se passe ? Le type d’interaction influe aussi sur le rapport entre les personnes et l’idée qu’un individu se fait de l’autre. On peut distinguer les interactions directes, typiquement « aux guichets » ou dans le cadre des auditions d’une part, et les interactions indirectes ou médiatisées qui se passent par écrit d’autre part. Selon la perception qu’un.e agent.e se fait d’un individu au travers d’une interaction, des facteurs tels que le genre, le statut social, l’apparence physique ou encore la manière de s’habiller peuvent jouer un rôle et vont contribuer à la construction de la réalité et des faits de ce qui devient un « cas ». Christin Achermann Professeure en migration, droit et société à l’Université de Neuchâtel 37 Poursuivre la réflexion • Achermann, Christin. 2019. « La Suisse exclusive : quelles pratiques à l’égard des personnes étrangères ‘indésirables’ ? » In Chroniques universitaires 2019, 44-56. Université de Neuchâtel. Version raccourcie et légèrement adaptée de l’article suivant : Achermann, Christin. 2018. Bureaucrates anonymes ou êtres humains agissant « en leur âme et conscience » ? Fonctionnaires dans les administrations de la migration. Terra Cognita 32 : 18-21. • Achermann, Christin, Lisa Marie Borrelli, Stefanie Kurt, Doris Niragire Nirere et Luca Pfirter. 2021. L’intrication croissante du contrôle des migrations et de l’aide sociale. Solidarité sans frontières. 3 mars. https://www.sosf.ch/fr/sujets/ politique-migratoire/informationsarticles/die-zunehmendeverschraenkung-vonmigrationskontrolle-und-sozialhilfe. html • Borrelli, Lisa Marie, Stefanie Kurt, Christin Achermann et Luca Pfirter. 2021. « Armut ist kein Verbrechen » – (Un)bedingte Wohlfahrt in der Schweizer Rechtsprechung. Décodage (blog), SAGW. 8 avril. https://www.sagw.ch/sagw/ aktuell/blog/details/news/armutist-kein-verbrechen-unbedingtewohlfahrt-in-der-schweizerrechtsprechung/ • Kurt, Stefanie, Christin Achermann, Lisa Marie Borrelli, and Luca Pfirter. 2020. Zuwanderungsund Aufenthaltssteuerung via Sozialhilfe ?. nccr - on the move, blog. 29 janvier. https://nccr-onthemove.ch/blog/ zuwanderungs-undaufenthaltssteuerung-viasozialhilfe/ Et en Suisse, comment ça se passe ? Distinguer les réfugié.e.s des migrant.e.s : pratique arbitraire ou simple application de la loi ? En Suisse, la procédure d’asile est un instrument central des politiques migratoires. Elle permet d’ordonner la mobilité des personnes précarisées et non européennes, en produisant une distinction entre « réfugié.e.s » et « migrant.e.s ». Les premier.e.s se déplaceraient sous contrainte pour des motifs uniquement politiques (persécutions ou guerres) tandis que la mobilité des seconds relèverait avant tout d’une stratégie économique. Alors que la recherche en sciences sociales a depuis longtemps montré la difficulté à démêler les causes politiques des motivations socio-économiques dans les dynamiques migratoires (Monsutti 2004), comment les autorités suisses distinguent-elles concrètement les « réfugié.e.s » des « migrant.e.s » ? Les décisions prises par le Secrétariat d’Etat aux Migrations (SEM) relèvent-elles du seul pouvoir discrétionnaire de ses agent.e.s ? Ou bien les agent.e.s ne font-ils.elles qu’appliquer la loi suisse sur l’asile de manière neutre et objective ? De récentes recherches réalisées sur les pratiques d’octroi du statut de réfugié en Suisse apportent un éclairage précieux sur ces questions. Elles montrent que les décisions ne sont ni arbitraires ni objectives, mais avant tout « situées », c’est-à-dire toujours liées au contexte politique, historique, administratif et social dans lesquelles elles sont prises. Si le cadre juridique auquel les décisions se réfèrent paraît clair, celui-ci fait, en effet, toujours l’objet d’interprétations. Or, ces interprétations varient en fonction d’enjeux de politique extérieure ou intérieure et d’intérêts 39 économiques. Certains travaux ont, par exemple, montré comment la Suisse a, pour des raisons essentiellement idéologiques, accueilli les Hongrois.e.s de manière extrêmement généreuse en 1956, alors même que beaucoup fuyaient leur pays pour des raisons autant économiques que politiques (Piguet 2019). Si leurs demandes d’asile avaient été étudiées par le SEM aujourd’hui, elles auraient fort probablement été rejetées. Depuis la fin de la guerre froide, la politique d’asile suisse favorise une interprétation au contraire très stricte du statut de réfugié.e. Les études réalisées auprès du SEM montrent l’existence d’un ethos institutionnel marqué par une attitude très légaliste et une logique du soupçon, qui tend à mettre systématiquement en doute la parole des requérant.e.s (Affolter 2021 ; Miaz 2017). Si les agents disposent d’un certain pouvoir discrétionnaire, celui-ci reste donc influencé par les orientations politiques plus larges du gouvernement. Il est également structuré par la jurisprudence interne au SEM ainsi qu’un ensemble de convictions et de « normes implicites » (telles que des stéréotypes sur les motivations des requérant.e.s selon leur nationalité) auxquelles les agent.e.s sont socialisé.e.s. Et en Suisse, comment ça se passe ? Mais le SEM n’est pas le seul à décider de l’avenir des requérant.e.s. L’issue d’une procédure implique en réalité un grand nombre d’acteur.rice.s : interprètes, médecins, psychologues, tous et toutes jouent un rôle dans la mise en forme du récit de la personne requérante et dans les chances que son récit aura de convaincre le SEM (Fresia & al 2013). Les œuvres d’entraide, qui fournissent un accompagnement juridique aux requérant.e.s, opèrent par exemple elles-mêmes un premier « tri » entre ceux et celles qu’elles décideront d’aider, et les autres dont les requêtes sont jugées comme ayant peu de chances d’aboutir. Elles peuvent soit renforcer les cadres normatifs en vigueur, soit au contraire, faire évoluer la jurisprudence vers plus d’ouverture lorsqu’elles décident de défendre un cas a priori moins solide (Rey 2013). Mais c’est aussi et surtout en amont de la frontière helvétique que se déploie un ensemble plus vaste de dispositifs de tri entre réfugié.e.s estimé.e.s « désirables » et « indésirables » : qu’il s’agisse des politiques de délivrance de visas très restrictives, des règlements Dublin, ou des pratiques de refoulement, de mise en camps et de détention aux frontières sud et est de l’Europe, toutes ces mesures permettent de garder à distance les « indésirables », tout en filtrant ceux et celles qui pourront poursuivre leur chemin. Loin d’être indépendantes du système d’asile suisse, ces pratiques, qui se déploient loin des regards, doivent être appréhendées comme lui étant intrinsèquement lié. Marion Fresia Professeure à l’Institut d’ethnologie, Université de Neuchâtel 41 Poursuivre la réflexion • Affolter, L. 2021. Asylum matters. On the frontline of administrative decision-making. Palgrave socio-legal studies book series. • Fresia, M. Bozzini et A.Sala. 2013. Les rouages de l’asile en Suisse : regards ethnographiques sur une procédure administrative. Etude SFM 62. http://doc.rero.ch/record/258654 • Miaz, J. 2017. From the Law to the Decision : The Social and Legal Conditions of Asylum Adjudication in Switzerland, European Policy Analysis Vol 3, n°2. • Monsutti, A. 2004. Guerres et migrations : réseaux sociaux et stratégies économiques des Hazaras d’Afghanistan. Institut d’ethnologie de Neuchâtel / Maison des sciences de l’Homme. • Rey, R. 2013. « Humainement je vous comprends mais juridiquement je ne peux rien faire ». Etude SFM 62. http://doc.rero.ch/record/258654 • Piguet, E. 2019. Asile et réfugiés. Repenser la protection. PPRU, Coll. Savoirs suisses. Et en Suisse, comment ça se passe ? L’asile en marges : les requérant.e.s d’asile face à la gestion migratoire Toute personne qui demande l’asile, en Suisse ou ailleurs en Europe, passe par une procédure juridique visant à reconnaître la légitimité de sa demande, sa qualité de réfugié.e, son besoin de protection, et à lui attribuer ou non un statut qui lui permettra de séjourner légalement sur le territoire national. Au cours de cette procédure, les requérant.e.s se retrouvent confronté.e.s à la gouvernance de l’asile, un ensemble complexe de textes légaux, d’acteurs et d’actrices, de pratiques institutionnelles et de technologies qui visent à réguler leur admission sur le territoire – en tant que sujets légaux –, ainsi que leur présence physique – en tant que corps à prendre en charge. Avec cette double condition, c’est aux marges de l’État que se retrouvent demandeurs et demandeuses d’asile en Suisse. Trois dimensions, en tout cas, caractérisent ces marges. La première dimension est temporelle. La demande ouvre une période d’attente indéterminée, un espace-temps pendant lequel la personne reste et vit sur le territoire national en attendant une décision des autorités. Ce temps est souvent vécu comme arrêté, rempli d’incertitudes, de doutes et de peurs, mais il est aussi investi d’espoirs, de désirs et de projets nouveaux et fait l’objet de multiples négociations et réappropriations. 43 « La vie en foyer est très dure, mais le plus dur c’est qu’on ne sait pas quand on va recevoir une décision ou être renvoyés. […] Toutes les semaines, on voit des gens qui partent, et on se demande quand ce sera notre tour. Pourquoi nous traitentils ainsi ? On a mis en danger nos vies. Ça, ce n’est pas l’humanité qu’on nous a promise. » T., requérant d’asile, 2016. La deuxième dimension est spatiale. La marginalité des personnes demandeuses d’asile renvoie aux frontières externes et internes de l’État. En effet, la procédure d’asile constitue un dispositif d’inclusion et d’exclusion territoriales : une réponse négative à la demande d’asile, ou une demande qui n’a pas été effectuée au « bon endroit », selon le système européen de Dublin, peut entraîner l’exclusion du territoire et le renvoi. Mais elle est également le théâtre de pratiques régulatrices qui assignent les personnes dans des lieux spécifiques – centres fédéraux, foyers, administrations spécialisées, lieux de détention, etc. Les requérant.e.s sont ainsi soumis.e.s à toute une série de techniques de gouvernement visant à assurer un contrôle sur leur présence. La marge renvoie donc ici à une mise à l’écart autant spatiale que sociale et à une précarisation des vies, mais elle peut aussi permettre l’émergence de nouvelles formes d’arrangement, de socialisation, de solidarités et de revendications. Et en Suisse, comment ça se passe ? « Aujourd'hui, nous sommes en Suisse, “ terre d'asile ” et nous sommes reconnaissants à ce pays et à ses habitants de nous accueillir. Pourtant ici aussi, on nous enferme. Nous vivons dans des abris PC, sous terre, entassés, sans fenêtres, sans air, sans soleil, pour certains depuis plus d'une année. […] Nous sommes des êtres humains. Nous avons besoin d'air pur, de soleil, comme tout le monde. De dignité aussi. Nous ne voulons pas continuer à être enterrés, cachés, tenus à l'écart de notre société d'accueil. » Mouvement Stop Bunkers (habitants des abris de la protection civile à Genève), Manifeste « Vous ne nous connaissez pas ? C’est normal ! Nous vivons sous terre », janvier 2015. Enfin, la troisième dimension est juridique puisque les personnes demandeuses d’asile n’occupent ni une position légale durable d’appartenance ni une position illégale de non-appartenance à l’État-nation. La procédure d’asile est alors moment de jugement, moment où les personnes sont progressivement placées dans des catégories, suivant un processus de décision mettant en jeu de nombreux acteurs – agent.e.s de l’administration, juges, spécialistes pays, conseiller.e.s juridiques, médecins, etc. Tout au long de ce processus, les récits et les corps sont analysés et interprétés, du savoir sur les personnes est produit pour rendre l’inconnu connu – juridiquement parlant – et prendre une décision sur la demande. La marge est ici celle qui s’ouvre entre le cadre légal et son application, entre l’abstraction des définitions juridiques et la complexité des vies. Il s’agit d’un processus composite et dialogique qui offre des espaces de négociation, des dilemmes et des choix, pour tou.te.s les acteur.rice.s impliqué.e.s. 45 « Vous vivez dans un autre monde. Quand je dis un autre monde, j’entends un monde nouveau, qui a été construit sous terre. Ils sélectionnent une catégorie de personnes pour y vivre. Ils imposent toute une série de règles pour les gouverner. Ce n’est pas juste sous terre. C’est un sous-système, des sous-lois. » M., requérant d’asile vivant dans un abri de la protection civile, 2015. C’est dans ces marges que prennent place le vécu et les expériences quotidiennes des requérant.e.s d’asile. Elles sont certes constamment investies par l’État au travers de différents dispositifs de gouvernement visant à réguler, trier et contrôler la population des demandeur.euse.s d’asile. Mais elles sont aussi le théâtre d’interactions entre ces dernier.e.s, différentes autorités et des acteur.rice.s non gouvernementaux, avec chacun leurs rôles, leurs intérêts et leurs objectifs. Chacune de ces marges représente ainsi un champ de contestation et de négociation, que ce soit pour contester les décisions, échapper au renvoi ou améliorer les conditions de vie. Il ne faut cependant pas se méprendre : si la gestion migratoire n’est pas implacable et ouvre toute une série de marges de négociation, elle reste un champ dans lequel les rapports de pouvoir sont hautement asymétriques. Non seulement parce que les autorités possèdent un large pouvoir d’appréciation dans l’évaluation des cas individuels, mais surtout parce qu’elles disposent du pouvoir de coercition pour faire appliquer leurs décisions. Raphaël Rey Coordinateur de l'Observatoire romand du droit d'asile et des étranger.e.s Et en Suisse, comment ça se passe ? Photo© Guillaume Perret 47 Poursuivre la réflexion • Affolter, Laura. 2021. Asylum Matters : On the Front Line of Administrative Decision-Making. Palgrave Macmillan. https://link.springer.com/book/1 0.1007%2F978-3-030-61512-3 • Akoka, Karen. 2020. L’asile et l’exil : Une histoire de la distinction réfugiés/migrants. Paris : La Découverte. • Brina, Aldo. 2020. Chroniques de l’asile. Labor & Fides. • de Coulon, Giada. 2019. L'illégalité régulière au quotidien : Ethnographie du régime de l'aide d'urgence en Suisse. Lausanne : Antipodes. • D’Halluin-Mabillod, Estelle. 2012. Les épreuves de l’asile : associations et réfugiés face aux politiques du soupçon. Paris : EHESS. • Eule, Tobias, Lisa Marie Borrelli, Annika Lindberg et Anna Wyss. 2019. Migrants before the Law : contested migration control in Europe. Palgrave Macmillan. • Kobelinsky, Carolina. 2010. L’accueil des demandeurs d’asile : une ethnographie de l’attente. Paris : Le Cygne. • Revue Vivre Ensemble (www.asile.ch) • ODAE romand (www.odae-romand.ch) Dans les coulisses de « Bienvenue à Heimatland ! » Critères d’intégration et règles implicites A Heimatland, la cohésion sociale est une valeur importante, et il est attendu des personnes étrangères qu’elles s’adaptent au plus vite aux règles explicites et implicites de leur nouveau foyer. Comme le dit la devise nationale : « Tous comme les autres, chacun pour soi ». Pour cela, les autorités assurent un suivi rigoureux de chaque personne nouvellement arrivée et peuvent les sanctionner lorsqu’elles estiment que leurs efforts d’intégration ne sont pas suffisants. Avez-vous remarqué qu’après chaque interaction avec un membre de l’administration, un point positif ou négatif était ajouté à votre livret ? L’évaluation de l’intégration par l’administration étatique repose sur cinq critères auxquels les événements que vous avez pu rencontrer durant votre parcours de migrant.e se réfèrent. Premièrement, les personnes étrangères doivent être indépendantes financièrement. Bien que Heimatland accorde facilement des aides étatiques et des prêts pour assurer à chacun un niveau de vie décent, l’intégration n’est jugée réussie que lorsqu’une personne est capable de ne plus dépendre financièrement de tels soutiens. Deuxièmement, la maîtrise de la langue est essentielle pour communiquer, travailler et se mêler à la population locale. Troisièmement, le travail est central à Heimatland et toute personne étrangère doit démontrer sa bonne volonté à s’engager professionnellement et se former. Quatrièmement, l’intégration ne peut être réussie que lorsqu’une personne respecte scrupuleusement la loi. Finalement, le respect des valeurs, us et coutumes de Heimatland est crucial pour toute personne qui souhaite s’installer à long terme dans le pays et un jour, peut-être, obtenir la nationalité. 49 L’évaluation de ces critères détermine les possibilités des étranger.e.s de rester dans le pays et d’évoluer vers un statut légal plus stable. De plus, les autorités disposent d’une certaine marge de manœuvre pour appliquer la loi. Il leur arrive donc parfois d’être influencées par des facteurs sociaux, culturels ou émotionnels qui permettront à certaines personnes d’être plus ou moins bien traitées lors de leurs interactions avec le personnel de l’administration. Vous l’avez peut-être remarqué si vous avez joliment décoré votre livret dans l’« Espace pour le développement de votre potentiel intégratoire » ! Laure Sandoz Docteure en anthropologie, chercheuse postdoctorante à l’Institut de géographie et au nccr - on the move Extrait du livret de jeu. Et en Suisse, comment ça se passe ? Une intégration « réussie » ? La fabrique de l’intégration En Suisse, l’obtention de la nationalité est régie par la Loi sur la nationalité (LN) depuis 1952. De manière très résumée, on peut dire que d’une période où l’obtention de la nationalité était conçue comme moyen de faciliter l’intégration des personnes étrangères en Suisse, l’on est passé à une conception où la personne doit d’abord faire la preuve qu’elle est intégrée pour obtenir la nationalité. Dans la dernière formulation de la loi (entrée en vigueur en 2018), les conditions de naturalisation sont fixées par deux articles. L’article 11 indique l’intégration comme l’un des critères matériels d’obtention de la nationalité ; l’article 12 spécifie les critères d’intégration : « une intégration réussie se manifeste en particulier par : (a) le respect de la sécurité et de l’ordre publics ; (b) le respect des valeurs de la Constitution ; (c) l’aptitude à communiquer au quotidien dans une langue nationale, à l’oral et à l’écrit ; (d) la participation à la vie économique ou l’acquisition d’une formation ; et (e) l’encouragement et le soutien de l’intégration du conjoint, du partenaire enregistré ou des enfants mineurs sur lesquels est exercée l’autorité parentale ». On peut donc noter que la maîtrise de la langue joue un rôle important, mais que ni la religion, ni la participation aux activités sociales et culturelles ne figurent explicitement parmi les critères d’intégration définis par cet article de loi. Au niveau fédéral, « l’intégration réussie » est donc légalement définie avec des critères stricts ; les cantons et les communes ont cependant une certaine marge de liberté dans l’application de la loi. 51 L’analyse approfondie d’une dizaine de dossiers de demandes de naturalisation dans le canton de Neuchâtel nous a permis de constater que le canton veille avant tout à ce que la personne soit économiquement indépendante, qu’elle se soit acquittée de ses impôts, et qu’elle n’ait pas fait l’objet de signalements suite à des infractions. Dans certains cas, des personnes ont été jugées peu intégrées parce qu’elles ont fréquemment changé d’emploi ou connu des périodes de chômage, qu’elles ont des impôts impayés, ou encore qu’elles sont « connues de la police » – par exemple pour des problèmes liés à la circulation routière ou de voisinage, parce que leurs épouses portent le voile, qu’elles ne parlent pas assez bien la langue du canton (même si elles ont appris une autre langue nationale). Par ailleurs, la procédure d’obtention de la nationalité prévoit que des personnes de l’entourage soient consultées pour juger de l’intégration ; dans notre recherche, des ami.e.s, des animateur. rice.s, des voisin.e.s estiment que la personne est « intégrée » si elle est sympathique, polie, ponctuelle, si elle se comporte « comme un.e Suisse », etc. Enfin, à Neuchâtel, des commissions communales examinent les candidat.e.s à la naturalisation avant de donner un préavis ; elles essayent ainsi de voir si la personne gagne sa vie, paie ses impôts, fait des efforts et se donne de la peine ; des critères de sympathie ou d’affinité entrent aussi en ligne de compte. Et en Suisse, comment ça se passe ? Les procédures de naturalisation sont longues (entre 18 mois pour une procédure simplifiée et 10 ans pour un dossier compliqué) et coûteuses (à Neuchâtel au minimum 1600 CHF pour une personne adulte). Pourtant les personnes s’engagent dans cette procédure, et ce pour des raisons différentes. Certaines, nées en Suisse ou immigrées depuis longtemps, ont accompli leur formation en Suisse, y travaillent, y ont leurs ami.e.s, y ont fondé une famille ; elles se « sentent » suisses, et souhaitent la nationalité pour valider ce sentiment d’appartenance. D’autres désirent participer à la vie civique – voter, pouvoir contribuer à un pays qui leur a « beaucoup donné ». D’autres encore visent à stabiliser leur situation, voyager ou travailler plus facilement, etc. Toutefois, dans certains cas complexes, les étapes de la procédure se mêlent à la trajectoire de vie, et l’exigence de faire preuve d’une intégration « réussie » a mis les personnes en difficultés financières ou sociales ; une personne nous a ainsi dit que la procédure l’avait « désintégrée »… 53 Tania Zittoun Professeure à l'Institut de psychologie et éducation, Université de Neuchâtel Pascal Mahon Professeur de droit constitutionnel suisse et comparé, Université de Neuchâtel Anne Lavanchy Professeure à la Haute école de travail social de Genève Flora Di Donato Professeure de philosophie du droit à l’Université de Naples Federico II Poursuivre la réflexion • Di Donato, F., Garros, E., Lavanchy, A., Mahon, P., & Zittoun, T. 2020. La fabrique de l’intégration. Lausanne : Antipodes. https://www.antipodes.ch/ produit/la-fabrique-delintegration/ • Labarthe, G. 2020. Les faiseurs de Suisses 2.0 – Face aux fonctionnaires, les candidats à la naturalisation peinent à faire valoir leurs parcours personnels. La Liberté, Octobre 27. https://www.reiso.org/articles/ themes/migrations/361integration-des-etrangers-leschoix-de-neuchatel • Emission radio en ligne : « L'intégration en Suisse ». RTS, Tribu. 12.01.2021, https://www.rts.ch/play/radio/ tribu/audio/lintegration-ensuisse?id=11868853 Et en Suisse, comment ça se passe ? La discrimination en Suisse : résultats d’une étude expérimentale sur l’embauche des jeunes descendant.e.s d’immigré.e.s Au-delà des différences de permis de séjour qui contribuent à créer des hiérarchies de droits entre personnes étrangères, la discrimination demeure un problème bien réel en Suisse. Fondée sur l’attribution de caractéristiques collectives considérées comme innées et statiques, la discrimination peut être basée sur divers facteurs tels que la nationalité, l’origine ethnique ou le sexe. Comme le montre la recherche scientifique, elle affecte non seulement les personnes immigrées appartenant à des groupes marginalisés, mais tend également à se perpétuer au fil des générations, favorisant ainsi un cumul des désavantages sur le long terme. Consciente de cet enjeu, une équipe du pôle de recherche nccr – on the move au sein de l’Université de Neuchâtel a mené une étude expérimentale sur la discrimination à l’embauche. L’expérience consiste à répondre à des offres d’emploi publiques dans le secteur de la vente et de l’électricité par l’envoi de candidatures fictives de citoyen.ne.s suisses présentant des qualifications identiques mais différant au niveau du pays d’origine des parents. L’analyse mesure la discrimination en comparant le traitement des candidatures des personnes nées de parents suisses à celui réservé aux dossiers de citoyen.ne.s naturalisé.e.s descendant.e.s d’immigré.e.s turcs, kosovars, camerounais, français (en Suisse romande) et allemands (en Suisse alémanique). Concrètement, l’équipe de recherche observe si ces candidat.e.s sont invité.e.s à un entretien d’embauche et calcule l’écart entre le nombre de dossiers que les candidat.e.s d’origine immigrée doivent envoyer pour être convoqué.e.s à un entretien en comparaison à leurs congénères de parents suisses. 55 Les résultats montrent que les probabilités d’être confronté à la discrimination varient entre les quatre groupes d’origine testés, faisant apparaître une forme de hiérarchie selon l’origine des parents : les Suisse.sse.s d’origine kosovare sont les plus exposé.e.s à la discrimination (ils.elles doivent soumettre environ 40 % plus de candidatures que les candidat.e.s de parents suisses pour être invité.e.s à un entretien d’embauche). Viennent ensuite les enfants d’immigré.e.s camerounais.e.s (30 % de candidatures en plus), suivis par les citoyen.ne.s suisses originaires de France et d’Allemagne (environ 20 % de dossiers en plus). Enfin, les Suisse.sse.s d’origine turque ont aussi moins de chances d’être invité.e.s à un entretien d’embauche que les candidat.e.s d’origine suisse (15 % de candidatures en plus), mais l’écart entre les deux n’est pas statistiquement significatif. De plus, l’ampleur de la discrimination varie d’une profession à l’autre. Elle est plus forte dans le secteur de la vente, où les contacts avec les client.e.s sont un élément constitutif du métier. Enfin, la discrimination à l’embauche est supérieure dans les zones rurales par rapport aux zones urbaines, mais elle est clairement une réalité dans les deux régions linguistiques, où l’on trouve une hiérarchie similaire. Au-delà de ces différences, les résultats de l’étude mettent en évidence une réalité encore peu débattue dans la société suisse : des inégalités de traitement affectent les jeunes suisses d’origine immigrée sur le marché du travail, même lorsqu’ils.elles sont détenteur.rice.s du même passeport et des mêmes qualifications linguistiques, scolaires et professionnelles que leurs contemporain.e.s d’origine suisse. Leur accès à un emploi Et en Suisse, comment ça se passe ? se trouve freiné par un traitement discriminatoire directement imputable à l’origine étrangère de leurs parents. Ces résultats mettent en évidence un cumul des désavantages pour les personnes issues de la migration sur le marché du travail suisse. Outre les discriminations à l’embauche fondées sur la nationalité ou le titre de séjour, des inégalités de traitement affectent les citoyen.ne.s suisses issu.e.s de la migration parce qu’ils.elles portent le nom ou la couleur de peau de leurs ascendant.e.s. Robin Stünzi Docteur en sciences sociales, responsable scientifique au nccr – on the move Pour certains permis, dans le jeu, les dés étaient pipés. Si l’égalité devant la loi est un principe, dans la réalité pratique les avantages ou les désavantages peuvent se cumuler. Photo © Guillaume Perret 57 Poursuivre la réflexion • Dahinden, Janine et Martine Schaer. 2020. Le double paradoxe des inégalités degenre en Suisse : la production des inégalités dans le domaine des migrations. nccr - on the move, blog. 11 juin. https://nccr-onthemove.ch/blog/ le-double-paradoxe-desinegalites-de-genre-en-suisse-laproduction-des-inegalites-dansle-domaine-des-migrations/ • Lavanchy, Anne. 2019. Racisme et racialisation – mettre en mots la discrimination raciale. nccr - on the move, blog. 28 mars. https://nccr-onthemove.ch/blog/ racisme-et-racialisation-mettreen-mots-la-discriminationraciale/ • Fibbi, Rosita. 2019. Minorités visibles. nccr - on the move, blog. 24 septembre. https://nccr-onthemove.ch/blog/ minorites-visibles/ • Emission Diversité du 01.12.2019, « La discrimination : un mythe ou une réalité méconnue ? », https://latele.ch/emissions/ diversite/diversite-s-2019-e-10? • Indicateur statistique sur la discrimination développé par l’équipe de recherche du nccr – on the move : https://nccr-onthemove.ch/ indicators/lessuisse%c2%b7esse%c2%b7sdorigine-immigree-sont-ils-ellesdiscrimine%c2%b7e%c2%b7s/ Photo © Guillaume Perret 59 Pour en savoir plus sur la migration en Suisse Site web du nccr - on the move www.nccr-onthemove.ch/ Blog du nccr - on the move https://nccr-onthemove.ch/blog/ Site web du Forum suisse pour l'étude des migrations et de la population www.unine.ch/sfm/sfm Indicateurs statistiques sur la mobilité et la migration en Suisse https://nccr-onthemove.ch/indicators/ Clips vidéo « Au-delà des idées pré-conçues » https://nccr-onthemove.ch/indicators/audela-des-idees-preconcues/ Partenaires impliqués « Bienvenue à Heimatland ! » est un événement dans l'esprit du Théâtre de la Connaissance entre arts de la scène et recherche scientifique visant à ouvrir un espace de réflexion autour du thème de la migration en Suisse et à déployer cet espace au-delà de l’université. Inspiré des recherches réalisées dans le cadre du nccr - on the move et de la Maison d’analyse des processus sociaux (MAPS) à l’Université de Neuchâtel, il s’est allié aux actions organisées lors de l’édition 2021 de NeuchàToi. Le Théâtre de la Connaissance désigne des événements proposés depuis 2014 par les Instituts faisant partie de la MAPS de l’Université de Neuchâtel et s’adressant à un large public. Il vise à développer des collaborations entre arts de la scène et recherche scientifique afin de diffuser et de créer des connaissances sur des thèmes centraux pour les sociétés contemporaines. https://www.unine.ch/ theatre-connaissance/ Le nccr - on the move est le Pôle de recherche national (PRN) sur la migration et la mobilité. Il rassemble des recherches issues des sciences sociales, de l’économie et du droit, visant à améliorer la compréhension des phénomènes contemporains liés à la migration et à la mobilité en Suisse et au-delà. www.nccr-onthemove.ch 61 NeuchàToi met sur pied, depuis 2006, des manifestations thématiques à l’échelle cantonale, ayant pour but d'encourager une meilleure connaissance et compréhension entre personnes suisses, étrangères et issues de la migration, entre personnes résidant de longue date à Neuchâtel et celles arrivées plus récemment. Pour se faire, NeuchàToi se propose de (ré)interroger régulièrement la population neuchâteloise sur les composantes de son identité, d’identifier ses valeurs et ses principes communs, tout en valorisant la diversité et le respect du pluralisme. www.neuchatoi.ch La version originale du jeu dont le projet s’inspire a été conçue par l’association bernoise ISA, Fachstelle Migration, sur la base de leur large expérience de travail dans le domaine de la migration en Suisse. www.isabern.ch Conception et direction du projet Laure Sandoz Chercheuse postdoctorante, Institut de géographie et nccr - on the move, Université de Neuchâtel Nicolas Yazgi Dramaturge et anthropologue Inspiré librement d’un concept développé en 2019 par ISA Bern (Fachstelle Migration, isabern.ch) Photo © Guillaume Perret Coaching & jeu Didier Chiffelle Etienne Fague Léo Moreno Sophie Pasquet-Racine Jeu Salomé Alvarez Chaves Sarah Blendermann Séverine Desarzens Jean-François Houmard Loanne Janin Nastasia Jeanneret Mira Plüss Meriem Zaimi Interlocutrices et interlocuteurs scientifiques Design, identité visuelle et communication Christin Achermann Professeure en migration, droit et société Dina Bader Chargée de transfert de connaissances et coordinatrice du projet pour le nccr - on the move Anne-Laure Bertrand Maître-assistante en méthodes quantitatives Gianni D’Amato Professeur en migration et citoyenneté Rosita Fibbi Chercheuse en sociologie des migrations Marion Fresia Professeure d’ethnologie Stefanie Kurt Professeure en travail social Raphaël Rey Coordinateur de l’Observatoire romand du droit d’asile et des étranger.e.s Laura Rezzonico Collaboratrice à l’Organisation suisse d’aide aux réfugié.e.s OSAR Robin Stünzi Responsable scientifique au nccr - on the move Nicole Wichmann Directrice administrative du nccr - on the move Jennifer Keller Rédactrice et journaliste reporter d’images Gaëlle Liechti Responsable d’événements Annique Lombard Chargée de communication Nando Luginbühl Responsable du Bureau presse et promotion de l’Université de Neuchâtel Yves Maumary Graphiste Gina Morelli Graphiste Guillaume Perret Photographe Inka Sayed Chargée de communication Administration et logistique Gina Fiore Walder Assistante administrative Adrien Rawyler Assistant étudiant Elisa Tanco Assistante administrative Karim Zayani Assistant étudiant Avec le soutien de nccr - on the move NeuchàToi Faculté des lettres et sciences humaines, Université de Neuchâtel Programme d’intégration cantonal (PIC), soutien de la Ville de Neuchâtel, de l’Etat de Neuchâtel et de la Confédération Société des Alumni, diplômés et amis de l'Université (SAN) Maison d’analyse des processus sociaux (MAPS), Université de Neuchâtel Institut d’ethnologie, Université de Neuchâtel Institut de géographie, Université de Neuchâtel Laboratoire d'études des processus sociaux (LAPS), Université de Neuchâtel Forum suisse pour l'étude des migrations et de la population (SFM), Université de Neuchâtel Bureau presse et promotion, Université de Neuchâtel 65 Pour la sixième édition du Théâtre de la Connaissance, organisée à l'Université de Neuchâtel du 17 au 19 septembre 2021, « Bienvenue à Heimatland ! » s'est inspiré des recherches réalisées dans le cadre du Pôle national de recherche sur la migration et la mobilité (nccr – on the move) et de la Maison d’analyse des processus sociaux (MAPS) sur le thème de la gestion migratoire. Au cœur de l'événement, un jeu de rôle théâtral participatif proposait une expérience immersive durant laquelle le public incarnait le rôle de migrant.e.s de différents statuts arrivant dans un pays inconnu. L’expérience invitait les participant.e.s à vivre pour un temps une réalité différente de la leur et à s’interroger sur les réactions et émotions que cette réalité produit. Dans un deuxième temps, un débriefing en petits groupes, suivi d’une discussion critique avec des chercheuses et chercheurs spécialistes du thème était proposée. L’événement, dans son ensemble, proposait une invitation à réfléchir ensemble sur un thème complexe au travers d’un format inédit. Cette brochure prolonge l’expérience en explicitant certains des éléments qui ont inspiré la conception de l’événement et en proposant des ressources pour poursuivre la réflexion. MAPS
https://openalex.org/W2774641923	https://ejournal.undip.ac.id/index.php/ijms/article/download/15094/pdf	English	null	Contribution of Terrestrial Runoff to Coral Disease Prevalence on North Bali’s Massive Porites	Deleted Journal	2,017	cc-by-sa	4,978	Abstract The widespread of coral disease may threatened Bali`s marine tourism which is the main asset for the nation prosperity. However, the disease prevalence is still unknown, in particular inshore coral reefs near to tourist spot areas. Therefore, the research aims to investigate the contribution of terrestrial runoff to coral disease prevalence and to examine the relationships between disease prevalence and environmental parameters (nitrate, phosphate, organic carbon and total suspended solids (TSS)) within the population of massive Porites on shallow north Bali reefs. Syndrome, diseases and healthy colonies of massive Porites coral were counted and noted within a 2 x 10 m belt transect at 3 sampling sites. The dominant disease observed was ulcerative white spots (UWS), while the syndromes were pigmentation response and aggressive overgrowth by macroalgae. The highest mean UWS prevalence was at site 3 which was the closest site to runoff (prevalence = 91%).This disease only affected one colony at site 1 and 2, respectively. Disease prevalence had strong relationship with TSS and nitrate, yet it showed weak relationship with phosphate and organic carbon. These results suggest that terrestrial runoff could contribute to the disease prevalence by increasing the TSS, nutrients and organic carbon loading to the inshore ecosystems. High level of organic carbon could severe the disease, particularly when combined with elevated TSS and nutrient, by reducing the coral`s immunity system. Keywords: coral disease, prevalence, terrestrial runoff, massive Porites, ulcerative white spots, environmental parameter, North Bali. The high level of nutrient is assumed to decrease the coral immune system, causing reduced defense mechanism against the overgrowth of opportunistic microorganisms (Voss and Richardson, 2006). An experiment with different concentration regimes of organic carbon and nutrients indicated that organic carbon had more adverse impact than nutrient to coral mortality by accelerating the disruption of coral and its microflora mutual partnership (Kline et al., 2006). Meanwhile, increased in suspended solids (TSS) associated with terrestrial runoff reduces the light quality and quantity for coral’s photosynthetic microalgae endosymbiont (Symbiodinium sp.), thus lowering the energy production in coral (Falkowski et al., 1990; Richmond, 1993). Coral produces mucus and moves the cilia in order to wipe away the excessive suspended solids which deposited on the coral surface (Furnas, 2003; Devlin and Schaffelke, 2009). As this mechanism costs a lot of energy, coral likely shifts its energy allocation (from grow to defense) making it more vulnerable against infectious microorganisms (Furnas, 2003; Devlin and Schaffelke, 2009). Contribution of Terrestrial Runoff to Coral Disease Prevalence on North Bali’s Massive Porites I Gusti Bagus Siladharma and Widiastuti Karim* Department of Marine Science, Faculty of Marine and Fisheries, Udayana University, Bukit Jimbaran Campus, Badung, Bali, 80361 Indonesia Email: widiastutikarim@unud.ac.id ILMU KELAUTAN December 2017 Vol 22(4):193-200 ILMU KELAUTAN December 2017 Vol 22(4):193-200 ISSN 0853-7291 Abstract Compared to the effects of nutrients, the relationships between organic carbon and TSS with disease prevalence is not clearly defined yet. Keywords: coral disease, prevalence, terrestrial runoff, massive Porites, ulcerative white spots, environmental parameter, North Bali. Introduction There was approximately 24% of coral reef population worldwide is in vulnerable condition due to the combination of human disturbances (Wilkinson, 2004) and climate change (Gardener et al., 2003; Bruno and Selig 2007; Veron et al., 2009). Among these factors, disease has emerged as one of the most substantial causes for coral reef declining (Rosenberg et al., 2007; Bourne et al., 2009). This pandemic was early documented in Caribbean reefs which affected more than 95% of its population (Aronson and Precht, 2001; Weil et al., 2006). However, studies lately reported that the outbreak has spread over other part of world reefs, including Indo-Pacific reefs (Raymundo et al., 2005; Haapkylä et al., 2007) in which may not be well documented. Despite the massive outbreak, the mechanism of disease virulence is still unknown. Field and experimental studies showed that the disease was strongly associated with nutrient enhancement (Kuta and Richardson, 2002; Bruno et al., 2003; Voss and Richardson, 2006), organic carbon (Kuntz et al., 2005; Kline et al., 2006; Baker et al., 2007), and sedimentation (Hodgson, 1990; Pollock et al., 2014). Received : 28-06-2017 Accepted : 29-07-2017 ijms.undip.ac.id DOI:10.14710/ik.ijms.22.4.193-200 *) Corresponding author © IlmuKelautan, UNDIP ILMU KELAUTAN December 2017 Vol 22(4): 193-200 even in dry season. Each site was separated at 1 km away three 2m x 10 m belt transects which placed randomly at 3-6 m depth. Diseased, syndrome and healthy colonies of massive Porites coral were counted and noted following the description of Coral Disease Handbook (Raymundo et al., 2008) and Underwater Cards for Assessing Coral Health on Indo-Pacific Reefs (Beeden et al., 2008). Identification of massive Porites was based on Veron (2008) and Suharsono (2008). Disease and syndrome prevalence were calculated by : This study provides preliminary information regarding the distribution, environmental stressors and prevalence of coral disease in north Bali island. As a world tourist destination, Bali is facing pressures from rapid economy growth, overfishing, climate change (Knight et al., 1997). Pemuteran village in north Bali was chosen regarding its location as one of few tourist spots in the north Bali (Figure 1.). Despite its beautiful underwater world, this village suffers from flood in almost every rainy season which hit the highway, villages and affects the surrounding coastal areas through terrestrial runoff and rivers (Gerogak district). In addition to flood, overfishing and coastal development for tourist facilities also threatened this area. Introduction The aim of this research is to investigates the contribution of terrestrial runoff to coral disease prevalence and to examine the relationships between coral disease prevalence, nutrients, organic carbon and TSS within population of Indo-Pasific reef main builder species (massive Porites) on shallow north Bali reef. Each 1500 ml of water samples (two replicates) were collected at ±1 m above the coral colonies and out of the belt transects for nutrient analysis (nitrate, phosphate), organic carbon and TSS. All samples were taken simultaneously with disease observations. Nitrate was determined by the brucine method and phosphate by the ammonium molybdate method, while organic carbon (total organic carbon/TOC) level was measured by titrimetric method (Strickland and Parsons, 1972). TSS concentration was measured gravimetrically from the weight difference between loaded and unloaded filter paper after drying overnight at 60°C.A 1000 ml of TSS samples were filtered to the pre-weighed 0.45 μm (125 mm diameter, Whatman). Sampling sites and environmental parameters Prior to investigation, reefs were selected based on its proximity to terrestrial runoff, river, human population and tourist facilities (hotel). The study sites were located in Selini beach, Pemuteran village, Gerogak district, Buleleng regency, north Bali island (Figure 1.). The sampling sites have similar geomorphology and ecology with fringing reefs along the beach. Sampling was conducted using SCUBA in the dry season (September 2016). Site 3 was the only site which has populated area, hotel and river. Moreover, there were three terrestrial runoff in this site which 2 of them were still actively flow into the sea Contribution of Terrestrial Runoff to Coral Disease Prevalence (I.G.B. Siladharma and W. Karim) Dynamics of coral disease and syndromes y y Survey observations indicated that some syndromes and disease occurred on massive Porites colonies at the sampling sites (Figure 2). The only dominant disease occurred on massive Porites was ulcerative white spot (UWS), while the syndromes were consisted of aggressive overgrowth of macroalgae, sediment damage, predation by snail, bleaching and pigmentation response. Among these syndromes, pigmentation response and aggressive overgrowth by macroalgae were classified as the most common syndrome particularly in massive Porites colonies. UWS has regular round shape, small white spots (diameter ±1 cm) that spread over the surface of coral colony. Pigmentation response was documented with the distinctive pink spot which bordered the dead coral skeleton. Another dominant syndromes observed was the aggressive overgrowth of macroalgae on the surface of massive Porites colonies. The outgrowth likely started from the bottom of the coral, and up to the coral surface. Silt accumulation on the coral surface was reported on some colonies mainly at Site 1. The affected tissue was lost and there was mucus secretion over the coral surface. Some colonies of massive Porites suffered from Drupella sp. snail predation. They left significant wide white patch as of tissue lost which commonly seen at the base of the colony. Additionally, few colonies of massive Porites showed stripe or patch of bleached tissue.These bleached tissue were still alive and demonstrated distinct border between healthy and bleaching tissue. The statistical results of regression analyses between these environmental parameters and UWS disease prevalence is shown in Figure 4. It showed that there was a significant strong relationship between disease prevalence (UWS) and TSS concentration (R2 = 0.99, P<0.05) and nitrate level (R2= 0.99, P<0.05). However, the relationship between UWS prevalence and phosphate (R2= 0.3, P>0.05) and organic carbon (TOC) (R2= 0.3, P>0.05) were weak. On the contrary, the disease prevalence between-sites showed that the prevalence of UWS were significantly different among sampling sites (P<0.05). The present study reveals that UWS was the only disease affecting massive Porites colonies at Selini beach, Pemuteran village, north Bali island. This disease had also been documented within massive Porites colonies in the Wakatobi Marine Park, south-east Sulawesi, Indonesia (Raymundo et al., 2005) and on Phillipine reefs (Haapkylä et al., 2007). The positively relationship between UWS prevalence and TSS suggests that high level of TSS could intensify the virulence of this disease. Previous study by Pollock et al. (2014) and Miller et al. Dynamics of coral disease and syndromes (2016) also demonstrated that increased turbidity due to high level of TSS significantly elevated the coral disease and compromised health prevalences. The high amount of TSS in the seawater column reduces the light intensity which is needed for coral`s photosynthetic endosymbiont. Consequently, it may diminish the supply energy from the photosynthetic algae for coral as the host. Also, the accumulated TSS on coral surface, may provide suitable media for the opportunistic microorganisms to grow (Hodgson, 1990). In order to remove the deposited TSS, coral secretes mucus on its surface. This mucus secretion consumes significant amount of energy that could be invested for growth, defense, and reproduction. Leads coral to be more susceptible to disease virulence. Although samplings were taken during dry season where the rainfall was only 2 mm (Indonesian Agency for Meteorological, Climatological and Geophysics), the level of TSS at site 3 was still relatively 3-fold higher than at site 1 and 2. The high level of TSS at site 3 was likely due to the two of three runoff and small river that The prevalence of disease and syndrome among sampling sites is presented in Figure 3. The highest disease prevalence of UWS was found at site 3 that reached 91% of the massive Porites colonies. In contrast, this disease had very low prevalence at site 1 and 2 with only 3% infection of one colony in each site. The pigmentation response was the most predominant syndrome found at all sites. The lowest prevalence of pigmentation response was indicated at site 1 (13%). The aggressive overgrowth of macroalgae was observed as the second largest syndrome at these sampling sites. Similar to UWS and pigmentation response, site 3 indicated as the highest prevalenceof this syndrome that was 46%. The total average number of massive Porites colonies is almost similar across the sites, with 40 colonies for each site 1 and 2, and 44 colonies for site 3. Statistical analyses One-way ANOVA was used to statistically assess the differences in disease prevalence between sampling sites. Furthermore, the relationships between disease prevalence and environmental parameters (nutrients, organic carbon and TSS level) were analysed by using simple linear regression. All the statistical analyses were conducted by using Statsoft. Figure 1. Map of the sampling sites at North of Bali island Figure 1. Map of the sampling sites at North of Bali island 194 ILMU KELAUTAN December 2017 Vol 22(4): 193-200 Results and Discussion According to the local diving operators, these sampling sites were no longer operated as diving spots and fishing areas since the underwater scenery was not appealing to tourists and suffered from overfishing (pers. comm. 2016). The level of phosphate at site 2 and 3 were as high as 11.6 µM, and lower at site 1. The organic carbon (total organic carbon/TOC) was similar at all sampling sites that reached 51 mg.L-1 in average. Similar level of nitrate was observed at site 1 and 2, but 5-fold higher at site 3. The same trend was found in TSS which showed approximately 17 mg.L-1 at both site 1 and 2, but 3 - times higher at site 3. Relationship between coral disease and syndromes prevalence and environmental parameters The summary of environmental parameters which assumed as the stressors of coral disease and syndromes at the sampling sites is shown in Table 1. 195 Contribution of Terrestrial Runoff to Coral Disease Prevalence (I.G.B. Siladharma and W. Karim) ILMU KELAUTAN December 2017 Vol 22(4): 193-200 (a) (b) (c) (d) (e) (f) Figure 2. Diseases and syndromes affecting massive Porites colonies at sampling sites. (a) Pigmentation response; arrows pointing at affected tissue. (b) Unusual bleaching pattern (c) Silt deposition on the surface of massive Porites. (d) Predation by Drupella sp. snail. (e) UWS disease (f) Aggressive overgrowth by macroalgae. (b) (a) (b) (a) (c) (d) (d) (c) (f) (e) (f) (e) Figure 2. Diseases and syndromes affecting massive Porites colonies at sampling sites. (a) Pigmentation response; arrows pointing at affected tissue. (b) Unusual bleaching pattern (c) Silt deposition on the surface of massive Porites. (d) Predation by Drupella sp. snail. (e) UWS disease (f) Aggressive overgrowth by macroalgae. were still actively loading particulate materials and sediments from the land to the surrounding coastal areas. In addition, low resuspension during dry season causes the high amount of TSS in water column. were likely the contributor of disease and syndromes appeared at these sampling sites. The finding in the present study is in agreement with Haapkylä et al. (2011) and Kaczmarsky and Richardson (2010) that argued nutrient enhancement promotes the coral disease prevalence, since nutrient is the limited growth factor for microorganisms. However, in relation to the high UWS prevalence at site 3, it is assumed that nutrient has to couple with other stressors in order to accelerate the disease severity. Kline et al. (2006) showed the effect of nutrients (nitrate, phosphate, ammonia) to coral disease were less harmful than organic carbon. The amount of nutrient (nitrate) in this study was significantly related to the disease prevalence. Although phosphate showed statistically no relationship with the disease prevalence, the concentration in all the sampling sites was significantly high. The concentration of nitrate measured at all the sampling sites was much higher than those in Kline et al. (2006) which the maximum concentration (6.5 μM) was the minimum concentration detected at site 1. Despite no strong relationship between disease prevalence and organic carbon at the sampling sites, the level of water column organic carbon in this study was 2-times higher than those in Kuntz et al. (2005). Relationship between coral disease and syndromes prevalence and environmental parameters Therefore, organic carbon alone could enhance the Likewise for nitrate, the maximum level of phosphate tested at experimental study by Kline et al. (2006) was 2-fold higher at nearly all the sampling sites. The high nutrients amount at all sampling sites 196 Contribution of Terrestrial Runoff to Coral Disease Prevalence (I.G.B. Siladharma and W. Karim) ILMU KELAUTAN December 2017 Vol 22(4): 193-200 Table 1. Environmental parameters along the sampling sites Station Km1 % UWS Nitrate (µM) Phosphate (µM) TOC (mg.L-1) TSS (mg.L-1) 1 0 3 11.3 8.4 50.6 17.2 2 1 3 6.5 11.6 51.8 17.8 3 2 91 48.4 11.6 50.6 53.9 Note.1Km is distance from runoff Figure 3. Mean prevalence of the most common disease and syndromes observed on surveyed sampling sites. Note. = Macroalgae overgrowth; = Pigmentation response; = UWS Relationship between UWS prevalence and TSS Relationship between UWS prevalence and TOC Relationship between UWS prevalence and P Relationship between UWS prevalence and N . Figure 4 Analyses of linear regression of relationship between UWS disease prevalence and environmental para 0 10 20 30 40 50 60 70 80 90 100 St1 St2 St3 % Prevalence Sampling sites R² = 0.9899 0 20 40 60 80 100 0 20 40 60 UWS Prevalence (%) TSS (mg.L-1) R² = 0.25 0 20 40 60 80 100 0 20 40 60 UWS Prevalence (%) TOC (mg.L-1) R² = 0.25 0 20 40 60 80 100 0 5 10 15 UWS Prevalence (%) PO43-. (µM) R² = 0.989 0 20 40 60 80 100 0 10 20 30 40 50 UWS Prevalence (%) NO3- (µM) ILMU KELAUTAN December 2017 Vol 22(4): 193-200 Table 1. Environmental parameters along the sampling sites Table 1. Environmental parameters along the sampling sites Table 1. Environmental parameters along the sampling sites Station Km1 % UWS Nitrate (µM) Phosphate (µM) TOC (mg.L-1) TSS (mg.L-1) 1 0 3 11.3 8.4 50.6 17.2 2 1 3 6.5 11.6 51.8 17.8 3 2 91 48.4 11.6 50.6 53.9 Note.1Km is distance from runoff Figure 3. Mean prevalence of the most common disease and syndromes observed on surveyed sampling sites. Note. Relationship between coral disease and syndromes prevalence and environmental parameters = Macroalgae overgrowth; = Pigmentation response; = UWS 0 10 20 30 40 50 60 70 80 90 100 St1 St2 St3 % Prevalence Sampling sites Station Km1 % UWS Nitrate (µM) Phosphate (µM) TOC (mg.L-1) TSS (mg.L-1) 1 0 3 11.3 8.4 50.6 17.2 2 1 3 6.5 11.6 51.8 17.8 3 2 91 48.4 11.6 50.6 53.9 Note.1Km is distance from runoff Figure 3. Mean prevalence of the most common disease and syndromes observed on surveyed sampling s Note Macroalgae overgrowth; Pigmentation response; UWS 0 10 20 30 40 50 60 70 80 90 100 St1 St2 St3 % Prevalence Sampling sites Figure 3. Mean prevalence of the most common disease and syndromes observed on surveyed sampling sites. Note. = Macroalgae overgrowth; = Pigmentation response; = UWS Relationship between UWS prevalence and TSS Relationship between UWS prevalence and TOC Relationship between UWS prevalence and P Relationship between UWS prevalence and N . Figure 4. Analyses of linear regression of relationship between UWS disease prevalence and environmental parameters. R² = 0.9899 0 20 40 60 80 100 0 20 40 60 UWS Prevalence (%) TSS (mg.L-1) R² = 0.25 0 20 40 60 80 100 0 20 40 60 UWS Prevalence (%) TOC (mg.L-1) R² = 0.25 0 20 40 60 80 100 0 5 10 15 UWS Prevalence (%) PO43-. (µM) R² = 0.989 0 20 40 60 80 100 0 10 20 30 40 50 UWS Prevalence (%) NO3- (µM) R² = 0.9899 0 20 40 60 80 100 0 20 40 60 UWS Prevalence (%) TSS (mg.L-1) R² = 0.25 0 20 40 60 80 100 0 20 40 60 UWS Prevalence (%) TOC (mg.L-1) Relationship between UWS prevalence and TOC Relationship between UWS prevalence and TOC Relationship between UWS prevalence and TSS p p R² = 0.989 0 20 40 60 80 100 0 10 20 30 40 50 UWS Prevalence (%) NO3- (µM) R² = 0.25 0 20 40 60 80 100 0 5 10 15 UWS Prevalence (%) PO43-. (µM) Relationship between UWS prevalence and P Relationship between UWS prevalence and N . Figure 4. Analyses of linear regression of relationship between UWS disease prevalence and environmental parameters. gure 4. Analyses of linear regression of relationship between UWS disease prevalence and environmental Contribution of Terrestrial Runoff to Coral Disease Prevalence (I.G.B. Siladharma and W. Relationship between coral disease and syndromes prevalence and environmental parameters Karim) 197 ILMU KELAUTAN December 2017 Vol 22(4): 193-200 degrade the reef ecosystem. The finding of this study was supported by previous studies that disease prevalence, particularly UWS, is strongly related to anthropogenic activities (Raymundo et al., 2005; Voss and Richardson 2006; Bruno and Selig, 2007; Kaczmarsky and Richardson, 2010). disease prevalence. The village and hotel may be the source of elevated nutrients and organic carbon to the sampling sites as their sewage effluents and wastewater flow into terrestrial runoff and river. Furthermore, the presence of organic carbon in the water column is indirect effect of elevated level of nutrients. As nutrient increased, the macroalgae population on the coral reefs overgrow and release organic carbon as the photosynthetic product (Smith et al., 2006) or even from dead tissue (Khailov and Burlakova, 1989). Acknowledgments This preliminary study with limited dataset presented initial report regarding the status of UWS disease in Indonesia which has not been fully investigated before, except the report by Haapkylä et al. (2007) in Wakatobi Marine Park, South-East Sulawesi. On the contrary, this infectious disease has been widely observed in the Phillipines reefs which share the same geographic region with Indonesia (Raymundo et al., 2005; Kaczmarsky, 2006). Studies by Arboleda and Reichardt (Arboleda and Reichardt, 2010) in infected UWS Porites cylindrica collected from the Phillipines showed that the agent of the disease was Vibrio sp. which is widespread where organic matter is abundant (Colwell, 1996; Reichardt and Jacinto, 2007). Additionally, the seawater temperature when the sampling conducted was 31°C, an ideal temperature for the Vibrio sp. to proliferate (Colwell 1996; Reichardt and Jacinto, 2007). Therefore, it is suggested that together with the poor water quality densed human population and seawater temperature would allow the disease agent to grow rapidly at site 3. We thank two anonymous reviewers for constructive comments on the manuscript. The authors wish to thank: Nidzar Muhammad Rafly, Putu Hernanda Krishna Ariszandy and Dika Madyawan for sampling assistances; Ni Wayan Gita Kanela and Naila Makfiya for TSS analyses. Marine Laboratory at Faculty of Marine and Fisheries, Udayana University for logistical supports. This study was funded by grants from Udayana University to I.G.B.S. and W.K. Conclusion This study provides preliminary information regarding the distribution, environmental stressors and prevalence of coral disease in north Bali island (study case: Selini beach, Pemuteran village). Ulcerative white spot (UWS) was the only disease affecting massive Porites colonies at the study sites. The present study reveals that terrestrial runoff could contribute to the disease prevalence by increasing the TSS, nutrients and organic carbon loading to the inshore ecosystems. This finding suggests that important consideration of water quality of terrestrial runoff and river which could deliver significant amount of disease drivers to the inshore areas could prevent the collapse of coral reef ecosystems and its social economic impacts, especially in Bali’s marine tourism. This hypothesis was supported by the large number of overgrowth macroalgae found at the sampling sites, in particular site 3. The overgrowth of macroalgae might be the result of overfishing of herbivore fish in this area since there were very few coral fish detected during surveys. Nonetheless, this study only tested for the level of total organic carbon, investigation on the effect of high concentration of particulated organic carbon (POC) and dissolved organic carbon (DOC) caused massive prevalence of atramentous necrosis disease (Haapkylä et al., 2011). High level of organic carbon could severe the disease, particularly when combined with elevated TSS and nutrient, by reducing the coral`s immunity system. Contribution of Terrestrial Runoff to Coral Disease Prevalence on North Bali `s Inshore Coral Reefs (I.G.B. Siladharma and W. Karim) References Arboleda, M.D.M. & Reichardt, W.T. 2010. Vibrio sp. causing Porites Ulcerative White Spot Disease. Dis. Aqua. Organ. 90:93–104. doi: 10.3354/ dao02222. Aronson, R. & Precht, W. 2001. White-band disease and the changing face of Caribbean coral reefs. 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https://openalex.org/W1542173032	https://www.frontiersin.org/articles/10.3389/fpls.2015.00430/pdf	English	null	Phosphoproteomics technologies and applications in plant biology research	Frontiers in plant science	2,015	cc-by	8,034	REVIEW published: 16 June 2015 doi: 10.3389/fpls.2015.00430 Edited by: Sabine Lüthje, University of Hamburg, Germany Reviewed by: Christof Rampitsch, Agriculture and Agrifood Canada, Canada Mohammad-Zaman Nouri, Rice Research Institute of Iran in Mazandaran, Iran Keywords: phosphoproteomics, enrichment, quantiﬁcation, phosphorylation site mapping, plant biology Phosphoproteomics technologies and applications in plant biology research Jinna Li 1, Cecilia Silva-Sanchez 2, Tong Zhang 3, Sixue Chen 1, 2, 3 and Haiying Li 1* 1 College of Life Sciences, Heilongjiang University, Harbin, China, 2 Proteomics and Mass Spectrometry, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, USA, 3 Plant Molecular and Cellular Biology Program, Department of Biology, UF Genetics Institute, University of Florida, Gainesville, FL, USA Protein phosphorylation has long been recognized as an essential mechanism to regulate many important processes of plant life. However, studies on phosphorylation mediated signaling events in plants are challenged with low stoichiometry and dynamic nature of phosphorylated proteins. Signiﬁcant advances in mass spectrometry based phosphoproteomics have taken place in recent decade, including phosphoprotein/phosphopeptide enrichment, detection and quantiﬁcation, and phosphorylation site localization. This review describes a variety of separation and enrichment methods for phosphoproteins and phosphopeptides, the applications of technological innovations in plant phosphoproteomics, and highlights signiﬁcant achievement of phosphoproteomics in the areas of plant signal transduction, growth and development. Correspondence: Correspondence: Haiying Li, College of Life Sciences, Heilongjiang University, 74 Xuefu Rd, Harbin 150080, China lvzh3000@sina.cn Phosphorylation is one of the most important post-translational modiﬁcations (PTMs) of proteins (Pawson and Scott, 1997). Approximately one-third of the proteins are modiﬁed by phosphorylation (Hubbard and Cohen, 1993). The kinase mediated covalent addition of a phosphate group to serine, threonine, and tyrosine residues in eukaryotes, and other amino acids such as histidine, aspartate, glutamate, lysine, arginine, and cysteine in prokaryotes and the subsequent removal of the phosphate groups by protein phosphatases constitute important signaling and regulatory mechanisms in living organisms (Batalha et al., 2012). Reversible protein phosphorylation regulates a wide range of cellular processes such as transmembrane signaling, intracellular ampliﬁcation of signals, and cell-cycle control. Protein phosphorylation often leads to protein structural changes that can directly modulate protein activity, and induce changes in interaction partners or subcellular localization (Jørgensen and Linding, 2008). The cascade of protein phosphorylation in a signaling pathway provides the backbone for complex signaling networks and regulatory processes in plant cells, including hormone sensing (Park et al., 2009), and environmental stress responses (Mishra et al., 2006). Thus, the analysis of signaling pathways in plants has often been focused on protein kinases. Traditional studies, however, described the phosphorylation of a single substrate by a particular kinase. Based on genome annotation, protein kinases were found to make up about 5.5% of the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000), which is nearly twice as many as in the human genome (Manning et al., 2002). This indicates high speciﬁcity and complex networks of phosphorylation events in plants Specialty section: This article was submitted to Plant Proteomics, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Plant Proteomics, a section of the journal Frontiers in Plant Science Received: 17 March 2015 Accepted: 27 May 2015 Published: 16 June 2015 Quantitative Phosphoproteomics Low stoichiometry of phosphorylated proteins and low ionization eﬃciency of phosphopeptide are two major challenges for protein phosphorylation detection. To reduce sample complexity, it is necessary to enrich the modiﬁed proteins and/or peptides before mass spectrometry (MS) analysis. Commonly used enrichment techniques were summarized in Table 1, with enrichment at the peptide level as a popular strategy. A successful phosphoproteomics study depends not only on the selective enrichment of phosphopeptides, but also on accurate detection and quantitation of the peptides, as well as precise mapping of the phosphorylation sites. Advances in these areas have been extensively reviewed (Batalha et al., 2012; Fíla and Honys, 2012; Kline and De Luca, 2014; Silva-Sanchez et al., 2015). Most of the technologies were developed in animal and yeast systems, and subsequently applied in plants. Here we brieﬂy describe the advancement of phosphoproteomics technologies in plant research (Table 2). Quantitative phosphoproteomics is aimed to enable a better understanding of phosphorylation regulated biological events. Comparative phosphoproteomics of wild-type and mutant plants or control and treated plants could be conducted in many ways. In general, the approaches can be grouped into gel-based, gel- free, stable isotope labeling, or label-free. Two-dimensional gel electrophoresis (2-DE) has been a widely used technology that resolves thousands of proteins by isoelectric point and molecular weight. Pro-Q Diamond is a ﬂuorescent stain that provides a convenient method for selectively staining phosphoproteins in acrylamide gels. The result shows a global map of the modiﬁed proteins and their relative abundances compared to non- phosphorylated counterparts when a total protein staining is used after Pro-Q Diamond staining. Diﬀerentially phosphorylated proteins in wild-type and snk2.8 mutant Arabidopsis plants were analyzed using 2-DE and Pro-Q, and putative substrates of SnRK2.8 were identiﬁed (Shin et al., 2007). Stable isotope labeling has been applied in plant phosphoproteomics successfully using a gel-free approach, for example, stable isotope labeling of amino acids in cell culture (SILAC).The ﬁrst SILAC in plants was done by introducing 15N in Arabidopsis suspension cells (Benschop et al., 2007) (Table 2). The methodology has been improved over the years and found more applications (Schütz et al., 2011; Stecker et al., 2014). Another labeling approach introduces multiplex isobaric tags to isolated proteins or digested peptides in vitro. Commonly used tags include isobaric tags for relative and absolute quantiﬁcation (iTRAQ) and tandem mass tags (TMT). Citation: Li J, Silva-Sanchez C, Zhang T, Chen S and Li H (2015) Phosphoproteomics technologies and applications in plant biology research. Front. Plant Sci. 6:430. doi: 10.3389/fpls.2015.00430 June 2015 \| Volume 6 \| Article 430 Frontiers in Plant Science \| www.frontiersin.org 1 Phosphoproteomics and applications Li et al. (Schulze, 2010). Many plant protein kinases have been identiﬁed to play essential roles in response to a variety of stresses including salt stress, cold stress, and pathogen invasion. Deciphering the molecular events occurring in stress responses will enhance our understanding of the biological processes in plants (De la Fuente van Bentem et al., 2006; Stecker et al., 2014). phosphorylated peptides, and ZrO2 tends to enrich singly phosphorylated peptides. A serial enrichment procedure with both TiO2 and ZrO2 can signiﬁcantly increase the eﬃciency of capturing phosphopeptides in biological samples (Kweon and Håkansson, 2006; Gates et al., 2010). Metal dioxide enrichment could also be coupled with other peptide fractionation methods. For instance, a combination of TiO2 enrichment and hydrophilic interaction liquid chromatography (HILIC) resulted in identiﬁcation of 2305 phosphopeptides belonging to 964 proteins in wheat (Yang et al., 2013). Electrostatic repulsion hydrophilic interaction chromatography (ERLIC), a variation of HILIC that uses electrostatic repulsion as an additional chromatography stationary phase, had also been used successfully for selectively enrichment of phosphopeptides (Gan et al., 2008; Loroch et al., 2013). The combination of phosphoprotein/phosphopeptide enrichment techniques, along with technological advancement in tandem mass spectrometry has been employed as a powerful tool to study protein phosphorylation and its biological relevance (Chen and White, 2004). In this review, a variety of separation and enrichment methods for phosphoproteins and phosphopeptides, their features as well as applications in phosphoproteomics research are described. Quantitative Phosphoproteomics The tags are designed to be isobaric during MS and fragment to reveal diﬀerential low mass ion reporters during MS/MS. Due to its capability of multiplexing up to 10 samples in a single experiment and the enrichment eﬀect for low abundance proteins, iTRAQ/TMT labeling has become popular in plant phosphoproteomics (Jones et al., 2006; Yang et al., 2013; Fan et al., 2014). Frontiers in Plant Science \| www.frontiersin.org Increased sensitivity due to complete deprotonation of phosphoproteins/ phosphopeptides at neutral pH. Elution at the physiological pH allow for protein activity and functional analysis. Uses 1,3-bis[bis(pyridine-2- ylmethyl)amino]propan-2-olato dizinc(II) complex as a selective phosphate binding tag in aqueous solution at neutral pH. Enrichment Strategies The most widely used enrichment method for phosphopeptides takes advantage of the aﬃnity binding between negatively charged phosphate and positively charge metal ions (Fíla and Honys, 2012). Immobilized metal aﬃnity chromatography (IMAC) is often coupled with strong cation exchange (SCX) for two-step phosphopeptide enrichment. For example, in a SCX-IMAC experiment, three times more phosphopeptides were identiﬁed when compared to the use of SCX or IMAC alone (Trinidad et al., 2006). The ﬁrst reported SCX-IMAC application in plants resulted in identiﬁcation of 283 phosphopeptides (Nuhse et al., 2004). In addition, Polymer-based Metal-ion Aﬃnity Capture (PolyMAC) is a variant of IMAC, also showed high selectivity. For instance, employment of complementary PolyMAC-Titanium (Ti) and PolyMAC-Zirconium (Zr) ion aﬃnity chromatography lead to identiﬁcation of 5386 unique phosphopeptides (Wang et al., 2013a). While both in vivo and in vitro label methods are limited by the number of samples, label free approaches enable quantitative phosphoproteomics of unlimited number of samples. There are two main methods in label free quantitation. The ﬁrst is based on precursor ion peak intensity/area, and the second is based on the number of MS/MS spectra acquired for a Metal dioxide especially titanium dioxides (TiO2) and zirconium dioxides (ZrO2) are gaining popularity for phosphopeptide enrichment. A comparison of the performance of TiO2 and ZrO2 performed with α-casein and β-casein as standard proteins showed that TiO2 tends to enrich multiply June 2015 \| Volume 6 \| Article 430 Frontiers in Plant Science \| www.frontiersin.org 2 Li et al. Phosphoproteomics and applications TABLE 1 \| Phosphopeptide/phosphotprotein enrichment methodologies. Enrichment method Description Advantage Disadvantage References Immunoafﬁnity enrichment Use of antibodies directed against pTyr, pSer, pThr, and more recently against the surrounding consensus sequences for pSer/pThr. Highly speciﬁc. Low efﬁciency, high cost, use of different antibodies for different phosphorylation motifs. Stokes et al., 2012 Immobilized metal afﬁnity chromatography (IMAC) Negatively charged phosphate groups on the phosphorylated amino acids interact with positively charged metal ions such as Ni2+, Fe3+, Ga3+, Zr4+, and Ti4+ that are chelated with silica or agarose through nitriloacetic acid or iminodiacetic acid. Good for both phosphoproteins and phosphopeptides. When used with peptides, it can enrich mono- and multiple phosphorylated peptides. Tends to bind strongly to monophosphorylated peptides, which makes it difﬁcult for elution. Non-speciﬁc binding of acidic peptides can occur. Enrichment Strategies Fíla and Honys, 2012 Metal oxide afﬁnity chromatography (MOAC) Similar to IMAC, the phosphate groups on the amino acids interact with positively charged metal oxides, e.g., titanium or zirconium that acts as anchoring molecules to trap phosphopeptides through the formation of multi-dentate bonds. Good for both phosphoproteins and phosphopeptides. When used with peptides, it can enrich mono- and multiple phosphorylated peptides. Tends to binds strongly to multiple phosphorylated peptides, which makes it difﬁcult for elution. Nonspeciﬁc binding of acidic peptides can occur. Gates et al., 2010 Phos-Tag chromatography, Uses 1,3-bis[bis(pyridine-2- ylmethyl)amino]propan-2-olato dizinc(II) complex as a selective phosphate binding tag in aqueous solution at neutral pH. Increased sensitivity due to complete deprotonation of phosphoproteins/ phosphopeptides at neutral pH. Elution at the physiological pH allow for protein activity and functional analysis. Mainly used to conﬁrm the phosphorylation state in relatively pure proteins, but not with complex mixtures. Kinoshita et al., 2006 Prefractionation by strong cation exchange (SCX) and strong anion exchange (SAX) In SCX, tryptic peptides often carry a charge of +2, except for phosphopetides with a net charge of +1, making them elute early in the chromatography. SAX retains phosphor-peptides, allowing separation based on the number of phosphorylated residues. Used for fractionation of highly complex mixtures, it can be performed on-line with mass spectrometry. Similar degree of unspeciﬁc binding as IMAC and MOAC. Leitner et al., 2011 Hydrophilic interaction liquid chromatography (HILIC) Phosphopeptides with polar phosphate groups are strongly retained on the HILIC stationary phase resulting in separation from non-phosphorylated species. Good for both phosphoproteins and phosphopeptides. When used with peptides, it can enrich mono- and multiple phosphorylated peptides. Similar degree of unspeciﬁc binding as IMAC and MOAC. (Yang et al., 2013) Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) ERLIC is a variation of HILIC using electrostatic repulsion as an additional phase to adjust selectivity by varying pH or organic solvents. Good for both phosphoproteins and phosphopeptides. When used with peptides, it can enrich mono- and multiple phosphorylated peptides. Similar degree of unspeciﬁc binding as IMAC and MOAC. Gan et al., 2008 Hydroxyapatite chromatography It takes advantage of the strong interaction between positively charged hydroxyapatite and phosphate ions. Good for fractionating mono-, di-, tri-, and multi-phosphorylated peptides when using gradient of a phosphate buffer. Developed with phosphoprotein standards, not tested with complex samples. Mamone et al., 2010 given peptide (known as spectral counting). Enrichment Strategies Both methods were used in plant phosphoproteomics (Reiland et al., 2011; and Weckwerth, 2006; Schulze et al., 2012; Minkoﬀet al., 2015). A triple quadrupole is typically used for the MRM TABLE 1 \| Phosphopeptide/phosphotprotein enrichment methodologies. Good for both phosphoproteins and phosphopeptides. When used with peptides, it can enrich mono- and multiple phosphorylated peptides. Good for both phosphoproteins and phosphopeptides. When used with peptides, it can enrich mono- and multiple phosphorylated peptides. Mainly used to conﬁrm the phosphorylation state in relatively pure proteins, but not with complex mixtures. Kinoshita et al., 2006 Uses 1,3-bis[bis(pyridine-2- ylmethyl)amino]propan-2-olato dizinc(II) complex as a selective phosphate binding tag in aqueous solution at neutral pH. Uses 1,3-bis[bis(pyridine-2- ylmethyl)amino]propan-2-olato dizinc(II) complex as a selective phosphate binding tag in aqueous solution at neutral pH. Similar degree of unspeciﬁc binding as IMAC and MOAC. Leitner et al., 2011 Used for fractionation of highly complex mixtures, it can be performed on-line with mass spectrometry. Good for both phosphoproteins and phosphopeptides. When used with peptides, it can enrich mono- and multiple phosphorylated peptides. Developed with phosphoprotein standards, not tested with complex samples. Mamone et al., 2010 and Weckwerth, 2006; Schulze et al., 2012; Minkoﬀet al., 2015). A triple quadrupole is typically used for the MRM measurement, in which the ﬁrst quadrupole (Q1) is set as a ﬁlter for the precursor ion with predetermined mass and Q3 is set to measure a speciﬁc fragment ion. The speciﬁc combination between a precursor ion and a fragment ion is called a transition and multiple transitions can be used to determine the relative and absolute (with synthesized peptide standards) levels of phosphopeptides (Schulze et al., 2012). given peptide (known as spectral counting). Both methods were used in plant phosphoproteomics (Reiland et al., 2011; Engelsberger and Schulze, 2012; Wang et al., 2013a). For instance, Reiland et al. (2011) characterized the function of a thylakoid- associated kinase STN8 in the ﬁne-tuning of cyclic electron ﬂow, which is regulated by the phosphorylation/dephosphorylation event. In addition to these large scale discovery phosphoproteomics approaches, multiple reaction monitoring (MRM) has been used for quantiﬁcation of targeted phosphopeptides (Glinski June 2015 \| Volume 6 \| Article 430 Frontiers in Plant Science \| www.frontiersin.org 3 Phosphoproteomics and applications Li et al. TABLE 2 \| Representative plant phosphoproteomics work in the past decade. Enrichment Strategies Plant Phosphopeptides/ Enrichment Quantitation Phosphorylationsite MS instrument References materials phosphoproteins method method mapping Arabidopsis plasma membrane 283 phosphopeptides IMAC None Mascot QTOF Ultima (Waters) Nuhse et al., 2004 Arabidopsis leaves 317 phosphopeptides Phospho- protein kit iTRAQ Mascot QTRAP (AB Sciex) Jones et al., 2006 Arabidopsis leaves 16 phosphopeptides None MRM MS3 de novo TSQ Quantum (Thermo) Glinski and Weckwerth, 2005 Arabidopsis suspension cells 1168 phosphopeptides TiO2 SILAC MSQuant LTQ FT-ICR (Thermo) Benschop et al., 2007 Arabidopsis leaves 111 phosphoproteins Pro-Q Diamond 2-DE Mascot QSTAR XL (AB Sciex) Shin et al., 2007 Arabidopsis plasma membrane 67 phosphopeptides IMAC Precursor ion intensity MSQuant LTQ (Thermo) Niittylä et al., 2007 Tomato leaves 48 proteins TiO2 Precursor ion intensity VEMS QTOF (Micromass) Stulemeijer et al., 2009 Arabidopsis leaves 3589 phosphopeptides TiO2 and FeCl3 Spectral counting Mascot Orbitrap (Thermo) Reiland et al., 2011 Arabidopsis leaves 3 phosphopeptides None MRM Previously determined TSQ Quantum (Thermo) Schulze et al., 2012 Arabidopsis leaves 5386 phosphopeptides PolyMAC Precursor ion intensity PhosphoRS Orbitrap Velos (Thermo) Wang et al., 2013b Arabidopsis leaves 1 phosphopeptide None MRM Previously determined 4000 QTRAP (AB Sciex) Prado et al., 2013 Wheat leaves 2305 phosphopeptides TiO2 and HILIC TMT Mascot Orbitrap Velos (Thermo) (Yang et al., 2013) Arabidopsis microsome 1229 phosphopeptides TiO2 SILAC Mascot Orbitrap XL (Thermo) Stecker et al., 2014 Cotton leaves 1315 phosphopeptides TiO2 iTRAQ PhosphoRS Q Exactive (Thermo) Fan et al., 2014 Arabidopsis leaves 14 phosphopeptides TiO2 MRM Mascot QTRAP 5500 (AB Sciex) Minkoff et al., 2015 LTQ linear ion trap; VEMS Virtual Expert Mass Spectrometrist; MRM multiple reaction monitoring; TOF time of ﬂight; FT ICR Fourier transform ion cyclotron resonance Please refer TABLE 2 \| Representative plant phosphoproteomics work in the past decade. LTQ, linear ion trap; VEMS, Virtual Expert Mass Spectrometrist; MRM, multiple reaction monitoring; TOF, time of ﬂight; FT-ICR, Fourier transform ion cyclotron resonance. Please refer to the text for other abbreviations. Frontiers in Plant Science \| www.frontiersin.org Applications of Phosphoproteomics in Plant Biology Research (MAP2K) and a MAPK, which activate in a sequential manner via phosphorylation (Figure 1). An activated MAPKKK ﬁrstly phosphorylates two serine and/or threonine residues (S/T-X3- 5-S/T) located within the activation loop of the MAPKK. Activated MAPKKs in turn trigger MAPK activation through dual phosphorylation of a highly conserved T-X-Y motif in the activation loop of MAPKs (Hamel et al., 2012). In a proteomic analysis of plasma membrane isolated from maize roots, four isoforms of Pto-interacting-like kinase 1 (PTI1) showed increased levels in response to low and high iron conditions (Hopﬀet al., 2013). Interestingly, a previous oxidative stress study in Arabidopsis demonstrated that interaction of a PTI1-like kinase (PTI1-4) with another serine/threonine protein kinase, oxidative signal-inducible 1 (OX1), mediates oxidative stress signaling. In addition, PTI1-4 was found to interact with MPK6 in the same protein complex (Forzani et al., 2011). These results imply that the PTI signals may function through the OXI1 and MPK6 signaling cascades. Recently, Hoehenwarter et al. (2013) developed a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide aﬃnity chromatography with phosphopeptide enrichment using TiO2-based metal oxide aﬃnity chromatography to enrich phosphopeptides from complex A. thaliana protein samples. The (MAP2K) and a MAPK, which activate in a sequential manner via phosphorylation (Figure 1). An activated MAPKKK ﬁrstly phosphorylates two serine and/or threonine residues (S/T-X3- 5-S/T) located within the activation loop of the MAPKK. Activated MAPKKs in turn trigger MAPK activation through dual phosphorylation of a highly conserved T-X-Y motif in the activation loop of MAPKs (Hamel et al., 2012). In a proteomic analysis of plasma membrane isolated from maize roots, four isoforms of Pto-interacting-like kinase 1 (PTI1) showed increased levels in response to low and high iron conditions (Hopﬀet al., 2013). Interestingly, a previous oxidative stress study in Arabidopsis demonstrated that interaction of a PTI1-like kinase (PTI1-4) with another serine/threonine protein kinase, oxidative signal-inducible 1 (OX1), mediates oxidative stress signaling. In addition, PTI1-4 was found to interact with MPK6 in the same protein complex (Forzani et al., 2011). These results imply that the PTI signals may function through the OXI1 and MPK6 signaling cascades. Recently, Hoehenwarter et al. (2013) developed a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide aﬃnity chromatography with phosphopeptide enrichment using TiO2-based metal oxide aﬃnity chromatography to enrich phosphopeptides from complex A. thaliana protein samples. The Phosphoproteomics of Subcellular Compartments Phosphoproteomics studies were often performed in a shotgun fashion, with the identiﬁcation of hundreds and thousands of proteins that lead to a very complicated set of phosphoproteins across subcellular compartments and organelles (Table 2), leading to a poor understanding of the networks that regulate the cellular activities (Jung et al., 2000). Compartmentalization in eukaryotes oﬀers a practical approach to study subcellular phosphoproteomics networks, with a reduced population of identiﬁed proteins. There are about 3000 proteins in the chloroplasts of Arabidopsis, but only four kinases were previously identiﬁed. It may be feasible to ﬁnd specialized kinases or families of kinases that can potentially show diﬀerential activities in the chloroplasts (Millar and Taylor, 2014; van Wijk et al., 2014). A meta-analysis of 27 publications of phosphoproteomics data sets in Arabidopsis comprises 60,366 phosphopeptides matched to 8141 non-redundant proteins. The phosphoproteins showed predicted subcellular distribution in the following categories: nucleus, secretory (containing endoplasmic reticulum, Golgi, plasma membrane, cell wall, and vacuolar), cytosol, other/unknown, intra-plastid, mitochondria, and peroxisome (van Wijk et al., 2014). The study of phosphoprotein compartmentalization supports the hypothesis that a ﬁne mechanism helps to maintain and regulate protein translation, post-translational metabolism, signaling, and traﬃcking through the cells (Millar and Taylor, 2014). Some studies have already started to focus on PTMs in subcellular compartments and here we describe a few examples. g g Abscisic acid (ABA) is a phytohormone that plays an important role in many aspects of plant life. For example, ABA is essential for regulating seed maturation and stomatal closure under abiotic and biotic stresses (Hubbard et al., 2010). Protein phosphorylation and dephosphorylation play a central role in ABA signaling. Multiple signaling components have been found to undergo phosphorylation/dephosphorylation regulation to control stomatal movement in response to ABA (Zhang et al., 2014). A simpliﬁed ABA signaling model consists of the soluble ABA receptors (members of the pyrabactin resistance 1 (PYR1) and PYR1-like (PYL) proteins, also known as regulatory component of ABA receptor (RCAR) family, and collectively referred to as PYR/PYL/RCAR), a subgroup of type 2C protein phosphatases (PP2Cs), and the SNF1-related protein kinase 2 (SnRK2) family (Umezawa et al., 2010). Umezawa et al. (2013) studied protein phosphorylation networks in ABA signaling using phosphoproteomics of Arabidopsis treated with ABA and dehydration stress, as well as snrk2 mutants to identify SnRK2-dependent protein components. Phosphoproteomics of Signal Transduction Protein phosphorylation in signal transduction is an important area of current plant biology research. Many key proteins such as kinases, transcription factors, and ubiquitin ligases function through reversible protein phosphorylation in the signal transduction cascade (Hunter, 2000). In recent years, it has become apparent that analysis of signaling networks is required for the understanding of the dynamic and complex mechanisms underlying cellular functions and outputs. Most of the studies in plants have often been focused on protein kinases and identiﬁcation of the phosphorylated substrates. The mitogen-activated protein kinases (MAPKs) constitute one of the most important signaling mechanisms in plants, and they play essential roles in linking the perception of diﬀerent stimuli with cellular adaptive responses. The MAPK signal transduction pathways are evolutionarily conserved in all eukaryotic organisms such as plants, yeast, fungi, insects, nematodes, and mammals (Mishra et al., 2006). A MAPK cascade is minimally composed of distinct combinations of at least three protein kinases: a MAPKKK (MAP3K), a MAPKK June 2015 \| Volume 6 \| Article 430 4 Li et al. Phosphoproteomics and applications FIGURE 1 \| A typical mitogen-activated proteins (MAP) kinase cascade. The MAPK cascades are generally organized as modular pathways, in which the activation of upstream MAPKKKs leads to the sequential phosphorylation and subsequent activation of downstream MAPKKs and MAPKs. FIGURE 1 \| A typical mitogen-activated proteins (MAP) kinase cascade. The MAPK cascades are generally organized as modular pathways, in which the activation of upstream MAPKKKs leads to the sequential phosphorylation and subsequent activation of downstream MAPKKs and MAPKs. activated proteins (MAP) kinase cascade. The MAPK cascades are generally organized as modular pathways, in which the s leads to the sequential phosphorylation and subsequent activation of downstream MAPKKs and MAPKs. particular, this study provided insights into the ABA signaling pathway by identifying potential substrate proteins of SnRK2s (Umezawa et al., 2013). method was successfully applied to identify MAPK substrates. A large number of novel phosphorylation sites and 141 MAPK substrate candidates (mostly novel) have been identiﬁed. For example, time for coﬀee (TIC) and non-phototropic hypocotyl 3 (NPH3), which are involved in circadian clock and phototropism, were found to be MPK3/6 substrates. The result suggests that plant circadian rhythm and phototropism may be regulated by the MAPK signaling network. Frontiers in Plant Science \| www.frontiersin.org Phosphoproteomics of Subcellular Compartments Recently, phosphorylation of Sec31 by a casein kinase 2 was found to control the duration of COPII vesicle formation, decrease its association with ER and promote ER-to-Golgi traﬃcking (Koreishi et al., 2013). Subcellular proteomics can address conserved mechanisms underlying plant responses to stresses. By analyzing the phosphorylation changes in proteins of microsomal fractions from A. thaliana and Oryza sativa, Chang et al. (2012) found similar phosphoproteins between the species including photosystem II reaction center protein H PsbH. Both Arabidopsis and rice showed an increased ratio for a diphosphorylated peptide (ApTQpTVEDSSRSGPR) of PsbH as a response to salt stress. Interestingly, the two phosphorylation sites (Thr2 and Thr4) are found to be evolutionarily conserved in many plants using sequence alignment. y gg g g Plant vegetative growth is important for biomass accumulation and potential biofuel applications. A recent phosphoproteomic study of Brachypodium distachyon as a model biofuel plant using TiO2 enrichment and LC-MS/MS has identiﬁed a total of 1470 phosphorylation sites in 950 phosphoproteins (Lv et al., 2014). Among the phosphoproteins, there were 58 transcription factors, 84 protein kinases, 8 protein phosphatases, and 6 cellulose synthases. Through bioinformatic analysis, a protein kinase and phosphatase centered network related to rapid vegetative growth was deciphered. For example, a MAPK signaling cascade might play an important role in leaf growth and development (Lv et al., 2014). This ﬁnding is very interesting, considering MAPK cascade is generally involved in plant stress responses (Mishra et al., 2006). Light plays a crucial role in the regulation of protein phosphorylation in photosynthetic thylakoid membranes. In Arabidopsis, the thylakoid Ser/Thr protein kinase 7 (STN7) and STN8 kinases are light regulated and participate in phosphorylation of thylakoid membrane proteins and stroma proteins. Ingelsson and Vener (2012) performed a thylakoid phosphoproteomics study using Arabidopsis wild-type and the STN mutants stn7, stn8, and stn7stn8. The results showed that STN7 is required for the phosphorylation of pTAC16 at the Thr451, and pTAC16 was found to be distributed between thylakoids and nucleoid. In addition, the results suggest that pTAC16 could anchor DNA to the thylakoid membrane, and it was proposed that STN7-dependent phosphorylation of pTAC16 may regulate membrane-anchoring functions of the nucleoid. Phosphoproteomics of Subcellular Compartments Comparative analysis between ABA treatment and dehydration stress revealed that dehydration stress induced multiple protein phosphorylation pathways in addition to the ABA-dependent pathway, supporting that multiple protein kinases are involved in dehydration stress signaling, including SnRK2s, MAPKs, and calcium-dependent protein kinases (CDPKs) (Umezawa et al., 2013). Further studies will be required for understanding how multiple kinases mediate dehydration stress signaling. It appeared that subclass III SnRK2s may be uniquely employed during ABA responses, and subclass II SnRK2s are the main subclass that regulates dehydration stress responses, although they are also activated by ABA. By integration of genetics with phosphoproteomics, it is possible to connect protein kinases with their in vivo signaling pathways. In Jones et al. (2009) performed a phosphoproteomic analysis of the nuclei-enriched fractions prepared from suspension cell cultures and seedlings of A. thaliana. The work led to the identiﬁcation of 416 phosphopeptides from 345 proteins. Two thirds of the proteins are known or predicted to be nuclear localized, and one half of the nuclear localized proteins have novel phosphorylation sites. Many phosphorylation sites and Frontiers in Plant Science \| www.frontiersin.org June 2015 \| Volume 6 \| Article 430 5 Phosphoproteomics and applications Li et al. regulators, respectively (Seo et al., 2006). Many protein kinases and phosphatases participate in ABA signaling to regulate seed germination. Recently, Han et al. (2014) used PolyMAC phosphopeptide enrichment and gel-free proteomics identiﬁed a total of 933 phosphorylated peptides corresponding to 413 proteins in rice embryos during early stages of germination. By quantitative normalization of phosphoprotein abundance and One-Way ANOVA testing, 149 phosphorylated proteins were found to be signiﬁcantly changed in abundance during germination. Among the phosphoproteins, seven brassinosteroid (BR) signaling pathway-related proteins were identiﬁed and three (BR signaling kinase 1, BR-insensitive 2, and BR-insensitive 1 suppressor 1) showed signiﬁcant increases in phosphoprotein abundance during the early stages of germination. In addition, treatment with brassinolide promoted the rice seed germination. These results suggest that brassinosteroid signal transduction plays an important role in triggering seed germination. kinase motifs were identiﬁed on proteins involved in nuclear transport (e.g., Ran-associated proteins), and on transcription factors, chromatin remodeling proteins, and spliceosome components. Surprisingly, many novel phosphopeptides from proteins involved in vesicle traﬃcking such as components of the exocyst complex (SEC10, SEC51, and SEC5a-like) were identiﬁed. How phosphorylation of these SEC proteins plays a role in vesicle traﬃcking is intriguing. Identiﬁcation and Functional Analysis of Novel Phosphorylation Sites The identiﬁcation of protein phosphorylation sites has been diﬃcult in the past. Nowadays, high throughput modern technologies such as tandem MS have promoted large-scale discoveries of new phosphorylation sites and phosphoproteins in recent years. Rao and Møller (2012) initiated a large-scale study of phosphorylation site occupancy in eukaryotic proteins. They analyzed the occurrence and occupancy of phosphorylation sites in a large number of eukaryotic proteins, and provided insights into protein phosphorylation and related processes. Phosphorylation probability was found to be much higher in both termini of protein sequences (much more in the C-terminus) than middle parts of the sequences. A large proportion (51.3%) of the occupied sites had a nearby phosphorylation within a distance of 10 amino acid residues. This proportion is very high compared to the expected value of 16.9%. More than half of the phosphorylated sites fall within a small number of motifs. Phosphoproteomics of Plant Growth and Development p Sucrose non-fermenting 1 related kinase (SnRK1) acts as a sensor of energy levels in plant development, and regulates plant growth by maintaining energy homeostasis during stress conditions (Tsai and Gazzarrini, 2014). It is activated by sugar depletion, energy depletion in the dark and hypoxia (Baena- González and Sheen, 2008). Trehalose 6-phosphate (T6P) is a signaling molecule involved in the regulation of embryonic and vegetative development, ﬂowering time, and meristem determinacy. An increase in the levels of T6P led to metabolic changes that promote plant growth. However, T6P regulates SnRK1 by inhibiting its activity. SnRK1 and T6P are global regulatory molecules that also interact with plant hormones, and along with ABA modulate several crucial cellular activities such as seed maturation and germination, ABA sensitivity and signaling, vegetable growth, and ﬂowering regulation (Tsai and Gazzarrini, 2014). Seed germination is known to be controlled by phytohormones, including gibberellins (GAs) and ABA, which play antagonistic roles as positive and negative A large phosphoprotein, the RNA surveillance protein UP- frameshift 1 (Upf1) in Saccharomyces cerevisiae, has only been partially characterized for phosphorylation sites, but the functional relevance of the phosphorylation has not been studied. Lasalde et al. (2014) used tandem MS and in vitro phosphorylation assays to identify novel phosphorylation sites June 2015 \| Volume 6 \| Article 430 Frontiers in Plant Science \| www.frontiersin.org 6 Phosphoproteomics and applications Li et al. in UPF1. A total of 11 phosphorylated residues of UPF1 were identiﬁed. Sequence alignment of UFP1 from lower and higher eukaryotes showed complete conservation of the phosphorylated residue Y-754. Residues corresponding to S. cerevisiae UPF1 T-194, S-492, Y-738, and S-748 were similar to those in the homologs of Homo sapiens, Mus musculus, Drosophila melanogaster, and A. thaliana. Since the phosphorylated residues in UPF1 were clustered in four small regions, each one was tested to determine its importance by independently deleting the four individual regions. The deletion mutant lacking phospho-motif- 4 was not able to complement the Nonsense-Mediated mRNA Decay (NMD) defect as revealed by Northern blot analysis. To test the role of phospho-motif-4 in translation termination eﬃciency, a well-established dual luciferase assay was used. The deletion-mutant lacking phospho-motif-4 was not able to rescue this defect, indicating that this motif has a role in translation termination eﬃciency. To dissect the sequences within phospho- motif-4 required for NMD activity, PCR-mediated mutagenesis was used to generate three additional deletion mutants (736–745, 746–750, 751–751). Phosphoproteomics of Plant Growth and Development The results revealed that deletion of residues 736–745 reduced NMD activity as measured by Northern blot analysis. To test the functional role of Y738 and Y742, site- directed mutagenesis was used to create phosphorylation mimic mutants. Interestingly, the Y738F and Y742F fully rescued NMD activity of a chromosomal UPF1 deletion-mutant strain, indicating that they are not compromised in their ability to function in NMD. These results provided strong evidence that UPF1’s ability to promote translational termination ﬁdelity is depended on the conserved C-terminal phosphorylation motif, which is important for its NMD activity. greatly improved over the years. For instance, combining the titanium (Ti4+)-based IMAC and the reverse phase (RP)- strong cation exchange (RP-SCX) biphasic trap column-based online RPLC is a great example of the advancements (Bian et al., 2012; Wang et al., 2013b). The recent development of speciﬁc labeling techniques has greatly aided the quantiﬁcation of phosphorylation proﬁles and their stress-induced changes. Especially, iTRAQ and TMT in vitro labeling and SILAC in vivo labeling have shown to be successful in combination with IMAC and MS (Isner et al., 2012; Yang et al., 2013; Zhang et al., 2013; Stecker et al., 2014). These studies have revealed novel nodes and edges in signaling pathways and regulatory processes that are dependent on phosphorylation. Despite many new insights gained from quantitative phosphoproteomics, improvements are required to enable a comprehensive description of total and PTM proteomes. Currently, LC-MS/MS based phosphoproteomic technologies have established as an indispensable tool in identiﬁcation of novel phosphorylation sites and signaling pathways. As large data sets accumulate, informatics tools will be indispensable, e.g., informatics has revealed phosphorylation probability to be frequent at the termini of protein sequences. Taken together, researchers have provided not only new insights into the complex phosphorylation regulatory networks in plants, but also important resources for future functional studies of protein phosphorylation in plant growth and development. Concluding Remarks Phosphopeptide enrichment and MS have been essential tools for studying protein phosphorylation. It is challenging to directly detect phosphoproteins in biological samples due to the low abundance and low stoichiometry of phosphorylation in diﬀerent biological processes. The enrichment methods of phosphoproteins/phosphopeptides from complex mixtures have Acknowledgments Research in the HL lab was supported by the National Science Foundation of China (Project 31471552: The response of antioxidant enzymes to salt stress in sugar beet M14, and Project 31401441: Identiﬁcation of root variation related proteins in sugar beet (Beta vulgaris L.) monosomic addition line M14 using iTRAQ analysis), the National Science Foundation of Heilongjiang Province (Project C201202: Comparative proteomics analysis of sugar beet M14 under salt stress), and the Common College Science and Technology Innovation Team of Heilongjiang Province. The paper represents serial 016 from our innovation team at the Heilongjiang University (Hdtd2010-05). Chen, W. G., and White, F. M. (2004). Proteomic analysis of cellular signaling. Expert Rev. Proteomics 1, 343–354. doi: 10.1586/14789450.1.3.343 Chen, W. G., and White, F. M. (2004). Proteomic analysis of cellular signaling. Expert Rev. 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Proteomics 5, 914–922. doi: 10.1074/mcp.T500041-MCP200 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. 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. Tsai, A. Y., and Gazzarrini, S. (2014). Trehalose-6-phosphate and SnRK1 kinases in plant development and signaling: the emerging picture. Front. Plant Sci. 5:119. doi: 10.3389/fpls.2014.00119 Copyright © 2015 Li, Silva-Sanchez, Zhang, Chen and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Umezawa, T., Nakashima, K., Miyakawa, T., Kuromori, T., Tanokura, M., Shinozaki, K., et al. (2010). Molecular basis of the core regulatory network in ABA responses: sensing, signaling and transport. Plant Cell Physiol. 51, 1821–1839. doi: 10.1093/pcp/pcq156 June 2015 \| Volume 6 \| Article 430 Frontiers in Plant Science \| www.frontiersin.org 9
https://openalex.org/W4362415107	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Table_4_from_Sixteen_Kinase_Gene_Expression_Identifies_Luminal_Breast_Cancers_with_Poor_Prognosis/22374626/1/files/39819866.pdf	English	null	Supplementary Table 4 from Sixteen–Kinase Gene Expression Identifies Luminal Breast Cancers with Poor Prognosis	null	2,023	cc-by	101,697	Supplementary table 4 : Expression data of the 435 kinase genes in 227 breast cancer samples. Probe sets ID Gene symbol * 10003 8525 3220 4814 8009 8406 9377 6137 6165 4700 5731 7264 7371 7641 7767 7809 4186cc 4568cc * Gene annotation based on Affymetrix, May 2007 5350 6238 10798 10639 11442 5771cc 5142cc 8862 2933 5520 6202 8524 10958 11485 11568 7004 5186cc 5901cc 9345 10959 12235 10090 10621 11756 11895 9077 9127 9725 9752 9941 9961 9966 8700 3401 6361 5257 7499 8600 12030 12698 9317 9737 9893 6453 6972 3824 6162 12635 13018 13045 7412 10339 10502 10980 11245 10797 11305 11350 8667 8772 8780 9992 7720 9507 9721 10181 7501 7647 10172 10314 11280 6142 6604 7729 8295 8847 9394 9983 9357 8796 9888 7810 7913 7942 7961 8781 8459 5342 8873 9059 9425 9622 10033 9840 9745 9349 9215 9207 8867 8794 8358 8188 7876 7676 5532 4641 10112 6010 12477 13035 9948 12478 12710 12423 7780 9822 13008 12854 12502 9296 12652 9358 8500 5836 13148 13180 10398 6823 9934 6244 13591 11348 11614 13209 13319 11041 10622 14029 10711 11029 9779 10729 13507 10550 10549 10517 13469 13784 3070 10982 2352 10684 3234cc 6231 9445 14663 2163cc 5397 11525 11911 11707 2661cc 9670 12521 9595 4402cc 4157cc 11604 3347cc 2730cc 12520 9063 8850 9091 11135 11979 12080 5521cc 12232 5754cc 8035 4981cc 6107cc 12115 9760 2227cc 5188cc 6345 12297 10552 11982 8581 11360 5119cc 14972 4593cc 3736cc 3737cc 5057cc 5437cc 5117cc 3764cc 10054 8595 8536 7420 5796 4630 3458 1007_s_at DDR1 873.49 1159.64 913.27 1696.75 1893.6 1248.59 975.66 1515.28 1348.24 1067.11 1438.3 1711.77 1000.8 1113.2 1093.54 460.7 1309.46 787.44 514.39 583.19 1175.55 1157.44 789.8 603.89 890.88 536.44 1260.27 1685.35 684.06 631.37 856.52 1805.52 970.95 2645.93 665.61 895.6 1048 1398.98 446.07 586.07 1235.32 1065.91 783.97 1177.91 1272.08 548.71 1440 1044.61 1542.75 1225.02 1453.36 1626.31 1160.19 1045.55 2040.07 942.32 965.94 1134.59 1459.82 1262.27 1114.78 884.13 1672.19 969.19 60.28 847.06 1303.33 329.69 941.09 889.04 667.72 797.42 826.54 674.73 1179.22 1085.6 978.7 649.82 924.06 575.34 1198.39 991.07 1465.3 1523.18 940.69 553.82 1212.92 629.56 602.96 1004.24 921.35 882.48 1091.83 551.36 727.55 683.3 1985.41 1320.08 768.28 647.26 1080.97 772.81 1137.02 779.51 937.9 940.55 1143.8 1351.43 2797.62 867.47 945.83 961.59 465.36 691.15 720.92 633.07 1719.63 1013.68 1323.13 1240.7 900.83 1127.93 426.58 1159.08 330.9 1295.59 967.85 594.24 516.13 751.96 513.74 1067.43 674.61 784.19 935.95 761.25 879.45 564.61 1322.27 682.46 669.97 957.33 634.72 1632.36 1012.23 863.93 823.97 785.62 1318.64 813.02 885.5 817.76 878.51 878.69 1235.67 1028.75 1102.51 373.66 976.47 729.6 1590.12 1007.66 755.56 1596.34 1440.32 1330.92 1272.95 776.95 852.1 1134.6 715.74 955.19 1106.38 853.1 452.35 1120.1 593.4 856.86 699.01 828.39 438.91 677.35 1557.95 812.94 1060.15 2209.08 1346.66 2140.04 757.45 1373.02 1227.89 568.9 896.44 1022.02 1023.16 975.17 1480.3 1483.97 986.73 629.91 2507.63 1117.44 889.48 647.9 1334.37 1253.18 752.12 670.91 952.98 1114.15 701.43 918.7 1593.26 887.6 870.06 1375.66 1311.56 619.65 993.15 1186.89 869.47 1465.74 1200.35 1071.9 981.3 1008.02 697.29 1552395_at TSSK3 85.09 70.35 77.18 91.44 61.93 83.46 88.56 78.31 86.31 67.06 63.56 65.19 61.57 77.44 67.43 87.48 102.2 58.9 60.76 67.87 79.74 69.42 5350 6238 10798 10639 11442 5771cc 5142cc 8862 2933 5520 6202 8524 10958 11485 11568 7004 5186cc 5901cc 9345 10959 12235 10090 10621 11756 11895 9077 9127 9725 9752 9941 9961 9966 8700 3401 6361 5257 7499 8600 12030 12698 9317 9737 9893 6453 6972 3824 6162 12635 13018 13045 7412 10339 10502 10980 11245 10797 11305 11350 8667 8772 8780 9992 7720 9507 9721 10181 7501 7647 10172 10314 11280 6142 6604 7729 8295 8847 9394 9983 9357 8796 9888 7810 7913 7942 7961 8781 8459 5342 8873 9059 9425 9622 10033 9840 9745 9349 9215 9207 8867 8794 8358 8188 7876 7676 5532 4641 10112 6010 12477 13035 9948 12478 12710 12423 7780 9822 13008 12854 12502 9296 12652 9358 8500 5836 13148 13180 10398 6823 9934 6244 13591 11348 11614 13209 13319 11041 10622 14029 10711 11029 9779 10729 13507 10550 10549 10517 13469 13784 3070 10982 2352 10684 3234cc 6231 9445 14663 2163cc 5397 11525 11911 11707 2661cc 9670 12521 9595 4402cc 4157cc 11604 3347cc 2730cc 12520 9063 8850 9091 11135 11979 12080 5521cc 12232 5754cc 8035 4981cc 6107cc 12115 9760 2227cc 5188cc 6345 12297 10552 11982 8581 11360 5119cc 14972 4593cc 3736cc 3737cc 5057cc 5437cc 5117cc 3764cc 10054 8595 8536 7420 5796 4630 3458 1007_s_at DDR1 873.49 1159.64 913.27 1696.75 1893.6 1248.59 975.66 1515.28 1348.24 1067.11 1438.3 1711.77 1000.8 1113.2 1093.54 460.7 1309.46 787.44 514.39 583.19 1175.55 1157.44 789.8 603.89 890.88 536.44 1260.27 1685.35 684.06 631.37 856.52 1805.52 970.95 2645.93 665.61 895.6 1048 1398.98 446.07 586.07 1235.32 1065.91 783.97 1177.91 1272.08 548.71 1440 1044.61 1542.75 1225.02 1453.36 1626.31 1160.19 1045.55 2040.07 942.32 965.94 1134.59 1459.82 1262.27 1114.78 884.13 1672.19 969.19 60.28 847.06 1303.33 329.69 941.09 889.04 667.72 797.42 826.54 674.73 1179.22 1085.6 978.7 649.82 924.06 575.34 1198.39 991.07 1465.3 1523.18 940.69 553.82 1212.92 629.56 602.96 1004.24 921.35 882.48 1091.83 551.36 727.55 683.3 1985.41 1320.08 768.28 647.26 1080.97 772.81 1137.02 779.51 937.9 940.55 1143.8 1351.43 2797.62 867.47 945.83 961.59 465.36 691.15 720.92 633.07 1719.63 1013.68 1323.13 1240.7 900.83 1127.93 426.58 1159.08 330.9 1295.59 967.85 594.24 516.13 751.96 513.74 1067.43 674.61 784.19 935.95 761.25 879.45 564.61 1322.27 682.46 669.97 957.33 634.72 1632.36 1012.23 863.93 823.97 785.62 1318.64 813.02 885.5 817.76 878.51 878.69 1235.67 1028.75 1102.51 373.66 976.47 729.6 1590.12 1007.66 755.56 1596.34 1440.32 1330.92 1272.95 776.95 852.1 1134.6 715.74 955.19 1106.38 853.1 452.35 1120.1 593.4 856.86 699.01 828.39 438.91 677.35 1557.95 812.94 1060.15 2209.08 1346.66 2140.04 757.45 1373.02 1227.89 568.9 896.44 1022.02 1023.16 975.17 1480.3 1483.97 986.73 629.91 2507.63 1117.44 889.48 647.9 1334.37 1253.18 752.12 670.91 952.98 1114.15 701.43 918.7 1593.26 887.6 870.06 1375.66 1311.56 619.65 993.15 1186.89 869.47 1465.74 1200.35 1071.9 981.3 1008.02 697.29 1552395_at TSSK3 85.09 70.35 77.18 91.44 61.93 83.46 88.56 78.31 86.31 67.06 63.56 65.19 61.57 77.44 67.43 87.48 102.2 58.9 60.76 67.87 79.74 69.42 67.76 95.84 82.14 82.62 82.81 73.58 81.45 76.83 70.28 81.79 84.9 64.29 89.31 102.46 79.98 57.02 63.94 73.59 62.98 57.35 69.03 64.97 65.2 59.23 87.32 61.59 115.63 72.99 88.72 57.96 65 63.21 69.8 77.85 80.1 89.09 58.09 97.01 95.5 95.23 86.74 58.7 61.78 64.9 51.46 54.48 57.33 55.56 63.7 53.41 61.57 53.75 63.68 70.49 66.25 63.24 78.57 74.57 75.03 70.4 67.99 69.76 65.97 59.57 76.84 60.96 64.66 59.42 71.32 67.3 57.98 58.5 64.48 48.22 83.91 72.32 50.51 58.24 76.95 61.09 82.77 61.64 51.74 66.54 53.53 63.56 64.52 86 45.55 42.67 58.4 51.18 47.93 53.43 55.38 56.31 46.68 44.71 60.01 54.45 72.41 76.35 53.07 57.25 51.96 46.2 51.48 51.02 44 52.8 57.26 61.75 47.93 49.51 66.1 56.71 61.18 72.22 59.75 68.2 58.33 62.61 58.72 67.16 66.18 64.77 55.16 54.39 64.56 68.02 71.4 64.26 56.01 56.71 57.79 54.98 55.83 66.55 63.44 64.63 76.47 71.45 86.08 75.34 64.35 74.25 79.65 67.47 54.65 51.88 55.15 59.93 77.04 60.69 51.24 63.89 36.69 52.55 68.03 71.76 64.65 62.14 61.95 65.04 66.1 61.32 68.99 58.86 58.48 65.49 50.67 48.32 47.67 51.81 48.69 46.91 41.71 42.83 48.65 44.94 45.69 70.86 55.17 53.46 48.98 67.88 53.37 51.16 57.08 48.07 57.23 51.09 57.58 55.91 61.48 65.31 65.16 59.99 51.65 57.5 50.2 56.18 50.59 55.61 67.87 1552504_a_at BRSK1 85.56 65.74 92.76 124.7 64.48 78.81 119.96 78.31 75 95.52 81.12 70.6 69.41 95.8 69.67 102.92 61.36 90.1 68.43 74.84 68.15 76.83 59.6 103.02 69.26 80.52 74.01 84.86 95.02 131.23 52.38 91.24 71.48 90.93 90.2 87.37 101.53 90.62 117.02 120.52 85.97 97.81 90.96 112.65 124.46 79.07 97.16 97 102.94 81.48 101.92 96.87 90.99 115.98 110.07 71.51 114.05 120.97 101.96 97.45 117.12 122.49 84.35 111.67 97.31 89.67 96.05 101.98 117.66 76.5 118.97 96.78 87.22 94.98 92.04 81.84 74.5 81.01 99.23 109.01 108.41 51.57 66.82 63.91 56.88 67.35 72.74 70.57 71.29 83.96 191.19 82.26 80.66 78.88 76.74 99.11 68.39 73.78 79.33 73.15 70.99 61.4 83.66 66.65 76.91 97.77 72.89 67.08 92.9 121.54 110 75.72 90.54 111.42 113.93 92.66 144.12 116.52 108.89 81.38 103.12 101.77 144.41 87.28 58.42 80.45 55.5 56.88 52.37 58.41 63.47 66.27 83.01 87.61 54.83 76.19 76.39 67.75 62.06 68.78 61.21 51.44 60.75 46.35 70.25 65.22 50.93 88.18 70.84 62.26 78.42 96.39 90.68 88.72 80.24 123.87 83.2 128.72 70.97 68.2 82.84 71.97 86.38 99.02 89.54 88.31 76.23 86.32 176.07 101.09 113.99 121.18 87.89 99.95 183.03 106.6 97.18 106.67 101.13 121.88 93.13 129.31 78.38 93.31 94.19 116.73 109.96 76.81 100.15 105.66 100.24 84.18 131.23 123.5 96.15 124.53 102.65 126.21 120.6 118.09 126.75 121.32 108.13 177.5 112.41 116.1 125.77 122.73 155.4 107.02 100 125.75 107.57 87.35 64.45 88.23 71.49 87.67 78.6 80.89 66.6 45.52 52.69 52.95 69.87 52.41 35.24 1552519_at ACVR1C 118.14 31.51 40.86 41.1 56.71 48.15 21.61 19.32 26.32 28.19 27.41 29.6 70.09 17.93 33.42 16.83 31.74 26.7 32.37 26.95 15.53 20.5 28.61 21.02 38.57 25.04 31.06 33.28 45.1 43.62 26.83 18.18 21.42 18.9 23.47 20.49 29.08 20.05 21.91 24.19 14.66 57.86 21.79 20.53 19.11 46.22 25.57 22.72 21.22 13.03 36.82 25.62 25.87 27.51 19.53 19.65 20.44 27.56 47.12 28.01 15 22.82 85.62 24.07 12.8 24.22 21.44 16.39 20.38 20.08 30.7 48.86 21.83 60.39 21.31 26.84 36.27 33.17 21.05 37 161.05 19.48 37.74 22.55 492.46 185.22 27.42 101.68 24.96 104.46 204.36 60.33 35.4 19.03 152.38 31.01 25.25 28.63 77.05 59.79 74.31 169.97 36.99 70.33 34.19 32.76 33.6 24.79 36.88 23.09 82.7 77.09 36.11 95.74 35.3 86.05 20.38 44.68 53.61 46.39 186.45 46.91 47.55 76.65 54.95 20.57 72 164.98 36.18 41.7 193.09 29.9 381.81 76.03 51.44 29.31 24.34 62.98 21.63 18.89 136.16 46.05 49.6 32 29.83 28.49 28.34 47.62 59.58 24.37 47.99 41.69 25.77 20.49 24.87 30.04 25.48 26.38 24.03 18.62 41.1 33.27 23.11 24.24 22.6 29.66 31.46 24.22 24.53 19.6 71.11 47.36 55.05 27.74 21.49 36.45 25.39 15.23 40 19.23 45.49 19.72 31.61 26.49 57.86 27.52 24.38 24.78 17.29 35.62 16.16 32.5 17.97 23.41 141.2 25.92 39.01 64.55 48.54 17.74 18.43 16.27 24.67 14.72 26.29 39.6 25.57 20.51 26.15 20.29 48.78 61.49 114.31 48.57 33.86 43.76 139.82 37.98 19.74 50.11 46.51 80.37 53.91 54.7 63.81 30.41 187.76 1552578_a_at MYO3B 25.75 5.64 7.11 7.54 5.91 44.85 6.34 5.35 5.77 5.68 6.31 5.71 6.23 5.91 5.52 5.44 7.99 8.85 7.7 5.7 6.12 5.69 14.06 5.33 6.61 5.55 5.66 49.67 8.05 6.46 5.85 7.27 5.57 12.57 6.37 6.06 5.74 8.7 7.28 11.3 15.14 7.05 6.47 6.57 9.6 6.01 8.54 6.15 13 6.16 9.05 7.65 7.37 9.17 6.24 5.52 5.86 23.92 5.75 7.07 6.2 14.91 11.33 8.47 6.48 6.67 6.67 6.45 12.8 6.06 93.53 9.77 7.75 6.37 7.04 7.37 7.39 7.42 11.94 8.08 10.12 230.17 8.26 6.98 5.88 5.45 5.45 41.22 5.36 6.83 7.01 5.83 6.53 5.9 7.18 7.08 7.04 8.43 6.53 16.33 8.76 7.08 6.51 6.7 9.91 8.07 8.82 9.9 9.53 39.89 16.79 8.67 17.33 9.08 7.9 12.21 68.4 9.33 8.71 9.72 8.42 12.25 9.79 9.58 6.63 6.18 6.41 6.35 7.55 13.24 6.22 7.3 6.39 7.52 6.88 6.13 6.14 7.57 7.98 6.92 6.1 6.77 6.61 7.7 6.4 6.56 6.73 7.16 7.44 75.73 7.56 8.1 7.4 7.13 7.23 8.28 7.64 7.44 7.35 7.22 7.48 9.73 117.83 7.02 7.15 7.98 6.87 7 7.42 8.23 8.3 7.7 7.09 7.34 9.12 8.1 7.97 7.56 10.93 18.21 8.56 9.34 8.18 8.07 8.72 7.91 16.6 7.92 9.46 14.03 7.76 10.13 6.01 5.67 7.27 7.12 19.35 8.46 6.94 6.4 5.99 7.72 6.55 5.39 11.26 7.93 6.95 7.97 5.81 7.14 22.85 7.95 6.65 7.01 9.67 6.69 80.53 7 6.93 6.29 18.96 37.2 7.01 6.95 15.66 7.94 9.25 1553114_a_at PTK6 154.63 201.15 110.69 173.93 591.46 174.8 333.59 107.41 136.21 194.88 65.08 67.41 114.98 189.85 109.91 83.89 107.8 84.41 67.66 71.95 106.68 89.01 101.67 103.48 96.21 82.06 195.82 72.3 90.48 244.22 78.55 123.36 141.83 203.61 145.51 87.76 81.63 80.2 102.26 112.04 131.97 120.14 155.07 189.52 165.84 78.28 100.11 90.8 108.92 313.61 109.88 112.55 262.56 119.34 244.75 300.7 106.7 79.31 200.83 200.86 166.75 85.43 104.58 203.25 112.79 156.21 70.08 65.61 87.55 64.38 93.03 110.1 142.09 180.86 142.49 138.57 84.87 95.66 225.94 179.77 180.73 178.12 114.98 264.93 120.46 150.52 95.52 108.41 139.97 170.98 170.76 101.29 254.55 172.75 183.02 126.35 193.58 292.45 152.09 98.36 63.87 142.97 128.66 158.78 111.05 166.71 105.73 113.13 296.3 291.6 67.75 58.46 77.39 140.35 156.59 108.17 238.71 92.34 101.45 226.96 140.44 102.19 101.76 93.46 81.28 115.78 172.47 61.41 146.42 78.69 85.43 224.31 110.35 118.51 111.66 136.16 104.03 161.82 180.32 130.25 124.09 120.85 100.52 134.88 116.01 193.39 157.86 115.53 138.94 124.1 70.59 121.18 241.45 87.08 140.77 107.78 134.83 223.46 99.97 94 199.68 112.4 145.42 214.01 158.18 248.76 119.07 115.59 318.47 180.34 248.37 141.75 222.71 101 102.27 235.6 107.27 132.54 58.94 115.2 78.25 201.35 144.31 180.14 117.42 162.05 134.45 132.55 243.54 202.22 156.79 89.08 197.54 439.68 81 144.96 83.38 128.73 520.38 161.91 208.5 166.97 115.06 150.36 98.33 135.65 72.81 101.98 143.87 101.55 147.54 185.04 91.38 178.93 220.06 142.01 112.45 144.62 130.68 96.26 146.36 108.76 136.77 100.75 168.85 92.22 219.2 1554112_a_at ULK2 36.88 64.48 48.51 56.15 50.12 94.3 80.85 230.1 69.44 77.32 58.62 113.22 68.83 147.96 25.88 65.23 24.83 23.05 41.92 152.22 27.5 36.35 92.72 27.61 50.04 31.46 73.97 21.98 31.49 73.68 47.54 13.41 106.41 158.58 21.29 34.68 23.38 30.14 31.17 25.4 45.97 37.1 31.68 44.59 28.67 40.11 47.06 62.95 126.31 66.6 37.41 47.75 73.81 37.9 179.04 88.33 24.5 81.66 22.02 29.84 135.62 30.44 52.11 24.94 17.04 35.86 24.34 24.27 57.25 37.62 30.48 24.52 24.69 176.72 38.98 20.04 186.2 29.35 30.28 26.09 33.84 35.51 39.35 41.74 47.09 69.26 32.14 45.67 34.29 21.96 22.19 39.84 21.23 29.59 18.35 14.44 24.69 31.85 30.1 36.59 42.62 31.89 32.07 19.97 25.24 58.91 49.61 68.78 41.84 31.1 25.78 41.26 60.69 41.58 35.87 30.71 33.84 21.11 21.73 28.71 35.1 32.59 58.61 19.77 28.13 83.22 73.03 34.94 18.02 25.62 21.6 36.32 27.61 25.36 20.07 32.07 83.26 20.05 35.79 22.71 32.67 17.89 28.72 78.04 29.88 24.55 24.56 109.98 29.06 93.73 62.08 43.23 23.19 48.76 67.68 62.27 57.52 23.58 48.5 32.59 71.82 41.13 100.02 42.32 71.93 47.83 81.15 28.01 29.06 31.57 32.31 26.65 36.69 28.33 16.23 31.48 22.16 39.59 19.8 90.28 23.92 25.22 41.41 29.38 19.54 23.73 24.69 94.38 22.93 52.51 21.75 38.62 42.27 25.3 34.56 27.62 55.27 29.61 28.83 22.74 49.92 29.57 34.07 30.13 25.62 55.57 28.31 29.08 29.69 30.61 24.73 25.68 56.14 21.05 77.08 45.19 28.75 25.19 40.26 84.63 35 26.55 27.99 52.24 28.91 55.92 39.74 1555310_a_at PAK6 129.44 108.05 77.08 218.57 90.74 24.95 134.05 63.67 154.16 68.64 58.89 102.12 19.64 195.33 51.12 27.57 117.06 18.88 168.19 92.26 20.88 53.78 64.7 29.39 172.69 31.5 41.78 92.34 42.73 56.67 65.43 60 24.49 173.79 41.96 101.41 39.32 42.23 23.79 33.61 55.75 58.24 73.75 67.12 42.92 55.27 46.64 64.3 66.57 48.75 64.45 58.32 26.17 122.25 122.92 132.97 76.32 48.44 44.77 29.95 192.73 133.53 46.89 30.31 18.29 90.87 73.07 60.81 79.24 53.74 205.25 22.55 28.27 118.23 95.56 32.47 106.43 31.97 49.49 79.27 70.25 221.64 26.95 94.84 55.37 39.43 60.66 45.45 54.22 58.83 60.05 31.52 100.95 75.53 89.6 61.67 83.33 70.07 18.33 79.82 174.49 84.1 60.62 40.56 37.93 144.76 42.92 68.07 335.81 92.24 74.6 63.85 26.37 56.6 74.74 199.99 182.64 26.52 55.97 80.29 52.75 117.26 172.63 58.53 40.2 20.3 44.42 38.59 71.43 80.13 23.65 82.71 114.55 55.53 53.18 105.82 173.92 123.99 80.67 169.71 115.32 44.22 127.22 86.06 26.65 34.19 141.08 54.91 62.97 167.17 71.47 47.01 155.33 20.68 20.3 109.64 103.74 54.16 49.83 72.46 93.86 53.83 121.67 60.02 66.25 76.54 51.84 66.47 60.2 201.36 25.01 66.81 119.32 96.23 75.72 71.3 50.62 87.15 63.13 274.08 38.77 36.88 141.22 60.46 31.55 55.44 29.08 42.05 48.08 76.04 49.59 77.36 61.79 155.39 63.49 68.16 191.21 97 99.07 106.34 115.92 93.74 64.67 72.96 186.87 102.43 79.51 58.41 52.55 63.49 88.02 39.67 152.78 85.77 181.06 25.2 39.36 72.2 36.77 81.45 49.32 84.88 148.04 81.95 66.69 116.67 60.65 1555935_s_at HUNK 28.99 38.19 68.08 35.37 27.48 34.52 146.42 27.62 36.07 24.66 30.76 19.8 24.33 19.02 32.32 141.33 30.37 23.1 40.82 32.57 33.07 22.21 23.92 29.54 26.61 78.58 53.57 26.84 53.21 34.22 77.01 28.79 35.54 25.68 31.08 21.73 29.35 60.75 31.98 31.98 95.94 19.4 28.39 33.13 29.72 66.23 66.13 18.13 44.24 139.81 50.27 225.47 87.04 38.25 17.08 57.29 26.75 49.05 38.26 35.11 141.24 303.54 42.07 34.51 23.22 32.07 57.89 28 20.3 36.35 42.88 40.18 23.02 52.13 75.68 70.23 23.29 31.41 41.16 84.36 74.66 41.97 26.19 27.61 55.29 62.89 33.57 46.34 27.06 50.81 31.72 45.42 31.26 30.44 58.13 29.21 57.81 90.65 44.96 60.3 26.08 40.76 51.72 16.31 35.91 173.21 41.2 39.27 32.25 38.7 38.04 53.25 21.21 30.85 27.31 63 46.64 26.59 94.54 39.74 41.42 60.83 33.95 42 60.56 95.28 45.51 43.36 35.14 26.19 32.8 90.75 44.54 40.1 32.57 28.2 33.98 46.61 126.93 80.27 45.09 31.69 68 28.9 29.84 32.45 75.26 34.1 72.65 38.34 37.02 35.79 40.38 37.58 29.15 31.07 40.1 26.38 38.05 28.97 51.71 43.97 35.68 30.73 82.74 28.91 31.61 66.01 29.68 55.62 20.62 21.96 46.91 18.33 45.59 20.34 31.86 25.4 44.48 83.15 26.45 22.84 53.31 41.15 48.9 40.58 39.19 21.54 18.4 22.24 138.09 49.69 31.08 37.8 63.64 40.83 34.63 48.51 51.93 16.74 20.64 48.49 35.74 28.28 24.67 37.85 20.76 22.83 43.02 32.26 25.44 24.06 57.68 34.94 25.67 20.54 34.52 89.26 79.61 20.48 64.97 70.14 35.7 56.51 67.46 25.77 55.35 1556043_a_at TTN 16.95 20.83 48.11 17.55 39.78 40.73 40.6 19.21 22.8 27.18 25.89 79.13 27.49 34.14 43.77 53.02 12.52 30.61 26.16 31.42 31.4 18.5 48.92 21.92 29.04 39.95 28.61 37.21 39.16 23.92 27.56 34.91 62.34 96.69 13.54 25.25 20.34 23.35 38.53 21.42 15.41 12.01 25.09 31.08 34.63 37.87 29.64 22.28 31.69 43.1 10.72 47.79 30.64 17.1 50.78 24.89 10.95 18.91 24.46 57.7 31.74 22.04 18.77 16.58 23.89 10.82 16.88 15.27 12.09 18.56 42.95 17.7 41.84 27.65 11.07 12.24 14.3 16.67 15.98 23.87 21.35 13.2 12.81 26.39 33.98 28.31 33.61 40.44 21.8 30.11 43.74 41.45 28.69 54.03 31.57 22.91 51.22 55.41 42.57 59.35 30.82 87.49 56.01 32.18 26.06 37.94 53.91 35.36 86.54 59.76 10.63 62.4 50.85 55.59 46.6 164.65 33.25 53.48 54.22 44 79.06 38.78 41.16 22.43 45.26 22.28 29.97 42.87 25.56 47.96 45 29.48 61.22 26.12 36.91 58.08 20.96 44.81 48 26.28 28.21 32.49 20.59 29.03 26.46 46.51 29.61 40.03 45.9 49.03 24.66 44.9 26.73 37.98 28.27 23.2 23.97 41.56 45.4 53.64 48.14 51.73 54.17 55.3 34.28 28.35 37.11 39.26 18.26 26.53 27.93 36.83 70.69 17.8 51.6 21 31.51 63.75 31.07 23.55 52.88 19.6 30.85 30.8 30.09 67.75 40.42 54.19 23.46 58.99 37.31 26.85 54.89 34.22 74.43 29.07 34.43 53.94 43.65 69.32 31.51 41.57 23.86 32.43 31.43 35.92 28.2 23.74 47.21 14.28 27.09 120.54 32.42 53.49 104.66 65.75 78.85 25.25 37.81 36.7 29.89 76.56 53.52 21.77 19.81 25.48 46.68 1556340_at MAPK12 60 43.98 56.42 85.37 44.44 47.96 51.31 71.12 54.89 52.74 38.73 27.19 30.48 45.27 53.41 60.89 64.23 53.38 32.12 32.11 54.99 48.44 37.13 84.94 56.09 50.1 54.61 53.32 55.9 77.54 41.34 62.23 47.73 46.39 55.76 61.55 57.25 59.49 77.46 68.36 49.98 55.44 60.18 54.48 70.41 57.89 56.55 60.15 58.64 47.44 62.04 41.21 63.88 49.87 78.96 32.82 121.39 93.77 54.15 78.9 63.57 61.42 70.74 50.48 41.76 53.23 44.01 38.35 59.75 35.13 72.75 56.49 50.08 43.37 47.48 51.35 42.72 58.14 66.16 68.98 65.67 65.4 55.89 71.78 57.38 46.23 72.65 59.54 63.12 44.75 120.55 43.45 50.17 35.69 54.71 49.73 63.64 51.35 48.91 54.12 56.26 55.84 63.77 43.13 31.84 51.57 66.61 38.3 56.54 130.58 55.13 40.38 43.22 45.57 64.91 63.81 56.2 54.61 47.75 64.37 68.44 62.28 70.11 71.02 57.14 60.25 50.61 50.82 57.85 61.12 44.66 59.32 73.64 71.09 47.76 68.18 84.58 63.83 68.51 64.86 55.76 69.13 53.37 68.69 69.99 70.33 68.39 54.29 48.26 41.14 66 68.4 76.64 59.94 55 80.11 80.36 50.78 52.69 65.3 48.21 54.06 73.24 87.11 82.01 70.38 57.96 72.33 71.44 59.85 67.31 71.25 53.51 54.5 93 78.88 56.08 55.18 43.12 58.01 47.2 88.25 74.06 45.67 76.11 71.3 67.1 47.53 76.56 77.95 42.78 53.45 63.74 58.47 46.72 56.92 48.22 58.51 42.79 53.57 66.4 40.21 45.04 86.09 60.52 53.49 42.2 51.38 77.19 48.1 54.54 53.37 60.38 71.78 61.37 79.22 69.02 71.16 86.03 63.48 78.66 65.05 73.46 76.69 64.15 80.58 71.36 1557073_s_at TTBK2 40.43 68.84 45.47 46.03 26.81 78.28 35.69 94.87 74.07 39.07 21.02 78.38 50.76 33.76 59.96 42.48 33.37 64.93 61.16 65.38 28.29 50.33 45.05 24.03 29.47 37.52 39.02 35.41 44.3 40.09 36.02 25.94 29.33 39.69 47.37 30.82 46.53 44.42 58.97 31.88 34.54 39.74 29.09 50.81 45.29 62.06 35.2 42.86 33.29 51.82 33.45 47.14 35.26 47.25 69.17 49.16 30.4 25.05 55.51 34.01 42.69 28.24 25.93 54.05 75.21 83.75 50.25 100.15 82.97 79.68 102.43 47.82 63.91 86.6 50.17 50.28 59.59 65.2 79.44 61.66 96.47 49.68 67.61 58.76 109.65 77.14 64.83 100.97 78.16 36.93 25.36 93.53 56.25 46.19 65.51 31.69 67.72 62.9 49.55 31.03 46.47 43.82 54.87 65.82 76.42 38.32 83.95 113.5 78.7 19.39 46.63 155.82 62.82 89.46 63.08 35.33 138.99 30.64 87.13 34.01 51.62 39.15 35.48 39.61 102.46 59.26 69.55 68.34 51.9 57.08 94.27 60.54 34.37 41.68 84.59 88.62 65.58 61.52 76.66 59.34 58.73 57.95 69.76 81.46 63.27 67.6 31.13 52.4 81.27 57.72 42.4 75.64 40.22 67.09 55.67 55.34 42.19 46.08 76.57 61.18 82.83 67.75 55 48.61 43.42 71.2 42.53 67.9 59.87 60.51 82.14 51.71 56.63 74 32.94 30.61 65.84 64.7 119.36 97.37 85.95 64.02 45.67 74.47 91.43 108.73 44.78 79.88 43.44 57.72 60.05 120.93 95.46 83.34 108.23 74.18 92.11 109.07 41.38 107.95 60.7 105.41 85.95 63.93 58.11 79.56 47.87 56.01 61.43 65.8 55.2 109.01 104.49 47.55 111.19 116.1 104.85 57.31 75.46 112.64 32.46 51.31 60.66 92.88 46.17 39.71 60.65 1557103_a_at LMTK3 14.35 22.27 26.56 42.26 16.52 25.34 22.85 19.61 15.01 17.98 15.06 17.49 13.4 101.74 33.02 18.16 21.56 10.59 14.08 14.5 19.67 12.62 10.94 28.9 12.7 16.53 45.48 22.58 20.85 26.99 33.14 33.74 23.39 18.33 18.15 16.53 21.37 19.02 12.38 46.42 23.57 12.75 25.1 33.53 16.89 19.07 17.14 18.36 35.85 23.4 20.15 22.97 31.42 94.54 57.53 89.83 21.47 36.06 101.29 25.08 52.55 32.23 21.59 65.17 24.71 15.1 15.07 13.09 15.11 14.62 15.23 11.62 11.41 88.85 16.02 20.01 15.6 15.79 38.58 18.08 65.37 56.15 39.26 32.43 16.6 27.52 18.55 19.63 26.77 16.87 74 16.6 26.27 15.01 14.04 17.23 18.89 19.17 21.21 17.67 14.96 72.64 52 21.94 16.38 65.08 45.63 43.91 167.32 164.02 24.22 19.76 14.12 20.24 28.76 15.43 39.51 23.99 17.11 67.47 49.86 30.45 23.58 34.52 14.72 27.94 20.76 21.6 59.31 23.81 19.8 75.95 17.39 68.59 170.19 18.51 19.06 17.39 25.45 25.8 18.97 126.79 20.2 22.47 90.64 30.8 22.86 29.65 88.36 13.67 17.93 18.91 81.15 61.92 29.27 44.93 48.23 41.34 21.24 27.33 97.8 25.82 22.82 18.4 19.53 19.85 14.9 41.09 36.2 24.52 39.76 42.9 28.82 35.25 32.99 62.5 18.37 24.61 14.44 51.83 20.45 30.18 18.94 25.12 20.43 25.58 102.76 52.08 32.38 34.58 21.06 18.06 118.28 51.25 32.1 75.22 82.18 30.69 153.52 29.72 203.28 282.3 37.44 100.27 36.82 28.68 25.85 115.49 45.53 80.48 33.52 47.33 157.34 325.22 62.73 28.75 35.33 251.9 32.67 45.14 19.58 30.55 19.23 21.82 31.53 34.89 72.78 1557170_at NEK8 50.28 77.03 42.84 38.92 59.05 88.56 121.6 65.12 56.46 96.32 112.87 84.31 110.47 95.3 123.73 79.18 42.15 96.4 70.45 67.2 62.04 75.31 67.09 58.95 45.39 52.56 72.81 62.18 62.9 39.1 42.79 59.91 224.46 79.6 54.81 53.55 48.22 40.39 78.56 53.65 47.34 43.74 36.65 41.08 32.08 43.29 69.74 51.4 62.86 51.99 49.37 67.69 73.4 46.67 52.59 62.46 42.61 31.16 187.27 73.08 73.65 71.62 41.71 81.53 146.38 51.52 70.36 87.04 41.87 49.75 41.92 59.51 67.57 60.39 55.38 234.68 51.47 72.92 79.3 55.83 62.31 50.21 122 86.55 74.82 74.48 87.69 72.45 148.74 76.28 266.45 82.45 104.82 78.35 85.28 66.78 93.9 65.75 103.25 65.48 98.98 56.83 57.75 99.86 88.39 80.46 51.81 64.08 79.57 583.56 65.38 77.27 73.25 80.23 67.54 69.14 182.59 153.03 137.5 73.95 82.86 74.98 88.13 68.39 75.16 75.4 229.18 84.36 47.82 93.48 115.22 86.17 106.11 102.18 88.54 84.8 73.55 80.33 191.64 87.41 127.1 182.57 81.96 82.75 77.74 267.42 252.04 42.11 85.08 55.53 42.37 91.14 47.01 53.78 56.49 52.61 56.22 49.86 48.49 96.12 133.86 67.18 58.47 94.88 69.45 76.59 89.81 131.83 380.16 82.05 69.43 76.51 118.48 71.19 333.28 85.26 68.4 96.79 83.61 97.52 80.54 69.51 225.1 137.52 84.39 110.31 90.08 77.86 81.21 99.79 100.71 66.04 94.51 96.78 71.71 47.92 84.9 80.85 66.97 80.89 82.86 87.71 70.95 151 71.64 87.12 100.45 66.22 84.7 65.22 95.74 116.49 102.03 69.45 125.41 93.24 75.72 67.35 66.9 127.5 45.72 47.42 79.88 49.28 53.49 60.8 55.35 1557316_at STK24 46.8 32.66 38.56 25.28 36.99 56.32 42.98 27.17 28.72 46.7 26.86 20.94 55.1 21.48 80.45 24.92 34.4 50.76 35.6 73.08 39.93 35.45 77.42 56.47 31.99 32.44 33.68 35.29 44.16 22.96 27.18 35.33 71.06 21.05 41.75 42.75 46.53 38.29 57.85 43.62 41.64 60.68 34.25 62.13 37.12 49.11 57.1 72.27 32.41 67.29 24.83 34.51 32.62 29.26 23.88 49.03 104.87 31.21 49.25 40.94 30.55 30.85 58.99 51.76 99.61 73.44 94.65 112.38 60.06 122.43 75.77 56.51 55.69 104.28 45.97 113.05 53.18 59.33 43.38 50.56 109.75 36.78 130.41 51.99 70.96 38.35 61.73 67.53 71.32 31.06 47.99 48.84 36.52 29.77 24.36 41.35 38.71 48.47 26.67 30.9 41.67 32.85 44.1 53.25 49.94 55.2 77.57 69.02 34.91 45.99 68.44 59.6 57.15 98.77 40.23 35.02 57.57 81.09 92.92 34.07 35.54 36.77 45.14 35.21 96.75 57.08 100.63 63.54 40.51 91.72 112.34 107.59 56.89 64.8 71.34 100.14 106.41 86.23 118.54 64.64 85.14 68.98 199.25 90.27 101.83 155.55 91.22 49.67 74.26 37.78 47.02 94.19 54.58 50.03 77.13 60.11 28.53 71.3 55.53 101.11 59.91 50 40.9 71.58 47.4 71.46 105.12 59.12 68.12 56.29 84.95 57.7 50.22 74.56 117.99 31.94 81.15 87.08 96.95 57.69 78 98.85 56.72 81.74 79.28 84.17 87.81 76.76 93.97 57.64 69.96 41.83 48.54 44.19 79.53 51.13 94.71 59.16 35.88 81.51 54.65 55.02 90.12 55.8 54.63 43.81 110.41 62.82 49 49.74 37.08 65.77 71.8 44.68 74.41 46.99 76.47 34.71 52.8 74.68 49.82 95.04 71.98 64.78 83.42 60.66 48.76 1557675_at RAF1 99.72 116.63 141.37 44.78 112.02 174.45 133.32 98.66 81.58 99.33 109.37 133.46 103.16 178.32 214.2 101.92 78.28 184.85 203.97 113.82 173.2 65.31 145.76 133.87 129.06 99.65 91.78 103.16 186.51 105.56 77.07 60.17 132.22 177.7 115.59 114.19 88.32 108.15 128.81 87.69 83.43 155.94 63.65 103.98 91.03 137.34 85.4 124.89 85.07 162.79 116.15 309.24 107.45 75.67 46.51 115.32 79.13 69.53 146.94 150.58 240.57 342.11 138.25 73.65 207.48 131.22 104.34 102.46 89.07 115.44 82.5 74.29 99.2 81.79 112.23 160.12 140.92 124.8 179.99 208.58 211.83 105.82 107.35 111.43 97.71 122.57 106.43 151.2 108.69 190.97 144.32 200.2 244.61 94.27 145.87 137.84 136.28 79.72 133.44 124.55 250.62 95.3 133.79 157.44 140.1 134.48 127.82 146.69 123.36 248.92 99.12 183.56 93.71 117.79 83.66 75.6 187.53 292.14 252.95 119.99 164.05 187.43 126.92 117.66 181.48 92.61 170.44 140.83 128.61 113.47 146.28 134.06 159.87 109.75 54.59 153.4 102.32 85.52 141.5 100.78 204.19 165.39 106.72 139.58 91.77 169.87 111.53 72.54 124.31 73.13 111.64 127.05 72.95 93.5 112.65 79.3 82.37 96.26 111.67 143.23 143.39 136.82 105.27 95.98 77.93 96.67 130.52 123.86 134.64 141.4 75.78 166.29 149.11 190.93 135.1 102.09 100.33 141.14 183.83 132.93 158.16 140.33 98.51 122.79 87.44 106.83 153.55 147.64 115.34 110.8 130.44 116.02 97.91 124.25 168.12 196.78 131.12 169.66 110.21 161.62 124.75 131.08 143.32 86.86 209.45 134.06 493.6 90.7 175.97 112.14 110.04 172.64 175.55 152.13 203.86 117.24 166.86 106.21 91.45 224.33 72.75 147.05 139.76 156.13 107.69 124.71 91.05 1558556_at CAMK1 26.02 25.38 35.77 38.28 22.26 65.13 30.63 35.55 33.18 37.17 44.5 45.96 66.7 33.83 90.18 36.77 25.32 75.61 41.33 39.08 75.21 27.04 42.13 57 43.22 30.49 28.57 36.11 57.07 35.24 25.18 28.35 27.41 32.64 33.13 38.66 41.51 36.01 45.52 29.73 29.09 40.99 28.57 35.99 29.95 45.89 26.32 39.55 27.97 56.02 43.06 69.98 44.67 27.51 32.12 31.81 31.83 30 55.18 48.47 52.66 83.22 45.15 59.53 185.07 37.56 52.28 37.73 42.62 52.33 43.91 33.46 39.78 35.11 53.99 44.82 24.29 33.74 50.93 55.89 59.22 41.27 48.75 48.39 44.92 57.98 43.32 56.72 69.8 77.1 58.18 66.06 59.47 44.3 63.51 45.15 37.46 38.33 50.52 45.76 41.55 23.88 45.37 75.98 31.53 47.81 34.45 59.77 44.57 130.72 37.99 84.7 59.67 50.4 43.4 24.78 105.48 85.24 80.14 35.93 36.83 46.78 57.51 59.49 87.68 54.48 68.2 70.5 44.94 56.5 68.52 55.51 41.02 60.43 39.87 96.39 66.43 47.5 53.88 58.01 137.68 38.69 56.46 69.09 55.1 57.38 58.22 40.68 54.32 39.03 52.06 50.79 33.58 45.87 53.23 46 47.61 44.38 47.82 56.51 53.47 48.51 41.74 48.8 42.73 45.09 56.05 37.21 81.46 68.88 51.03 65.75 64.48 47.97 112.06 38.85 53 70.23 90.86 55.2 93.64 37.5 65.33 68.83 67.03 86.1 53.58 56.56 41.94 65.39 41.74 56.67 55.19 51.37 95.09 52.54 65.31 85.81 28.6 101.22 69.16 58.58 60.69 72.18 83.21 86.1 148.05 49.53 54.86 60.19 77.01 88.38 78.05 31.76 122.04 50.31 51.98 40.65 42.06 107.1 36.57 43.47 69.58 38.61 42.11 57.99 43.39 1559394_a_at ROR1 11.24 12.25 47.22 10.4 22.95 31.39 18.99 11.95 11.75 11.73 13.03 12.91 27.56 15.17 59.7 17.05 12.64 19.71 51.63 21.64 15.79 12.85 10.84 64.89 13.92 34.23 20.78 14.17 15.36 45.91 13.11 13.12 17.54 13.2 14.07 28.82 29.92 66.97 48.12 13.7 11.43 26.44 15.84 33.55 26.75 39.76 24.17 19.55 20.34 16.52 18.41 22.02 13.52 23.6 11.54 20.06 12.76 14.73 21.35 48.57 21.45 50.11 73.9 11.97 24.78 107.61 136.63 18.09 13.28 35.97 24.22 64.19 67.7 42.28 16.94 24.57 12.18 20.15 15.94 15.51 25.71 16.49 12.89 17.7 29.97 27.1 28.6 38.07 13.97 40.07 26.39 43.78 17.76 54.16 35.88 17.08 19.23 20.22 18.7 43.33 19.18 24.72 19.65 34.23 38.91 33.17 27.35 24.52 34.57 35.62 63.17 96.83 49.14 195.74 32.79 51.88 44.92 219.68 89.73 49.94 67.79 225.7 19.65 23.68 154.48 15.94 24.57 92.4 19.46 100.62 95.16 22.87 78.78 37.93 22.3 29.84 72.85 32.3 44.7 37.17 23.41 22.18 11.89 27.8 35.87 42.06 30.55 24.23 86.23 14.24 214.38 34.27 21.2 25.68 15.74 101.26 16.46 39.51 31.57 19.38 21.68 89.63 19.27 25.44 19.82 20.41 14.82 37.13 24.34 18.76 17.58 28.41 37.54 24.77 142.98 25.84 58.47 29.57 78.73 12.01 61.94 27.79 12.74 28.96 85.15 26.07 23.81 20.74 22.09 59.98 76.85 39.85 62.21 19.14 81.35 27.93 178.71 63.63 43.17 66.51 16.77 29.07 46.21 18.08 34.43 53.98 76.01 27.65 44.41 11.62 50.15 63.41 45.08 57.95 29.94 26.3 41.22 33.52 36.52 50.54 44.86 164.69 31.43 57.7 97.22 59.03 36.02 1561190_at CDKL3 33.19 69.97 52.18 32.31 43.76 97.87 128.65 59.09 48.8 73.83 71.48 59.94 95.61 96.79 135.59 67.73 30.15 68.26 68.17 51.34 58.64 53.58 43.94 44.29 50.76 35.16 52.26 51.73 49.32 47.49 41.22 37.79 49.19 87.38 39.15 47.43 30.49 48.89 52.07 30.88 35.83 36.83 30.2 41.11 36.37 47.66 74.13 47.24 45.21 56.83 45.12 49.97 48.62 36.66 33.84 58.61 36.78 36.61 58.03 96.11 64.09 61.54 39.89 57.01 108.84 51.75 43.94 63.8 42.38 67 60.89 44.84 62.62 52.48 45.99 60.01 31.74 47.72 83.8 43.76 97.53 39.19 51.3 52.01 49.77 62.69 37.73 46.2 62.8 111.52 101.78 77.33 72.97 109.32 54.15 55.84 129.67 70.24 75.96 53.13 63.95 35.75 62.2 83.92 99.32 87.79 54.06 62.65 72.07 141.36 39.55 59.03 44.75 48.15 35.39 48.73 82.76 101.94 66.76 49.87 51.9 55.25 51.24 37.46 55.67 62.28 60.34 60.2 66.58 55.67 61.7 52.24 47.05 48.86 75.2 93.47 73.97 56.94 57.21 46.25 104.54 56.54 65.52 58.61 53.05 66.26 53.08 49.65 62.77 42.95 31.63 48.2 37.59 55.16 46.51 52.47 52.97 36.1 63.33 62.49 87.03 54.83 52.99 91.19 58.58 46.04 57.46 61.72 157.88 80.81 36.07 56.93 44.45 38.43 107.43 54.39 53.22 73.17 67.53 49.93 54.68 37.41 57.3 54.64 52.39 81.06 64.21 53.7 39.9 66.88 109.89 49.08 72.28 68.19 72.87 43.69 53.9 77.37 31.15 60.75 55.33 58.43 56.13 74.87 48.58 55.47 95.1 47.06 71.87 49.43 41.61 58.1 68.63 37.43 115.37 63.05 36.98 39.71 42 86.16 27.88 39.22 50.45 37.89 39.23 32.04 36.62 1562031_at JAK2 27.71 13.29 21.84 7.79 8.66 38.4 12.89 9.93 12.99 13.24 10.69 9.71 18.6 6.82 33.67 11.39 15.22 99.17 22.7 67.74 17.23 41.53 17.54 15.77 29.66 62.95 17.4 9.08 21.77 13.88 26.59 15.94 11.51 11.14 19.91 33.78 25.39 55.94 86.79 26.41 119.84 71.76 21.04 51.26 14.22 28.42 12.78 55.2 15.72 28.23 13.62 18.35 10.38 17.28 26.28 13.47 15.95 9.26 100.99 53.36 17.19 13.42 21.58 40.05 113.13 178.88 5 353.41 39.06 48.27 53.57 67.2 118.92 36.56 20.25 41.37 29.06 49.26 20.21 269.52 30.81 24.08 76.88 48.41 62.9 48.59 35.14 144.84 69.1 19.59 18.31 68.96 21.26 26.61 14.59 66.8 20.7 31.46 48.08 12.09 8.75 12.86 33.5 33.69 20.47 17.53 34.62 65.54 11.65 13.59 32.13 58.33 36.09 56.14 66.39 10.84 46.75 29.74 142.55 24.04 27.9 18.3 16.08 29.1 79.48 44.1 64.68 36.62 33.26 161.67 151.58 65.41 8.96 30.1 65.38 35.58 41.31 62.05 58.08 73.18 45.82 28.03 47.28 42.72 173.12 68.96 32.08 10.65 46.49 19.23 31.29 33.93 32.9 44.61 52.14 50.61 12.99 83.09 36.81 103.38 56.59 71.62 25.81 29.95 34.91 60.35 40.52 86.34 26.38 21.72 33.73 20.41 34.5 54.79 20.18 11.83 118.13 40.65 105.69 44.41 88.42 39.72 31.66 44.28 88.98 43.58 28.85 81.44 40.72 28.67 79.43 31.02 25.62 39.26 58.74 23.25 27.92 29.85 10.77 58.37 38.62 58.89 117.23 22.21 51.82 26.39 54.91 50.32 54.29 36.79 49.27 98.03 30.53 21.68 61.06 26.86 108.61 60.39 27.91 24.11 65.1 41.51 30.68 38.42 38.5 50.6 58.98 1565627_a_at LRRK1 66.17 52.26 88.47 57.82 79.11 66.13 52.03 42.46 56.83 71.16 56.48 55.16 87.75 62.66 108.52 70.88 50.91 120.69 147.15 79.55 57.71 80.92 58.42 57.43 65.61 127.09 45.56 73.91 103.35 57.92 59.03 47.5 40.65 56.63 86.34 118.66 82.07 80.38 153.97 233.7 70.97 98.49 59.84 58.64 64.15 66.75 62.66 94.46 82.91 79.12 175.8 71.6 70.17 89.6 73.04 49.18 110.06 78.23 90.61 101.86 53.34 144.72 82.66 87.47 444.55 116.49 131.61 273.04 78.54 86.41 117.62 124.29 74 102.19 107.37 188.62 145.31 120.36 105.99 107.35 81.17 99.62 122.58 104.65 113.51 52.63 110.69 188.59 126.44 173.62 126.44 184.21 109.88 160.96 68.72 201.36 67.63 89.46 77.39 146.28 84.11 79.19 70.25 182.13 57.4 73.23 78.37 80.85 67.05 102.88 107.57 102.91 103.62 169.01 152.39 60.28 120.82 274.52 379.85 106.09 133.26 166.24 208.37 177.08 185.26 103.21 149.11 171.16 79.88 159.62 240.57 117.44 172.82 146.61 111.41 110.07 118.65 97.5 80.4 103.86 81.33 108.62 58.52 84.55 134.47 127.43 100.92 70.33 51.81 47.16 81.24 100.79 79.74 85.06 74.92 61.85 45.4 127.56 75.05 113.01 90.13 92.72 84.81 60.96 48.86 100.52 56.43 83.86 80.87 54.42 71.54 87.97 100.51 104.43 88.84 80.81 149.33 108.58 91.47 63.82 197.04 132.97 74.22 101.2 126.53 99.57 67.12 66.78 136.4 174.67 136.99 81.4 148.32 131.26 138.85 108.33 116.54 133.41 173.1 272.63 142.41 84.64 146.82 147.34 82.85 87.33 80.86 128.93 238.51 62.49 380.87 204.16 123.49 84.53 98.09 79.63 109.99 72.25 65.39 139.09 91.83 91.01 89.94 65.94 93.6 157.59 107.44 1569522_at LAT1-3TM 205.79 154.65 175.84 292.65 157.04 166.87 188.8 170.55 206.07 134.31 158.03 147.03 150.1 148.97 140.92 175.08 262.61 181.09 137.97 171.63 167.88 138.41 143.31 235.04 182.65 207.84 208.75 183.68 232.37 180.37 151.73 183.15 145.08 143.86 215.84 263.05 226.44 219.97 210.4 281.72 158.74 184.86 196.65 206.27 222.53 155.98 222.7 242.06 224.54 211.59 185.52 178.33 172.7 225.19 178.98 160.86 218.39 243.05 166.07 255.54 217.4 223.32 263.08 210.43 179.42 149.33 202.33 182.07 200.21 149.39 163.21 199.79 177.98 192.02 174.47 178.13 155.92 169.96 166.3 204.69 145.59 198.15 193.73 189.17 155.75 193.42 232.85 187.9 244.43 155.75 299.01 134.47 161.98 126.1 148.06 172.57 150.78 159.73 143.32 152.71 183.8 164.27 180.33 111.75 115.51 212.7 148.67 154.11 181.38 262.03 176.61 126.04 163.08 148.44 175.61 252.42 186.54 180.66 179.92 177.28 191.12 183.7 272.39 251.19 139 157.5 136.11 162.12 160.39 160.82 127.52 142.41 179.17 272.32 145 155.11 191.56 168 164.45 166.66 189.2 196.07 195.9 178.53 180.21 211.15 185.47 191.54 181.46 153.91 186.6 200.02 213.71 170.96 168.6 163.89 179.75 229.7 152.76 271.94 163.77 193.65 205.26 293.98 231.31 187.96 206.91 189.23 234.33 193.99 179.17 193.44 139.94 162.93 183.72 213.52 163.66 163.79 83.06 141.93 166.21 196.84 194.97 157.06 224.1 183.72 195.96 169.96 194.18 174.57 135.14 166.31 175.21 170.2 133.79 169.14 157.25 158.23 147.86 132.86 188.17 143.95 219.8 328.47 189.94 167.6 198.23 173.54 200.38 198.44 190.35 196.46 160.13 176.2 148.88 181.03 175.53 221.16 237.56 160.12 223.04 207.29 139.85 210.19 159.82 223.2 203.16 1570439_at MAP3K4 17.96 24.77 21.67 13.83 12.92 33.07 22.22 12.45 13.84 19.28 19.16 28.61 48.26 12.73 94.85 12.83 17.16 27.92 21.47 47.02 22.64 17.49 15.45 24.95 23.66 17.33 17.71 80.95 14.23 20.01 16.71 24.66 32.97 15.16 15.93 22.6 16.19 28.3 30.13 15.21 13.07 19.41 20.95 19.17 19.65 27.55 16.58 22.82 16.47 27.38 17.02 19.7 20.96 15.93 11.33 24.58 16.83 11.97 33.92 17.91 59.06 41.38 21.66 17.44 35.89 21.61 21.54 20.94 14.75 27.51 18.94 15 34.93 22.31 24.77 29.99 29.74 24.3 37.83 37.19 84.97 11.8 13.6 13.86 19.42 17.38 16.73 24.32 27.51 22.17 20.03 27.73 17.01 25.48 16.52 23.13 17.04 31.26 13.8 10.92 11.3 9.74 16.62 27.09 38.64 15.81 38.95 46.93 19.76 23.34 45 38.5 29.95 68.76 21.36 13.29 67.08 42.69 30.87 16.63 17.55 23.19 13.66 19.26 49.77 19.79 53.67 33.42 22.4 22.26 48.23 27.55 11.96 24.5 24.45 24.37 17.44 38.17 38.2 22.71 28.34 28.44 20.14 19.37 24.93 44.9 20.13 13.77 31.5 13.6 16.24 21.54 19.87 18.73 24.71 23.05 14.38 20.86 29.76 23.56 24.58 29.12 16.29 20.36 15.31 19.39 22.61 24.84 36.26 17.73 23.24 27.39 31.49 20.94 41.5 14.2 23.7 32.92 85.01 18.52 33.47 23.49 24.15 29.45 17.52 22.32 28.13 25.67 16.21 24.48 48.93 27.13 30.04 23.38 39.51 14.92 18.83 50.4 15.78 47.18 18.49 23.08 25.2 17.47 23.75 31.03 119.27 26.63 30.27 16.91 28.46 27.38 30.68 20.37 31.21 28.02 25.32 17.45 16.73 44.15 18.55 31.95 30.32 35.15 33.32 14.37 26.08 200979_at MAP3K15 112.38 121 72.32 83.06 148.3 98.41 75.85 109.39 163.81 166.56 66.79 73.24 82.61 106.38 66.97 131.94 158.13 96.5 138.74 235.16 160.08 72.29 75.07 86.1 235.86 94.5 85.11 80.87 106.98 96.59 64.15 68.83 100.54 89.76 169.41 82.54 96.72 68.9 55.3 87 91.26 96.37 193.3 134.38 95.57 58.01 53.03 53.68 89.89 63.16 90.83 81.99 118.67 186.98 95.8 105.34 64 56.55 94.58 169.61 39.38 94.49 135.46 46.89 52.2 100.81 49.48 93.91 94.1 67.18 100.05 79.01 127.66 64.65 60.66 52.56 34.57 81.55 35.73 67.79 70.53 73.04 71.12 75.78 79.08 64.46 74.38 81.83 67.18 63.53 48.15 65.86 51.5 116.32 53.2 70.35 148.63 59.12 74.21 96.72 80.12 107.69 74.23 82.63 74.67 67.69 63.31 67.33 142.84 33.53 104.22 77.17 91.28 120.11 114.34 154.49 54.94 57.66 109.76 130.51 62.78 67.31 79.09 83.52 93.25 111.93 74.04 69.91 96.32 73.6 67.62 66.29 90.8 78.92 67.12 89.9 93.78 75.16 61.56 75.93 36.98 43.64 75.28 76.03 65.56 39.81 77.93 85.99 75.94 89.37 101.4 81.59 64.88 69.79 60.23 76.87 99.01 66.47 66.63 102.32 73.34 75.28 93.08 57.18 86.91 78.58 62.04 84.48 68.52 66.22 62.74 56.49 58.53 117.86 40.63 90.55 59.93 61.73 62.97 92.7 54.33 163.62 59.21 37.8 55.67 49.43 145.91 186.07 210.58 88.29 39.77 53.27 66.63 151.14 95.63 66.89 85.6 105 77.9 283.67 82.38 64.77 71.31 52.81 139.47 53.7 58.93 124.25 33.98 76.27 78.99 55.19 77.7 72.15 74.7 92.04 101.37 190.51 95.57 78.18 170.43 97.78 85.29 124.48 132.71 123.35 112.11 200990_at TRIM28 980.56 1218.36 963.08 753.92 1132.58 900.03 1042.76 1444.14 958.28 635.48 1007.39 959.52 1146.7 1336.22 856.15 1440.56 1507.43 1308.01 1782.68 1325.46 1340.33 568.92 643.53 1101.58 1316.09 664.06 759.48 1131.98 1394.88 1021.26 692.45 827.29 768.52 1253.77 1244.27 1530.29 720.25 718.4 770.03 734.33 1021.13 1841.09 1087.59 1056.22 584.72 780.21 1032.48 551 708.91 504.11 822.63 577.64 1651.3 1329.93 2014.58 1189.52 1222.15 806.69 990.14 946.61 1196.96 4041.62 728.36 757.36 570.87 1141.91 1855.6 1073.87 566.71 646.8 756.7 996.15 912.72 659.55 751.49 559.8 743.89 647.94 729.7 884.53 871.96 1008.47 869.43 1058.08 791.05 996.3 689.1 804.9 909.1 494.64 743.43 719.61 634.7 475.11 344.63 301.22 970.02 1187.56 346.69 493.1 1071.56 837.23 821.18 443.52 853.3 2046.81 1001.48 1364.51 1783.85 1068.26 1152.18 935.07 572.8 1013.7 556.26 268.9 1077.6 601.16 610.95 1102.89 802.01 1163.37 1042.58 1327.54 425.05 378.7 551.81 483.88 335.16 676.76 393.79 511.06 691.66 696.15 312.56 1274.25 965.38 253.74 437.19 391.01 347.23 647.87 809.82 618.97 548.37 747.11 601.89 1402.79 1200.94 798.22 734.31 875.36 883.94 855.55 859.33 2348.41 882.4 810.57 1007.97 961.44 1210.69 1016.99 624.48 858.67 949.2 767.02 1088.15 957.27 1003.12 1549.81 799.25 844.32 782.33 480.62 950.86 684.22 512.91 582.58 739.23 1266.01 553.09 786.75 820.83 1153.19 742.61 691.9 928.12 962.75 921.86 932.54 636.44 541.17 979.74 963.28 962.48 957.62 1140.04 1503.44 680.34 793.67 1434.48 824.62 1472.88 1238.88 990.04 802.24 612.52 771.05 678.12 577.8 756.58 1106.24 1512.24 947.1 1082.89 1066.01 849.61 1128.35 846.15 976.7 1002.71 881.26 1163.4 1111.93 1350.74 1071.57 845.59 201234_at ILK 377.62 565.84 789.18 605.19 467.67 526.92 354.88 567.24 532.61 431.5 534.01 482.15 509.56 574.63 511.16 760.87 429.72 413.91 462.98 433.26 557.99 409.1 299.27 506.99 376.27 507.03 623.3 343.32 500.43 466.12 603.43 561.7 501.56 443.94 495.23 452.39 416.73 578.46 615.12 586.39 498.08 425.99 511.42 446.29 533.24 691.54 575.88 560.16 683.44 647.46 593.05 711.81 721.5 871.28 474.55 732.95 524.71 469.15 564.02 545.54 664.82 900.91 904.32 369.24 284.56 357.16 551.84 467.58 357.57 460.42 425.42 435.75 581.97 427.76 274.87 384.75 424.06 585.53 413.93 559.13 544.61 815.18 499.31 588.46 597.63 594.37 595.76 542.15 459.28 322.92 490.87 450.83 296.11 318.51 265.16 331.19 468.7 621.18 246.22 318.46 383.26 483.73 536.46 311.05 529.45 544.26 623.74 569.63 780.19 619.76 568.65 654.36 487.81 571.44 461.61 164.2 422.39 424.63 527.17 480.3 794.06 575.59 377.83 779.93 750.63 398.02 399.71 429.41 383.99 299.65 410.63 361.77 548.15 461.72 388.42 293.38 257.7 389.5 399.21 459.23 271.41 365.49 391.69 339.63 400.13 334.29 269.75 584.92 492.9 523.21 331.59 563.88 585.61 579.36 549.84 474.37 292.26 540.55 629.4 349.41 490.81 571.61 528.88 380.42 449.5 476.89 540.24 502.6 279.3 435.88 526.46 779.42 549.75 328.12 175.99 448.79 524.68 379.04 423.48 622.47 601.94 444.23 416.95 590.13 581.67 500.71 420.96 515 363.12 414.62 474.63 585.99 585.11 365.27 589.16 472.52 642.15 468.49 447.43 324.76 286.42 593.28 457.56 189.73 244.89 509.94 293.77 383.95 485.76 446.54 429.63 436.2 624.59 545.13 415.4 421.63 414.71 454.43 539.27 497.75 624.3 509.81 443.67 526.41 550.86 485.36 666.94 201314_at STK25 233.08 158.73 161.8 103.2 198.17 265.57 146.37 233.38 209.78 204.18 130.34 250.74 193.85 248.44 233.06 267.73 175.49 180.24 180.79 178.71 182.36 131.13 187.84 162.56 175.91 161.47 159.31 142.41 199.6 165.47 145.71 206.77 237.41 179.65 197.26 132.57 151.78 205.24 185.01 184.88 137.55 233.41 214.12 187.83 126.07 172.25 167.64 199.93 159.51 155.25 214.77 178.56 189.08 294.36 264.28 174.91 198.87 136.06 185.98 177.46 120.07 340.97 201.52 95.16 129.76 197.74 212.62 173.25 94.36 154.08 156.7 193.07 163.57 122.02 165.01 151.1 146.9 119.97 113.26 165.99 191.58 164.98 116.69 139.43 145.4 134.19 134.94 126.65 123.79 200.63 132.37 167.5 108.02 127.72 142.33 118.79 144.6 195.89 146.88 178.33 171.29 122.4 139.97 212.96 140.86 183.08 152.17 170.78 112.87 154.22 177.35 158.83 172.74 152.74 99.85 65.48 248.04 220.19 74.26 152.57 159.9 228.71 98.04 155.12 122.21 88.47 95.34 117.53 88.12 139.35 135.46 97.08 158.33 120.44 113.02 116.12 101.71 77.53 109 142.73 104.73 72.77 123.38 129.55 80.38 124.19 118.31 201.41 179.63 157.67 210.82 162.87 93.44 159.2 131.32 136.32 68.43 133.87 168.72 126.54 209.66 191.45 86.57 83.94 134.78 120.92 188.36 133.88 175.51 162.89 131.48 152.55 171.13 125.12 107.71 74.94 89.21 130.96 151.9 189.71 127.15 162.19 137.91 155.39 140.69 131.62 160.93 132.88 178.78 131.63 131.38 135.62 155.19 108.72 149.8 160.28 133.23 169.84 152.71 202.23 126.04 89.09 177.47 180.05 159.98 136.74 110.16 137.31 78.87 121.4 130.36 190.89 168.23 149.26 179.88 144.01 172 138.99 140 214.79 157.09 253.86 194.88 221.2 195.67 91.29 162.41 201460_at MAPKAPK2 320.98 315.42 502.21 631.09 434.54 467.85 409.66 364.66 330.08 544.09 721.74 478.46 411.52 499.11 300.56 284.47 648.33 352.18 361.06 304.77 447.64 269.59 307.45 356.35 363.3 326.26 434.84 397.01 304.61 318.64 235.77 511.5 1215.36 278.85 394.01 509.6 422.86 259.25 400.95 384.92 446.28 430.4 554.79 339.63 638.04 289 534.67 489.66 391.26 368.48 324 297.5 806.8 643.06 907.5 487.66 581.05 323.58 414.8 361.77 492.09 438.7 734.2 389.23 359.02 353.74 269.37 282.45 435.07 253.04 319.54 332.71 346.57 329.04 570.04 409.81 291.97 398.39 279.19 451.89 397.82 431.34 222.85 372.84 321.1 441.01 398.08 354.33 384.55 420.95 221.57 472.93 667.61 205.37 330.99 503.34 365.76 795.43 581.68 416.69 260.03 357.8 440.24 354.37 407.66 546.49 454.36 385.9 590.41 210.09 549.96 216.23 289.49 312.46 324.51 190.38 149.28 279.37 266.73 448.34 361.73 337.32 408.38 363.45 203.9 475.4 265.2 233.97 345.3 446.13 355.65 427.93 332.73 378.34 205.57 249.87 262.81 232.84 236.71 918.86 442.44 274.25 432.89 371.35 482.01 565.46 222.76 437.6 375.93 487.06 479.18 484.43 422.26 271.38 323.51 352.11 480.57 428.92 346.75 326.03 681.09 368 311.42 497.77 442.72 459.88 700.07 428.9 444.26 364.07 885.9 468.22 464.95 447.81 362.81 492.37 444.23 698.31 462.35 358.78 446.98 320.32 441.61 504.5 412.73 338.62 346.42 780.26 330.86 323.95 465.51 329.75 352.42 447.99 257.41 494.77 402.48 785 426.15 422.41 834.47 539.76 483.25 391.89 472.69 314.73 317.22 404.57 288.5 404.17 304.58 366.93 431.91 335.85 790.25 362.72 377.64 369.08 295.41 561.35 472.67 698.89 312.94 559.17 376.54 455.41 369.12 201587_s_at IRAK1 1268.98 866.54 1152.37 745.53 853.79 630.94 1075.61 998.16 931.42 1262.6 796.52 588.94 773.76 727.63 531.65 773.28 1722.88 1371.79 2308.99 1792.01 725.48 630.84 2161.71 1085.42 3013 760.68 594.61 908.25 1728.08 1762.69 1602.23 829.97 981.28 960.18 1109.86 1634.55 973.68 1451.28 645.42 1014.74 1338.78 1957.94 1728.15 733.95 1268.86 717.25 686.93 870.25 1493.21 644.59 852.85 640.89 991.13 982.16 655.67 558.28 1259.6 1720.28 841.49 1471.7 631.95 987.06 1559.96 787.21 772.2 1556.14 1491.53 991.35 3257.38 990.05 942.21 1109.51 858.45 902.96 1302.96 588.41 2182.99 597.35 543.74 2120.12 633.95 509.4 329.05 554.21 426.3 620.36 542.3 532.64 606.76 1148.23 189.73 698.41 1016.18 893.83 1114.15 771.31 1053.42 1051.68 1053.92 1970.8 687.36 767.88 776.76 745.52 853.96 1036.41 721.82 648.73 1539.65 373.34 1206.33 467.92 874.51 986.42 1642.98 1187.42 752.12 661.04 686.48 1191.23 846.47 1289.36 1637.79 1145.54 576.85 817.56 529.48 585.98 437.39 1400.09 651.68 839.7 588.34 557.59 660.49 544.36 1104.79 432.16 486.3 581.44 333.59 659.43 667.01 533.83 549.75 412.97 859.09 604.03 762 480.42 2291.6 773.28 1003.48 572.13 580.47 648.59 666.65 952.05 517.54 1076.37 801.53 562.65 722.92 533.69 529.93 894.05 532.88 476.75 365.84 506 690.07 522.49 668.38 1644.43 326.97 1145.54 1402.2 612.81 648.36 367.11 586.94 2725.25 671.31 672.65 866.97 556.15 603.82 645.61 3564.06 351.66 592.86 482.12 560.69 796.66 507.8 1048.43 1478.56 784.4 645.7 646.19 1252.42 621.83 674.85 423.91 1046.49 338.33 1060.2 576.46 587.4 461.41 500.44 573.07 567.7 458.12 496.02 371.19 472.36 673.63 464.99 431.31 1483.91 1125.87 465.89 943.15 784.62 1138.97 787.11 201648_at JAK1 939.21 1239.37 1408.35 969.02 1369.27 1702.08 631.61 1767.42 1051.01 807.47 1266.73 1177.85 1136.6 1107.19 981.51 918.87 1744.33 1300.26 1296.83 943.77 1423.97 1212.45 883.95 1106.11 864.51 1840.98 1119.8 1634.49 963.62 899.49 1080.23 1489.38 1043.47 850.77 788.67 1165.46 1109.57 1220.78 1490.24 1146.66 1034.97 1075.61 945.84 1305.26 1132.16 1310.48 1162.55 1258.42 1545.79 725.92 1068.85 1173.89 1903.33 1370.73 1278.91 1203.87 1093.26 1379.84 1260.58 963.63 767.4 2989.64 1444.29 1121.08 2115.51 909.82 902.33 1204.3 1395.03 1061.99 909.88 1533.23 1065.83 967.41 756.12 1367.36 831.76 1467.51 664.23 1286.4 731.79 1094.83 1582.86 1167.15 1622.27 1342.76 817.26 1551.05 1128.23 578.69 470.05 859.04 490.25 337.85 432 605.13 549.44 954.75 702.26 602.26 1115.51 1592.69 1142.48 595.86 547.72 1477.22 1984.97 986.15 1161.18 187.28 886.72 1160.13 1182.54 907.24 991.84 837.48 928.99 479.05 1118.01 1191.63 1459.77 1037.13 761.52 1048.12 1427.09 1213.11 909.44 1449.97 525.55 897.65 1290.94 874.45 1718.12 1296.29 1084.78 831.37 1129.65 1239.13 1115.98 855.3 436.9 757.01 921.45 646.7 1614.34 622.43 633.7 1178.7 1160.03 1016.39 783.64 1464.65 946.33 1295.15 1367.82 859.21 635.3 1293.96 1348.59 1330.97 834.76 1075.2 611.33 785.11 671.88 1057.48 904.36 1304.32 329.92 436.42 915.61 1111.55 1144.28 1062.96 185.78 711.54 1945.83 1014.82 1340.78 412.27 1521.51 1024.12 765.06 1165.24 942.24 778.38 891.55 915.32 971.87 1184.64 885.02 1213.06 1102.42 855.55 891.45 1047.85 711.72 877.87 1342.79 919.11 688.02 855.29 1266.59 251.29 977.01 995.84 562.97 713.9 842.72 747.86 1004.21 1120.83 819.2 689.67 1171.36 644.53 575.82 625.73 698.33 843.51 1459.74 948.57 1059.92 654.36 884.76 1015.88 925.58 201739_at SGK 1159.58 592.71 649.92 749.03 596.38 731.95 418.44 477.59 740.29 1052.34 556.38 396.22 476.41 205.15 1171.38 315.23 903.36 724.11 780.54 401.55 958.53 872.72 1374.33 1343.69 549.8 1643.54 1109.52 4375.54 1044.63 474.33 825 541.07 777.77 213.43 1104.47 615.77 774.27 962.67 1138.07 994.84 777.65 889.71 607.74 855.38 1831.73 1118.46 2001.77 724.47 733.36 689.82 656.93 558.53 453.62 1308.85 129.88 785.37 516.38 888.07 728.5 947.76 767.18 930.69 906.02 381.88 428.2 424.67 493.24 848.33 543.21 472.92 726.96 448.6 792.89 735.58 606.15 361.43 638.48 1229.5 362.96 1626.21 241.18 1211.12 374.17 1056.1 795.61 535.51 673.1 928.66 470.01 698.58 75.22 906.8 315.07 468.95 777.91 819.96 443.59 278.56 630.39 644.94 306.15 1489.05 592.62 598.64 536.48 479.89 812.27 544.42 389.58 45.7 880.1 924.99 777.83 737.96 1261.17 1091.27 175 629.33 549.37 746.34 969.14 502.17 512.14 639.47 740.15 396.72 367.17 513.49 501.67 320.19 444.83 319.84 258.7 774.42 310.59 212.7 419.39 688.17 291.69 420.1 488.89 591.8 160.42 401.31 433.97 324.55 157.36 381.32 736.76 563.72 419.53 776.88 1047.56 751.34 526.38 326.62 249.71 1659.21 350.92 878.41 372.25 773.94 666.24 202.44 216.16 631.7 367.11 918.46 80.85 368.44 712.53 458.8 781.46 470.6 57.37 582.86 713.6 705.75 204.51 221.2 548.48 751.42 325.03 195.7 446.51 224.02 228.33 343.23 285.51 211.75 475.52 435.77 213.18 742.94 402.88 689.12 462.4 207.98 592.52 727.05 133.14 459.55 431.95 52.23 795.9 484.41 568.58 567.93 1110.36 424.54 286.77 412.39 325.67 560.07 462.19 227.66 1097.47 549.66 520.94 588.73 846.34 807.55 254.68 837.97 980 863.05 584.89 201745_at PTK9 445.28 505.18 106.45 365.54 455.12 208.56 258.2 285.07 499.61 274.5 442.59 597.96 296.27 381.55 98.43 305.62 706.3 192.68 346.74 473.99 351.84 698.58 303.9 123.73 391.74 448.33 427.68 415.38 269.58 395.88 392.2 288.65 442.29 341.01 281.03 60.02 248 120.49 205.23 121.12 359.91 257.6 220.13 153.24 200.42 169.2 140.21 294 176.66 179.07 486.51 287.53 192.92 404.91 686.91 290.21 174.65 105.83 193.58 211.73 156.74 349.98 195.62 291.7 105.02 228.02 273.76 257.57 218.32 311.48 270.81 165.16 227.79 220.25 279.13 289.8 865.25 349.28 297.39 282.15 227.65 432.64 414.91 441.57 399.61 445.39 400.06 254.64 236.38 246.01 29.57 281.34 344.59 406.57 498.35 390.81 483.95 352.67 651.52 337.96 149.04 279.39 274.83 267.65 386.34 171.28 292.64 153.9 108.18 9.04 145.78 169.16 153.49 227.69 281.53 358.37 131.05 54.66 115.75 168.91 141.41 130.1 183.09 250.08 149.47 228.27 288 215.82 426.35 589.12 197.56 267.99 45.25 120.07 345.03 248.54 225.3 301.22 217.16 244.74 323.88 341.46 370.17 266.16 267.25 104.92 211.78 219.8 241.68 483.63 157.25 119.47 296.26 199.19 341.03 248.13 315.45 384.83 282.29 203.06 286.42 326.44 391.16 203.83 391.67 351.07 386.29 118.54 91.08 252.86 220.81 92.61 270.65 245.45 81.03 149.23 225.44 140.41 111 198.58 180.32 85.07 313.04 244.05 153.71 134.1 219.33 281.98 133.83 317.53 310.79 376.17 205.29 166.71 210.74 223.47 468.82 172.34 178.52 140.77 217.15 264.1 333.22 211 265.02 173.55 205.45 463.51 126.45 293.74 152.88 227.79 190.16 192.71 218.8 285.27 320.74 177.94 396.22 215.41 194.85 188.75 181.83 196.86 261.68 190.52 179.27 201895_at ARAF 187.18 290.64 319.53 261.78 337.31 386.34 149.71 337.27 317.96 293.21 336.24 271.55 275.1 305.63 263.5 270.78 361.27 209.23 210.71 245.47 361.89 227.49 523.57 271.96 133.48 294.3 297.76 210.94 207.74 213.14 255.06 362.96 371.81 400.16 338.21 445.06 374.16 246.78 312.07 400.41 258.42 391.27 361.05 572.22 295.91 299.43 244.38 300.19 422.75 273.07 201.94 279.61 352.08 302.06 385.6 301.43 997.55 300.04 383.75 290.22 417.65 311.38 297.26 205.83 207.67 182.12 284.18 183.91 131.58 251.12 211.88 391.73 176.95 237.24 159.39 253.86 174.63 218.1 209.92 239.24 134.77 306.11 292.78 304.6 268.68 291.19 343.9 284.73 305.26 222.25 228.22 295.17 284.16 149.25 177.48 229.56 241.32 256.62 176.89 197.52 277.53 253.82 302.31 207.42 265.93 304.88 242.91 243.26 360.23 160.06 278.37 235.9 238.1 287.76 296.12 125.72 294.15 257.61 240.71 320.79 227.01 253.54 353.16 252.05 176.72 174.71 287.13 173.33 165.32 156.91 209.34 239.08 230.04 215.83 181.71 189.02 243.91 161.96 183.61 178.35 128.23 191.83 178.29 223.75 162.62 166.18 242.3 335.01 376.22 341.78 282.15 412.09 248.71 268.82 306.18 363.27 376.11 304.8 259.6 334.45 435.88 257.26 280.58 321.19 343.31 412.17 355.19 323 197.83 247.02 226.53 172.53 238.44 241.08 145.86 348.31 194.31 175.26 183.58 146.25 202.62 191.07 521.94 188.39 184.17 181.04 202.41 243.82 228.56 188.66 179.02 180.83 239.96 240.87 187.25 242.5 150.2 255.16 227.94 159.42 912.64 202.48 188.66 230.92 266.1 233.79 107.6 185 144.18 194.5 264.54 220.26 478.06 305.76 256.9 269.12 229.72 309.52 273.84 225.76 275.71 203.92 302.95 207.24 205.81 165.64 298.67 201939_at PLK2 193.89 522.4 381.18 693.04 323.14 1641.2 272.09 124.87 209.68 157.41 577.69 265.24 386.2 858.15 684.3 340.79 323.22 112.1 197.48 617.84 343.75 846 102.66 214.94 265.85 354.16 355.42 88.05 383.65 334.67 237.55 220.75 205.66 732.99 148.68 262.13 139.79 163.78 263.79 200.82 123.25 131.21 137.36 204.52 277.97 529.01 633.95 515.89 167.14 1095.09 163.21 287.73 189.14 459.48 1156.15 201.9 203.1 140.24 454.9 216.16 598.76 114.76 317.75 321.53 44.38 204.68 274.92 146.82 93.58 238.81 207.61 173.41 331.74 362.31 118.2 242.78 320.65 263.32 406.42 91.69 206.79 1470.24 545.45 513.91 622.51 282.44 297.18 313.22 126.07 652.85 80.57 586.32 202.54 1294.82 365.22 385.34 192.92 146.72 239.57 241.29 121.92 396.28 356.66 432.47 991.16 355.74 437.16 311.38 406.15 24.63 125.59 910.01 143.29 407.68 120.1 324.17 1068.06 143.49 232.14 144.8 390.54 122.33 103.52 390.41 168.08 104.02 176.21 190.52 538.76 125.82 584.07 156.23 520.96 335.78 830.76 89.32 164.42 158.65 458.15 317.28 303.6 167.5 74.19 921.5 473.79 239.75 275.22 741.49 366.64 235.42 136.8 159.46 298.89 447.73 803.17 198.27 473.75 97.57 552.5 900.24 242.8 420.62 390.51 505.64 182.91 108.49 1523.7 564.05 128.58 115.04 440.23 439.14 467.72 200.15 316.5 190.69 125.04 132.88 294.61 207.57 293.67 257.56 212.71 464.21 101.86 162.78 856.53 199 318.42 215.28 510.78 529.88 270.59 188.09 365.91 245.88 397.98 163.08 227.44 366.03 392.03 384.61 642.48 23.46 139.73 435.1 172.11 75.39 275.6 473.83 75.98 132.5 134.46 282.04 255.35 119.23 453.37 80.98 522.39 483.16 148.61 187.86 432.44 354.28 335.23 136.71 367.69 202009_at PTK9L 195.21 110.76 153.36 188.15 104.63 123.46 156.29 132.4 180.8 163.5 154.06 115.33 167.1 121.39 135.16 205.36 140.91 157.43 183.43 152.46 146.77 158.41 151.45 134.43 162.88 162.59 179.35 127.54 134.41 213.97 152.48 156.3 152.6 132.02 151.45 174.57 148.66 156.69 142.8 242.81 180.65 173.53 153.7 128.06 162.93 134.99 141.67 116.36 183.5 170.86 139.36 146.68 167.5 211.9 199.25 155.85 192.77 209.39 142.66 157.06 113.87 108.89 122.9 138.73 152.79 141.32 149.18 169.2 185.42 166.24 203.86 176.31 151.32 149.75 153.6 151.73 179.73 181.18 169.87 194.61 163.52 134.92 144.45 176.26 139.87 122.04 124.83 157.66 130.35 131.98 165.19 138.21 129 125.23 136.4 160.84 135.9 150.77 139.15 123.83 90.29 103.65 158.85 114.1 133.93 161.02 158.8 152.57 154.93 284.76 176.48 130.27 173.77 145.59 167.3 119.79 114.05 120.96 147.06 116.12 138.63 206.19 142.89 129.09 121.7 213.78 122.9 132.5 171.18 143.12 167.55 127.17 118.22 161.6 118.95 134.23 109.43 135.32 128.73 152.35 105.02 100.02 140.88 125.1 128.18 109.25 111.39 165.47 146.06 189.89 154.2 113.61 147.47 192.56 154.2 183.4 178.16 213.45 147.75 156.61 108.36 135.42 114.25 145.39 134.19 128.11 123.42 164.14 175.6 132.5 176.57 169.56 119.81 261.48 210.52 156.06 188.71 156.57 132.24 151.75 162.64 152.52 107.97 146.36 144.86 131.13 151.18 139.52 128.62 148.9 99.04 145.7 133.21 148.01 120.62 178.21 131.72 137.1 129.57 112.83 177.51 151.15 126.79 117.91 106.47 103 90.71 111.57 117.99 120.16 123.13 111.83 175.46 129.41 90.87 99.83 128.19 133.21 129.22 94.03 181.62 142.53 130.45 168.16 150.42 131.98 133.5 202030_at BCKDK 135.94 219.18 199.67 208.57 202.22 289.38 196.64 180.34 252.05 261.15 286.08 201.44 158.73 278.05 107.63 241.12 239.36 132.62 129.43 151.29 153.44 150.49 165.9 157.16 92.95 138.65 172.19 145.21 146.91 259.37 158.89 139.34 164.46 316.8 168.68 244.43 196.32 195.79 154.57 230.91 140.42 164.12 128.04 164.25 228 108.77 149.7 157 220.2 175.34 154.34 217.92 237.99 251.26 275.18 230.31 173.27 216.57 330.19 151.8 201.75 146.18 152.09 240.65 156.37 157.93 243.94 142.09 280.12 114.87 133.78 122.23 191.47 599.99 239.12 232.49 194.12 134.89 223.78 222.49 159.52 242.33 120.49 336.8 163.99 225.04 162.71 165.78 176.04 135.53 84.3 172.7 295.3 158.63 248.14 196.56 293.75 269.78 159.19 164.94 139.87 148.83 198.82 162.93 227.28 236.02 349.56 212.6 354.44 207.6 185.78 140.54 133.61 171.46 206.03 78.58 209.27 110.41 150.95 172.6 257.43 319.89 408.89 205.53 112.68 226.13 126.43 120.94 194.28 123.49 109.03 199.38 172.27 126.02 142.51 127.97 204.08 118.87 141.85 130.05 122.21 171.68 139.63 197.97 119.21 171.76 150.15 252.44 203.2 189.73 229.68 153.05 246.66 174.02 173.23 255.23 231.68 201.23 168.46 142.36 161.09 134.17 145.6 188.81 153.25 262.96 197.07 132.03 122.22 176.47 184.78 211.36 256.09 205.03 134.81 261.91 132.27 165.77 171.01 177.75 136.79 257.03 155.08 146.18 159.67 166.98 158.34 200.06 289.78 265.16 188.59 148.58 227.35 223.79 166.6 219.62 167.55 358.06 278.86 151.95 384.41 275.11 221.62 234.79 233.58 179.26 88.5 157.56 176.5 226.59 126.42 185.48 215.46 162.59 157.1 208.42 193.23 213.89 235.89 147.71 269.94 203.99 189.5 223.13 270.28 248.18 164.38 202123_s_at ABL1 193.37 356.1 781.76 431.6 494.77 443.35 195.5 538.9 354.78 326.01 460.3 371.36 336.54 314.95 525.77 629.9 331.52 389.97 378.92 364.43 669.3 370.25 411.16 344.1 324.33 384.54 383.75 421.89 268.62 520.62 289.65 408.4 407.12 473.75 305.02 410.35 194.36 417.72 555.93 341.32 195.86 291.15 304.58 445.97 429.56 601.66 701.03 247.92 454.32 475.62 258.18 426.7 554.78 445.98 425.54 330.41 352.7 340.91 427.86 446.04 514.68 515.78 775.93 328.51 229.65 473.8 347.72 293.23 286.36 412.96 463.21 530.81 263.72 284.43 278.54 361.05 238.09 332.64 183.77 330.36 332.61 453.68 306.03 366.83 454.8 433.91 488.64 426.49 360.47 390.21 265.38 379.24 370.1 363.11 502.83 262.41 331.02 333.82 283.68 256.67 692.77 393.19 425.5 372.63 342.65 666.13 533.48 491.92 633.53 215.22 692.88 515.53 347.56 354.65 384.57 53.26 364.37 420.13 289.23 463.69 625.75 432.81 462.03 302.59 480.03 223.77 375.54 385.71 212.75 200.23 355.5 340.65 474.12 463.83 351.16 308.95 304.88 253.36 292.1 469.18 204.12 273.14 286.55 304.26 260.72 304.92 292.86 535.74 492.96 330.26 327.45 610.46 355.7 450.75 349.95 619.72 270.5 288.69 408.2 257.41 398.16 496.31 260.26 472.56 292.32 467.75 327.59 423.46 351.3 359.59 333.16 557.5 414.13 313.25 853.16 343.08 420.36 281.17 718.61 496.64 435.91 274.59 438.17 494.94 356.08 382.63 387.39 367.94 195.54 487.56 448.16 414.56 367.23 529.48 448.4 636.06 290.9 421.96 429.12 327.29 381.89 405.42 402.81 344.13 533.39 415.04 226.18 253.69 275.37 332.78 392.58 338.58 526.31 450.4 394.36 275.48 317.34 357.43 255.5 406.15 348.3 454.21 515.68 319.65 437.26 407.77 415.3 202126_at PRPF4B 278.41 287.93 111.43 294.62 269.85 196.57 278.44 283.47 249.93 237.55 336.13 248.23 292.04 195.46 90.59 271.89 338.89 178.23 369.74 650.61 317.61 492.04 174.52 278 225.53 302.58 273.18 238.53 178.1 304.62 389.23 278.31 363.89 207.87 385.29 325.3 229.28 199.72 169.42 152.97 406.54 308.93 314.58 196.62 180.3 249.75 207.39 169.79 181.92 196.69 150.11 291.85 182.66 250.45 149.28 273.52 226.19 224.95 199.32 90.29 170.2 424.3 192.81 153.69 236.48 272.73 292.76 180.42 272.54 273 200.21 221.64 238.81 259.57 198.58 184.59 340.83 392.3 268.91 164.9 86.84 398.88 270.39 318.46 306.08 389.53 246.11 259.16 182.24 315.16 19.32 223.29 282.65 286.15 352.42 319.29 206.65 227.04 339.29 260.75 246.75 301.45 230.69 186.78 315.59 268.52 258.83 202.53 97.53 10.49 129.68 265.99 240.87 250.22 338.77 426.51 193.96 69.79 142.23 128.57 144.84 178.15 128.66 321.23 190.32 277.24 234.08 222.98 414.64 200.84 210.43 152.98 107.76 269.96 243.94 241.99 220.66 364.73 239.27 278.09 256.33 221.11 244.38 316.15 209.21 59.61 341 224.17 165.45 339.32 258 124.06 161.11 292.52 245.2 347.09 232.32 164.83 246.12 242.33 158.35 233.04 246.59 135.54 207.18 197.08 249.72 273.97 32.47 256.46 202.32 84.43 209.01 292.23 33.04 178.1 194.48 223.34 204.43 132.43 242.63 90.03 327.22 183.44 130.05 251.66 185.49 264.35 111.57 320.18 163.21 370.61 164.73 309.54 265.64 241.58 316.98 137.77 201.23 96.61 149.49 224.25 271.6 24.43 140.59 259.96 256.96 237.07 116.17 391.98 192.19 154.13 163.22 105.33 172.56 183.18 231.55 305.67 222.78 105.23 306.89 252.02 251.09 284.12 302.22 350.31 282.31 202130_at RIOK3 626.68 685.24 376.96 403.49 416.45 498.68 739.66 549.32 388.28 568.27 380.8 790.6 522.3 526.68 455.57 574.85 548.78 443.93 1022.28 745.88 390 501.15 563.17 456.27 995.4 487.18 398.36 554.87 652.83 646.47 840.8 431.44 302.58 408.74 503.24 581.55 522.72 432.56 459.18 536.01 779.72 488.74 401 532.25 659.61 377.89 523.94 481.28 525.7 392.09 577.63 548.58 488.15 961.2 608.04 432.22 478.18 513.76 428.27 357.51 432.98 238.78 1027.71 289.68 339.8 458.53 515.24 397.57 478.34 1193.23 489.32 282.54 629.66 645.54 411.45 262.65 1322.7 543.75 385.12 404.16 281.85 574.88 461.4 294.68 468.67 454.94 289.01 492.99 256.89 524.19 124.44 385.95 423.07 570.53 451.79 525.96 586.44 506.03 364.38 476.18 642.77 597.45 388.61 380.23 422.42 597.24 521.28 623.12 497.4 102.85 619.12 478.45 765.38 510.44 646.87 879.9 520.83 427.71 371.27 406.55 388.4 538.15 262.23 640.22 385.73 331.89 259.54 466.32 392.95 641.81 414.98 415.85 212.42 378.27 479.24 309.62 375.42 469.05 284.94 453.01 227.02 342.97 395.75 405.66 329.04 249.81 209.22 394.45 422.86 710.63 647.42 413.42 614.04 489.41 540.21 502.91 885.27 767.59 563.49 870.78 392.3 507.33 538.5 356.4 274.71 455.47 486.45 377.88 290.58 272.76 335.56 247.05 315.11 402.42 182.53 285.16 288.27 551.46 276.45 253.55 370.36 411.39 214.1 330.63 344.73 176.57 306.96 317.75 457.39 264.3 334.43 396.15 358.11 271.41 353.79 398.11 540.35 255.92 311.98 328.88 242.61 469.27 313.92 280.16 489.39 340.25 707.81 434.04 316.69 268.75 428.5 436.29 237.66 270.61 250.73 340.87 380.11 307.71 273.23 334.92 549.47 395.82 519.92 639.78 562.16 967.49 420.95 202140_s_at CLK3 158.55 131.11 196.54 128.08 238.64 224.42 209.73 144.87 217.49 188.69 213.92 192.02 192.55 241.46 193.48 264.21 208.18 214.06 195.02 171.96 214.96 158.49 120.62 212.91 115.53 198.88 139.93 138.74 187.37 162.78 129.2 136.6 217.67 265.17 205.34 159.99 141.25 152.3 193.03 177.71 210.31 158.59 205.69 242.24 203.18 171.86 137.51 180.96 186.24 178.42 192.27 150.57 186.93 197.11 256.81 161.29 189.7 176.86 183.81 218.48 254.27 189.68 140.09 142.25 288.56 159.11 191.6 161.69 147.79 136.41 152.64 217.61 128.45 145.44 138.01 140.53 323.27 170.14 118.15 111.01 152.6 138.42 130.12 149.64 112.92 104.24 120.69 143.85 104.08 75.98 255.2 127.7 128.22 59.3 66.57 93.93 148.01 40.73 79.95 115.89 204.45 158.44 121.24 143.52 110.46 166.01 157.09 151.32 177.63 316.32 134.39 148.93 157.38 154.9 141.48 95.33 203.96 190.83 118.25 178.64 156.8 169.11 138.86 163.89 128.59 155.95 167.68 109.94 132.03 106.56 157.83 126.32 195.64 150.67 142.2 126.02 102.38 96.35 79.19 112.43 72.7 131.15 87.03 101.04 114.59 181.18 129.74 249.88 184.96 183.91 189.66 206.54 179.8 130.04 158.08 148.11 139.81 166.17 160.11 154.72 145.56 148.6 118.01 149.43 129.91 166.11 133.46 204.71 250.97 176.48 173.39 169.37 159.63 150.16 183.55 177.68 169.77 108.63 111.72 161.18 151.68 204.51 135.07 158 128.18 184.25 208.04 164.37 201.35 94.98 120.6 123.75 138.75 163.19 140.94 137.1 134.56 175.6 181.26 112.03 181.42 147.48 136.27 188.17 148.85 117.88 98.97 146.16 149.25 178.9 202.5 149.33 159.4 254.61 137.85 150.54 160.24 231.81 148.11 162.09 185.57 154.85 169.03 157.07 168.32 185.53 145.42 202161_at PKN1 103.66 224.54 224.18 287.86 204.94 285.18 237.36 276.25 298.51 325.59 323.83 189.99 262.4 427.92 134.53 305.91 78.94 161.35 161.99 320.99 184.79 89.94 360.41 192.33 205.74 160.09 130.83 213.79 158.41 134.96 193.48 108.83 139.4 360.43 296.54 304.39 287.17 253.85 238.33 226.06 250.27 214.56 290.63 310.23 159.36 195.32 144.83 215.02 262.89 279.38 166.67 245.63 328.74 270.14 494.03 220.17 292.12 140.26 251.63 212.1 207.02 410.47 210.56 158.84 195.59 194.53 389.99 282.67 193.93 196.28 212.62 234.36 169.94 272.07 187.02 195.88 500.05 122.58 169.73 213.16 274.15 125.19 214.17 225.64 194.58 235.03 276.16 225.38 215.17 126.18 211.88 192.94 147.53 105.24 109.88 110.07 166.06 322.03 101.29 136.43 123.32 89.83 183.9 108.81 143.82 289.11 203.08 244.97 267.91 272.16 269.26 157.99 217.33 220.27 156.17 45.13 349.97 188.89 194.44 97.17 229.13 526.1 394.71 236.52 134.77 150.04 208.89 141.25 117.22 74.43 147 149.16 151 115.15 109.16 132.3 103.98 75.1 123.88 120.62 75.98 70.7 135.94 117.2 97 150.51 166.69 217.52 322.86 245 273.11 320.13 171.37 137.37 230.79 189.92 132.83 233.35 249.84 135.63 249.52 144.22 225.07 149.8 233.41 250.02 209.93 202.54 443.31 213.1 275.1 176.71 151.13 230.01 113.98 122.8 186.38 164.68 162.53 338.13 131.31 140.85 125.97 154.49 128.01 182.64 91.88 235.45 157.35 111.97 160.86 133.17 188.27 178.14 125.29 125.33 193.87 255.67 120.92 169.04 224.07 238.18 167.31 118.33 165.04 156.23 101.19 92.08 106.92 145.9 199.6 160.72 254.09 99.49 207.01 215.9 142.69 136.26 174.81 236.35 270.56 195.71 287.89 201.56 207.73 261.58 167.38 202178_at PRKCZ 304.35 136.47 104.64 92.76 183.08 299.02 284.08 191.62 85.08 287.66 150.92 415.74 193.73 164.86 206.98 114.1 372.01 159.57 239.54 292.37 180.56 129.28 190.85 168.98 215.39 141.88 139.73 245.44 163.47 156.62 105.64 211.5 242.41 168 303.88 164.99 103.98 105.15 75.04 171.88 157.86 140.27 182.54 129.41 88.95 116.39 117.54 139.49 125.31 79.1 100.48 191.81 182.69 160.59 347.26 174.61 281.91 25.35 215.45 89.04 106.04 193.89 40.68 88.69 306.58 113.48 173.86 59.84 166.85 134.25 46.44 165.46 106.2 88.43 169.08 107.47 136.04 93.59 60.74 135.33 271.78 163.24 242.27 172.39 135.62 106.78 99.9 72.16 138.09 215.1 356.29 176.56 168.03 120.51 110.85 168.39 132.51 395.05 270.06 198.9 185.82 173.32 241.29 341.61 190.96 205.45 180.28 179.29 273.69 359.35 152.48 158.81 192.66 233.24 124.76 126.07 296.12 100.89 209.13 103.05 96.01 396.07 216.02 171.25 69.28 141.22 113.4 106.6 58.59 169.72 128.54 142.38 167.82 136.08 168.56 167.23 129.41 81.2 103.37 150.96 41.79 91.37 195.75 138.81 192.97 214.04 111.55 278.26 116.98 236.19 157.07 168.61 151.39 151.2 194.36 153.91 74.8 93.73 127.66 199.26 271.55 154 70.46 194.29 52.99 131.02 142.41 166.84 234.96 127.28 138.63 232.54 216.38 115.1 436.42 245.18 147.31 103.91 144.54 239.08 120.28 285.07 80.59 196.05 103.57 80.76 142.62 160.16 345.99 93.7 97.22 103.67 136.35 82.52 119.33 232.68 205.67 92.75 119.95 213.64 256.84 151.49 138.3 111.1 121.93 109.78 63.14 144.56 112.15 117.96 247.32 115.03 146.58 174.81 166.65 97.43 182.41 174.47 151.02 185.72 186.18 134.76 384.61 327.47 233.4 126 187.2 202193_at LIMK2 1273.66 154.47 367.89 124.49 135.98 178.64 139.99 200.72 195.04 379.86 223.2 441.86 225.86 188.96 391.15 200.48 331.19 204.67 219.69 199.26 93.47 195.35 114.86 215.67 375.86 348.52 267.72 158.82 291.71 140.93 182.86 230.82 101.65 613.3 140.03 306.18 297.69 221.72 299.26 142.65 365.64 170.18 173.53 194.35 244.07 235.51 126.55 235.15 285.08 408.2 461.17 176.07 238.72 375.83 225.83 111.01 219.04 304.85 196.11 254.86 307.88 92 725.42 119.6 108.58 508.14 255.54 187.75 309.7 500.33 420.78 149.03 174.12 275.01 168.91 274.47 130.51 91.57 290.94 122.66 256 313.33 191.69 113.02 113.6 92.58 306 107.93 133.46 290.1 65.4 303.01 364.02 185.86 495.18 590.83 258.98 448.26 269.94 800.96 539.68 336.67 195.8 499.26 258.85 180.87 307.32 572.68 214.54 112.16 304.03 362.79 140.98 265.25 376.29 600.9 130.66 497.46 419.38 319.69 354.43 222.97 104.74 285.04 172.93 179.48 168.13 226.99 294.21 221.7 211.31 423.09 326.2 227.46 359.48 139.44 183.64 305.43 265.58 292.52 217.44 388.9 156.31 232.07 152.36 204.16 263.97 123.89 358.48 390.92 187.38 227.42 211.01 259.82 245.97 451.84 107.9 189.16 249.34 219.73 135.61 249.2 146.4 126.46 126.98 154.38 131.2 226.1 235.66 87.83 173.49 145.31 188.48 728.14 232.19 205.53 214.73 148.59 365.03 309.67 166.27 322.23 102.72 196.36 613.98 205.53 253.57 183.04 386.95 308.11 190.4 271.32 130.18 474.45 256.61 374.81 512.38 477.8 637.03 424.88 211.15 310.31 201.16 61.96 258.68 216.84 255.1 177.91 176.64 269.79 516.93 267.42 238.29 296.87 178.96 227.24 342.98 230.18 145.71 248.72 321.01 310.97 319.49 888.95 340.67 223.2 242.62 202200_s_at SRPK1 600.36 406.38 255.6 392.49 384.85 250.65 363.18 483.41 232.03 704.56 539.76 352.67 315.42 363.79 205.81 508.35 1013.2 647.51 864.7 1431.96 527.65 657.31 534.51 701.5 721.5 368.42 574.07 575.65 590.01 515.33 327.28 515.74 504.52 367.87 426.91 876.95 808.82 696.22 208.37 392.69 430.32 587.91 692.08 359.06 535.18 235.77 432.79 411.49 888.78 294.39 577.47 274.55 227.45 443.72 266.12 288.45 493.79 477.71 284.8 279.74 306.44 1235.68 502.54 275.08 307.2 725.29 385.89 658.62 645.38 783.95 480.41 608.35 617.47 557.41 536.73 399.06 607.13 255.74 225.09 182.98 189.52 222.45 261.36 210.95 177.89 220.94 330.64 162.64 238.92 313.07 56.15 248.55 214.9 270.23 336.8 555.67 253.26 543.56 322.23 545.22 346.33 368.66 323.68 201.1 434.63 386.94 201.81 275.85 168.36 45.22 573.15 169.92 403.54 352.9 559.82 702.96 423 187.76 359.51 348.5 189.22 402.7 231.72 507.72 200.25 558.29 285.81 322.87 326.58 771.44 262.98 296.2 140.67 387.93 348.64 325.42 697.11 260.26 450.01 388.99 249.2 523.65 500.82 434.11 290.26 152.58 542.65 137.88 253.33 314.94 659 184.87 454.87 358.62 244.21 570.41 192.83 252.28 207 348.19 161.44 261.49 221.42 148.34 245.38 244.25 245.96 327.62 169.76 317.74 136.73 139.84 276.1 498.17 50.73 244.5 194.14 313.15 240.76 336.36 243.41 568.53 290.63 142.89 242.79 353.48 279.86 289.45 604.13 505.7 262.18 279.89 178.69 437.65 261.56 268.77 647.88 351.35 288.29 470.06 269.76 253.26 336.07 120.16 757.05 188.23 560.63 798.13 292.77 361.79 293.51 250.83 143.83 100.45 280.17 208.99 362.48 254.83 205.57 207.47 345.99 686.31 196.96 778.61 455.2 403.55 179.36 202240_at PLK1 162.11 143.48 60.22 98.22 163.11 31.57 149.43 128.31 122.17 138.55 63.67 36.64 40.4 172.15 29.68 83.06 344.63 313.29 194.91 179.44 184.51 143.82 164.97 157.13 159.49 100.58 94.61 181.69 195.19 264.2 158.28 105.4 217.25 147.03 192.26 532.15 153.73 278.31 50.01 184.88 174.07 356.1 137.29 171.35 149.03 51.89 147.43 108.59 166.99 111.28 106.4 68.1 177.18 138.87 135.5 113.04 240.35 205.88 188.1 108.84 113.39 538.96 239.74 77.3 98.08 188.91 327.9 368.23 403.7 139.25 189.57 116.33 115.35 92.48 164.25 107.68 238.11 44.44 51.47 100.73 53.26 75.03 59.24 62.64 27.85 68.67 70.85 62.8 122.29 40.35 64.83 41.13 47.47 51.34 45.05 65.22 58.85 92.4 70.33 133.84 85.62 65.44 73.53 35.7 90.77 154.61 37.18 51.34 149.19 68.02 153.07 24.7 59.12 165.83 125.24 31.27 128.52 61.98 62.79 87.52 62.77 269.14 78.43 126.53 26.67 138.17 62.63 44.18 83.13 162.7 39.2 61.5 50.05 74.54 55.47 85.15 120.82 53.39 47.13 59.35 38.51 84.98 147.29 70.79 52 55.11 74.37 85.05 84.63 51.93 404.04 79.71 151.45 77.16 50.28 348.08 102.72 250.83 48.06 209.38 75.02 40.04 76.04 54.61 67.33 68.82 62.68 67.81 50.24 141.99 70.02 74.24 86 512.06 59.99 87.45 105.38 62.4 37.67 75.2 52.74 161.78 78.03 57.76 69.29 64.25 79.45 74.86 192.15 73.84 49.31 32.74 57.47 168.85 45.33 77.97 132.08 127.67 69.11 114.06 52.18 62.04 70.68 63.32 145.27 24.5 84.49 86.79 52.53 61.31 90.3 42.55 79.73 52.7 81.66 67.85 66.07 200.08 94.2 52.17 152.55 130.31 41.3 160.9 217.51 241.26 43.61 202241_at TRIB1 1096.58 1227.1 873.14 1038.71 1405.27 951.41 1208.5 1020.34 541.87 953.88 949.41 1923.98 1186.6 1623.93 740.49 742.73 355.13 839.12 1663.11 2529.43 1786.27 1264.69 2523.99 1499.68 844.17 1745.5 784.06 553.72 1598.08 762.5 1183.12 1775.33 2265.84 2449.68 803.51 1318.58 2374.42 1554.16 709.83 747.6 494.69 831.13 987.49 836.36 385.48 937.19 1099.24 836.29 773.52 2668.38 1114.78 712.39 1003.36 839.57 1043.6 1294.87 3504.12 1105.03 836.69 2246.29 2226.17 1035.69 509.16 1300.18 2089.59 494.31 808.13 966.6 1770.87 827.53 955.59 1289.92 1296.04 1972.64 751.99 1262.32 463.23 1801.14 2790.86 994.21 1070.36 2279.44 965.27 3032.47 656.67 1299.45 714.5 944.79 1330.45 1048.52 1006.36 1513.02 2112.7 591.52 1072.31 1405.32 428.13 1184.31 2721.85 1104.23 1400.89 721.15 459.74 1291.45 520.56 657.36 412.87 1770.79 849.75 221.28 802.48 798.61 444.37 964.15 959.71 602.86 254.41 399.54 564.31 741.53 1051.43 926.1 786.22 721.91 701.87 1416.96 3627.42 1084.94 1140.7 649.78 734.69 617.3 870.51 957.59 818.64 416.69 488.41 704.72 1176.34 818.59 2040.84 2815.29 662.49 640.18 532.86 855.35 286.74 700.38 1625.13 951.42 805.17 1232.98 3158.67 609.08 1010.37 728.49 3294.83 1480.1 350.57 1498.87 905.15 735.97 1891.35 643.75 523.32 379.78 567.14 1119.45 495.47 1344.06 846.75 679.84 1188.42 628.24 505.36 624.44 632.91 680.06 403.48 2136.54 1142.98 247.72 579.72 311.98 795.9 1061.45 562.29 755.41 384.36 1828.86 908.07 405.58 328.98 631.01 489.86 568.35 896.13 386.43 1483.94 1022.37 800.24 2357.87 589.25 242.02 739.73 1597.92 1101.17 784.04 754.05 999.68 626.71 406.23 466.81 503.82 1322.15 393.63 534.28 844.7 364.6 1316.02 736.68 2108.97 265.86 1705.35 1111.46 1921.49 612.65 202246_s_at CDK4 848.78 710.97 584.91 828.84 610.39 466.6 490.8 541.37 701.77 666.47 692.53 498.34 636.55 435.68 410.01 967.1 1795.95 868.21 621.3 825.03 1069.06 791.51 789.58 754.23 940.57 483.02 916.39 787.14 1140.1 1093.93 2608.63 776.75 621.07 751.56 784.21 1282.86 1067.51 1265.27 576.25 650.8 1090.64 662.47 1188.47 462.31 613.85 577.25 855.5 375.86 901.07 647.96 948.73 761.35 2588.08 649.84 1294.06 894.36 889.02 1144.79 1158.27 605.13 741.39 1760.91 1037.29 936.38 347.15 653.05 797.97 695.89 720.42 708.74 539.14 1177.4 611.06 887.61 956.48 656.99 715.34 610.03 584.18 787.24 837.94 692.62 496.82 470.88 520.07 699.45 605.12 562.43 534.2 225.05 304.97 322.14 326.02 195.28 192.61 275.66 492.81 623.25 271.52 280.44 436.88 493.96 380.21 239.59 415.83 670.72 574.09 553.51 567.2 230.56 581.38 496.84 413.96 676.59 510.23 406.25 406.21 375.37 417.87 663.14 362.53 642.68 1267.85 820.64 346.2 728.85 324.22 393.01 512.14 496.42 400.26 574.01 359.94 493.45 468.82 498.08 514.19 482.34 394.09 524.93 325.38 498.37 587.18 475.52 440.51 318.97 3943.49 745.41 698.46 796.45 569.93 446.26 607.53 604.5 508.84 872.06 482.8 466.48 605.02 395.89 668.1 590.01 502.72 730.86 489.23 513.31 536.73 462.3 213.58 605.67 488.66 513.69 634.18 554.01 433.29 596.51 488.18 322.44 401.44 596.04 441.11 881.16 360.05 632.1 647.28 468.18 397.4 454.15 970.59 512.01 374.95 542.37 598.9 566.92 644.45 548.94 733.91 719.6 648.74 577.11 618.62 553.2 480.48 434.32 677.11 424.98 413.23 495.55 432.76 796.99 715.27 408.13 482.09 519.71 448.56 367.5 374.93 488.76 541.18 263.12 516.66 534.05 462.09 618.04 720.95 686.21 478.12 202273_at PDGFRB 209.8 510.62 1348.57 398.73 696.24 834.46 347.07 655.71 438.42 462.13 529.25 294.42 401.28 349.22 710.22 484.3 288.95 229.73 484.35 271.89 638.88 309.42 178.25 453.64 290.49 411.96 590.19 437.94 357.1 391.77 526.68 379.24 538.62 339 344.5 212.04 277.99 502.17 861.58 240.6 261.17 274.19 268.46 526.58 489.8 943.11 684.04 236.63 664.19 677.93 330.57 615.07 909.4 538.04 354.51 1189.62 285.81 299.34 370.49 573.11 554.67 270.81 985.74 304.76 171.41 362.72 151.77 297.51 171.06 511.85 555.08 370.19 340.38 487.6 252.17 410.47 190.59 518.44 178.88 636.77 371.62 436.15 204.55 497.11 633.76 459.84 572.74 579.81 238.69 353.07 320.37 565.97 336.08 351.47 479.86 216.5 553.91 415.58 291.13 326.49 546.12 394.71 457.06 537.64 456.46 447.69 709.62 601.43 1185.09 202.75 394.5 663.19 596.93 495.58 378.89 179.17 660.13 491.74 363.91 709.19 819.18 341.89 286.03 405.75 910.21 218.4 588.67 1047.4 351.07 222.57 759.5 525.34 836.15 444.75 527.12 418.02 422.25 560.32 633.01 655.43 288.87 357.27 200.72 429.68 653.44 338.87 248.87 596.17 768.46 305.17 386.21 877.37 600.45 676.52 336.65 461.91 336.53 388.16 720.94 303.29 583.55 794.99 443.65 599.81 553.93 637.51 547.93 668.08 213.72 374.73 396.01 804.94 475.61 205.36 180.46 540.07 637.19 426.33 1238.49 257.12 651.04 504.97 448.24 930.01 695.37 606.61 380.34 462.06 215.54 355.44 739.63 718.39 812.3 451.48 652.49 583.19 235.25 645.89 480.37 436.43 412.09 392.18 516.25 135.93 432.76 586.82 180.17 321.39 510.91 332.85 548.88 596.33 762.07 445.32 548.17 395.39 514.51 327.17 512.27 551.94 322.42 469.61 768.1 377.4 624.1 173.14 407.24 202288_at FRAP1 90.87 96.16 79.19 63.41 86.39 95.46 91.55 128.27 70.03 85.5 65.49 166.08 86.41 68.1 101.31 77.46 147.11 91.82 197.29 113.91 78.43 99.08 78.79 80.25 82.27 85.88 74.05 86.53 101.92 74.05 69.48 74 120.45 73.43 106.82 131.65 57.39 62.42 70.17 60.75 86.7 94.32 86.2 69.13 67.37 66.86 83.4 47.43 80.12 170.69 70.93 80.74 100.9 87.38 105.97 73.76 112.3 113.75 98.43 66.84 56.68 106.76 87.64 58.92 83.4 73.75 67.89 85.62 134.67 136.08 71.53 131.42 48.09 66.09 81.46 50.61 50.73 36.1 39.19 42.58 48.24 80.58 73.99 87.02 62.65 54.6 56.97 51.11 63.68 51.47 61.36 55.55 57.73 55.31 51.15 69.19 72.31 101.98 52.64 49.68 82.75 103.7 79.93 60.44 61.73 74.05 73.3 71.86 110.62 65.22 48.42 51.49 61.86 66.72 45.4 52.21 70.43 59.07 46.52 63.19 53.42 69.71 69.18 53.42 58.52 79.67 64.07 81.35 57.73 79.61 81.6 66.64 76.17 73.75 88.34 65.86 96.34 78.93 64.99 71.28 36.63 75.35 97.77 90.24 78.7 65.31 55.44 85.51 78.91 79.67 123.9 76.34 72.77 72.61 85.42 76.81 54.36 57.93 78.84 95.09 73.25 70.5 43.34 47.84 49.3 72.5 71.18 80.78 60.62 54.28 86.08 81.32 79.94 93.18 60.42 92.48 57.2 80.04 91.19 72.67 60 53.97 42.73 71.33 67.93 44.8 63.18 76.08 54.78 62.7 56.36 55.57 61.8 69.3 66.18 97.11 50.18 78.69 64.73 65.23 74.91 72.54 74.7 52.97 73.16 58.79 56.34 74.14 58.3 55.2 65.74 68.88 58.26 77.93 91.65 68.59 113.86 83.31 70.98 73.71 97.82 84.82 104.32 85.39 74.75 82.85 78.33 202315_s_at BCR 374.21 145.66 284.41 207.06 208.23 166.64 170.09 224.66 248.24 305.89 209.5 264.71 218.45 147.91 181.96 111.94 387.94 321.46 303.75 199.21 168.24 106.78 401.89 116.85 209.06 208.1 198.39 263.84 160.81 142.6 420.69 142.22 269.13 314.08 214.79 356.9 230.14 266.02 183.73 169.71 294.53 500.44 367.17 281.48 272.58 230.2 96.66 185.63 245.89 283.63 166.66 172.97 337.4 351.72 478.86 179.53 610.15 488.75 238.88 158.08 253.64 186.64 292.12 191.1 194.83 163.99 219.35 351.12 500.72 160.13 193.39 137.98 206.67 193.58 402 233.42 89.4 112.03 120.61 400.54 204.2 253.53 175.68 160.44 127.71 137.3 238.87 164.05 240 159.37 66.6 159.46 144.9 50.16 113.05 160.79 143.41 95.81 79.32 216.37 193.55 272.59 141.63 93.15 154 205.59 181.73 209.48 192.44 60.52 219.25 140.62 115.62 198.4 120.1 69.93 131.6 302.74 122.3 259.81 208.01 293.62 376.4 169.01 124.13 105.78 109.94 142.52 155.38 211.91 115.15 235.18 165.09 198.07 144.47 117.66 82.97 104.92 92.85 138.08 78.12 173.87 81.99 167.2 80.4 153.38 145.62 253.44 227.93 158.94 332.6 270.88 183.99 170.85 188.36 234.84 163.32 256.01 144.61 201.85 140.65 170.53 78.35 119.31 119.41 174.77 122.18 227.32 143.71 113.26 278.45 103.77 107.91 146.69 200.48 195.74 147 55.8 144.64 154.13 86.83 187.17 108.84 104.33 181.61 155.34 140.61 396.46 211.04 137.22 129.2 102.01 87.76 186.46 157.72 247.16 182.23 262.42 239.11 166.64 246.27 203.23 232.63 75.21 133.26 123.23 171.1 121.52 90.84 108.45 155.73 139.94 263.76 199.4 130.9 268.75 222.58 225.02 156.9 176.68 257.23 167.55 211.41 367.15 211.18 177.26 178.49 202329_at CSK 275.09 271.76 515.78 580.89 518.24 454.5 581.38 393.32 399.56 565.83 393.77 374.92 356.61 365.91 288.79 389.31 476.38 827.4 507.35 354.7 396.87 440 224.97 605.62 334.56 617.15 302.26 450.48 506.15 354.91 248.82 261.79 421.61 566.07 623.27 465.86 329.07 375.04 550.13 714.1 464.6 685.03 357.44 376.72 351.9 325.17 380.95 343.33 466.42 292.75 259.83 424.55 594.96 435.25 482.72 303.92 447.21 385.88 569.82 368.31 331.05 515.92 393.03 421.59 1398.71 224.33 631.08 423.55 357.32 275.54 416.4 513.56 280.41 365.15 138.29 167.49 302.38 338.43 174.37 215.31 259.79 385.54 288.19 336.95 279.68 280.68 362.93 430.99 402.14 834.38 353.48 357.85 495.78 272.4 599.07 381.42 291.67 109.09 427.07 452.68 328.23 360.71 296.94 343.87 233.24 451.93 283.01 371.37 525.88 196.91 391.46 292.39 463.44 388.81 623.25 273.42 496.97 496 441.54 414.8 432.43 449.9 516.29 520.87 211.36 384.36 245.51 292.46 206.8 284.52 421.6 275.79 335.42 386.32 296.68 261.68 321.7 196.2 176.92 310.98 142.55 208.45 206.98 216.4 249.71 404.07 311.95 396.26 478.06 297.93 412.75 623.67 299.37 341.16 388.12 421.17 260.14 493.8 296.42 562.76 422.89 326.76 212.37 403 230.34 445.45 255.57 633.57 544.8 308.74 324.05 336.98 314.48 335.48 390.97 259.32 353.97 206.23 236.38 390.95 314.66 307.57 273.81 257.08 341.67 347.64 314.14 306.94 395.99 217.94 261.41 200.62 303.23 305.08 231.04 366.34 242.04 414.4 391.54 334.35 447.11 337.08 336.14 375.62 396.55 224.78 270.32 246.42 277.41 275.35 403.22 400.64 385.55 304.85 382.51 249.81 406.66 442.12 348.32 315.15 679.38 618.67 438.75 453.94 489.73 564.61 503.56 202332_at CSNK1E 230.49 173.25 250.31 182.54 258.89 274.76 173.67 528.12 203.35 286.26 236.98 322.72 288.23 235.84 254.25 287.11 270.06 289.46 460.89 349.78 306.02 203.41 304.6 229.54 630.32 172.75 193.5 180.64 210.08 203.09 130.1 240.4 170.38 400.42 124.29 149.8 283.77 195.94 185.79 136.45 208.78 210.31 353.48 299.56 238.53 214.37 217.75 134.75 165.21 198.75 323.05 188.99 271.81 248.03 421.33 168.45 293.36 121.74 234.39 279.83 409.53 340.39 238.09 157.57 244.47 442.73 393.22 276.57 337.06 359.49 424.37 188.9 178.6 157.55 354.82 321.49 168.69 147.08 179.59 174.38 417.16 205.67 231.33 185.06 193.59 181.43 254.95 214.05 248.51 380.82 429.41 357.95 232.47 268.1 539.96 157.1 311.01 380.79 209.95 467.52 967.3 223.89 273.63 342.46 327.15 208.31 312.06 290.68 262.1 384.47 302.79 361.89 220.95 309.65 179.59 212.38 704.78 254.59 256.42 413.48 303.5 501.32 465.21 230.23 230.65 112.67 198.36 189.67 153.44 151.06 219.18 140.96 496.21 164.53 189.05 272.77 161.46 134.81 191.89 246.15 163.99 170.35 124.45 256.35 151.94 249.58 203.78 260.09 315.27 284.75 304.23 352.67 250.14 189.28 247.24 235.52 169.05 170.74 219.43 263.54 205.6 327.72 190.4 173.8 196.39 185.44 174.87 405.59 431.73 122.44 362.76 235.06 249.65 407.47 758.02 276.3 308.08 171.75 426.17 507.29 230.78 269.98 177.6 288.17 342.93 174.28 151.83 360.91 263.19 229.24 306.55 240.74 198.68 183.61 283.03 282.44 273.42 210.9 282.84 522.22 207.83 211.03 149.71 270.05 178.42 230.81 263.64 204.94 152.18 176.85 242.86 159.04 203.87 270.93 354.36 237.8 211.84 150.51 169.05 631.95 212.48 250.98 417.5 352.39 271.39 303.59 185.08 202424_at MAP2K2 636.52 738.05 583.48 680.23 634.11 593.87 500.16 568.7 715.71 848.63 648.64 748.04 656.55 738.53 535.24 974.32 689.69 591.43 635.42 682.71 492.18 550.27 896.53 680.78 728.03 424.71 503.1 407.08 455.07 549.4 413.33 343.49 470.78 867.18 675.41 954.19 672.75 677.53 584.33 643.48 883.28 1108.77 1339.1 561.47 458.78 584.62 565.08 420.08 748.54 537.6 288.78 523.5 942.55 633.31 1106.83 583.02 904.49 439.02 717.2 816.86 552.57 617.6 486.66 454.81 554.96 410.55 594.42 640.17 503.28 508.88 586.37 687.25 542.59 562.45 1027.42 676.11 510.27 342.91 344.15 367.5 750.88 609.12 462.63 574.66 354.39 428.74 602.33 450.08 510.44 248.26 556.97 481.25 481.16 265.98 200.13 335.91 385.96 403.57 230.24 524.72 491.59 561.92 644.36 320.18 418.12 785.67 561.6 479.23 700.61 795.07 705.77 483.46 438.57 704.19 491.38 116.32 532.06 458.85 622.28 886.94 595.08 463.71 888.43 611.71 288.04 296.93 273.75 318.23 268.84 336.6 494.94 420.05 529.58 315.49 326.97 310.8 313.92 252.05 348.11 534.04 150.44 250.38 585.76 324.88 243.81 445.25 477.07 540.4 698.08 614.83 526.77 862.12 801.69 569.64 656.5 756.69 749.86 727.29 511.8 759.47 728.51 723.32 673.11 661.25 699.88 943.65 765.44 717.52 881.9 423.31 697.62 680.03 554.81 668.81 1029.47 802.91 420.27 311.2 426.41 852.09 470.15 569.36 424.6 567.08 534.69 437.5 405.31 430.79 596.26 608 512.98 421.57 730.77 694.54 624.4 856.62 526.06 806.9 745.73 577.87 672 810.78 541.92 610.19 773.6 575.18 445.91 524.64 453.58 386.26 626.06 645.09 807.53 800.62 611.23 607.4 500.58 563.94 654.51 668.49 700.57 530.68 598.22 613.17 499.84 881.07 589.57 202478_at TRIB2 386.82 351.98 1363.3 640.29 720.91 745.69 435 387.18 528.94 486.99 473.39 562.66 696.33 272.82 1014.43 1367.54 201.31 598.45 534.37 342.4 478.07 843.7 464.53 703.64 506.55 698.3 372.29 206.25 610.75 385.7 470.63 1055.2 430.42 281.49 422.11 817.29 640.89 955.78 672.69 563.16 375.36 993.49 436.24 989.04 506.95 730.83 945.73 660.93 764.93 648.09 169.47 521.34 646.63 586.73 359.02 478.94 746.65 286.05 803.03 813.09 765.06 181.67 493.55 205.04 2089.87 201.5 450.73 208.9 169.36 509.97 642.11 539.13 537.01 465.6 103.7 288.49 112.91 478.46 130.6 189.77 746.77 257.54 188.28 661.85 410.07 364.97 872.58 346.6 479.11 522.86 179.62 460.58 404.87 335.94 788.3 510.48 249.21 527.31 277.41 397.96 276.23 547.22 947.35 262.12 240.69 331.52 277.36 340.36 664.25 83.94 251.74 372.85 420.77 933.65 90.19 408.72 523.26 338.4 455.24 258.64 639.66 915 167.98 486.43 259.71 173.19 169.68 1483.83 115.67 70.33 309.91 174.49 321.24 404.13 349.77 172.94 533.54 290.61 113.95 1089.39 219 312.14 131.97 322.7 235.78 153.81 262.04 306.87 501.44 255.81 293.91 350.28 1108.2 365.57 368.51 306.67 147.68 325.29 433.97 331.75 273.6 506.56 437.42 212.85 1917.71 562.15 435.84 269.84 323.63 960.46 232.97 397.12 305.54 165.63 224.37 162.7 360.73 269.46 376.91 326.83 324.21 117.86 292.04 251.43 419.36 215.91 229.55 169.78 63.06 390.3 237.68 397.35 170.55 109 236.13 232.01 202.93 199.86 236.21 230.44 201.97 275.92 785.86 54.03 179.41 158.55 242.92 243.03 105.02 249.57 187.98 323.98 402.09 583.9 184.98 200.24 1275.25 276.21 232.24 201.72 363.47 529.8 1413.48 429.51 529.11 106.92 348.09 202530_at MAPK14 359.12 349.9 285.61 304.93 322.9 282.97 347.54 464.53 260.69 255.38 282.06 246.36 240.4 366.12 219.71 232.32 397.44 271.06 343.77 551.13 357.23 333.26 229.51 348.64 274.21 332.41 400.72 363.99 394.55 328.71 323.23 312.17 278.32 397.82 278.29 375.38 332.54 360.67 292.56 257.57 344.61 322.09 211.96 300.07 289.45 317.41 336.25 272.09 583.81 277.62 336.44 374.11 338.08 356.54 346.65 343.87 264.52 295.29 343.36 280.95 238.19 343.96 551.78 264.28 245.6 450.65 257.58 283.05 211.98 323.02 298.39 363.77 265.64 278.29 321.71 294.86 364.49 323.14 315.21 225.19 205.11 397.52 316.97 303.79 306.48 307.48 325.11 325.58 278.31 455.02 71.55 262.58 390.45 325.79 481.05 358.55 332.37 293.12 638.57 503.9 298.14 317.01 332.89 303.24 289.8 316.33 290.54 301.6 299.94 39.01 342.76 342.62 466.94 274.78 430.41 438.31 364.48 327.63 290.92 331.68 273.94 276.07 205.86 367.86 391.09 390.83 323.66 339.79 354.81 314.39 355.33 321.31 291.37 367.03 307.89 298.32 481.68 275.14 288.37 329.15 247.7 366.65 356.01 300.77 313.24 170.22 292.86 278.82 337.83 344.83 329.07 373.16 243.44 331.33 317.82 419.31 263.47 429.19 296.24 345.15 291.76 271.14 227.8 212.03 225.67 343.87 332.28 355.19 62.76 369.39 238.86 266.25 313.7 322.19 48.52 217.64 237.98 214.9 316.11 252.45 304.68 252.89 315.63 206.56 266.55 336.52 275.88 319.86 252.3 226.29 243.46 275.77 213.44 371.45 266.87 245.32 318.39 293.71 366.81 244.25 272.05 321.47 310.61 61.98 356.72 265.03 230.18 312.08 270.54 277.02 256.8 221.91 274.56 238.37 262.39 245.83 306.76 223.12 224.07 214.12 395.38 522.18 322.52 363.77 332.55 168.19 282.5 202545_at PRKCD 386.27 330.84 312.38 185.07 341.25 248.31 149.46 358.4 512.05 177.65 334.72 261.83 274.54 199.26 248.58 113.34 523.52 295.81 188.94 151.84 329.4 193.29 320.84 223.52 287.09 289 662.3 301.03 134.14 407.57 173.93 209.51 225.15 232.28 146.88 289.77 227.93 149.1 189.53 336.32 201.59 336.28 207.07 172.97 174.62 183.36 78.35 173.28 227.33 215.57 92.62 156.73 721.59 377.08 1671.03 303.62 320.47 199.78 423.13 320.7 546.18 102.34 189.81 250.47 195.6 168.01 105.19 270.18 223.11 208.3 235.78 168.12 123.92 255.45 102.41 123.32 83.08 259.91 296.49 223.13 365.68 249.92 269.49 333.21 437.84 253.59 387.81 276.3 307.63 61.56 220.94 231.1 206.33 92.45 74.71 135.13 231.16 226.3 138.85 87.74 170.06 182.86 148.24 103.63 128.77 346.78 283.91 443.86 325.69 210.87 201.2 124.59 169.21 275.13 215.52 40.44 368.12 187.44 164.64 403.6 155.99 410.79 198.54 182.18 88.73 224.09 463.63 101.72 276.34 111.57 167.81 144.16 102.6 191.39 162.08 160.84 61.81 71.33 179.54 129.64 137.49 233.77 130.25 94.28 111.56 240.85 57.98 445.87 206.74 296.32 136.11 238.81 275.94 192.03 291.36 191.53 555.02 356.16 226.51 192.07 167.24 140.45 277.4 456.97 397.49 213.19 296.47 250.62 327.3 426.46 238.23 335.49 159.84 170.05 235.66 108.43 141.4 210.47 74.98 135.87 127.81 117.05 165.76 200.24 80.56 242.51 306.3 369.43 119.91 371.99 135.76 79.43 130.6 119.8 90.58 145.62 125.7 198 118.02 118.29 306.3 300.4 265.99 179.87 78.28 131.14 79.09 110.55 162.99 203.96 108.29 182.69 540.99 270.8 134.15 196.52 514.2 241.12 191.54 206.8 285.98 214.4 344.83 274.23 162.23 126.74 204.95 202573_at CSNK1G2 343.3 400.63 429.55 474.81 556.7 489.78 509.64 518.84 417.12 512.31 414.97 376.8 418.71 467 399.24 698.89 396.74 486.84 389.37 489.88 488.06 470.15 269 464.97 381.75 389.67 377.57 430.48 362.54 386.78 295.37 219.4 361.8 512.87 341.85 370.61 395.32 306.71 459.72 255.99 563.74 529.08 249.59 486.12 332.34 399.68 693.59 293.34 454.19 333.49 177.52 333.74 551.8 367.49 363.39 348.34 591.43 306.29 487.64 344.4 352.48 407.19 382.05 288.25 473.68 334.76 369.8 467.65 334.38 341.12 306.82 267.16 391.13 381.94 232.63 327.42 329.76 268.57 180.86 231.75 285.88 222.44 233.88 310.4 217.67 261.34 211.29 264.95 271.41 567.35 472.43 424.15 344.92 345.96 456.16 427.53 292.85 302.5 444.28 543.1 454.64 341.56 331.86 605.52 384.9 449.31 376.99 331.72 371.28 543.32 426.97 330.59 355.6 477.04 430.74 426.93 615.94 445.94 457.9 705.15 462.51 398.02 393.82 427.02 276.58 255.01 384.98 325.93 180.39 277.9 408.17 408.03 345.73 293.27 317.03 297.66 264.85 307.26 280.98 285.73 244.01 177.12 187.13 318.04 239.21 352.55 527.66 316.51 614.19 301.61 373.1 519.05 405.54 422.06 365.84 414.31 288.41 418.32 280.52 401.69 459.22 382.71 345.06 404.29 327.16 316.2 346.9 491.3 470.9 233.76 423.73 284.49 468.41 420.74 560.39 427.3 367.59 334.23 443.8 347.23 387.15 443.15 318.51 387.28 361.65 341.21 309.68 298.74 441.08 216.3 519.5 295.92 360.6 438.31 321.18 329.09 296.78 432.41 458.57 416.68 360.51 354.29 277.67 270.75 385.43 271.44 286.01 320.39 314.39 251.95 280.55 444.56 295.55 306.68 397.51 336.05 283.85 257 333.79 410.68 431.73 479.11 432.2 445.15 420.46 579.6 348.09 202590_s_at PDK2 58.9 128.81 99.69 122.01 101.92 129.1 162.94 196.94 205.29 106.97 164.57 89.2 96.98 169.76 97.69 156.09 63 72.98 68.8 98.7 243.26 66.25 150.85 88.41 45.39 73.88 112.18 56.98 110.38 68.64 192.92 169.32 92.98 203.75 78.75 109.08 63.69 75.96 83.59 128.24 115.68 91.59 77.75 46.93 70.68 93.49 110.41 108.47 179.99 131.54 70.67 126.43 165.8 113.64 205.98 131.46 107.46 109.78 92.55 109.05 83.25 93.79 91.2 57.39 64.88 57.15 95.83 86.5 31.91 70.85 55.71 72.88 64.96 51.67 59.44 49.71 73.8 75.97 99.68 64.55 60.9 100.82 177.99 138.62 225.53 141.16 86.83 233.26 149.34 65.23 124.25 96.89 109.28 56.64 97.06 51.78 150.92 49.31 62.66 76.12 146.41 81.99 56.64 92.17 83.67 121.29 140.54 203.99 253.51 147.45 87.88 178.87 97.67 78.47 88.83 67.86 135.61 75.72 122.01 90.68 105.21 87.68 95.91 85.93 100.75 100.04 136.38 64.74 63.83 28.77 60.39 84.11 134.85 69.78 82.32 93.66 73.39 67.35 80.7 41.74 64.89 224.11 41.85 113.51 72.21 63.09 70.53 144.33 382.61 71.92 192.29 121.16 57.82 81.04 78.86 77.78 156.31 68.75 121.29 74.2 100.13 90.5 139.67 151.41 83.76 170.19 151.43 94.51 65.7 147.01 81.16 115.99 98.83 81.37 468.91 79.97 64.67 115.28 90.86 113.72 102.99 91.91 104.38 95.55 73.79 125.79 75.51 103.21 87.15 174.21 70.92 109.61 117.99 113.75 167.67 64.82 187.19 189.74 95.5 108.8 681.65 135.75 95.96 56.93 70.33 179.74 64.61 67.55 89.86 170.25 122.12 148.82 197.02 84.85 124.03 160.35 131.07 108.51 79.28 109.52 84.37 55.76 120.03 68.63 79.79 108.43 109.05 202606_s_at TLK1 545.74 584.43 217.64 544.68 355.69 479.14 244.92 379.38 516.4 468.76 623.62 765.46 481.5 307.86 332.72 389.65 314.02 325.38 511.06 321.79 576.17 383.8 750.04 262.62 309.81 516.74 405.67 537.7 406.87 480.01 628.94 444.09 553.57 745.83 503.95 438.03 231.11 259.04 275.62 396.21 430.51 404.77 320.14 296.74 215.03 424.55 273.41 226.27 188.01 227.18 417.94 476.58 272.77 489.47 422.62 395.48 393.18 373.87 377.13 208.59 373.26 422.79 588.88 119.74 313.05 341.5 285.45 240.21 446.66 280.31 544.82 346.06 304.86 301.89 336.2 398.54 255.66 519.27 432.88 372.63 218.37 835.12 473.34 553.82 361.92 494.42 287.43 294.55 336.02 702.91 36.92 443.09 445.11 642.38 672.56 472.84 401.89 427.13 501.97 420.47 397.6 789.1 342.12 433.38 464.86 465.44 526.68 448.09 459.24 40.55 316.88 395.22 377.22 794.55 546.36 551.63 467.6 192.9 280.77 266.34 281.82 463 490.72 345.33 223.95 266.57 301.61 317.19 410.75 289.76 317.03 212.31 150.11 302.18 413.45 333.52 384.45 316.64 333.81 451.16 452.96 419.8 378.03 254.43 219.16 103.51 261.1 422.03 299.98 361.27 549.79 251.26 346.24 327.28 410.82 229.63 356.01 378.25 428.8 369.38 362.69 445.69 309.39 284.33 343.33 253.1 502.84 346.51 67.94 423.9 339.68 149.85 407.47 336.33 144.14 214.33 305.41 309.33 330.94 382.7 378.15 285.58 444.71 449.41 246.98 255.63 312.49 332.98 302.65 366.41 373.29 483.56 258.31 318.47 367.76 333.91 364.82 253.24 291.54 337.32 303.89 345.48 426.31 55.08 338.13 443.32 446.68 244.95 225.1 352.97 207.25 341.6 289.19 235.12 246.85 311.72 385.98 289.81 237.45 285.4 315.69 305.82 429.33 257.46 270.6 208.72 363.33 202625_at LYN 501.62 166.39 123.28 353.41 125.44 130.59 179.5 166.14 134.83 166.07 83.19 49.1 96.9 57.23 46.04 125.53 232.99 297.03 412.58 170.49 146.21 318.1 140.65 207.05 147.42 701.39 110.16 152.33 226.23 258.12 168.51 86.19 114.23 56.95 252.85 327.73 491.6 317.92 333.92 326.38 662.33 479.3 483.36 262.28 198.71 147.05 121.19 364.01 418.71 130.2 77.26 157.12 96.2 399.22 20.39 85.61 487.58 237.36 269.15 323.7 48.01 185.22 290.07 88.46 984.92 513.32 159.46 401.76 508.78 248.64 135.26 333.01 386.63 255.57 62.26 130.67 117.15 245.19 33.44 81.9 33.84 149 103.51 107.71 150.62 174.5 100.86 191.1 132.1 185.18 12.79 170.61 66.91 149.66 179.19 215.47 86.59 47.98 106.64 200 211.69 178.77 133.53 54.91 81.02 136.5 103.76 76.94 82.25 8.86 176.13 94.64 212.37 213.93 417.58 200.38 28.73 74.5 197.21 178.02 156.86 88.88 131.22 237 122.73 150.32 50.46 106.81 85.04 179.88 170.65 106.05 83.5 133.36 112.08 55.9 132.47 96.41 69.93 144.3 43.19 75.35 56.16 97.61 131.14 40.21 38.64 75.52 133.17 146.84 71.99 186.69 209.93 139.1 139.19 127.45 55.82 522.21 90.73 335.24 93.48 103.84 114.78 32.8 64.81 194.2 67.47 227.37 14.84 59.37 129.34 80.51 114.07 284.22 9.59 82.08 217.66 105.59 66.52 39.72 139.71 121.99 50.53 57.71 296.49 87.73 77.31 62.14 136.73 24.91 111.06 115.19 98.56 162.14 69.62 160.77 132.55 119.42 164.27 103.84 48.18 165.74 271.01 22.13 192.65 60.57 127.08 129.41 150.19 169.39 129.26 153.15 90.39 102.38 78.27 83.21 131.52 118.89 194.77 78.82 421.56 266.48 76.16 377.04 252.2 289.3 188.71 202642_s_at TRRAP 385.18 370.18 353.24 288.48 542.84 463.75 379.29 504.07 214.77 303.84 339.28 422.66 242.41 365.67 309.36 362.51 415.79 502.35 480.61 312.61 457.48 336.86 351.35 346.53 398.24 359.76 338.32 345.97 386.35 315.8 211.8 274.03 550.4 336.43 369.4 450.1 297.27 336.53 284.13 351.72 429.3 609.82 360.12 521.09 356.48 323.97 431.81 153.48 444.78 257 367.64 291.77 270.96 371.11 362.8 289.32 220.43 288.42 366.47 339.84 446.21 369.66 449.73 211.62 366.94 318.66 519.33 527.24 369.44 272.89 602.07 391.73 309.4 288.89 479.44 294.44 157.9 194.52 132.76 266.58 236.69 191.42 237.23 284.13 229.56 197.9 213.55 234.43 252.12 392.83 154.14 313.43 365.19 280.39 350.82 352.75 395.04 438.66 344.68 276.2 488.79 335.46 308.47 335.86 431.51 398.43 334.29 392.17 439.12 192.6 361.88 344.06 343.28 291.49 328.35 453.52 567.43 279.72 400.49 410.49 384.69 633.11 336.35 374.12 177.54 192.25 230.48 238.52 175.49 217.69 208.5 301.57 223.19 216.09 175.5 233.33 341.88 136.76 247.88 248.88 128.69 134.51 230.96 217.72 226.65 249.5 145.65 294.72 336.05 201.83 458.01 295.82 316.79 278.34 329.15 440.49 235.12 404.1 293.26 381.67 456.17 275.36 203.65 180.31 285.5 224.75 211.62 352.38 208.1 276.15 271.27 215.27 251.93 227.9 208.89 173.95 199.45 161.38 267.8 216.07 215.9 226.14 211.79 203.18 214.49 186.77 224.23 157.65 203.09 175.56 224.86 163.12 219.87 252.43 190.23 222.94 261.55 223.18 261.71 252.74 368.47 218.86 250.89 144.81 355.56 149.62 240 298.09 158.03 122.58 199.1 219.17 235.81 224.58 274.82 177.17 282.44 301.94 194.22 247.65 444.24 570.59 343.68 345.41 317.48 306.6 326.83 202670_at MAP2K1 347.21 423.86 336.44 354.74 319.61 226.94 399.23 428.87 503.99 378.09 335.96 353.85 266.78 327.71 174.49 504.97 615.04 369.85 458.62 333.05 251 433.27 296.69 377.4 303.02 527.76 315.24 350.59 472.06 322.47 363.66 284.11 322.56 474.85 526.36 330.01 456.02 637.43 336.99 529.2 532.91 768.38 387.75 277.52 382.95 306.9 286.43 516.7 445.74 501.74 394.82 378.65 489.78 406.18 308.87 488.5 511.22 342.98 403.14 347.23 225.04 421.83 455.86 343.88 423.26 463.6 542.71 507.45 438.88 267.23 369.62 301.82 244.79 507.64 266.99 216.85 693.37 382.66 251.17 252.93 148.2 379.85 307.67 270.78 295.1 286.78 330.56 349.61 216.21 286.32 65.33 280.93 455.64 379.51 328.09 417.4 404.06 236.33 431.47 388.83 215.25 443.85 204.49 203.32 244.23 302.64 244.84 224.01 319.28 65.5 302.24 264.08 308.33 334.99 412.12 336.61 254.45 237.77 394.41 370.62 302.66 412.46 228.36 382.72 257.05 465.71 301.17 226.17 365.65 248.5 285.47 275.53 170.74 326.16 300.07 232.69 240.49 204.36 179.89 268.12 168.61 337.62 200.72 196.9 241.01 166.51 256.35 684.92 378.91 382.48 460.75 331.03 534.59 267.21 309.88 344.76 451.53 590.99 261.06 419.11 247.54 266.77 353.92 191.3 302.03 300.46 203.68 318.33 77.69 363.85 267.36 200.37 306.01 251.75 28.94 226.21 273.12 236.04 174.87 256.92 284.12 343 264.91 238.42 279.23 320.71 343.29 417.32 467.43 194.01 208.35 276.87 245.96 368.22 222.34 306.6 388.23 307.93 267.1 289.54 331 282.7 266.59 72.45 333.18 258.76 227.17 287.95 263.57 416.58 187.56 257.47 242.74 183.05 223.74 267.97 225.49 255.04 326.08 199.05 528.62 329.52 202.47 475.62 546.94 348.02 400.38 202686_s_at AXL 322.93 570.91 1061.02 622.27 452.71 419.1 225.56 495.58 435.93 376.79 340.01 307.37 319.43 461.85 433.73 315.19 263.71 369.86 468.17 174.51 587.01 558.17 189.39 360.65 223.65 670.25 480.72 288.97 408.57 233.33 547.84 261.09 422.59 185.82 341.5 296.24 271.84 582.81 421.1 626.37 534.53 429.24 273.75 394.91 369.68 694.85 591.63 470.94 635.79 516.03 285.36 521.58 579.02 535.82 140.71 482.53 330.2 348.02 505.67 402.61 328.47 111.56 681.64 287.55 31.59 455.76 198.1 569.24 175.07 428.97 405.42 329.82 321.29 420.41 187.19 359.78 220.91 495.37 253.42 612.32 328.09 426.39 431.45 429.21 479.84 597.03 524.76 601.3 371.06 366.51 168.5 633.65 274.46 526.34 925.07 458.24 489.87 237.71 468.32 332.44 266.96 439.42 442.39 270.51 379.49 640.86 588.95 468.06 625.51 27.81 305.34 465.26 566.04 427.9 619.98 34.6 133.24 284.73 391.53 801.02 831.68 281.85 298.68 514.46 651.2 149.41 351.83 525.08 288.79 264.83 639.72 366.49 454.62 315.39 403.71 342.97 357.29 333.91 399.62 530.83 285 333 183.52 412.75 356.64 171.82 187.87 345.53 322.67 207.24 190.47 619.7 321.68 551.95 350.93 269.15 178.13 702.32 579.09 314.7 405.34 347.07 447.27 283.45 315.01 675.83 323.17 404.35 50.33 276.68 300.55 588.65 411.73 264.65 23.92 399.07 492.33 366.26 459.51 122.58 462.11 247.97 250.44 398.08 381.37 373.83 345.96 290.97 154.61 185.74 471.32 458.39 723.36 393.2 283.86 433.18 209.68 395.61 537.32 182.86 205.89 549.08 418.02 28.18 145.75 389.06 169.85 179.43 524.94 355.55 396.88 435.33 527.19 370.51 317.47 275.33 345.1 250.68 416.27 261.38 536.89 459.82 419.36 278.73 444.68 185.39 451.98 202693_s_at STK17A 107.52 211.02 190.75 209.7 161.87 8 106.02 105.76 77.3 146.66 164.27 59.21 93.49 18.19 75.97 78.33 436.2 247.52 190.08 129.32 87.75 294.15 190.68 188.75 162.83 550.19 216.25 283.32 288.35 123.04 229.6 231.09 131.71 41.23 298.12 131.03 72.93 141.99 205.97 131.77 266.28 260.73 119.07 119 92.77 153.28 84.37 193.57 119.07 154.09 226.04 142.65 74.48 236.75 33.26 115.15 85.13 189.51 213.11 265.22 71.25 41.18 136.39 119.38 275.24 136.45 158.02 235.17 188.92 126.53 95.97 237.63 111.94 310.82 114.79 198.68 173.01 441.81 232.53 97.94 48.12 211.35 101.05 181.1 136.89 187.78 98.44 210.6 229.34 270.62 13.17 163.08 139.52 157.37 105.89 283.74 171.07 142.44 326.32 278.37 24.06 123.11 108.64 83.46 146.29 165.95 80.2 83.58 70.79 8.64 141.49 75.4 277.17 102.41 187.19 137.58 25.49 117.71 392.44 75.92 97.13 52.72 89.83 342.03 59.74 87 196.18 63.26 88.44 130.08 100.59 169.96 113.73 37.07 185.45 93.77 43.65 50.73 109.3 74.88 99.66 54.11 96.5 287.15 107.25 159.29 79.61 59.73 84.12 174.6 135.65 236.54 256.76 305.35 221.22 262.28 72.87 256.57 426.23 110.2 256.01 105.31 135.15 79.25 72.76 33.57 207.56 93.83 340.52 22.37 32.54 117.77 77.99 59.91 144.69 12.2 66.67 199.71 54.52 29.22 17.8 268.28 57.48 61.27 144.51 181.91 40.37 63.06 81.2 30.41 26.78 47.69 94.65 77.66 93.38 82.13 210.55 94.46 98.06 266.56 109.17 55.37 161.65 203.08 12.88 46.38 94.74 137.64 86.37 109 174.16 105.37 151.75 46.88 66.55 51.8 95.17 143.32 192.75 147.12 68.89 338.52 214.84 41.87 123.5 137.51 99.09 167.21 202696_at OXSR1 574.77 409.4 310.66 297.49 334.22 373.77 241.29 497.49 380.13 263.69 322.93 251.69 293.28 398.87 233.49 346.55 481.58 402.69 418.72 638.77 427.73 521.35 654.52 341.53 422.83 388.67 292.94 496.65 531.68 429.83 393.22 396.1 401.11 376.53 349.19 494.95 315.59 304.66 222.2 523.22 302.18 667.14 501.79 617.64 256.24 287.24 387.12 378.46 497.83 402.04 453.34 249.99 385.87 358.68 426.43 377.71 420.51 346.3 238.37 281.47 360.66 362.77 1000.26 222.91 306.76 430.67 507.62 301.95 394.1 389.14 273.15 349.74 198.64 274.84 302.88 470.41 406.01 363.21 280.56 234.62 170.33 311.11 292.62 291.73 294.8 282.38 280.86 325.96 190.15 371.54 101.82 289.8 290.22 366.81 431.93 315.5 384.64 272.99 317.69 496.38 353.84 360.85 390.78 418.59 362.95 334.36 400.79 369.99 461.28 100 280.59 283.51 237.97 386.34 279.3 383.91 183.8 271.49 212.86 341.97 292.6 395.94 205.22 264.89 242.76 245.71 240.82 301.01 298.13 256.15 196.04 181.69 161.61 288.15 179.99 239.63 175.94 175.8 165.18 246.79 207.08 241.85 254.82 206.15 210.01 143.79 174.46 427.12 265.79 309.01 623.54 239.32 309.31 305.37 298.45 309.26 356.76 348.44 313.59 442.11 176.85 250.33 390.85 197.87 263.48 263.58 222.36 275.23 104.53 321.04 298.39 274.21 341.28 374.77 78.29 180.76 266.57 299.51 259.29 242.15 293.05 370.79 250.56 257.76 213.82 205.79 282.2 290.16 327.56 316.03 183.03 268.27 213.58 311.76 266.32 311.2 307.25 275.26 217.06 216.27 258.39 237.58 228.13 103.7 181.59 238.84 174.07 270.54 175.65 368.66 188 230.36 272 239.18 211.04 203 340.65 270.77 223.87 208.8 340.38 302.93 278.5 350.01 518.19 644.85 238.71 202741_at PRKACB 180.25 243.13 312.57 671.79 359.6 284.1 278.79 290.08 421.76 289.94 3213.44 292.14 244.23 46.42 219.18 402.68 275.4 432.38 345.47 436.95 414.61 689.35 274.13 324.39 167 645.92 485.26 833.32 382.68 261.36 554.82 129.82 884.29 99.4 225.94 328.8 490.25 268.8 538.27 431.4 394.71 313.84 123.73 308.84 365.43 482.71 248.09 372.75 260.68 1185.69 753.44 348.13 409.46 606.24 478.94 374.03 131.58 378.61 477.62 160.83 81.68 198.12 539.57 286.01 566.96 197.45 196.04 228.28 184.18 308.87 356.57 201.34 340.04 373.81 432.86 273.14 377.63 896.84 80.53 183.51 165.49 282.26 200.88 585.5 544.74 450.97 352.45 603.02 405.21 616.22 33.98 1260.19 486.46 584.63 617.49 493.44 444.98 157.91 651.52 402.09 244.84 1169.77 654.68 336.89 416.21 453.35 422.85 364.33 395.41 24.87 235.39 464.34 612.75 365.81 1001.79 426.89 253.44 112.16 594.67 814.92 385.38 284.13 282.67 596.28 543.66 4351.17 3080.24 413.06 419.95 252.3 572.97 257.66 207.44 575.14 453.16 236.16 392.72 388.3 388.94 380.03 148.13 334.08 267.74 333.52 300 125.79 343.33 182.53 348.36 4676.58 262.56 453.34 400.73 579.87 699.69 284.04 165.51 474.57 402.53 386.51 213.17 430.96 157.95 200.3 448.01 437.83 789.43 524.64 39.36 97.33 391.22 1720.78 360.18 328.61 19.72 341.59 437.06 101.78 297.51 48.19 598.74 305.49 236.37 214.99 491.29 138.81 450.53 228.79 267.86 490.81 573.39 596.36 193.93 357.33 353.71 490.67 316.57 264.73 385.03 304.03 154.73 394.72 429.83 77.6 122.37 147.55 75.8 275.27 171.43 280.07 151.36 213.04 275.34 394.72 82.8 276.2 248.1 281.25 321.79 100.28 595.97 406.72 321.76 174.19 278.25 92.76 566.93 202762_at ROCK2 395.65 229.85 242.05 202.73 348.69 135.2 234.31 217.53 268.41 281.53 185.58 193.14 180.74 66.34 158.01 399.19 384.29 182.95 479.61 171.78 242.62 231.74 237.37 212.11 319.72 254.68 258.39 239.86 219.57 185.46 263.34 76.1 278.83 74.1 263.38 138.11 168.25 354.01 181.41 202.98 250 304.56 179.57 584.7 360.54 274.48 158.92 202.02 247.61 159.37 361.75 166.45 193.42 455.68 198.33 190.25 251.27 280.02 174.5 179.66 152.43 147.14 335.02 269.26 168.43 197.59 329.77 192.11 362.84 316.91 217.08 270.85 355.58 212.05 130.86 136.08 224.45 158.63 177.2 222.95 233.86 144.23 176.81 192.1 208.12 123.36 173 238.94 91.6 243.14 94.14 211.02 194.07 179.18 244.69 165.21 284.33 253.4 246.75 331.19 428.24 361.18 229.47 266.48 177.23 251.46 274.87 223.51 433.85 79.73 337.05 364.92 305.11 530.57 365.79 483.23 143.48 203.16 259.7 359.67 379.81 651.21 209.33 285.99 440.7 165.44 180.38 252.98 166.69 308.54 125.34 191.39 257.43 152.39 126.5 190.82 259.62 269.33 232.79 211.82 93.53 171.51 169.56 187.52 200.76 116.68 408.43 237.78 246.55 65.75 109.23 125.27 231.99 239.37 112.23 279.37 132.55 186.53 289.54 181.46 192.07 240.53 265.95 140.76 225.98 148.03 187.99 203.52 71.25 141.45 118.42 157.68 193.89 218.27 52.95 153.37 193.94 131.79 179.29 24.71 199.15 197.18 113.1 197.48 203.59 99.61 176.46 215.24 180.07 160.76 123.24 212.38 162.56 179.33 179.41 150.22 303.34 133.03 189.85 451.29 119.68 115.52 114.22 46.58 90.79 160.94 159.5 168.6 128.25 128.61 277.21 132.84 135.98 189.06 89.09 106.01 166.18 257.74 167.04 149.91 184.06 234.54 77.24 257.38 195.79 103.13 214.14 202786_at STK39 95.99 311.64 126.87 639.86 196.31 502.13 248.88 343.04 510.5 389.88 1447.66 511.25 447.6 499.6 182.84 424.36 151.38 145.73 171.36 128.05 327.43 177.88 49.04 76.8 105.74 215.67 116.61 488.01 120.96 199.59 295.02 352.6 326.54 600.34 308.7 87.67 124.41 193.66 80.95 94.25 103.09 278.55 151.02 306.58 107.66 157.04 85.12 229.8 68.61 62.19 260.99 508.36 300.67 191.13 540.09 276.62 106.26 105.76 295.92 54.69 154.29 736.62 377.52 143.73 447.5 155.95 121.05 58.45 237.56 81.09 207.87 91.17 81.87 129.89 782.87 1002.43 56.15 336.89 334.42 167.64 274.89 243.98 603.9 354.19 219.24 181.49 422.43 144.58 279.52 408.31 94.26 232.96 704.96 541.73 349.42 455.03 210.73 219.35 805.17 237.57 522.68 126.56 261.02 619.58 292.79 493.18 240.89 520.07 194.47 38.92 122.82 171.16 111.61 146.6 123.46 382.31 292 96.89 176.44 62.03 100.51 119.57 197.72 135.02 153.35 312.91 107.47 189.91 273.56 133.76 197.94 374.48 160.18 123.23 154.04 356.67 157.03 133.42 126.14 305.16 367.74 183.05 405.92 282.1 222.47 132.87 306.91 365.03 157.86 586.03 223.49 190.67 232.02 102.62 427.4 44.03 713.76 543.74 260.42 137.53 238.84 388.53 177.57 256.06 279.32 281.97 402.47 219.48 420.91 612.04 447.36 195.02 286.29 90.55 44.94 157.11 185.19 160.56 174.11 512.94 203.8 123.19 247.7 231.45 113.86 296.07 482.06 303.43 168.15 90.48 301.16 372 100.62 143.22 167.11 161.55 201.74 191.36 170.39 201.62 381.2 212.99 373.25 214.16 155.67 317.05 278.65 68.44 145.67 329.79 80.45 132.2 293.29 60.38 205.74 569.05 286.39 169.19 168.4 231.57 87.64 85.36 651.78 149.04 126.01 97.67 117.92 202788_at MAPKAPK3 346.6 212.8 181.73 295.14 198.28 217.27 248.9 285.38 394.76 419.94 340.59 179.26 353.76 208.77 266.48 242.04 364.28 325.71 373.88 287.24 366.92 239.13 302.08 278.04 415 252.11 413.55 247.15 328.56 266.57 264.43 236.89 218.81 301.69 340.21 415.13 209.01 206.46 243.71 588.37 360.17 328.07 331.26 185.97 151.9 258.8 156.7 263.45 441.72 455.52 236.18 227.83 408.49 327.53 434.26 331.98 303.6 449.71 279.55 782.37 511.72 213.98 277.29 229.69 141.67 165.43 223.52 247.67 189.34 247.44 251.63 228.87 235.94 224.67 262.96 174.42 289.59 263.12 287.19 238.92 283.3 350.74 352.43 374.7 263.93 262.71 286.35 332.43 265.73 211.05 160.33 321.68 205.31 184.39 247.38 269.71 238.78 162.92 285.75 237.08 108.19 175.29 297.73 189.37 188.76 390.58 300.64 499.07 269.27 244.45 289.81 246.81 209.97 265.41 308.84 196.53 114.82 210.84 214.48 241.72 266.15 323.19 254.5 342.21 199.7 346.35 207.75 293.09 337.18 261.42 258.12 261.91 172.45 407.56 194.78 246.98 206.22 218.03 217.72 185.26 233.69 207.72 234.94 155.59 255.46 167.29 232.14 353.01 342.59 445.36 213.41 286.41 411 442.96 320.05 276.34 433.98 790.75 274.83 346.71 178.02 305.26 427.74 304.47 416.15 232.67 185.97 296.39 331.23 376.8 412.26 320.69 172.25 412.19 396.47 261.46 321.95 280.54 262.78 292.49 343.4 338.58 236.12 232.24 199.93 300.48 834.96 261.29 368.65 402.56 224.88 318.45 223.81 257.03 263.4 232.65 311.52 287.69 172.63 138.57 442.77 326.4 252.67 321.24 265.49 170.65 84.51 214.73 183.14 384.87 200.2 175.35 281.85 160.7 265.59 255.45 639.4 337.07 312.47 148.85 320.64 269.01 223.04 223.05 361.35 353.04 312.54 202801_at PRKACA 89.8 131.86 139.29 220.84 156.28 155.1 164.01 168.78 191.1 218.97 171.71 134.84 113.97 185.62 152.58 171.92 128.53 101.07 162.55 89.73 105.92 75.15 105.07 145.19 103.48 118 216.63 113.41 140.06 157.38 95.9 213.24 84.41 171.61 194.02 118.99 177.83 130.68 135.37 177.23 117.02 156.46 140.64 171.71 192.91 166.85 121.5 123.29 159.85 177.83 101.19 148.53 209.03 162.09 248.18 169.2 212.09 81.22 208.8 201.57 201.86 224.18 281.58 103.49 103.59 172.06 151.66 98.7 92.75 82.16 157.73 95.69 97.53 95.32 154.28 111.01 153.48 106.17 65.46 118.87 144.24 153.02 153.37 175.14 128.57 120.14 114.48 104.71 131.19 92.72 187.4 145.28 132.34 129.31 76.57 98.07 142.33 205.44 132.1 113.8 184.6 172.79 198.6 112.34 125 178.63 107.6 155.64 201.09 131.64 101.52 108.36 103.25 117.1 119.12 107.35 111.76 77.69 116.61 264.36 185.59 226.35 117.42 134.32 144.98 80.48 120.71 137.69 107.86 96.9 106.25 133.93 208.89 135.56 95.13 108.14 131.4 120.69 88.3 120.98 91.3 104.03 156.2 112.97 110.03 147.2 135.18 139.65 180.02 165.39 196.36 169.59 148.57 118.5 125.5 178.78 115.67 98.57 192.96 105.93 152.95 137.92 123.99 185.47 161.82 145.3 148.62 155.68 183.36 157.21 176.33 177.22 141.3 162.37 203.86 227.2 111.24 106.37 142.4 196.03 138.59 117.97 141.59 144.08 156.54 140.77 123.04 152.77 136.6 120.56 166.36 145.86 200.22 225.09 226.99 253.32 261.74 302.01 239.88 339.49 356.34 241.93 258.97 328.6 252.6 214.64 161.35 149.21 142.94 188.14 205.64 229.51 230.22 251.34 199.84 212.09 218.39 137.88 159.77 219.44 205.97 140.31 223.41 189.32 183.27 202.41 201.31 202848_s_at GRK6 124.84 104.95 112.2 161.26 128.23 131.82 96.63 175.51 159.49 110.9 148.24 94.98 111.66 151.99 99.04 143.9 176.26 182.33 156.03 252.56 132.95 115.5 100.16 164.68 383.97 120.21 126.56 119 130.82 95.84 155.89 104.43 132.12 148.45 119.95 143.55 185.76 120.8 146.45 153.16 117.78 153.35 134.73 162.78 115.58 134.3 142.48 148.13 350.79 106.37 116.74 140.27 128.86 134.33 169.8 115.31 161.05 171.85 164.48 103.73 99.72 135.61 169.01 113.29 202.66 107.49 173.39 168.05 137.51 98.97 116.51 155.18 107.09 139.03 113.04 125.04 295.59 125.13 104.35 142.46 115.36 98.72 99.46 116.64 93.95 119.59 128.64 142.91 186.11 148.65 162.23 116.98 101.57 94.16 96.47 171.11 109.5 138.52 128.86 114.83 73.77 107.61 108.02 114.91 122.08 163.93 107.04 125.68 122.77 142.38 166.26 121.03 151.18 135.75 170.07 106.41 152.42 126.31 172.24 121.89 126.95 100.1 215.83 154.81 68.36 169.7 93.62 78.66 100.58 85.53 122.85 108.17 91.85 106.9 93.68 87.65 138.6 81.76 83.65 85.99 74.17 84.14 168.83 97.63 87.59 100.19 110.74 159.12 129.61 135.06 225.28 157.01 146.79 150.65 121.32 153.48 132.85 192.41 87.73 165.18 118.69 108.22 96.86 89.77 95.56 150.08 114.56 144.55 135.24 137.27 151.05 133.11 118.18 159.92 166.74 147.89 164.24 98.71 89.74 167.14 161.43 155.71 118.29 163.27 129.63 95.25 127.71 113.83 133.24 139.33 117.46 94.6 79.33 142.39 105.96 155.89 130.49 135.87 127.47 117.79 128.42 127.46 151 145.96 179.22 91.91 98.86 109.42 125.1 147.9 115.35 117.16 126.71 88.56 95.92 114.56 142.18 122.31 111.35 120.3 131.11 95.79 72.85 85.41 79.77 134.25 95.61 202894_at EPHB4 449.93 259.28 382.87 456.29 421.66 176.02 389.36 118.58 144.17 633.85 482.6 330.82 342.57 380.1 382.62 626.21 350.86 166.96 605.16 327.99 571.54 402.33 427.13 272.05 394.58 191.32 937.6 469.36 339.69 710.74 224.23 590.84 388.22 428.97 423.99 322.63 250.31 419.23 308.35 309.04 393.53 491.15 508.95 887.19 578.49 471.19 450.3 187.98 636.31 392.19 643.57 395.2 358.57 473.74 196.34 503.57 453.73 229.55 517.43 332.05 746.79 253.65 583.77 325.24 111.75 368.66 457.38 176 381.72 327.95 422.41 276.18 293.44 710.64 434.9 468.35 298.62 190.25 162.57 319.03 209.56 227.49 183.88 469.13 241.46 229.23 469.88 226.69 155.86 292.17 318.32 257.78 271.06 290.91 276.84 346.87 209.9 557.33 240.31 249.98 408.46 647.78 256.72 274.4 303.41 336.16 420.14 374.25 560.19 368.58 433.95 350.34 337.64 272.76 402.5 196.12 281.08 254.87 407.86 559.53 467.06 692.72 339.25 243.19 227.71 207.3 221.62 376.95 230.8 384.3 236.94 220.48 421.1 270.2 187.85 157.54 306.42 284.22 235.47 328.03 161.74 238.42 277.41 178.82 196.02 139.52 231.96 264.42 483.63 283.6 530.73 386.24 762.18 479.08 232.77 549.31 230.02 547.71 378.87 370.61 402.63 340.85 389.29 228.42 608.52 220.4 172.76 633.97 232.45 304.05 126.54 340.93 240.61 223.97 509.45 421.8 264.66 247.66 386.67 269.67 194.32 331.63 346.87 305.85 214.27 241.89 349.33 184.45 338.32 315.1 354.54 221.8 393.37 334.35 358.6 453.52 456.47 351.33 606.06 485.63 486.37 277.66 264.61 104.62 361.65 338.33 357.16 321.86 142.28 276.96 370.07 263.28 252.05 169.05 354.99 123.32 249.1 254.41 277.37 276.3 440.17 773.53 831.14 593.81 555.85 560.29 388.24 202932_at YES1 586.34 303.42 111.1 57.06 128.22 69.03 94.53 140.97 115.53 224.6 175.58 170.93 157.74 102.77 85.18 140.08 710.42 228.68 277.8 755.19 194.22 369.78 933.89 203.58 307.01 195.77 251.88 476.08 589.71 203.57 234.62 242.73 166.04 34.12 167.02 174.95 305.08 389.35 109.15 321.13 309.99 277.69 618.06 407.19 232.8 260.38 389.13 137.71 296.53 85.23 375.38 180.97 147.24 277.75 74.19 200.44 551.41 554.5 43.43 162.06 149.61 253.13 1030.36 91.41 14.87 431.61 166.55 130.57 767.92 393.85 268.9 370.52 459.67 254.75 139.19 307.85 609.28 201.74 227.26 123.01 124.5 194.52 283.94 99.65 208.93 198.23 140.17 149.73 73.75 126.41 24.48 146.58 98.4 186.37 154.08 372.06 162.93 258.57 136.57 248.55 319.69 183.73 75.3 53.41 145.74 144.41 104.31 168.14 147.86 23.2 280.5 247.67 193.37 249.43 268.03 251.08 174.15 157.06 65.42 159.15 103.68 370.61 159.69 214.97 172.45 203.47 129.36 137.69 300.42 396.35 142.87 168.76 74.82 126.51 188.94 83.99 415.69 224.97 140.88 153.43 116.83 276.39 247.92 164.69 127.88 82.65 262.44 204.74 120.83 48.76 746.11 87.36 521.24 199.41 193.17 198.53 125 208.38 245.44 282.08 117.56 262.71 116.57 132.13 120.98 117.5 111.07 154.71 62.76 78.93 46.29 82.16 113.16 388.6 27.93 192.35 107.67 110.66 130.09 102.22 139.81 187.66 75.07 123.1 136.01 24.73 122.64 103.74 271.12 251.88 119.18 182.7 167.25 174.82 216.06 140.72 659.52 141.55 116.27 172.7 169.31 185.72 102.5 33.79 454 136.96 297.82 288.57 76.59 174.32 155.86 74.94 95.26 89.84 72.48 101.31 100.65 393.9 120.32 160.08 225.44 511.33 145 157.09 298.41 584.4 226.38 202951_at STK38 1019.31 619.13 745.1 576.47 684.18 693.28 720.1 648.68 485.87 663.84 619.01 741.83 710.68 1342.55 780.54 721.03 778.42 1132.52 1006.72 1194.55 575.29 751.33 650.06 771.98 611.4 869.97 647.67 1501.26 624.8 638.91 436.45 644.6 393.95 647.95 833.25 1091.24 979.68 717.32 912.81 531.5 724.72 517.34 630.25 612.88 706.39 481.3 486.89 579.93 1369.47 394.19 1042.44 779.1 433.54 1357.09 321.78 659.3 348.32 622.09 614.94 581.75 542.19 1144.55 807.78 482.21 472.33 777.68 810.29 595.46 322.81 1532.54 458.12 865.18 509.06 446.14 335.17 370.57 646.15 635.19 285.88 349.88 857.06 432.53 319.07 533.92 447.46 420.81 471.49 590.1 556.49 586.95 509.13 449.87 404.98 412.48 459.27 687.65 343.44 596.07 481.34 723.54 833.62 436.53 549.01 558.59 525.46 556.44 512.46 480.69 305.02 202.8 1014.46 561.66 951.02 755.02 588.28 752.39 631.39 628.21 801.22 408.84 566.36 856.65 589.52 669.6 750.79 558.34 338.36 674.47 234.98 1049.01 614.17 678.75 450.07 565.45 512 419.3 415.94 473.43 659.12 502.72 361.78 478.5 654.64 378.6 636.64 518.24 541.81 335.04 562.59 379.26 665.94 608.89 390.83 468.76 392.54 1065.76 209.7 473.19 549.14 696.44 646.72 601.44 232.46 289.84 267.14 424.73 434.03 615.78 573.75 413.31 234.34 364.46 445.88 606.38 168.42 241.35 389.66 279.44 377.85 459.61 392.97 564.4 338.36 270.92 411.87 551.7 280 270.13 737.97 380.86 395.27 328.27 333.57 335.1 317.96 276.21 621.14 311.92 432.57 592.8 291.33 403.07 453.52 305.03 393.01 318.08 613.15 439.95 317.36 245.82 561.77 507.84 295.31 322.66 417.48 379.31 427.47 425.11 282.49 424.01 669.71 1475.53 258.83 739.98 782.28 392.76 455.44 202971_s_at DYRK2 135.07 130.63 288.74 91.35 169.96 128.92 12.05 109.36 92.37 168.19 133.84 89.42 95.25 80.38 110.51 123.86 83.22 137.86 349.23 103.35 211.3 144.77 231.63 133.69 269.69 231.27 66.47 166.39 109.21 97.28 665.79 93.37 172.07 86.26 77.49 89.06 148.55 84.65 147.61 77.55 133.05 147.93 151.8 175.06 86.24 164.33 116.1 76.97 120.81 104.01 119.43 165.97 98.89 130.34 54.1 158.23 65.14 32.31 143.04 60.62 84.12 330.99 246.62 136.27 224.59 240.99 263.18 267.2 255.73 381 392.54 642.68 189.97 321.33 294.69 289.84 179.88 381.21 93.99 264.13 88.55 251.63 158.18 212.73 283.71 303.97 210.2 362.84 353.75 369.33 54.61 344.13 182.79 266.14 443.81 268.91 197.4 174.64 235.63 216.36 218.16 163.08 267.27 181 232.6 193.07 226.97 151.93 261.31 19.83 96.53 189.16 379.18 244.2 251.03 31.01 388.31 275.02 395.93 198.2 451.38 213.2 250.1 399.07 302.81 147.11 416.8 403.58 155.96 150.99 453.86 234.14 112.8 154.91 270.97 358.8 225.44 319.15 340.35 343.25 174.2 114.68 109.16 233.64 229.29 101.3 198.21 176.25 255.03 128.28 165.71 329.8 168.43 276.19 247.46 122.01 97.41 138.44 164.34 266.37 260.55 268.35 151.76 151.26 106.24 260.17 158.39 135.48 34.36 151.82 118.1 124.05 177.75 134.44 37.73 112.88 233.24 121.37 199.6 84.3 277.51 410.02 159.76 314.76 346.13 139.47 227.06 153.52 484.65 133.49 221.99 170.93 126.15 89.04 116.04 155.8 119.85 203.11 149.89 127.08 156.97 137.02 201.52 42.87 108.47 112.84 298.8 162.76 186.24 174.63 167.7 222.76 267.09 143.74 173.44 206.75 316.02 504.77 1008.27 206.61 240.78 253.74 258.65 290.79 253.43 141.65 195.33 203104_at CSF1R 372.6 218.65 351.89 652.05 394.44 413.3 204.05 164.58 287.32 261.35 192.62 88.04 219.19 139.58 162.42 242.13 301.45 404.58 400.92 195.19 318.64 420.98 129.7 353.17 191.04 1009.7 192.28 225.76 462.68 243.43 297.89 219.34 230.34 125.1 556.99 281.61 536.3 681.13 790.03 658.06 676.21 464.09 201.81 310.69 330 457.13 414.65 588.45 812.2 372.4 337.06 297.45 262.59 711.05 63.47 291.33 529.2 476.99 625.84 361.57 221.25 91.83 344.58 142.37 507.19 321.12 274.43 794.5 259.01 310.44 224.1 190.67 323.36 353.31 173.4 157.33 256.54 661.73 200.8 239.76 145.59 415.3 251.42 322.95 354.01 405 256.25 424.76 259.85 154.76 48.25 400.05 173.45 256.04 245.66 288.6 204.29 106.6 212.85 278.87 163.59 347.22 332.58 120.65 248.94 414.82 430.87 278.53 290.18 50.69 498 314.06 463.39 360.5 500.28 58.87 110.51 310.64 508.93 423.83 542.75 190.4 291.65 466.71 754.07 207.29 222.91 316.48 188.35 264.39 557.38 238.69 293.52 498.19 141.79 196.15 438.17 227.01 197.35 368.24 140.45 150.78 104.82 246.84 453.08 118.24 135.38 195.69 365.86 929.07 230.62 844.31 255.93 429.88 441.9 360 194.62 938.22 293.12 440.42 497.97 238.87 256.78 118.02 278.23 591.25 221.32 641.31 55.71 246.18 517.46 337.5 421.07 480.49 59.27 258.05 778.02 700.82 484.61 130.71 603.24 420.03 262.7 183.94 340.89 433.69 267.11 169.5 263.8 137.98 428.89 385.68 392.04 433.21 122.54 396.02 196.8 279.94 413.58 200.23 170.12 470.35 620.6 82.89 200.03 244.39 203.96 261.84 432.87 261.4 405.95 401.25 419.13 222.88 379.24 276.67 352.64 296.16 473.52 284.02 913.71 454.21 388.28 340.75 368.02 256.25 594.95 203110_at PTK2B 66.49 52.64 145.59 91.1 88.28 77.21 85.91 45.05 64.59 97.85 53.06 39.75 86.8 86.21 85.11 50.43 52.8 206.61 111.62 64.68 83.66 89.94 70.72 149.33 77.42 165.63 65.94 66.04 118.51 81.67 70.96 169 64.39 137.31 127.5 149.71 113.07 122.24 172.7 241.44 137.64 181.12 106.57 101.06 73.38 89.88 99.93 126.71 110.99 80.89 57.02 106.23 100.66 138.5 92.84 152.97 131.02 108.26 130.76 203.51 180.99 47.19 60.55 48.9 227.9 82.02 70.84 130.51 54.2 53.89 52.53 139.24 89.61 56.51 49.81 58.9 37.65 67.95 53.22 38.1 56.36 83.07 56.55 59.33 65.1 61.49 47.97 105.83 102.09 70.39 72.63 88.15 69.42 43.65 52.92 75.76 52.28 57.55 57.56 67.73 60.18 70.18 89.99 53.78 34.64 131.34 110.74 109.57 112.76 183.17 155.19 92.87 157.63 122.16 182.25 85.08 50.21 154.48 169.02 114.57 135.38 135.88 186.02 195.03 53.74 88.12 46.87 59.92 44.43 45.74 93.81 55.54 57.33 95.37 49.36 56.94 76.38 54.36 53.74 51.12 46.26 47.83 43.9 50.85 56.73 80.74 32.3 83 103.44 119.62 98.06 182.48 109.69 98.22 99.6 73.27 76.81 189.91 50.17 149.6 86.92 59.95 103.22 62.42 107.02 114.35 71.01 141.71 60.86 82.56 96.11 89.24 90.07 98.44 133.35 80.35 131.58 82.83 88.04 150.67 133.87 79.03 76.77 86.63 115.1 82.64 105.49 70.95 68.69 107.34 96.12 68.57 74.93 120.3 75.56 101.07 72.74 135.81 124.63 109.18 69.38 123.01 168.54 112.67 105.72 74.95 68.21 90.17 152.19 78.85 85.42 169.97 115.18 89.05 97.87 87.39 96.41 83.04 75.7 134.94 146.66 64.18 109.49 48.49 79.71 57.41 85.37 203131_at PDGFRA 648.31 912 1531.27 901.45 761.52 490.6 859.57 302.84 342.19 648.49 323.05 306.99 626.28 163.4 818 1362.32 355.19 410.92 550.19 350.77 704.22 941.03 504.37 679.12 284.62 1249.26 760 667.37 770.92 382.1 663.88 225.38 684.24 147.18 569.88 193.36 919.41 898.31 2298.02 361.35 390.9 247.7 348.19 809.4 952.49 1236.95 825.89 294.31 799.15 457.86 656.23 803.15 787.02 1133.57 199.55 869.44 412.12 512.5 371.13 298.72 249.09 267.63 1102.26 311.56 108.85 583.34 305.79 592.98 362.25 591 619.15 322.27 381.6 540.84 198.57 384.77 442.75 914.02 105.53 949.06 587.45 341.18 140.53 294.73 1223.34 960 1131.32 944.99 328.59 1017.23 222.84 1134.83 339.11 453.95 1643.91 578.5 513.99 388.95 696.83 902.38 637.89 1233.38 587.33 794.04 550.37 470.97 1126.87 594.1 744.17 23.15 629.88 1692.01 917.04 672.38 1042.87 467.74 32.31 741.09 653.97 566.03 620.26 436.51 399.49 421.09 1710.45 97.66 550.21 1655.33 618.75 362.37 1090.68 1109.37 1085.1 632 757.49 595.41 434.81 859.65 1132.35 867.66 277.62 607.76 282.02 1010.21 1180.86 334.16 246.48 682.45 675.7 543.05 441.71 786.19 1048.64 1059.24 220.67 437.24 181.06 652.07 1045.47 251.52 489.99 1231.32 302 442.45 322.11 1101.92 433.71 901.87 97.47 232.66 698.92 754.17 552.23 362.71 27.36 390.15 1041.74 281.37 1692.76 81.29 1388.19 432.38 527.68 964.48 953.67 551.2 519.57 388.4 271.6 426.98 609.58 1370.07 1324.34 646.72 1407.11 819.06 1116.04 503.15 813.74 654.94 314.39 459.8 653.63 19.85 351.05 979.28 280.71 291.2 1529.25 373.82 457.83 1100.58 923.76 654.15 324.11 433.96 589.96 542.02 507.89 430.2 631.53 685.26 263.57 556.7 702 275.56 893.38 203139_at DAPK1 458 201.1 215.03 361.25 197.3 234.31 164.29 357.15 268.35 253.93 124.15 118.36 89.33 89.62 105.25 82.09 71.09 337.31 630.76 558.53 304.56 205.73 286.38 266.41 245.33 566.98 466.6 1134.58 367.23 224.09 178.21 237.24 219.14 113.2 244.41 619.41 1206.08 566.47 479.64 325.73 678.29 248.85 355.46 310.79 374.47 277.8 332.51 759.62 519.65 214.73 695.7 315.25 290.56 712.99 164.21 162.12 681.23 255.61 315.92 186.73 142.29 1606.12 468.62 104.24 125.46 424.74 313.98 575.44 129.73 269.96 479.03 859.36 166.29 280.56 71.72 208.82 122.18 187.24 97.26 173.07 82.33 175.43 82.27 170.96 182.98 166.94 173.24 212.36 217.4 212.73 28.94 211.44 102.88 260.29 379.55 366.01 221.39 64.91 457.51 428.37 3059.18 219.66 676.62 131.94 214.44 237.16 232.67 133.98 285.57 44.84 213.58 128.88 289.92 524.43 556.4 423.22 137.42 288.26 235.42 307.37 257.12 194.57 400.31 446.48 237.35 168.62 337.94 342.47 145.43 374.7 393.17 142.48 245.79 308 215.1 183.44 756.37 241.36 120.47 251.62 106.39 152.64 127.2 160.66 258.77 101.04 295.57 58.08 173.58 145.22 1024.45 186.72 82.36 431.64 154.51 564.08 81.58 412.91 85.94 232.83 148.89 159.97 110.98 42.84 75.92 252.3 95.97 291.46 25.83 93.01 179.22 157.77 178.56 243.4 265.6 163.36 346.83 207.83 144.85 63.27 216.41 512.2 186.42 63.46 361.85 125.13 103.03 73.83 557.38 63.43 221.12 139.38 194.52 289.7 80.51 280.51 382.52 122.97 210.19 381.94 65.51 257.26 237.54 38.71 218.69 132.46 479.9 124.09 329.54 150.25 292.35 207.57 182.87 200.41 116.19 127.78 253.72 360.76 199.27 109.22 271.8 369.99 106.5 325.98 450.58 839.11 201.15 203198_at CDK9 97.57 108.21 173.33 110.46 119.7 145.96 39.88 166.64 158.04 89.99 146.18 129.87 91.45 137.72 129.72 87.04 162.14 107.93 101.48 107.79 159.31 124.32 162.64 73.29 134.58 141.64 144.31 96.15 112.85 127.67 126.73 142.36 143 119.29 99.92 101.35 92.66 113.95 108.22 126.88 99.61 144.27 88.08 111 133.58 111.16 94.82 90.55 163.09 147.05 59.97 135.21 130.96 181.43 138.42 107.12 85.47 56.11 116.53 111.83 124.01 101.66 198.28 55.69 59.43 69.04 114.12 89.53 66.91 79.03 73.72 121.11 71.39 94.76 136.72 88.87 89.81 106.35 77.26 89.38 78.73 144.14 94.53 100.86 114.42 135.27 81.33 110.47 115.69 107.4 33.79 57.73 124.51 103.05 113 78.73 79.98 77.6 68.64 51.69 72.04 89.79 51.72 69.71 81.65 95.05 105.12 193.74 70.97 53.03 80.55 72.62 72.29 60.67 65.16 12.63 47.63 63.34 53.96 66.4 68.99 64.85 46.22 54.59 38.47 39.27 35.99 45.31 38.95 39.25 36.97 41.51 51.22 53.1 42.56 34.8 35.23 36.06 39.18 50.82 32.88 58.71 41.49 42.8 34.87 48.78 54.31 118.3 87.14 110.46 75.02 86.49 54.7 95.95 86.89 75.12 70.27 105.94 75.98 91.06 82.18 74.99 78.08 71.79 91.03 84.7 94.72 66.01 51.55 78.79 55.11 68.41 58.19 39.49 50.18 38.52 49.25 47.4 40.81 48.02 47.69 30.21 41.99 36.48 40.48 43.22 51.12 60.37 24.38 49.84 39.49 41.23 63.75 52 57.64 65.58 51.67 46.5 72.89 46.11 62.45 57.72 73.59 49.5 48.52 46.37 39.34 37.79 32.34 58.61 35.41 48.87 71.18 47.57 64.02 53.28 56.76 54.89 41.8 109.26 59.62 72.93 69.19 61.12 57.7 59.72 86.28 203213_at CDC2 240.31 533.85 35.06 1093.72 388.87 13.55 599.61 371.27 292.86 328.28 407.11 67.91 139.49 588.25 19.82 287.57 801.93 409.81 754.45 749.81 508.72 452.67 881.16 321.87 485.87 356.26 396.51 273.51 383.28 539.8 418.23 269.82 626.93 393.98 953.85 356.08 280.87 417.26 56.57 279.04 801.13 265.26 436.38 404.08 226.35 167.4 255.06 487.59 233.27 527.41 148.37 266.64 212 212.44 569.57 258 434.06 233.36 293.88 106.88 297.32 408.55 143.9 201.7 134.03 353.75 730.43 282.31 390.56 250.31 193.47 386.4 394.42 257.8 440.92 359.58 1191.25 239.98 194.57 256.64 297.89 232.42 333.55 145.6 21.66 85.49 87.56 107.17 195.58 133.13 35.59 88.53 156.75 319.4 136.77 247.97 74.02 308.42 316.47 594.65 95.82 181.53 276.57 22.41 285.55 468.74 69.55 92.66 153.52 15.98 299.3 10.09 150.79 347.42 199.55 447.45 159.46 45.93 152.35 92.16 86.47 123.71 102.05 189.22 20.46 595.68 144.37 68.03 448.05 310.95 47.41 129.59 28.78 198.37 134.27 246.15 167.35 153.48 124.86 197.09 201.26 310.26 601.26 160.38 102.37 110.93 275.96 162.45 132.4 155.94 670.15 52.91 320.38 285.85 172.33 710.64 740.36 307.85 52.27 288.63 104.84 63.23 652.12 115.47 124.58 78.3 218.01 132 126.95 544.61 75.53 64.84 206.5 488.93 131.84 68.22 164.32 50.6 42.17 286.02 49.59 216.49 292.12 129.32 95.91 98.07 178.13 160.94 262.65 130.63 107.7 17.35 71.86 347.97 104.92 138.34 361 123.08 164.95 294.21 189.14 211.58 194.08 147.61 304.47 13.62 423.17 558.37 89.5 244.3 103.75 117.99 68.06 38.29 123.64 136.63 68.46 157.39 241.42 95.6 197.84 186.98 65.33 193.07 358.6 388.75 54.09 203218_at MAPK9 223.22 457.75 213.74 415.26 295.32 189.29 710.83 404.08 412.95 229.61 599.24 319.42 228.2 458.25 97.91 209.07 344.5 230.54 317.52 407.78 302.35 187.64 510.11 302.01 150.94 246.95 320.18 175.31 145.68 419.85 447.98 101.81 386.01 467.21 235.7 202.07 395.58 167.63 167.49 253.92 251.46 189.85 162.72 420.98 291.82 179.65 157.1 251.71 416.86 218.82 130.92 353.8 213.5 200.87 224.07 238.24 203.34 183.07 214.57 154.2 228.36 155.85 247.85 168.08 198.4 247.89 297.07 243.65 292.1 96.81 95.15 147.64 229.67 406.58 119.87 266.89 394.03 447.58 514.26 489.62 144.64 253.71 510.58 247.63 236.91 354.07 235.12 176.95 269.66 175.86 28.18 284.34 230.95 313.65 281.09 646.39 432.69 387.55 344.63 347.14 112.43 447.7 262.2 246.21 407.35 589.14 253.61 159.43 168.7 57.47 246.25 156.5 211.42 179.6 178.06 332.68 379.98 48.27 182.91 243.5 271.09 86.61 157.62 178.05 152.24 661.77 413.18 258.48 408.87 219.09 292.34 551.35 127.89 211.67 243.06 186.77 354.85 216.62 171.26 242.71 307.33 764.23 525.87 225.81 238.21 169.28 220.58 265.4 250.58 395.64 244.54 124.91 328.1 273.85 209.43 197.18 382.79 277.97 250.1 213.09 243.52 212.06 368.65 112.65 346.64 385.34 483.6 225.23 91.13 219.56 233.69 214.98 460.56 214.98 36.66 212.16 304.68 313.26 176.95 311.31 233.25 193.01 335.12 588.83 207.15 192.54 531.29 242.5 209.43 164.66 479.56 248.68 302.26 334.72 166.69 312.13 163.01 458.9 384.71 162.63 168.6 348.1 308.24 34.34 283.7 283.73 124.96 159.87 143.7 725.5 150.58 162.08 127.33 160.25 391.75 224.8 117.24 333.44 206.71 211.95 274.34 150.44 131.67 194.32 169.68 223.84 199.69 203229_s_at CLK2 413.48 536.39 804.31 274.81 805.9 738.72 605.25 424.32 391.92 345.52 966.31 604.98 620.94 812.4 811.8 554.64 524.27 848.01 735.8 817.25 702.25 411.07 717.55 961.34 579.89 561.24 467.17 581.23 541.05 996.43 401.5 693.05 576.15 876.7 582.95 851.41 918.09 562.79 664.97 499.6 483.21 1304.99 734.21 812.59 642.41 576.03 997.6 580.62 579.86 702.93 289.46 505.42 821.91 514.21 340.98 593.72 733.65 660.3 664.65 530.33 1057.13 1313.68 641.27 315.67 570.85 397.84 510.05 426.62 458.51 349.51 362.32 571.04 442.95 819.89 488.4 506.99 385.72 367.72 427.35 510.94 730.3 357.31 338.72 392.61 417.27 508.25 481.03 387.71 388.01 375.54 590.4 467.75 523.71 304.43 315.35 380.95 590.46 597.71 406.91 468.14 875.28 581.5 690.9 714.95 896.32 1478.24 924.81 822.15 683.21 1271.62 634.9 545.97 558.42 576 559.81 680.38 605.71 1189.29 474.89 656.78 838.15 944.96 721.94 530.93 348.29 315.92 698.02 338.9 401.29 563.05 626.17 498.67 478.28 343.35 303.44 367.79 408.01 314.54 446.31 382.42 636.78 293.49 361.4 335.49 303.35 509.55 582.12 526.2 611.3 408.66 663.08 612.19 506.99 484.21 749.8 1025.61 379.82 422.94 517.91 390.28 1022.94 633.41 481.3 532.73 588.04 460.13 432.86 493.38 757.54 1260.67 262.32 594.67 680.35 450.02 515.51 440.44 508.52 773.11 740.35 785.34 560.19 591.23 670.93 1043.57 472.62 660.05 1004.5 491.59 530.57 814.25 950.51 384.18 629.63 483.89 505.76 479.88 373.32 713.74 815.2 640.35 457.55 586.49 527.72 641.86 658.54 838.68 778.48 792.07 415.42 579.12 668.53 950.31 670.87 936.91 795.64 426.95 387.58 435.83 378.22 501 590.54 752.85 758.83 622.98 828.26 687.21 392.71 203266_s_at MAP2K4 172.87 81.73 128.24 161.22 330.11 344.46 160.1 196.23 196.19 158.69 283.44 159.28 183.53 561.75 114.41 194.56 169.96 171.9 136.72 315.49 246.87 164.2 335.96 141.22 109.69 194.97 214.38 182.94 164.38 135.26 199.24 142.57 338.63 759.31 154.96 218.63 113.98 120.57 143.31 133.03 125.69 171.37 113.49 163.86 131.42 160.6 210.62 119.44 276.19 52.44 97.28 168.35 205.55 143.6 154.7 255.22 81.73 221.09 90.2 122.92 415.27 109.88 137.97 48.9 165.79 138.93 95.91 104.35 87.82 167.3 129.14 123.17 205.07 223.82 136.67 143.54 118.34 213.72 179.73 150.84 199.16 302.77 163.85 313.71 176.49 227.43 135.52 190.19 138.44 74.6 53.31 140.76 76.38 81.17 92.24 76.62 55.53 136.44 92.97 108.89 253.89 237.93 164.56 61.99 150.74 152.05 287.88 156.33 171.4 91.66 106.17 221.74 164.88 178.64 166.99 154.2 151.37 96.11 123.69 138.68 182.73 167.18 130.22 116.51 172.83 281.05 150.73 169.87 187.09 152.56 153.11 275.35 133.88 183.01 183 200.06 135.31 105.33 155.5 150.71 368.76 146.22 28.73 142.34 143.53 96.84 195.91 190.35 203.7 553.46 153.1 134.69 167.61 153.66 278.55 202.98 324.33 151.16 101.77 137.89 214.25 209.86 216.39 98.45 248.14 83.36 237.59 203.44 51.78 146.63 217.94 106.95 236.84 111.09 49.55 127.61 119.09 257.5 161.02 433.04 225.79 135.13 200.77 114.37 96.93 85.17 138.23 299.05 132.91 329.93 125.16 220.38 183 262 251.47 202.56 234.03 152.52 156.3 124.22 163.72 177.16 221.92 90 128.04 277.93 97.09 241.65 80.58 182.66 100.38 122.23 154.34 94.68 446.23 48.17 152.71 136.26 113.78 400.58 162.84 149.43 133.68 180.94 186.52 138.05 171.53 203379_at RPS6KA1 117.12 75.56 94.96 109.43 133.4 83.84 87.23 78.87 85.68 95.84 88.94 91.1 137.45 64.27 92.55 77.72 220.82 140.4 175.65 97.34 131.84 106.11 102.91 149.78 66.58 144.25 189.53 89.01 158.37 193.85 66.94 91.82 84.27 93.73 145.97 146.48 144.32 218.41 149.83 196.8 219.25 185.44 142.26 149.72 114.3 86.99 102.85 141.26 140.3 72.83 114.06 162.31 147.29 197.3 240.74 106.61 193.79 133.2 156.09 122.9 192.65 143.51 80.45 54.79 215.31 144.09 113.24 148.02 191.1 138.07 102.31 147.17 83.14 92.21 93.8 85.37 111.78 133.43 43.2 63.86 111.8 140.34 116.88 114.36 98.59 80.56 58.45 90.61 101.97 78.15 79.82 110.22 48.24 27.03 81.81 101.91 63.09 205.49 28.77 61.91 78.1 158.83 109.07 78.81 42.33 131.04 138.46 76.66 99.92 76.85 93.8 92.02 128.53 197.1 131.49 94.96 146.4 88.46 127.92 148.46 102.06 150.15 179.06 143.92 45.91 95.67 58.72 85.77 65.87 105.36 124.2 58.03 74.34 125.59 76.61 60.18 83.54 64.32 46.07 51.66 26.16 44.82 72.81 41.46 82.35 79.63 58.91 97.41 89.63 206.27 151.58 150.27 124.59 95.4 125.06 108.2 50.25 161.46 80.17 163.8 107.2 94.39 68.82 41.3 65.02 92.43 87.26 131.14 108.73 39.36 151.12 94.62 123.44 95.65 117.57 89.63 146.01 127.55 78.29 139.27 125.15 96.31 44.06 113.63 105.82 60.05 91.74 47.18 104.09 78.61 76.29 80.99 81.64 84.39 89.13 231.41 200.53 137.1 135.09 109.34 72.63 163.7 169.95 81.89 127.7 120.06 100.04 121.98 80.81 97.54 79.67 93.83 82.41 126.39 103.12 105.84 76.17 123.09 76.15 132.94 217.69 113.11 89.47 148.65 154.86 150.94 147.39 203415_at PDCD6 1157.31 601.5 371.58 422.45 594.36 511.93 549.6 366.92 575.8 557.17 827.13 1108.71 546.46 357.26 404.47 471.69 1413.53 513.35 549.56 475.76 569.09 721.04 432 428.98 564.18 487.96 807.14 604.75 685.51 947.32 987.71 646.16 744.85 822.87 572.9 901.48 561.2 276.21 312.23 481.35 1113.07 630.98 659.8 406.66 398.4 492.34 487.34 554.56 583.42 350.9 616.39 850.45 606.36 380.92 808.88 783.53 970.37 470.58 514.17 442.87 627.16 664.49 380.9 553.9 227.19 490.11 892.76 313.86 396.27 490.11 437.71 413.84 612.56 370.64 544.51 539.72 736.06 564.95 569.17 433.39 686.18 604.22 456.71 436.91 414.96 403.51 322.11 363.73 273.81 146.43 138.94 264.63 217.51 264.15 194.22 263.94 567.81 377.76 212.77 301.46 415.98 483.48 457.28 218.76 424.13 410.62 473.53 488.82 296.56 175 503.85 436.13 562.4 311.39 275.86 374.07 327.67 497.93 330.05 340.39 334.46 524.35 288.51 452.59 186.7 500.73 396.84 281.11 521.74 813 336.79 606.7 172.79 339.78 453.54 249.39 186.18 675.4 288.97 516.96 322.86 611.87 557.55 618.21 428.11 235.94 376.46 544.76 520.97 743.52 519.67 320.23 534.41 417.64 498.68 328.02 958.21 614.21 499.29 363.57 516.78 388.8 552.99 354.84 550.72 566.14 652.78 338.92 263.06 753.98 539.32 377.71 341.65 324.88 82.87 391.29 356.16 340.66 255.49 412.53 321.35 402.76 490.44 353.73 296.49 363 445.62 640.75 524.94 840.74 364.29 391.74 251.87 330.54 351.46 462.06 626.72 537.44 536.75 885.29 559.69 392.76 454.34 237.67 367.56 330.91 201.69 303.9 354.61 570.92 264.95 230.21 314.8 296.79 300.13 417.9 425.04 336.21 551.33 301.01 409.86 412.43 366.24 335.85 430.19 651.13 448.27 203468_at CDK10 80.6 72.58 109.26 53.17 95.89 109.85 304.49 60.14 59.44 115.43 128.46 93.7 136.55 170.58 142.89 153.34 74.63 119.76 93.89 63.49 151.19 67.31 72.72 126.65 163.22 74.36 126.46 91.01 93.7 60.3 96.03 66.48 183.98 138.44 76.17 128.33 89.3 82.8 132.27 75.77 81.36 98.31 59.91 100.32 74.8 105.9 55 42.09 69.9 76.35 79.79 83.37 129.99 52.19 61.9 116.6 64.45 51.48 97.16 63.52 97.52 104.64 119.34 55.39 118.96 46.24 112.71 72.91 53.51 88.51 78.44 75.73 84.12 81.84 97.85 63.12 66.54 53.02 34.27 57.02 108 39.19 44.91 49.49 58.17 53.75 56.91 58.59 55.59 186.16 289.77 122.24 157.08 59.77 120.8 115.21 112.1 190.56 87.86 79.83 177.72 107.3 73.91 228.34 212.61 88.77 55.33 61.62 54.05 292.25 50.9 54.22 50.72 63.56 60.5 45.22 188.61 114.22 59.72 73.51 63.32 68.88 51.7 58.06 76.29 34.63 74.03 80.58 36.54 89.11 115.71 69.98 150.33 66.44 53.6 106.65 218.18 59.84 70.09 67.72 52.41 72.06 58.59 93.22 71.96 91.77 78.7 145.25 86.13 86.01 96.96 92.21 52.06 95.78 73.35 69.23 31.17 72.16 87.77 109.18 179.25 113.67 57.59 78.1 92.27 89.38 76.37 100.26 192.02 136.7 73.47 58.45 86.37 59.44 294.61 89.3 80.22 35.79 83.88 134.11 56.69 108.6 57.14 122.5 66.33 137.5 107.56 77.63 124.27 81.45 127.14 46.27 96.7 69.14 121.52 61.9 158.62 132.55 90.29 108.69 113.07 81.76 71.93 113.87 174.59 88.53 101.6 80.26 108.34 59.23 204.26 129.21 111.64 136.59 88.5 143.34 104.44 59.49 86.02 101.61 75.26 125.81 89.51 142.13 127.87 79.88 73.41 203499_at EPHA2 36.68 38.76 71.66 61.45 46.91 40.31 52.68 26.91 42.66 57.5 33.23 30.66 56.16 43.59 67.25 377.68 214.17 39.98 58.42 26.43 41.74 51.77 41.89 55.35 44.91 56.61 60.41 36.37 68.08 89.12 35.9 28.35 30.69 40.28 75.19 100.44 36.62 59.63 46.86 54.33 86.66 73.05 60.4 81.15 64.97 75.39 110.29 62.43 70.1 48.77 44.57 50.23 57.76 73.68 40.12 64.44 176.24 60.59 49.2 86.49 41.16 26.9 72.96 36.33 30 55.17 26.89 38.64 76.59 56.88 84.78 78.63 38.12 44.86 24.19 19.41 26.5 26.65 19.98 23.29 35.46 51.19 18.73 26.99 54.29 36.72 31.9 40.5 33.98 57.3 41.45 33.14 26.2 39.6 34.81 45.22 21.12 26.9 25.64 47.92 39.26 66.59 18.35 47.47 20.22 26.78 22.79 21.42 29.24 18.29 21.78 52.85 30.41 65.55 27.8 36.95 20.68 24.35 43.06 32.51 60.3 50.39 26.21 17.33 57.75 33.7 18.38 46.37 27.19 32.21 23.88 30.04 80.05 39.24 26.62 23.16 31.95 42.77 22.11 29.58 16.46 29.18 20.07 20.85 30.95 27.09 29.92 34.16 75.16 32.89 58.55 57.25 127 28.5 26.6 50.23 26.04 104.08 27.94 193.12 32.85 46.44 26.91 18.59 23.31 27.7 29.41 54.31 35.69 25.61 29.54 42.3 34.87 36.44 42.55 28.22 55.25 28.93 33.7 56.89 33.31 28.68 25.33 32.44 43.7 31.45 35.41 36.38 24.07 37.96 26.16 33.55 27.96 28.26 37.38 38.21 22.99 24.9 32.73 41.04 25.15 24.57 34.99 28.3 50.92 38.75 28.05 28.32 22.67 26.08 34.69 29.79 35.66 34.3 28.22 20.59 28.63 47.32 54.28 31.96 43.43 61.46 45.5 52.88 66.2 48.97 54.36 203510_at MET 674.56 74.3 86.09 51.87 325.99 69.28 48.32 82.1 34.1 11.79 55.94 25.04 87.5 24.92 102.18 102.47 700.08 108.35 680.93 126.13 200.69 32.81 457.82 188.34 699.15 145.09 43.68 79.01 297.87 113.06 45.37 38.28 102.81 33 166.02 86.88 68.66 139.32 174.24 52.22 443.56 27.96 292.16 113.35 102.77 196.27 77.69 284.09 455.06 55.52 699.77 125.45 53.28 287.39 177.34 154 289.57 295.32 103.88 381.79 32.73 104.01 1009.6 35.42 18.92 260.07 62.15 28.9 207.01 403.83 674.99 227.32 227.2 251.66 15.7 150.3 55.54 90.99 11.97 23.87 28.15 90.97 231.15 38.08 217.45 143.69 107.01 125.52 38.93 244.64 35.08 145.94 95.33 55.5 220.39 417.65 22.58 89.55 120.69 426.59 108.82 271.2 33.48 207.3 55.63 75.66 91.48 76 144.84 13.9 218.1 232.83 81.3 183.88 147.81 131.7 133.03 90.02 87.3 172.22 81.65 635.98 75.12 135.3 204.53 43.26 82.5 317.23 98.91 476.2 68.91 40.36 197.29 98.81 48.47 224.25 75.34 218.38 107.46 24.24 64.16 34.32 59.93 69.53 173.5 68.97 40.09 255.31 182.66 57.67 150.21 78.09 1066.6 70.25 131.18 106.04 57.56 981.39 200.19 1205.46 82.55 273.01 8.75 70.75 21.02 83.73 33.71 204.34 33.18 29.31 23.71 34.67 48 61.81 12.05 389.16 161.91 21.09 159.26 24 77.5 15.14 36.56 68.76 39.28 63.21 60.56 77.53 10.93 9.14 33.79 163.5 44.18 154.69 187.94 114.22 159.28 97.74 143.64 173.13 65.75 49.56 71.61 34.24 49.59 128.12 84.68 68.07 53.05 75.12 623.55 46.59 162.71 90.01 43.04 58 142.71 331.58 77.28 160.89 774.32 273.73 99.35 145.39 389.92 293.58 281.06 203552_at MAP4K5 224.5 275.85 288.74 139.66 269.52 289.45 165.04 287.63 249 175.27 196.36 353.59 192.78 232.23 189.81 231.73 114.26 108.9 177.38 243.02 218.46 162.74 295.21 134.13 235.87 207.02 178.1 190.9 513.28 178.73 193.81 162 275.32 36.51 131.08 96.32 175.1 310.05 186.41 72.2 171.98 101.86 129.53 187.31 163.8 116 72.62 156.77 135.78 177.75 209.78 127.42 168.02 231.99 151.51 210.15 90.81 94.65 130.48 162.89 198.99 121.47 406.05 106.05 99.34 158.6 84.43 98.13 102.94 176.48 141.77 136.75 110.72 189.53 159.82 186.35 380.97 220.32 108.1 248.03 126.74 233.16 164.35 202.88 208.26 214.34 194.22 193.23 95.49 247.14 24.99 254.74 193.41 293.72 300.52 200.79 183.78 231.18 209.69 271.73 299.6 213.44 256.3 198.4 273.89 139.93 224.1 203.22 121.15 19.73 99.07 212.96 211.39 286.21 172.61 129.18 331.43 143.62 167.55 130.11 253.41 221.26 100.71 127.07 216.4 79.2 212.71 264.22 256.2 207.66 207.88 174.8 149.5 139.87 220.22 183.08 214.35 226.76 252.18 269.14 263.19 182.67 195.44 183.69 147.83 176.41 232.23 177.69 95.68 176.76 139.94 123.66 126.47 218.79 148.43 136.86 103.07 111.71 300.72 123.22 155.09 239.61 156.78 152.62 173.52 150.54 296.04 131.06 30.15 187.66 125.2 200.31 258.24 167.33 35.26 165.49 188.25 118.95 201.93 110.85 233.03 161.78 199.14 205.01 203.59 171.53 166.05 204.1 204.12 205.14 353.18 368.1 319.23 152.24 173.84 169.99 670.72 255.7 208.22 252.61 141.46 191.32 215.47 24.63 141.15 254.23 257.76 162.5 216.73 173.07 289.95 238.55 155.98 227.33 162.57 173.59 198.07 203.35 263.74 231.98 103.61 155.47 114.25 192.75 224.2 199.84 111.92 203575_at CSNK2A2 157.99 148.25 184.08 131.27 144.79 113.29 185.75 126.67 180.59 238.21 72.85 88.15 118.29 118.46 121.1 292.55 201.12 184.32 157.47 182.27 168.59 102.32 123.69 184.37 544.97 150.05 210.7 133.71 170.56 119.38 128.62 87.44 141.6 114.06 120.9 141.2 97.41 164.05 132.87 262.67 148.81 195.89 217.82 108.84 176.17 141.81 112.87 123.56 189.57 100.76 162.25 131.38 160.78 223.79 158.66 194.34 269.1 336.3 161.91 238.44 72.46 96.36 213.95 139.1 186.88 116.33 161.07 116.56 148.62 105.56 251.33 145.86 149.09 151.54 193.46 115.63 158.16 96.47 52.84 113.43 158.4 113.01 96.49 96.56 167.12 115.1 133.89 143.48 119.49 188.31 131.17 205.76 189.74 166 201.23 164.85 148.47 210.96 210.76 186.74 224.49 207.15 118.84 228.4 209.53 222.09 168.63 157.96 197.29 104.51 142.54 290.76 149.65 269.67 184.6 158.62 142.92 104.79 188.87 244.22 178.18 143.66 401.76 171.67 196.68 140.09 98.7 166.12 137.65 117.93 180.07 118.5 252.48 150.65 109.85 184.09 131.56 160.09 108.94 89.88 55.81 156.36 115.77 109.25 127.19 113.12 141.01 231.99 169.22 166.67 88.28 190.75 253.21 146.41 91.4 246.35 106.12 125.63 183.05 182.41 156.03 143.07 116.81 89.31 225.13 146.77 90.02 161.82 110.74 141.43 211.95 137 117.11 132.98 300.2 187.46 151.86 72.42 156.87 230.78 151.89 250.24 73.04 168.7 276.67 127.15 164.25 129.22 278.59 126.78 108.93 159.14 117.67 129.62 199.41 134.54 207.71 172.15 155.65 128.19 143.19 108.5 101.69 83.04 323.35 165.89 62.92 115.49 104.57 81.33 161.75 94.54 187.19 116.43 60.39 104.13 157.63 182.81 133.7 99.95 226.13 209.95 122.49 245.46 218.75 137.03 214.13 203628_at IGF1R 2893.6 737.23 586.54 436.7 1875.16 1511.59 330.69 1470.87 1186.92 137.84 1621.04 1094.09 1237.42 3402.97 1079.32 656.8 370.84 73.83 572.18 201.34 3321.23 66.92 234.25 298.06 271.89 132.55 323.44 385.76 357.32 81.25 452.57 245.82 1412.36 2517.61 43.83 37 229.73 319.83 405.96 114.01 391.87 74.04 216.5 128.79 139.74 653.14 370.37 424.61 193.86 275.49 2026.64 137.38 12665.65 380.45 3664.8 497.22 602.8 261.78 84.94 77.97 720.81 1007.16 171.05 1105.33 164.04 235.95 560.65 473.67 964.38 1022.37 671.95 419.41 289.63 262.38 3274.85 2010.56 671.71 1515.95 2035.37 2423.7 1286.92 2102.54 2208.75 3598.5 3294.24 1801.13 797.39 7497.09 540.95 1303.04 1481.13 2524.96 3428.15 771.11 1503.07 200.25 3060.18 293.45 1128.19 389.4 1494.54 342.81 116.76 1346.61 572.51 1093.44 1254.51 832.89 1819.94 1471.88 312.94 1359.6 372.6 91.82 100.55 48.6 635 1038.97 1275.61 736.79 536.19 484.14 182.13 534.83 730.15 1092.33 2263.64 678.31 730.48 104.38 479.94 493.51 764.37 223.66 373.94 1800.31 184.74 279.16 1022.51 370.49 1708.77 522.17 785.77 603.1 1292.03 972.04 88.94 7991.72 455.67 922.58 41.64 1247.69 283.97 259 663.09 211 1660.94 97.09 1144.05 115.32 1743.07 791.85 962.2 1906.69 1144.07 475.28 1043.17 344.52 959.85 2033.26 2508.14 1506.56 700.06 348.85 52.23 230.89 246.69 1371.1 2248.05 1335.78 983.16 425.72 1782.8 2124.94 392.66 1263.07 1680.02 3550.79 350 284.05 1229.33 745.02 843.04 225.26 1326.23 214.53 502.46 1023.61 323.83 884.37 2257.8 295.61 722.43 2805.44 321.27 870 775.04 67.77 581.61 330.53 2145.87 578.42 636.22 750.53 1842.64 1821.5 435.36 462.34 1137.28 1916.78 108.96 287.92 1870.15 904.61 290.23 1923.09 339.56 203652_at MAP3K11 139.61 194.45 238.79 280.74 216.79 204.93 314.55 140.68 132.27 417.1 231.48 216.1 224.26 196.96 201.52 209.57 257.19 272.82 168.29 115.81 232.08 204.4 278.8 265.51 132.72 206.83 191.71 287.98 201.02 115.25 131.13 201.39 154.15 366.47 196.88 259.94 422.04 225.74 293.88 223.19 259.18 256.48 221.86 378.44 291.86 230.91 206.18 189.85 286.95 207.94 175.79 256.9 370.24 279.63 306.9 197.93 319.92 236.31 365.92 265.27 368.27 335.14 238.41 128.61 219.27 148.56 161.67 177.82 187.57 181.4 229.68 176.65 181.43 118.77 87.52 157.01 113.58 163.81 127.51 215.41 173.61 188.52 160.6 164.04 170.94 153.28 212.95 198.29 154.71 271.19 409.7 359.76 212.7 186.41 175.75 241.17 189.83 363.7 269.94 296.67 184.96 178.38 208.66 304.12 242.52 356.65 222.05 251.83 368.57 397.97 268.11 163.33 177.98 187.09 260.81 122.65 233.42 292.78 250.73 395.48 279.7 373.92 353.75 273.19 189.48 211.01 170.01 183.02 117.79 253.51 187.57 220.32 384.33 244.29 185.67 103.43 193.78 148.5 151.65 158.84 105.08 154.61 203.28 120.77 159.19 209.64 170.91 270.73 469.72 214.35 237.58 387.34 277 183.93 250.21 258.4 124.56 311.61 187.25 205.21 343.23 230.5 163.32 234.06 168.45 223.91 152.19 316.06 413.33 153.86 281.16 319.18 429.03 202.48 405.93 255.87 251.96 165.56 269.02 401.98 218.89 256.46 146.94 247.03 228.58 228.35 222.31 239.58 283.49 195.02 220.18 160.5 283.36 284.72 227.28 363.5 234.96 285.14 311.05 463.24 305.2 270.38 210.77 412.66 381.98 324.8 189.46 176.2 182.39 209.88 237.63 271.78 323.31 356.98 219.07 181.49 205.1 210.61 213.4 270.16 255.59 213.9 237.46 155.12 206.51 187.39 200.93 203709_at PHKG2 112.59 149.95 123.94 95.37 156.31 246.58 157.77 134.29 159.86 198.1 167.87 185.17 164.1 259.12 155.54 118.87 212.82 223.68 90.09 95.03 155.65 140.8 150.83 122.18 99.72 139.6 143.94 109.67 134.26 147.97 112.79 126.57 260.04 190.09 152.21 214.85 138.73 124.14 127.24 158.9 110.18 131.78 93.89 131.32 126.93 101.11 115.69 170.58 126.92 155.05 118.19 197.9 141.14 156.16 123.9 148.64 110 137.57 267.08 176.8 230.17 129.16 130.01 154.54 102.18 143.18 160.11 90.58 118.62 78.68 107.87 128.37 110.51 77.85 140.89 117.3 106.75 119.05 133.51 135.19 175.19 131.99 85.59 159.71 93.87 152.15 110.72 103.66 128.93 93.09 249.2 118.25 111.67 70.72 68.64 96.51 142.7 184.7 92.08 80.56 203.11 109.1 133.09 131.46 170.51 195.13 160.55 119.85 175.3 370.33 172.92 105.03 87.41 130.52 140.04 78.27 201.96 224.91 118.19 129.5 159.61 230.04 141.71 132.33 65.76 106.36 106.33 81.97 57.15 84.51 117.47 165.13 98.53 112 132.82 97.75 111.21 70.32 76.61 96.67 115.39 97.69 136.96 97.11 78.55 183.67 97.64 202.24 156.25 154.61 179.46 122.64 124.58 109.29 135.09 181.64 133.06 140.93 129.62 162.32 159.08 111.27 89.42 127.6 127.74 119.98 171.89 127.62 131.41 145.82 174.43 212 324.76 153.14 228.11 158.52 137.83 160.71 130.85 201.62 114.29 162.17 116.3 110.7 81.26 99.64 170.75 131.19 152.52 87.33 163.76 84.8 138.36 121.84 88.37 140.92 130 229.46 150.03 151.25 240.64 159.05 153.61 270.84 236.03 92.99 117.12 90.86 115.5 143.65 113.43 167.77 130.84 209.56 124.6 148.24 124.49 131.2 130.46 149.72 142.39 128.43 166.73 131.27 134.36 131.26 93.43 203755_at BUB1B 301.26 284.42 63.72 402.16 287.12 15.01 329.52 336.49 268.99 205.27 196.7 43.13 63.6 301.11 24.54 211.41 429.27 372.67 405.38 686.82 331.88 323.86 526.98 208.06 383.09 216.77 123.65 391.38 288.07 369.68 172.71 147.94 363.8 259.4 568.45 333.61 319.65 266.71 36.25 83.59 258.06 419.82 266.09 277.57 164.25 107.63 223.45 284.85 131.73 226.08 128.76 139.36 182.32 227.79 300.43 126.34 369.29 157.31 209.33 134.32 189.33 498.98 252.04 117.29 147.84 315.65 232.08 464.37 338.47 218.02 442.78 216.34 214.3 364.65 199.54 109.1 391.28 91.57 67.12 127.89 239.46 143.88 173.08 59.2 18.74 57.88 58.54 65.13 128.25 111.08 20.08 85.26 110.7 118.57 93.5 171.48 110.74 134.71 175.38 147.65 193.49 124.79 136.15 24.1 163.09 269.95 54.87 81.55 287.7 47.67 181.52 14.41 95.19 205.9 254.58 335.05 298.1 85.24 107.92 148.87 77.81 277.96 175.99 247.32 26.54 382.33 155.45 60.45 185.94 225.91 78.94 77.96 24.97 123.53 156.88 249.35 232.8 166.11 107.12 215.35 129.85 229.73 387.69 194.84 96.72 164.46 326.71 77.87 60.65 89.36 520.35 89.88 336.42 126.93 115.31 373.66 160.72 380.2 37.3 355.32 112.88 80.2 207.16 87.83 74.21 98.73 139.9 173.67 103.92 417.34 156.3 129.17 206.01 461.97 75.4 80.89 188.61 69.54 88.91 258.51 74.86 286.24 283.7 104.69 61.51 205.52 92.93 198.6 318.58 151.05 54.33 11.42 109.3 456.86 84.01 232.45 360.28 164.4 172.59 359.64 122.25 147.18 152.88 187.01 320.71 17.86 309.57 348.88 70.41 206.86 152.01 101.23 144.27 38.7 257.45 105.33 92.68 290.2 187.09 74.18 140.1 238.48 70.76 217.56 276.49 333.94 37.38 203777_s_at RPS6KB2 109.9 213.6 109.13 123.94 455 113.14 160.88 229.85 115.37 123.35 124.48 112.38 103.87 316.02 111.19 105.32 145.99 110.16 104 108.82 117.09 130.67 197.53 120.7 115.86 87.57 120.59 107.73 109.25 87.2 142.63 114.4 112.63 142.67 103.75 127.09 128.42 96.88 116.62 104.48 118.66 127.29 144.28 161.87 99.23 93.8 91.05 101.57 138.58 118.12 138.61 151.37 133.41 127.06 172.37 292.14 108.66 100.06 143.44 124.06 98.22 128.43 113.45 135.03 100.53 70.66 107.69 85.75 120.41 114.35 123.18 103.8 114.07 125.49 153.35 116.5 98.24 105.4 79.74 121.02 110.24 121.1 83.47 111.67 86.59 68.42 76.2 95.08 113.11 131.29 152.66 160.6 136.41 93.76 125.25 112.45 112.17 195.81 147.98 128.76 107.09 93.24 144.54 118.79 128.18 106.26 86.23 99.58 108.08 146.06 130.05 75.82 98.14 118.65 114.68 75.98 144.46 113.61 114.76 151.99 62.9 82.16 208.99 122.45 70.91 108.45 58.91 77.23 67.14 83.01 78.72 127.44 76.01 82.75 84.48 76.36 75.46 81.86 92.82 78.76 47.96 68.44 88.73 73.9 74.34 87.34 86.51 108.52 165.6 105.93 111.26 111.94 154.32 88.06 85.19 113.77 94.51 128.53 93.65 95.76 153.59 104.21 83.17 125.8 76.4 104.35 111.71 112.43 148.19 67.95 145.9 151.24 188.18 95.02 208.1 138.36 103.79 101.45 96.34 182.8 98.13 146.84 73.1 129.86 109.97 89.46 126.89 120.02 151.74 89.23 100.54 88.44 113.76 109.96 93.54 142.28 104.74 125.93 119 111.56 118.23 108.97 100.96 167.32 170.33 83.13 107.07 101.6 88.44 100.22 102.11 98.06 111.29 131.47 138.81 69.52 107.41 111.56 109.76 82.14 139.68 128.23 114.23 96.29 132.39 109.29 110.97 203794_at CDC42BPA 299.26 254.65 463.98 201.62 291.6 344 396.78 292.86 375.39 392.41 330.95 270.24 270.63 353.5 249.24 393.62 261.11 105.89 342.6 530.41 254.92 160.73 332.69 249.4 420.81 204.62 203.33 353.79 186.62 265.26 268.37 182.41 229.69 389.94 265.71 286.16 423.08 335.01 241.12 273.17 302.55 270.37 315.04 606.03 372.42 294.59 287.35 421.48 436.28 298.65 388.46 533.2 398.63 394.46 461.93 273.8 345.2 140.83 114.45 248.67 436.48 165.65 308.07 132.11 71.45 220.42 239.3 239.19 230.78 287.16 201.73 160.35 296.07 197.52 275.31 319.08 148.65 92.36 278 238.86 405.34 95 287.3 163.51 238.59 154.2 405.42 162.03 309.84 174.68 221.86 156.91 255.52 304.43 289.06 311.56 278.4 268.63 228.37 299.04 334.23 748.95 275.96 338.02 348.45 481.75 489.99 379.34 592.25 236.01 404.68 334.47 281.45 255.99 109.17 603.65 397.31 374.59 177.02 353.92 537.86 341.5 197.35 277.36 365.2 206.65 233.3 267.11 324.69 199.66 272.57 253.59 330.48 257.6 127.99 188.24 304.41 152.34 258.26 211.72 263.04 203.2 170.03 238.88 188.75 96.01 200.02 223.12 429.18 81.89 276.54 294.65 278.94 197.15 138.12 430.83 297.92 164.55 313.22 197.81 353.49 317.65 360.12 186.53 271 265.8 90.51 187.64 333.44 285.41 72 239.23 241.47 345.1 92.22 218.28 351.58 338.52 225.07 797.79 265.19 284.7 255.97 384.86 258.65 403.72 299.26 588.67 270.43 268.66 234.04 414.68 398.95 241.9 301.9 327.65 363.32 254.1 211.39 88.08 260.56 109.2 164.9 222.91 285.32 668.59 153.2 271.87 214.17 226.02 272.2 202.74 538.41 301.58 344.36 309.89 309.45 352.36 344.33 265.06 244.53 383.03 180.69 460.09 242.35 221.06 208.69 203825_at BRD3 344.48 508.57 1043.55 1345.15 1225.79 1367.02 730.08 924.6 609.66 984.25 1420.23 1183.32 1204.41 1785.19 1117.13 963 390.28 1127.38 877.86 452.03 1251.37 726.81 795.77 637.36 941.36 542.82 751.23 1141.45 772.68 648.62 533.56 638.43 822.08 1166.63 341.82 785.93 680.66 612.81 704.83 506.5 381.44 552.65 524.23 518.74 781.41 849.61 903.27 406.48 821.32 797.4 386.53 812.37 757.02 871.94 1054.08 573.87 786.66 365.71 1230.75 535.09 1042.15 1230.93 618.42 809.86 576.98 683.96 880.21 668.99 527.13 1397.03 857.64 726.52 502.65 506.36 637.74 789.04 528.9 1054.83 658.35 548.12 900.89 735.58 715.55 936.57 818.44 526.51 720.9 784.69 796.37 580.65 1379.55 778.54 586.14 437.68 514.22 389.68 557.75 853.26 379.29 426.84 1609.79 664.86 1047.59 482.14 629.19 1269.43 1072.68 995.98 754.97 1083.17 1424.08 901.58 620.62 962.21 881.22 127.55 1941.27 754.29 813.94 1105.13 1238.26 1072.43 1173.96 780.04 735.03 642.87 1152.25 728.81 425.88 847.17 1334.15 703.86 1168.72 1064.21 928.26 1183.09 712.9 659.49 828.75 2546.49 709.35 1367.35 757.63 989.56 861.45 1614.16 926.89 1360.66 1117.4 557.47 655 907.49 561.1 701.75 788.83 709.95 393.52 420.54 749.48 558.75 1222.58 870.56 546.78 1203.19 676.42 938.24 1277.36 1062.22 1531.97 885.09 1078.87 1301.94 1579.04 1015.5 2737.38 990.98 886.86 1014.82 1434.73 1389.32 914.14 779.92 1176.37 1236.21 1011.05 1239.39 1294.14 1160.98 764.15 854.44 1579.29 890.24 800.89 797.37 934.15 1302.63 800.84 1096.24 990.57 1139.28 1173.51 883.07 1160.58 1737.28 794.03 1222.72 921.93 607.34 684.74 819.74 1124.72 959.24 1394.64 1139.49 945.78 632.15 1200.79 603.7 773.78 1490.18 629.64 744.24 1351.4 674.4 611.35 833.6 697.15 203837_at MAP3K5 102.52 147.17 216.94 200.63 223.11 166.8 146.02 124.85 131.94 160.25 74.43 57.48 209.72 72.23 169.26 84.12 517.19 191.14 186.08 226.55 97.48 272.75 129.27 147.39 146.45 376.99 255.52 349.96 233.94 165.63 128.62 277.1 80.78 149.7 148.09 100.12 84.04 217.49 236.18 151.99 182.55 126.9 87.47 171.3 173 210.95 96.97 161.77 161.66 193.91 150.83 78.69 126.61 177.27 50.38 79.14 148.38 189.32 163.87 127.83 158.87 135.61 290.2 61.85 275.6 92.51 148.65 145.15 126.54 94.43 109.25 129.49 134.34 172.78 87.62 259.44 117.48 215.87 133.43 122.31 91.79 162.13 127.28 102.74 190.04 152.33 137.83 141.2 175.85 266.08 50.47 167.65 84.83 93.32 214.87 261.27 93.57 240.26 191.82 185.42 92.92 485.81 139.12 157.25 145.17 162.27 131.83 93.77 80.76 21.15 143.81 155.57 125.5 147.68 166.34 448.62 101.52 94.14 162.93 152.41 149.42 203.93 112.18 181.27 177.04 86.86 179.56 250.09 118.97 180.79 202.95 85.69 179.21 169.66 218.07 87.66 96.43 336.63 101.14 587.23 164.71 147.26 101.51 151.31 197.66 82.03 181.2 84.76 166.45 223.28 59.68 119.77 235.01 171.79 133.56 73.42 74.82 267.98 95.37 164.88 126.68 165.93 110.4 46.96 206.31 126.03 60.43 192.91 57.12 55.39 108.38 66.16 90.3 90.69 65.69 379.54 155.54 130.78 102.86 117.25 143.58 87.28 64.03 60.53 116.96 74.88 85.83 157.11 75.96 137.48 101.36 124.38 53.35 184.47 104.02 155.99 130.53 86.2 237.9 109.93 38.56 86.18 113.43 34.86 76.75 91.18 139.8 82.33 99.81 67.78 230.7 87.45 85 108.38 126.19 121.68 125.35 188.08 144.98 129.36 284.24 208.34 179.08 119.42 194.59 166.34 239.74 203856_at VRK1 180.74 165.55 34.32 94.87 129.32 52.29 473.18 121.51 61.02 61.2 129.58 73.33 69.71 226.98 41.86 233.75 240.48 127.87 281.31 215.11 151.56 115.23 204.61 115.45 110.1 151.56 78.71 83.41 92.91 179.72 92.45 124.9 108.47 91.4 234.93 141.96 94.4 69.05 57.3 109.69 176.06 206.27 129.77 79.83 81.14 60.47 60.47 107.18 60.38 46.51 65.86 69.03 101.34 163.07 73.2 75.84 109.81 56 155.24 61.95 95.09 261.74 30.56 107.29 198.08 128.89 153.67 271.41 159.52 108.55 116.34 89.12 125.37 180.75 108.61 117.58 453.77 158.53 73.34 72.41 66.48 87.74 115.01 96.52 77.42 79.75 65.18 74.07 95.65 45.85 22.78 50.99 71.06 85.91 48.99 99.49 50.81 106.53 105.46 108.29 80.92 71.92 111.05 73.93 129.71 76.2 24.78 43.2 29.1 12.94 90.63 66.62 70.03 171.63 130.97 177.88 176.36 48.36 123.21 46.34 54.95 56.78 108.9 152.49 34.06 122.83 52.54 42.8 146.62 125.83 66.57 51.84 27.16 52.66 64.57 40.92 128.38 66.67 44.31 76.03 81.33 77.83 180.93 66.13 108.29 72.91 129.83 89.85 53.26 125.09 153.5 37.66 163.3 87.06 75.23 122.07 73.2 125.59 64.95 141.39 69.7 56.12 92.03 54.17 69.33 96.94 56.04 76.78 55.22 167.32 99.95 31.48 83.45 100.82 34.94 105.38 77.54 28.98 31.58 117.09 74.31 91.93 53.73 40.07 41.38 23.62 50.94 32.59 125.02 22.26 71.81 63.51 45.11 115.45 92.42 135.22 109.27 43.46 113.28 59.72 50.24 85.86 73.82 28.37 87.32 36.11 142.25 130.78 41.53 105.99 64.47 44.7 24.43 32.02 93.88 62.71 31.44 178.31 71.27 50.4 120.05 97.25 46.11 100.7 116.26 145.2 62.56 203891_s_at DAPK3 128.81 84.65 115.69 106.82 105.19 87.08 116.82 69.03 81.12 129.73 123.28 135.98 108.09 133.56 96.13 121.49 97.38 87.37 94.25 93.65 105.56 95.56 130.93 101.45 83.52 67.15 105.37 70.28 56.85 114.36 108.05 101.66 120.86 109.18 79.54 112.34 86.5 99.22 105.75 68.23 109.38 119.26 162.84 113.78 94.46 90.55 112.1 52.97 102.9 109.61 64.51 121.11 149.95 102.08 125.75 95.99 86.73 59.2 101.2 103.83 90.37 62.5 107.99 69.82 47.37 62.62 55.92 57.33 82.65 67.87 76.39 87.53 88.92 102.85 101.13 80.85 66.48 47.5 75.06 59.14 135.9 67.38 66.21 71.5 58.81 61.06 91.58 85.14 65.09 86.3 144.34 69.44 61.94 65.73 58.77 75.61 81.82 96.1 92.82 92.09 92.29 64.4 92.53 91.15 129 89.87 112.12 72.26 112.1 139.62 94.08 75.85 66.19 125.83 62.25 38.67 98.46 86.12 70.01 112.72 91.88 65.75 67.14 80.13 75.16 64.63 77.21 77.32 64.53 68.46 97.77 104.01 97.23 82.19 81.31 52.3 56.43 67.56 80 107.76 55.2 55.11 72.65 69.2 61.4 70.27 76.32 82.31 87.39 90.34 92.45 99.03 119.49 94.28 89.71 123.64 100.19 102.76 71.74 69.21 108.5 98 106.69 117.11 77.99 95.2 91.9 101.2 135.91 58.53 94.04 104.25 89.03 92.35 137.58 125.41 98.46 88.41 84.69 108.67 85.5 103.42 76.64 109.95 84.49 86.13 69.28 58.32 102.15 94.34 100.41 76.99 92.9 108.73 60.88 139.12 93.19 117.61 119.01 87.9 105.22 104.39 71.44 103.56 94.63 92.14 68.35 79.01 86.27 71.01 85.55 95.25 99.05 94.91 82.68 70.02 77.6 74.87 107.83 80.1 97.55 101.1 93.78 134.71 129.36 64.62 66.6 203934_at KDR 72.98 76.38 200.76 140.47 198.26 72.56 63.71 140.99 92.98 49.59 82.97 54.69 107.87 86.01 112.65 73.81 111.65 55.54 161.14 127.67 98.56 177.16 45.59 60.2 82.5 95.16 160.54 63.31 96.88 63.38 98.94 69.8 59.98 82.19 56.37 64.82 124.38 117.03 225.62 62.79 60.97 113.93 57.65 98.86 188.91 293.57 32.83 73.99 117.73 113.97 69.27 116.65 84.49 119.97 61.92 66.14 67.31 50.57 73.01 128.29 43.64 47.8 119.51 76.8 49.16 47.16 86.9 99.07 53.14 113.89 94.95 98.07 99.33 112.27 58.86 83.1 52.14 178.56 41.1 52.09 81.35 202.75 33.81 102.46 341.34 101.72 119.27 243.55 70.02 139.18 104.62 128.32 57.46 103.43 120.36 151.34 126.16 101.33 105.56 89.9 108.16 295.71 71.42 98.59 67.49 122.05 131.31 140.74 154.63 45.87 56.55 127.72 69.23 65.71 57.79 95.11 92.59 59.27 52.37 80.99 158.56 69.9 49.45 70.24 255.78 181.84 157.67 261.65 114.2 65.77 154.32 105.77 389.46 134.66 143.42 87.11 415.23 220.76 169.2 103.53 94.18 111.39 84.37 123.49 270.79 116.48 109.4 141.44 185.14 87.92 87.31 145.86 82.03 123.26 85 116.8 39.9 104.58 86.5 98.23 72.09 165.15 62.68 93.11 104.14 127.06 111.18 131.34 56.53 66.69 95.42 141.51 131.47 118.75 33.19 149.03 132.02 129.36 150.48 86.06 142.98 158.43 155.93 164.61 205.61 181.62 146.04 290.2 93.54 121.4 119.17 109.52 129 147.63 206.63 116.28 98.32 152.61 113.92 112.89 117.53 123.64 116.75 43.29 153.42 87.58 63.06 113.02 68.64 88.25 141.09 165.99 133.58 160.19 135.36 85.55 112.74 128.15 131.63 113.82 180.88 226.29 192.53 188.51 175 77.21 194.64 203935_at ACVR1 569.89 528.73 622.68 359.58 395.31 294.73 324.77 373.71 393.45 325.38 477.56 340 289.75 308.37 225.66 289.64 250.77 167.12 293.77 157.41 349.13 422.89 458.26 253.11 474.09 497.8 732.37 677.44 248.47 334.01 711.1 403.17 301.62 151.49 386.16 109.94 255.55 316.51 367.12 247.28 310.31 236.21 342.51 299.5 297.96 525.05 300.86 188.59 376.01 337.68 679.93 593.81 301.74 526.29 403.47 379.62 311.62 258.51 223.98 342.63 204.3 128.68 841.23 144.07 98.56 215.72 273.4 191.32 203.41 482.17 669.22 269.99 436.9 603.01 362.82 482.92 181.04 413.19 259.84 391.79 157.01 450.25 237.6 301.71 528.11 447.17 384.72 364.63 179.66 437.94 28.57 394.76 200.9 454 774.62 388.1 394.27 253.74 416.51 340.66 222.82 645.29 268.16 410.16 326.32 296.95 393.83 253.61 461.68 20.51 276.73 480.25 382.7 386.32 436.61 505.14 122.09 135.29 161.69 455.44 507.14 293.59 170.36 574.64 375.11 130.76 273.89 379.8 369.37 255.31 368.45 279.14 263.19 369.18 507.89 222 162.51 708.98 354.31 392.16 218.82 388.71 166.19 370.66 297.95 80.24 263.19 378.24 299.71 387.92 341.69 279.64 356.22 396.12 208.64 169.58 189.04 248.62 482.01 206.37 259.47 457.43 325.79 165.67 255.48 392.16 387.02 344.29 58.09 141.18 236.85 228.91 284.24 162.33 23.53 257.9 388.8 245.74 328.07 101.75 366.96 114.44 278.81 324.42 572.34 217.41 277.46 240.19 104.2 213.77 280.81 535.73 350.87 252.22 383.37 382.71 237.96 250.24 354.12 148.4 162.57 281.24 325.01 25.44 289.02 443.25 148.15 215.09 265.44 362.57 314.37 296.78 276.98 239.6 241.66 191.76 396.5 460.03 300.12 232.21 328.92 489.52 314.53 394.79 462.73 131.95 406.58 203942_s_at MARK2 85.22 114.53 167.52 159.77 134.83 127.02 41.78 76.16 71.4 138.02 147.01 190.05 110.51 76.38 110.27 70.95 153.04 200.73 139.06 71.87 183.1 99.18 178.39 127.66 160.53 116.25 95.3 88.97 100.07 88.21 77.08 182.19 159.31 213.23 102.57 117.26 135.25 140.58 76.89 132.19 137.87 128.26 189.24 201.32 209 109.92 80.97 110.8 118.8 132.41 86.84 97.91 179.66 211.05 227.39 146.19 153.36 113.53 159.15 129.58 179.95 169.48 102.15 117.35 108.94 110.83 86.98 71.78 158.31 135.65 172.73 96.16 103.74 101.13 70.54 71.74 124.06 71.22 86.8 105.75 108.3 116.22 64.58 57.45 71.86 75.93 89.89 77.07 94.16 64.22 152.26 62.78 54.48 53.85 23.12 74.71 87.64 221.48 42.71 78.4 232.7 128.58 134.57 53.44 100.84 180.76 121.19 163 363.64 187.26 201.14 131.57 112.04 141.62 137.32 44.71 125.33 160.06 107.59 295.91 125.98 317.15 174.64 97.47 62.73 51.27 58.49 66.04 57.74 115.76 78.9 88.99 72.64 107.91 85.83 54.03 106.91 59.26 59.52 67.59 27.07 46.97 80.16 109.04 75.24 135.52 74.16 144.02 127.18 79.85 143.08 187.61 95.23 84.53 49.8 129.3 54.43 89.27 93.99 84.93 115.43 93.3 73.21 111.52 96.2 114.28 77.92 91.98 140.2 82.87 121.03 148.49 83.52 130.34 384.27 141.77 98.46 95.3 109.49 95.37 76.72 153.88 79.02 121.96 112.61 112.71 129.61 143.42 178.1 128.75 102.66 63.11 99.21 156.46 99.47 188.59 97.88 120.74 179.84 231.55 129.4 108.51 80.69 121.9 179.25 106.8 86.02 80.05 106.03 89.18 125.7 100.21 193.94 214.73 177.95 79.34 150.18 113.63 92.9 128.66 125.95 162.34 146.77 91.55 131.59 130.72 121.8 204061_at PRKX 456.77 85.24 69.26 68.36 61.6 49.56 73.39 30.01 45.15 271.44 62.83 99.1 89.02 65.21 76.4 95.04 173.1 339.27 264.28 376.95 161.64 99.87 140.88 281.84 118.76 200.09 210.82 217.94 147.08 163.14 41.18 48.55 44.81 48.33 104.31 411.37 238.5 96.33 108.99 107.09 240.97 197.81 223.22 261.45 83.39 85.03 84.22 535.65 259.52 41.33 346.59 63.18 40.39 221.09 22.29 55.89 92.73 218.98 107.75 71.75 50.58 256.17 161.61 50.04 169.2 204.1 153.11 118.88 313.88 98.91 156.87 595.55 174.42 58.02 28.54 106.05 142.11 90.31 23.33 42.74 76.21 47.93 52.58 42.55 106.74 83.17 153.1 88.55 77.9 34.03 32.07 42.46 22.99 24.51 29.29 46.67 21.66 65.74 45.31 157.06 412.07 103.7 32.38 35.24 40.37 115.53 50.23 85.46 30.73 30.98 137.86 85.89 112.32 132.34 138.52 61.47 166.98 76.15 141.61 59.28 54.67 95.02 270.67 275.6 64.48 77.24 50.99 63.85 49.63 136.3 92.15 49.74 45.04 89.92 58.84 18.74 120.22 52.05 56.63 71.47 46.72 51.36 27.3 52.86 66.93 37.61 248.79 100.59 73.2 39.97 875.97 117.74 51.41 102.05 108.27 446.05 26.11 110.06 71.74 150.27 58.61 88.1 30.23 53.39 25.88 71.81 37.81 124.97 65.53 89.13 58.61 44.41 63.94 195.53 79.95 35.67 98.46 38.17 50.47 138.66 96.66 205.68 56.77 52.62 79.17 79.39 66.1 29.88 293.06 200.64 43.76 42.82 45.95 61.03 87.81 58.16 242.81 116.7 87.55 145.62 37.16 50.74 106.6 39.49 89.6 41.7 138.4 157.49 50 82.79 105.65 105.73 78.84 51.83 39.88 39.65 83.6 39.77 43.16 57.03 265.34 133.76 53.79 219.25 107.16 241.29 68.35 204068_at STK3 742.22 550.07 183.21 645.94 397.41 251.13 469.47 348.58 521.84 487.42 283.25 575.38 248.95 337.19 184.71 305.78 315.34 215.63 522.78 679.45 164.05 476.16 391.22 242.58 459.68 308.49 489.99 230.11 446.74 916.64 847.72 187.49 456.2 410.78 151.59 297.51 241.9 296.55 109.31 161.12 445.68 213.6 514.11 246.03 309.6 246.82 127.6 202.23 333.67 295.28 299.02 248.18 237.62 552.67 566.95 304.6 321.59 437.07 269.04 406.27 401.49 96 258.38 290.3 160.18 323.35 244.41 141.52 592.64 302.65 332.29 279.62 291.58 401.23 297.99 329.21 582.26 242.13 628.22 1014.17 372.51 363.39 224.15 284.99 177.68 350.96 275.88 134.61 161.24 306.91 60.02 217.08 957.49 446.73 456.97 446.12 487.67 314.37 696.58 790.81 217.61 276.51 284.63 310.76 392.75 210.51 199.06 411.13 311.65 45.8 175.98 199.33 189.19 263.82 291.89 162.53 102.23 97.7 239.77 162.4 179.67 141.18 102.58 163.31 167.4 354.29 288.8 219.4 636.67 210.14 157.73 486.85 157.96 177.07 235.59 222 351.05 176.12 457.56 172.52 233.66 542.95 368.69 509.18 131.54 146.45 141.56 168.94 319.58 264.77 246.07 207.69 538.12 217.18 163.23 444.07 676.2 273.27 244.61 189.88 216.97 257.68 175.97 305.57 178.38 324.19 237.48 161.51 212.38 361.13 102.8 135.02 254.13 351.4 83.04 432.64 279.71 81.19 131.37 283.08 124.11 169.94 225.51 197.59 322.07 272.61 140.41 161.93 208.6 345.49 211.05 233.17 181.92 142.39 173.79 208.03 520.51 167.75 237.92 140.53 330 336.57 221.19 86.25 132.2 209.44 241.32 288.71 271.63 298.32 263.25 136.9 286.31 217.01 195.45 201.67 231.87 267.89 139.4 171.78 252.81 248.01 243.34 749.48 369.89 255.1 241.08 204090_at STK19 266.88 534.56 394.5 190.85 332.12 280.43 328.99 274.08 294.53 373.49 360.74 336.59 336.81 308.55 303.9 380.98 343.26 275.47 255.45 470.72 301.08 430.27 324.38 334.93 260.12 281.52 358.79 245.2 277.55 304.62 309.56 431.42 284.68 355.12 341.75 331.93 265.68 249.1 293.05 346.06 289.68 266.01 249.46 376.57 310.66 243.35 353.46 320.63 401.39 310.84 248.58 368.53 303.98 319.6 239.19 299.92 204.5 222.5 1383.02 368.31 613.36 277.89 227.09 175.13 179.22 259.57 266.28 178.81 143.63 196.82 149.64 305.83 225.38 220.66 426.03 440.01 389.86 205.24 242.22 241.08 184.09 243.98 229.28 277.36 219.09 219.2 327.7 236.29 174.31 109.27 265.17 221.81 183.07 188.37 213.63 224.13 380.19 305.14 202.76 218.4 239.35 147.11 253.37 239.86 339.91 541.71 313.5 354.41 330.34 697.8 223.1 206 197.57 202.64 186.63 136.16 257.33 471.44 249.36 138.68 136.68 266.69 213.29 191.28 190.92 222.32 305.86 231.39 259.72 217.96 263.62 285.22 139.88 228.64 249.86 158.98 214 205.86 300.69 217.14 237.44 229.41 282.98 268.15 167.09 249.48 158.39 219.82 288.5 437.02 265.31 328.04 242.46 266.78 404.3 294.49 310.16 222.72 323.03 267.73 276.69 226.69 234.49 268.76 284.58 254.59 345.94 220.12 365.92 312.65 219.9 290.23 327.61 193.54 219.68 235.6 283.69 300.33 228.16 337.36 245.96 317.2 203.61 199.03 255.03 265.07 317.18 232.93 282.57 267.89 423.14 229.93 300.45 269.79 255.15 265.38 305 395.02 359.77 169.13 209.61 434.64 319.8 248 264.16 250.04 162.28 244.05 277.13 376.32 245.74 339.92 236.45 111.51 258.13 229.4 289.25 643.41 258.87 304.83 216.38 332.73 257.61 283.41 256.05 241.69 232.5 204106_at TESK1 381.41 184.85 360.22 282.86 320.17 266.82 258.58 283.86 220.09 370.72 281.73 313.35 231.44 360.92 253.3 203.71 209.62 285.81 265.33 183.79 256.15 256.93 329.93 233.7 650.89 203.98 477.36 195.92 179.17 295.13 192.89 180.89 240.05 397.59 154.93 374.88 181.07 148.42 215.02 245.99 182.53 366.31 309.31 255.94 258.85 209.72 138.81 207.13 295.75 222.39 172.72 278.89 264.99 286.94 287.7 176.16 188.36 196.27 244.09 252.03 191.31 203.97 443.9 220.54 212.77 237.46 222.47 132.09 197.08 238.66 252.25 276.29 174.91 183.78 268.46 242.81 136.09 193.22 172.08 377 341.23 188.95 243.42 226.35 187.46 196.98 226.24 241.44 223.21 303.91 481.43 279.62 238.49 252.21 274.26 267 192.22 365.75 333.11 304.12 321.14 259.16 341.99 414.28 271.78 261.14 235.93 316.51 422.63 536.87 200.97 176.2 156.97 194.65 243.46 128.81 229.56 150.22 248.45 243.49 224.05 275.99 342.83 173.15 188.44 210.36 274.74 185.07 253.93 166.9 215.85 285 251.05 182.31 222.82 179.69 174.25 129.62 214.02 272.94 142.57 206.31 357.54 187.26 168.8 307.02 158.39 182.3 259.31 191.4 212.92 224.41 190.1 237.42 224.78 340.94 176.97 259.21 171.91 177.42 245.11 217.35 177.86 151.08 149.32 344.42 188.01 224.05 341.15 176.34 229.13 293.16 196.18 232.81 434.31 295 159.65 101.41 204.7 301.23 147.94 219.32 127.05 230.88 267.92 198.57 206.75 218.96 222.4 249.13 201.34 147.86 201.55 132.6 162.99 221.22 145.5 244.68 163.63 204.2 262.6 177.54 169.98 391.27 155.32 172.59 165.96 190.69 210.13 191.23 211.96 207.13 250.5 354.41 219.5 174.69 250.69 216.97 235.9 340.13 348.01 262.31 269.5 240.86 311.25 233.7 205.42 204247_s_at CDK5 255.5 96.09 89 221.18 142.02 161.67 114.27 317.2 346.42 116.16 127.97 93.7 126.77 115.63 67.1 102.31 133.44 104.27 134.49 120.44 102.35 95.07 130.37 88.78 108.17 76.04 123.62 71.27 113.12 127.95 85.43 100.94 143.77 166.69 78.41 159.62 94.65 148.92 92.54 145 376.02 139.92 122.08 58.1 255.25 54.78 114.5 65.76 189.69 120.87 94.57 119.28 128.18 114.06 785.21 135.21 49.24 146.06 135.56 83.77 132.71 63.21 189.15 117.3 57.81 133.45 106.58 123.29 167.22 69.61 179.65 94.76 96.51 133.81 158.31 91.35 251.19 89.68 104.04 218.52 116.26 84.22 95.06 116.56 90.59 112.53 80.84 81.63 160.4 65.18 49.67 66.41 99.74 37.75 57.67 95.69 153.72 110.13 109.82 71.26 50.32 111.2 110.79 40.7 89.04 205.15 85.65 100.53 200.52 167.68 182.68 55.66 80.71 74.94 104.11 25.56 95.34 33.57 105.73 121.84 79.83 132.37 157.01 167.23 49.89 238.57 69.61 52.35 86.21 89.12 49.91 297.03 67.44 68.35 84.55 105.16 127.98 52.89 100.98 85.8 70.55 78.21 161.85 135.53 97.06 119.91 70.68 207.72 55.76 292.29 251.28 85.52 198.24 85.5 115.03 196.85 184.28 128.96 87.86 88.16 141.2 89.1 87.87 38.97 148.64 185.73 96.86 92.09 60.49 104.55 173.23 109.38 66.3 108.72 96.23 234.64 69.96 80.98 57.68 192.17 53.7 232.77 93.38 109.79 93.25 86.43 106.75 95.29 284.56 160.67 49.75 70.88 117.39 104.67 74.22 133.65 144.12 94.08 98.02 95.18 195.75 157.8 103.66 134.59 135 61.58 47.46 124 60.21 97.88 63.53 72.41 84.8 72.27 85.83 134.56 118.08 141.86 93.13 72.69 185.62 110.09 85.4 117.81 105.72 146.76 115.42 204252_at CDK2 176.58 259.47 169.54 167.57 181.79 86.89 203.48 178.05 188.49 205.2 307.91 144.09 125.04 280.76 112.06 177.76 336.76 231.26 204.92 437.28 235.36 194.11 230.93 156.36 168.91 149.56 174.66 306.53 158.62 229.83 676.91 247.12 263.84 301.7 314.33 215.29 337.3 189.84 128.98 239.97 270.85 217.33 161.24 234.35 118.48 105.27 275.31 192.98 276.72 263.25 221.32 139.78 143.64 163.81 179.68 187.11 237.37 201.64 255.78 151.65 260.05 756.09 376.94 234.31 182.58 245.42 159.25 160.22 119.8 143.43 110.74 152.87 152.36 124.81 146.92 121.43 186.08 85.86 76.5 113.76 140.37 120.87 126.63 96.89 51.53 109.54 71.08 111.29 119.47 104.13 82.48 94.53 95.78 164.3 102.35 91.06 112.37 126.43 145.31 192.02 171.56 121.05 108.61 98.69 169.02 139.03 70.44 57.61 108.04 52.89 139.6 86.33 80.08 191.23 76.07 272.11 81.51 110.98 98.99 109.98 87.63 132.81 108.77 151.16 41.21 89.47 56.74 54.67 38.37 58.95 35.79 56.92 54.83 80.95 64.4 66.05 74.14 73.83 61.86 68.7 65.62 87.88 142.29 53.64 51.08 54.39 65.4 115.7 102.76 120.35 170.81 59.32 101.89 88.66 62.28 145.84 66.79 83.82 86.03 144.21 121.02 69.97 84.79 67.67 91.6 46.1 128.89 74.32 92.81 115.64 41.28 62.16 88.09 98.18 31.67 47.8 55.76 46.62 37.91 63.88 41.87 62.89 53.31 45.04 52.88 35.87 51.4 35.76 45.65 54.46 39.61 38.5 43.53 73.12 45.16 51.36 94.34 54.94 65.32 61.98 49.06 44.83 58.14 128.81 73.56 48.62 82.8 58.85 54.76 62.55 38.73 76.1 69.24 55.52 67.01 74.6 52.7 72.37 112.04 52.15 111.34 114.11 53.2 155.85 181.03 188.61 59.57 204269_at PIM2 146.23 84.69 436.69 255.79 127.17 169.12 111.63 106.81 74.66 436.86 78.14 35.9 117.31 61.8 102.25 88.1 218.39 379.66 523.8 172.39 94.85 291.56 115.14 295.87 132.83 314.73 162.64 248 302.59 143.29 90.23 112.14 97.1 194.38 175.89 182.88 500.44 120.87 652.27 475.18 230.71 418.09 295.88 190.26 227.8 115.94 106.74 240.24 179.11 84.07 75.36 131.11 134.29 368.13 112.89 76.51 301.22 189.97 269.62 228.09 64.55 147.37 56.44 197.85 312.22 187.3 169.17 196.52 171.16 103.26 123.58 202.26 47.56 176.71 79.98 44.48 52.11 112.39 66.71 64.08 60.04 84.71 80.73 177.35 86.67 79.81 100.04 158.56 353.84 140.36 82.72 130.49 100.08 82.63 104.7 181.05 59.9 66.59 113.16 165.77 56.89 93.21 99.65 79.89 67.81 158.62 68.91 78.47 83.49 62.56 166.13 64.85 185.76 181.37 214 121.91 44.85 132.69 154.68 83.01 113.31 62.11 291.33 163.31 64.8 99 86.41 55.31 80.41 106.37 140.15 52.04 93.14 140.6 57.96 59.7 71.75 60.52 79.32 53.01 62.34 64.24 73.05 79.14 77.23 87.81 45.99 93.52 147.07 95.15 118.34 182.2 67.99 120.82 160.45 95.11 118.18 297.79 72.16 161.48 76.85 86.38 50.29 55.99 79.9 111.39 66.05 214.12 59.86 80.33 182.89 84.53 71.79 134.45 77.17 96.74 198.04 69.19 65.24 76.7 99.1 91.91 64.45 67.7 122.89 71.03 78.93 71.81 81.1 59.5 55.03 81.87 61.26 202.5 96.28 189.3 70.31 96.19 171.5 80.19 163.76 78.06 117.79 83.82 305.61 62.84 79.35 139.51 222.96 94.89 78.89 109.04 145.02 96.25 64.85 87.57 167.1 206.3 76 91.91 256.89 127.02 70.57 46.67 50.3 93.76 161.26 204310_s_at NPR2 145.41 87.69 192.95 75.97 105.63 64.74 63.19 51.17 50.18 67.1 57.21 69.65 75.3 97.68 98.51 194.13 41.49 47.37 119.19 29.9 68.95 35.88 88.56 67.38 43.38 82.24 66.95 60.35 165.14 44.25 55.69 45.55 75.11 75.1 50.79 150.05 67.33 72.92 91.96 64.16 43.77 53.79 66.27 84.98 59.99 96.07 61.12 80.86 74.58 89.07 157.5 81.12 85.39 220.39 49.82 83.69 66.86 46.05 67.69 138.03 63.8 65.99 143.75 50.18 61.59 56.35 64.98 53.64 42.38 851.77 72.67 71.46 54.04 62.89 67.55 63.9 29.78 75.37 39.14 69.14 101.53 61.05 40.8 57.45 104.54 67.95 68.02 92.74 38.63 77.4 112.37 105.98 53.59 58.12 75.99 42.38 61.53 53.64 60.67 74.38 130.59 100.69 72.28 61.29 55.95 88.11 149.98 98.73 116.99 121.96 63.26 108.31 94.41 101.47 69.81 91.45 54.72 49.63 96.88 95.45 124.86 193.74 174.98 96.06 168.23 58.69 90.52 124.91 49.72 41.14 120.42 74.82 190.31 69.47 60.41 68.92 45.15 55.37 68.41 67.14 47.06 43.56 51.34 63.74 62.1 56.74 49.4 77.47 109.56 63.4 211.07 121.42 82.61 82.3 67.98 70.21 50.33 60.81 120.93 61.85 102.98 115.06 66.43 64.67 60.17 95.54 94.51 90.68 69.23 55.53 66.33 98.16 69.77 62.89 108.11 55.01 77.17 65.71 160.82 85.98 88.79 60.79 43.48 83.51 93.39 75.93 55.88 67.15 66.21 55.28 75.94 125.77 125.29 73.94 165.17 114.93 250.53 110.54 94.41 101.62 82.3 85.77 92.73 83.34 66.13 126.09 77.69 52.76 89.87 50.77 123.31 82.76 183.29 112.85 77.46 63.63 72.21 51.83 69.58 76.97 120.49 110.09 140.08 479.68 119.31 35.45 131.31 204357_s_at LIMK1 106.3 73.74 133.93 103.13 81.24 69.79 87.53 70.3 77.55 121.15 86.15 80.88 73.28 59.54 96.11 68.65 109.18 109.3 104.52 45.17 77.09 76.32 89.63 95.37 112.46 131.26 154.73 85.01 80.81 121.78 60.24 86.92 75.95 85.01 110.96 103.18 91.64 130.77 81.2 104.47 88.92 120.26 116.17 180.21 120.2 74.76 53.79 86.06 99.46 79.81 78.05 100.25 100.01 187 123.06 78.4 99.27 109.74 122.72 189.58 114.56 65.2 117.66 61.68 86.04 85.76 66.17 63.65 73.13 58.02 88.08 111.8 70.06 102.52 59.86 81.69 46.1 56.67 39.87 81.16 89.61 62.03 44.66 48.98 48.08 59.16 91.29 77.15 70.62 48.24 84.44 61.46 53.9 65.3 68.05 88.34 66.11 67.54 51.62 69.31 72.04 107.91 84.49 67.53 56.02 80.7 99.05 81.53 109.77 131.26 88.02 54.45 88.86 90.84 112.63 83.8 57.76 82.7 88.97 220.49 136.01 122.26 116.34 76.62 60.44 78.96 69.54 71.18 48.76 70.55 97.18 81.14 79.76 92.59 70.16 62.39 74.56 63.93 67.39 63.41 55.58 66.37 51.48 60.43 70.78 69.22 57.99 73.92 88.48 72.63 93.27 93.72 167.34 80.57 87.71 97.59 77.05 168.34 91.4 81.67 98.62 101.35 59.4 58.7 79.21 89.41 75.45 94.68 117.02 75.85 97.78 100.21 88.05 90.62 141.42 114.94 119.19 100.84 102.7 102.23 93.62 91.2 62.55 107.81 106.13 85.56 99.57 74.51 75.13 97.25 97.24 84.75 89.15 78.96 55.66 140.59 56.06 94.67 124.6 97.86 96.13 83.53 85.52 82.3 107.57 66.73 76.41 84.19 80.2 67.71 90.51 75.35 101.92 139.48 92.3 57.29 69.61 117.17 65.97 85.52 96.06 75.66 66.11 56.52 78.21 61.52 59.75 204379_s_at FGFR3 53.32 547.29 29.26 447.72 175.73 463.04 1251.11 338.87 74.19 300.24 716.92 178.02 133.91 1569.78 231.54 553.94 594.76 34.58 14.32 69.69 1364.53 128.75 222.58 28.03 14.63 43.35 292.19 2181.41 104.18 126.78 156.92 125.52 910.99 2336.01 93.19 29.87 15.93 38.52 30.29 198.7 38.38 269.19 28.78 269.22 15.49 87.4 136.82 337.34 146.69 61.54 22.26 161.22 302.95 80.49 584.79 441.83 140.01 12.46 154.66 25.75 86.04 501.35 89.68 149.95 20.8 29.34 261.84 15.24 22.29 383.97 55.82 50.07 471.67 14.15 19.07 53.29 10.79 87.25 448.44 24.52 235.47 170.52 387.71 884.39 140.03 787.24 120.2 59.39 454.61 304.51 378 54.88 290.68 408.82 111.3 53.26 873.51 842.36 546.77 649.84 50.76 24.6 105.66 492.23 131.94 193.39 170.22 319.74 344.01 317.18 20.6 27.99 541.99 101.49 443.01 342.01 31.03 468.27 363.31 25.68 110.96 255.14 16.34 100.75 228.06 95.79 94.92 226.56 43.12 15.42 22.03 58.85 86.98 116.57 47.71 283.71 30.33 596.14 233.63 286.7 216.21 40.23 247.43 257.11 54.83 171.31 882.57 675.72 188.52 42.89 20.78 362.73 30.73 20.74 1249.71 514.34 276.28 19.48 28.47 219.88 310.87 18.62 23.73 204.27 99.67 86.32 267.49 154.43 274.47 572.6 676.62 525.39 223.16 348.71 590.5 138.68 269.5 303.66 130.92 817.09 86.87 20.45 648.8 257.39 108.51 231.82 239.13 3872.69 16.47 21.95 194.99 28.11 233.84 107.94 39.76 562.78 106.68 41.12 30.56 63.33 3778.78 107.6 535.83 167.12 51.18 204.87 65.45 45.75 63.34 25.68 133.47 57.54 352.16 140 856.53 893.61 136.37 100.42 443.86 565.89 85.68 43.79 187.62 711.76 395.01 138.08 37.61 204396_s_at GRK5 106.94 136.44 172.9 106.81 132.84 142.55 73.35 97.98 96.27 130.99 83.12 53.56 116.68 68.66 134.6 387.58 77.22 111.82 135.26 87.72 58.45 116.06 43.21 93.74 137.68 183.43 80.19 52.86 86.03 64.8 63.9 186.33 38.59 83.13 65.53 45.96 84.39 76.63 266.18 79.61 108.15 99.16 47.57 69.01 197.97 200.16 38.19 69.14 114.74 75.6 51.32 103.45 89.16 144.39 51.43 135.4 75.14 48.21 115.04 43.85 134.18 60.61 125.11 42.34 767.68 76.54 100.58 139.53 66 194.89 101.69 96.83 63.29 90.04 63.58 33.25 79.26 113.14 74.87 45.3 40.28 130.05 67.41 142.26 183.33 133.14 81.81 177.8 122.36 346.42 117.94 183.36 123.15 439.84 336.84 106 99.88 130.19 161.58 199.35 97.28 135.42 117.93 304.72 99.92 101.98 78.83 125.21 74.02 51.71 250.83 143.37 108.36 64.37 163.14 101.47 95.15 203.06 258.53 102.47 194.01 68.34 132.8 137.36 331.04 73.44 57.29 197.95 58.47 39.42 146.28 83.33 340.98 60.64 88.33 58.49 137.63 102.32 68.18 50.84 61.18 47.21 51.98 75.9 121.45 74.35 58.86 116.14 213.43 85.73 36.46 154.95 242.58 99.1 68.81 113.43 33.5 104.83 100.02 144.42 77.28 119.56 189.02 62.86 90.07 137.95 98.23 152.08 28.19 130.73 73.47 66.67 86.27 88.52 31.1 69.52 117.5 86.11 113.34 55.36 113.26 74.18 55.16 90.86 149.61 88.37 65.42 101.15 66.54 171.1 71.52 94.9 90.69 67.96 103.14 73.04 180.45 135.87 106.59 191.58 73.53 113.86 112.22 59.68 70.93 58.51 63.02 55.92 83.94 38.3 92.84 146.87 136.69 82.54 59.87 99.69 85.27 59.11 63.04 108.36 181.34 202.88 102.19 124.64 99.72 72.62 161.88 204468_s_at TIE1 45.39 70.99 156.42 120.24 150.9 183.34 62.7 62.7 93.25 68.1 127.85 68.98 120.94 110.27 191.67 961.38 125.09 52.93 128.52 80.99 113.83 78.51 59.21 71.49 73.48 62.03 116.1 61.07 51.39 52.13 57.43 48.17 58.39 132.99 63.68 51.43 70.31 73.14 409.29 58.17 45.37 73.19 36.18 68.1 166.64 274.26 55.41 45.26 132.18 96.16 39.56 77.52 128.46 140.26 34.49 131.28 68.89 55.36 111.55 76.06 81.79 36.93 142.09 56.02 53.24 61.22 27.28 62.49 25.37 75.57 57 67.39 34.32 61.13 31.73 35.67 23.95 75.58 14.88 30.67 58.16 85.53 24.69 43.63 159.87 113.41 90.54 116.05 50.14 44.64 156.12 86.08 45.7 42.53 38.16 22.55 74.9 72.35 39.48 47.97 95.69 102.82 54.71 140.24 64.91 153.21 122.48 165.85 68.71 43.47 61.15 104.53 55.48 63.67 54 73.32 85.3 92.55 94.84 135.18 255.81 100.68 68.01 62.16 246.64 87.29 78.67 201.85 35.04 31.78 78.18 57.4 318.43 113.39 60.15 43.03 99.29 74.12 78.83 32.27 46.45 39.42 37.5 65.1 92.55 97.64 44.6 189.18 362.61 98.06 36.34 238.61 67.05 102.94 44.69 113.76 37.05 77.39 84.11 87.35 80.83 143.33 35.11 60.86 115.51 128.07 96.58 157.62 67.04 55.38 70.14 104.74 94.97 96.69 37.02 117.61 78.17 53.77 143.8 100.49 93.62 112.68 65.53 126.76 91.43 136.17 108.81 157.02 54.53 116.61 83.35 96.85 119.99 119.31 141.96 79.05 53.68 220.52 131.58 175.53 120.14 80.17 82.58 70.67 80.42 52.23 25.83 65.58 46.36 45.69 129.53 165.68 218.72 116.31 101.24 67.65 54.23 55.81 96.76 181.22 113.48 122 180.52 113.51 124.82 72.25 180.58 204510_at CDC7 138.08 169.9 52.14 122.66 126.74 28.16 224.12 344.21 131.42 131.95 103.47 95.26 65.06 210.18 42.53 164.59 199.15 248.48 335.26 730.93 107.72 238.02 184.43 130.16 138.12 101.81 32.21 391.9 307.36 99.82 87.62 97.26 117.66 357.94 64.11 257.39 95.99 126.12 104.23 49.23 95.23 132.47 180.12 190.92 123.66 50.04 244.95 168.82 83.16 54.06 272.36 76.44 56.99 67.48 29.83 88.35 60.42 76.55 138.39 67.73 86.04 388.85 333.1 56.96 102.46 86.49 319.37 172.83 220.09 117.44 155.73 65.66 106.51 64.96 170.24 84 94.68 56.21 32.95 66.58 70.37 41.92 112.72 34.8 22.01 29.3 51.02 62.35 63.19 100.5 19.04 66.08 67.53 64.93 85.17 68.65 41.08 181.44 135.47 315.48 73.46 47.91 185.75 28.1 107.42 132.82 20.49 45.14 110.67 12.11 191.49 31.31 56.25 154.82 100.66 373.35 221.41 39.67 86.2 226.17 53.46 108.94 82.14 83.94 27.35 113.08 46.3 52.42 34.59 205.43 68.99 27.02 25.53 62.16 113.62 152.72 327.95 61.92 124.89 84.25 38.11 78.51 256.74 42.05 51.7 80.6 200.22 75.76 56.03 136.67 180.89 47.88 55.56 108.55 83.84 181.29 56.55 114.34 35.62 165.11 77.57 30.59 65.83 27.75 40.73 72.43 60.29 90.82 35.43 87.1 69.61 27.69 169.81 139.91 41.85 26.29 102.54 27.16 30.25 271.38 59.49 115.82 71.26 36.18 81.04 31.89 67 68.06 130.56 100.93 76.98 61.67 69.86 81.93 73.87 63.07 217 50.98 84.6 250.63 48.29 143.67 64.66 143.08 344.71 19.17 285.79 189.42 31.28 75.11 170.08 83.85 41.11 74.22 205.27 94.21 30.44 87.95 111.35 61.42 99.86 307.9 33.09 109.61 234.18 357.98 52.29 204549_at IKBKE 106.06 67.71 84.24 96.84 89.6 77.29 73.57 70.65 85.65 109.42 84.55 51.12 81.95 73.53 76.8 141.97 120.49 149.68 104.66 66.54 74.06 96.44 84.11 141.11 72.34 192.46 81.96 82.2 82.09 72.45 51.87 97.21 104.64 81.04 109.5 113.33 161.9 74.02 87.45 149.6 338.76 311.8 162.21 69.8 170.14 69.19 185.44 117.12 103.9 97.28 68.27 69.53 92.18 89.36 89.98 75.34 295.2 149.38 124.4 92.53 134.04 64.35 83 60.81 155.78 117.3 82.26 193.5 128.47 85.41 86.66 150.76 102.06 77.29 99.82 71.68 77.51 110.35 59.64 87.73 108.5 90.84 70.13 82.3 58.14 86.7 89.15 110.4 94.16 84.11 101.15 83.67 84.18 59.77 59.36 96.57 70.33 56.76 89.65 95.61 55.5 78.64 115.4 63.19 57.11 83.92 83.11 86.46 100.01 111.89 139.43 55.37 105.66 88.99 137.02 103.78 74.31 139.59 107.16 98.45 106.96 106.3 83.85 161.36 69.91 91.86 79.08 75.41 90.11 126.18 110.45 91.5 79.22 84.07 51.24 68.18 81.36 79.49 58.5 80.7 91.6 76.77 67.3 80.74 92.17 195.56 65.04 87.05 106.1 89.92 120.13 164.4 144.81 93.21 83.95 102.99 76.52 150.33 86.76 122.57 102.36 92.77 126.69 97.11 111.59 124.25 80.19 143.13 114.39 212.76 108.76 79.52 77.63 116.98 126.03 73.83 106.44 107.77 65.98 118.24 96.12 132.23 89.93 96.44 116.58 102.99 85.28 80.2 120.7 87.4 121.15 74.23 99.79 86.18 84.76 84.58 84.93 130.89 99.33 93.07 158.46 103.86 112.22 105.98 154.91 81.61 89.72 97.85 96.41 127.05 99.14 128.78 106.66 87.39 88.59 86.83 111.48 96.21 94.22 65.95 141.4 133.12 60.44 102.89 59.31 149.96 68.33 204569_at ICK 49.57 66.73 49.99 56.71 99.55 47.72 56 52.98 49.32 46.32 84.88 40.52 40.36 53.86 39.08 37.8 49.95 30.17 54.52 203.51 41.48 57.86 51.74 35.99 69.14 53 56.39 69.97 44.75 39.32 408.81 37.04 52.16 52.65 41.98 38.95 40.76 83.5 37.38 39.17 51.7 37.19 47.95 75.24 33.81 44.89 36.51 31.09 39.69 34.35 70.42 42.8 35.82 61.68 74.04 58.6 33.75 46.42 33.78 32.69 20.96 51.71 44.53 32.72 28.37 29.21 24.3 29.65 50.35 54.61 46.12 46.92 65.73 33.83 52.85 21.57 73.57 38.84 45.16 25.38 16.51 59.17 58.9 50.91 86.41 55.64 56.42 55.16 36.29 64.18 9.44 61.87 55.62 74.8 65.25 109.8 48.84 39.69 72.05 59.56 68.71 47.82 31.97 55 37.77 18.79 26.01 20.08 33.87 6.48 28.27 35.65 105.97 36.02 77.27 25.42 75.59 17.23 23.22 38.14 27.4 19.66 17.63 22.28 35.93 44.76 33.55 35.07 56.93 33.32 30.36 31.5 21.32 28.32 29.52 34.41 37.25 42.46 33.3 29.88 59.95 87.96 37.57 36.97 38.13 17.97 45.75 34.68 32.29 48.89 37.92 25.54 21.91 24.52 38 39.85 34.86 14.88 36.66 26.36 34.66 43.84 45.07 31.67 41.5 29.73 42.1 29.83 22.19 33.66 19.3 23.44 32.14 53 14.56 25.35 36.43 60.31 43.53 49.33 39.8 26.26 54.23 41.35 33.2 48.88 33.68 71.9 35.61 37.07 43.75 52.75 35.15 63.78 46.98 34.18 81.6 39.42 41.87 40.68 59.49 40.55 42.93 25.35 53.13 52.48 31.62 48.22 46.37 66.88 69.07 33.84 28 19.96 53.78 91.46 46.25 47.71 49.58 44.19 50.78 87.14 32.43 53.43 58.5 23.95 40.17 204579_at FGFR4 37.26 32.2 43.53 63.86 529.11 137.67 242.9 234.23 23.94 270.52 58.48 728.95 45.31 28.61 648.81 45.83 38.9 44.95 29.02 233.84 92.27 145.38 28.21 54.11 33.07 24.37 59.43 13.09 25.43 25.03 16.22 24.34 39.41 36.7 26.77 54.09 39.15 19.84 18.03 22.34 29.72 38 34.84 126.99 18.82 72.28 36.36 13.96 33.86 19.74 26.49 204.56 39.79 37.3 36.62 25.36 53.38 36.06 462.45 26.83 24.42 61.1 26.89 155.81 17.61 21.74 29.38 23.65 32.85 21.6 35.3 22.5 22.8 52.63 28.5 38.35 20.03 10.76 18.19 11.66 39.64 23.78 35.6 16.92 158.78 13.96 133.42 24.51 15.22 24.64 74.51 13.91 21.86 21.64 30.84 71.3 476.74 64.37 26.45 22.58 22.35 82.53 254.53 48.5 108.81 35.02 17.13 33.3 21.37 33.75 20.82 20.76 19.53 18.3 32.61 18.36 162.17 17.9 181.33 124.6 38.78 23.77 21.89 26.47 13.28 64.38 38.78 74.55 47.01 25.86 39.23 61.02 144.1 66.53 62.69 89.65 34.03 32.62 211.56 201.3 14.95 196.73 48.47 41.24 34.05 44.11 174.07 14.55 68.24 15.12 35.56 23.48 24.93 157.39 134.51 30.39 22.73 13.27 23.59 19.78 30.98 30.41 16.35 20.93 18.7 91.19 23.71 199.46 32.47 27.03 48.94 42.98 21.27 30.89 188.6 93.16 19.11 14.14 22.88 27.75 16.49 25.65 26.59 32.76 23.68 56.85 38.14 15.84 23.51 28.33 22.04 18.73 26.63 254.47 26.35 162.46 25.79 86.48 279.42 16.92 489.65 67.6 91.22 152.93 56.18 18.86 20.65 31.24 26.47 40.54 20.5 29.17 114.91 356.13 31.95 18.47 42.35 29.47 44.31 28.59 20.29 39.58 284.57 38.94 30.36 41.46 50.03 204589_at NUAK1 89.88 497.77 685.11 486.4 249.5 318.66 785.63 360.05 266.17 324.82 391.74 466.87 427.34 301.39 326.23 753.8 186.92 78.5 200.77 93.22 414.24 166.76 90.66 160.91 176.29 213.09 395.81 356.92 122.85 341.25 328.75 160.5 254.18 332.24 263.81 74.51 219.49 236.66 686.91 315.66 96.62 217.12 309.38 322.57 265.03 380.78 770.22 92.49 289.19 304.66 133.43 610.83 424.93 346.87 510.32 265.25 122.78 194.78 217.28 172.76 417.04 316.88 297.65 298.66 144.11 370.81 189.31 217.43 243.38 342.48 559.05 242.23 412.08 482.67 231.43 436.5 163.8 254.56 275.28 910.08 544.85 344.82 138.77 290.73 325.25 366.96 480.66 413.07 140.48 440.5 299.7 409.46 436.75 588.19 604.47 336.6 534.29 658.37 400.07 459.39 201.43 263.27 403.86 442.53 621.32 258.71 421.3 307.99 512.36 130.17 284.09 230.28 404.09 242.64 217.31 249.21 199.99 353.9 151.25 520.37 717.19 151.39 248.59 172.46 441.16 115.82 276.38 617.68 354.05 260.76 360.06 358.63 713.77 226.83 383.82 715.17 183.55 357.58 361.92 338.62 221.96 359.73 125.23 354.9 228.29 173.06 161.56 390.1 376.66 195.43 226.03 228.28 369.49 364.18 180.33 147.24 74.54 186.16 506.5 121.51 265.95 176.36 215.65 190.09 245.77 277.51 269.97 237.53 322.64 153.39 231.22 331.48 452.71 59.65 269.73 436.94 286.76 280.01 283.47 157.72 316.99 191.05 177.8 220.01 250.24 262.72 217.27 200.95 83.94 227.67 548.17 624.01 869.28 270.04 184.04 411.14 127.41 415.72 252.67 151.55 196.87 321.47 184.04 123.4 81.87 321.15 148.98 95.43 505.97 348.24 169.7 318.79 304.86 296.42 328.12 236.24 390.96 164.07 350.18 335.38 127.32 528.78 224.57 277.25 478.76 119.49 241.72 204600_at EPHB3 556.2 157.91 515.06 266.69 270.19 249.16 354.71 364.71 224.21 256 763.63 234.1 253.97 356.68 384.56 1062.3 175.53 281.26 199.48 220.42 212.47 296.22 419.65 437.14 324.63 234.54 250.57 148.69 587.38 1403.37 268.36 231.55 152.11 189.92 424.8 600.42 862.15 1195.02 222.06 305.66 238.83 216.94 440.77 310.16 285.73 292.16 316.05 891.3 264.19 479.93 1423.28 331.4 270.26 676.32 333.39 195.72 247.08 306.24 284.78 915.25 514.4 544.41 442.31 196.78 163.83 345.07 135.41 204.26 368.25 747.79 352.45 349.82 886.03 150.82 250.25 388.41 181.25 156.06 181.05 142.08 159.97 115 233.17 110.41 251.03 177.19 316.61 245.61 172.65 202.79 262.95 295.24 259.7 245.9 417.99 162.03 302.5 483.01 210.64 352.87 1118.53 211.22 287.59 291.26 221.79 274.61 232.55 210.42 291.9 179.47 159.89 421.61 310.56 387.09 475.72 331.1 210.87 366.48 406.76 168.62 265.28 456.32 265.97 272.63 237.78 199.06 147.07 223.62 170.53 443.48 207.22 170.88 395.79 193.2 213.68 143.92 350.33 195.96 202.57 252.81 158.82 110.72 118.38 152.75 138.37 168.7 257.22 296.56 218.13 416.31 891.1 237.59 242.58 343.05 157.95 592.99 172.08 109.43 274.73 292.44 222.86 676.7 336.77 232.22 679.14 191.27 218.55 361.23 266.44 244.8 171.88 210.64 132.23 259.6 299.1 109.66 183.88 163.45 265.67 279.21 166.35 424.4 158.29 243.86 659.46 147.72 199.69 211.98 469.51 161.51 185.62 197 263.04 228.95 842.26 216.62 993.15 328.68 266.15 647.8 210.5 257.84 168.53 283.97 517.33 278.45 716.69 473.91 185.77 468.76 311.36 256.91 215.64 185.63 431 245.42 183.51 148.61 227.37 218.13 487.85 324.85 634.88 581.39 650.42 441.05 176.35 204604_at PFTK1 83.01 341.3 311 267.58 100.31 218.61 163.37 583.04 102.3 226.72 443 324.13 143.96 143.33 130.64 378.35 67.9 100.06 181.5 244.74 254.14 151.08 104.59 84.84 79.96 174.28 157.5 212.42 189.08 96.01 188.16 251.16 175.61 46.11 178.54 62.17 84.25 290.53 165.43 137.95 390.96 111.4 101.32 215.06 150.9 203.67 230.85 133.7 124.94 143.92 368.37 133.7 181.28 172.17 291.37 93.25 183.2 199.96 76.54 160.67 282.03 61.47 317.5 80.56 178.54 176.88 140.46 95.91 114.64 144.31 459.4 128.11 112.5 103.24 99.99 247.2 79.83 157.39 238.37 254.85 78.57 141.82 140.59 149.57 212.08 193.66 196.55 159.55 260.19 182.17 49.75 164.84 76.66 230.55 256.89 138.67 127.13 100.25 95.76 96.87 131.37 149.9 220.07 146.13 123.24 174.98 222.57 162.09 180.39 10.36 302.84 166 190.89 167.76 162.65 159.92 120.69 69.34 102.1 181.06 222.3 260.88 120.02 146.19 157.32 152.5 162.33 178.11 189.59 198.61 135.25 122.03 136.98 102.88 220.24 190.6 184.86 77.19 203.09 248.31 156.2 104.11 229.12 139.5 79.51 70.37 87.25 150.05 119.85 244.63 178.2 145.07 421.53 242.41 90.48 173.87 366.38 173.51 208.38 570.45 126.64 112.62 188.74 87.7 226.58 148.97 107.77 159.21 44.12 64.4 126.44 105.2 108.32 68.27 49.86 186.18 109.97 193.03 223.67 261.48 142.7 56.96 113.16 140.64 117.97 110.45 114.04 95.29 28.58 106.94 253.71 216.42 247.27 189.18 126.15 153.44 208.43 134.02 112.56 145.45 56.45 132.54 168.79 41.65 148.89 181.76 152.49 50.47 159.14 111.14 96.88 166.95 148.91 125.84 182.3 134.84 94.11 225.47 150.87 121.63 126.62 172.74 166.35 132.27 160.67 121.71 177.53 204632_at RPS6KA4 234.91 152.06 226.76 308.55 183.34 179.03 224.35 160.92 168.73 216.87 240.7 166.34 171.28 178.79 155.19 168.57 266.24 364.15 164.16 135.67 197.02 157.85 224.66 212.27 186.11 189.99 220.6 241.46 191.29 205.23 169.71 214.3 194.74 189.67 230.02 256.19 296.36 225.63 273.65 306.62 360.05 345.91 232.42 276.34 227.44 191.03 256.84 244.66 275.34 220.3 155.66 215.94 260.17 362.06 257.25 194.9 404.85 275.15 261.06 266.86 162.29 262.62 244.95 166.04 172.43 145.36 187.84 220.11 267.39 137.24 265.98 261.98 210.09 249.94 136.32 183.75 122.78 147.02 112.05 191.95 155.81 146.98 147.76 154.26 148.05 171.18 188.84 155.94 175.15 183.88 297.6 154.18 170.71 147.39 144.51 176.56 194.49 235.26 162.93 200.79 165.19 182.77 199.4 119.4 122.83 344.05 246.43 288.06 341.98 261.74 232.8 166.49 193.3 225.36 243.12 183.36 192.78 210.59 235.77 273.79 220.02 274.46 323.78 229.16 132.17 128.79 124.27 149.2 115.48 196.11 169.55 142.13 156.08 215.34 146.01 120.98 186.26 154.32 136.12 158.56 131.15 120.34 144.75 134.04 144.18 185.57 138.91 212.22 239.88 219.98 232.5 295.89 294.56 270.17 196.25 298.81 224.89 473.06 206.11 258.24 296.73 230.73 218.08 256.31 206.56 288.95 197.6 383.52 268.94 196.23 225.42 267.53 166.49 223.91 162.47 278.63 234.44 158.75 143.78 151.44 203.91 265.28 162.57 187.23 207.52 201.71 197.26 153.38 247.62 181.45 143.78 156.55 279.18 288.33 193.71 331.08 238.42 256.35 214.82 220.9 220.63 211.57 279.2 233.32 471.77 167.1 206.33 180.56 179.36 233.13 275.79 272.22 250.71 224.76 173.14 195.66 211.54 220.75 219.64 183.76 332.79 247.95 176.72 209.16 249.6 249.35 275.31 204634_at NEK4 184.83 229.91 72.12 120.9 166.69 181.05 255.01 327.53 189.8 108.97 259.94 128.88 175.95 193.46 93.61 95.08 224.38 148.09 274.41 525.21 134.99 177.07 254.96 76.25 198.92 149.85 182.99 179.33 141.09 173.31 194.41 76.38 292.88 289.75 74.4 40.33 92.32 99.63 69.01 33.71 150.64 154.45 56.14 80.12 46.26 93.13 63.08 105.11 69.9 160.89 120.04 94.5 110.63 118.97 231.3 106.16 95.1 151.66 87.08 77.66 228.06 185.72 137.36 120.07 103.53 96.99 78.18 187.4 102.72 98.8 24.43 103.27 63.48 90.6 239.97 67.46 194.01 167.84 181.15 66.89 124.5 306.69 190.37 239.82 176.68 169.62 133.68 100.57 107.06 161.02 19.4 203.75 153.22 231.99 309.2 212.06 157.63 123.38 292.4 124.95 160.95 176.65 157.18 165.39 140.2 195.24 123.72 108.99 75.45 15.83 68.44 105.38 91.1 81.34 78.51 117.58 120.74 24.72 83.72 41.37 81.51 102.75 61.91 67.24 74.79 204.22 137.04 91.17 263.51 90.42 68.06 69.93 44.29 78.4 90.21 175.33 59.92 59.5 100.84 116 268.09 141.63 280.17 91.14 119.47 58.56 112.06 153.55 80.94 278.63 107.55 47.56 71.57 106.64 152.12 83.76 340.78 75.71 134.11 78.58 56.01 103.51 251.04 93.6 213.52 99.42 96.46 89.99 52.29 192.88 163.15 67.21 78.75 93.71 32.16 82.04 43.35 122.34 112.72 137.91 83.06 60.01 81.32 131.66 29.28 122.9 144.55 158.16 88.2 198.98 88.23 155.55 68.4 171.46 113.39 61.43 123.63 84.03 67.26 52.75 199.16 132.13 178.72 129.71 116.48 153.26 124.53 128.79 74.35 159.06 57.06 75.36 80.24 80.27 133.18 164.49 145.31 121.25 126.49 79.56 58.78 82.77 96.18 80.22 76.63 56.73 91.46 204635_at RPS6KA5 26.77 130.31 47.87 91.9 107.36 143.05 112.77 134.24 58.7 34.34 119.68 145.69 186.02 121.78 133.13 88.12 32.38 48.31 108.63 82.64 121.63 109.93 25.39 55.05 98.48 75.23 50.73 86.15 49.74 55.65 61.86 126.24 47.46 92.07 44.03 23.93 57.18 37.33 61.97 38.74 77.6 80.82 49.08 73.67 30.57 73.32 34.46 69.01 42.73 87.58 59.61 100.79 62.76 125.51 139.26 79.93 39.74 40.87 100.18 37.96 201.34 130.66 31.12 28.06 130.01 61.07 61.77 71.88 45.56 72.29 36.58 64.96 90.15 65.47 43.45 48.61 115.89 241.34 97.21 63.49 23.26 204.81 147.77 201.99 215.51 103.33 78.87 96.75 101.61 146.76 21.2 132.21 112.18 189.62 228.09 156.41 69.28 77.63 102.94 154.08 104.44 137.1 195.33 121.95 187.35 79.74 50.36 81.95 59.1 32.85 43.56 189.16 67.42 105.89 227.69 59.16 285.22 54.81 119.98 61.81 84.34 33.63 66.94 125 101.49 46.88 117.1 59.06 70.11 35.35 92.73 50.82 63.58 74.65 66.32 68.4 318.39 131.77 80.73 68.66 156.04 100.52 288.43 73.36 102.01 77.52 161.91 159.04 62 143.63 37.46 67.31 52.73 99.39 60.66 74.37 58.88 39.72 86.33 64.86 61.35 77.46 123.71 131.31 83.28 97.92 90.98 77.82 23.78 90.95 56.9 74.42 81.48 75.09 23.26 178.14 60.47 70.58 101.79 46.39 120.57 97.85 122.1 67.88 41.43 53.84 160.25 96.42 164.12 101.41 92.53 112.94 40.39 68.13 228.15 104.91 85.01 57.24 68.14 62.76 49.49 116.43 128.52 27.76 26.08 133.85 124.46 75.91 57.36 80.97 164.27 72.53 79.39 126.4 57.8 77.19 62.94 79.46 62.95 110.68 81.45 65.33 101.58 72.75 59.72 38.55 115.89 204641_at NEK2 256.59 207.51 33.87 494.74 160.98 7 446.84 129.05 146.68 116.84 187.64 21.66 55.03 251.54 15.83 95.26 536.24 182.81 373.16 400.87 107.79 194.7 145.7 223.6 197.48 105.32 67.52 152.54 158.08 218.18 146 185.31 354.9 126.94 298.95 288.95 198.06 117.9 9.37 52.99 240.57 168.93 192.5 91.57 100.68 27.73 200.13 231.71 155.04 116.59 65.32 98.78 199.07 171.67 201.65 178.2 137.05 49.54 63.66 44.63 131.48 166.63 146.63 106.29 22.12 217.08 214.28 148.61 248.1 115.09 74.95 94.47 114.14 99.99 236.29 100.9 359.58 48.41 51 49.56 138.26 103.43 84.75 51.96 6.69 64.17 54.65 41 167.09 40 9.07 13.54 50.66 34.97 22.11 38.04 29.92 147 85.53 106.69 45.64 39.7 61.83 9.78 79.23 219.21 24.37 17.26 77.98 13.83 146.41 6.68 31.38 49.19 58.39 311.44 61.17 25.5 32.9 20.5 29.44 47.37 34.46 81.2 8.46 167.16 54.43 12.44 185.44 170.18 14.48 39.54 7.19 37.65 22.7 67.94 125.07 51.94 46.52 365.8 80.43 88.4 144.07 79.77 34.24 23.47 85.53 39.82 21.68 38.06 254.38 40.84 144.7 44.51 42.54 234.2 173.56 118.69 12.77 76.43 67.66 22.83 133.37 15.96 114.81 43.9 76.05 40.22 31.91 291.82 18.03 22.33 39.15 299.37 26.38 30.48 67.77 24.44 14.72 152.95 9.4 101.81 63.62 49.64 25.52 37.06 59.87 90.88 118.25 96.43 78.36 9.39 29.37 117.51 17.69 34.34 204.94 129.53 47.79 41.95 64.1 48.75 47.76 67.62 110.04 8.43 81.7 106.22 16.81 70.1 58.22 26.12 54.68 8.19 140.16 49.35 50.78 61.16 78.98 19.99 83.34 157.12 47.51 191.14 131.62 391.32 15.94 204648_at NPR1 71.85 63.27 105.77 97.78 63.02 68.83 91.34 59.63 76.44 83.84 97.89 75.21 98.32 65.46 85.81 85.38 121.48 91.72 76.89 81.74 87.02 88.8 67.69 124.18 83.61 76.53 83.98 76.54 73.33 114.22 84.87 130.2 84.83 63.05 83.38 86.61 103.38 99.56 111.96 93.76 94.77 118.04 90.41 101.85 124.74 96.16 84.54 88.28 105.08 81.46 109.99 104.05 115.73 87.68 104.23 88.53 166.08 112.2 81.73 103.4 72.33 87.31 169.72 104.96 90.84 65.08 85.42 134.73 103.44 81.35 95.03 118.88 83.2 90.34 85.22 71.6 74.86 87.04 111.01 97.43 104.23 77.91 73.41 69.93 101.06 72.61 67.87 71.32 72.2 79.68 196.01 65.73 74.7 69.84 68.21 71.79 70.76 76.42 86.86 69.3 98.89 87.09 89.86 74.77 64.21 93.14 91.09 82.13 93.64 167.52 114.02 81 64.37 80.01 96.01 67.13 80.09 95.18 83.08 93.95 93.17 103.68 108.61 77.06 102.84 105.05 71.34 82.03 74.58 70.66 74.9 74.82 116.42 91.19 61.55 59.14 86.16 85.21 67.86 63.17 65.64 48.66 67.13 57.63 74.04 69.58 69.39 119.78 118.63 75.33 89.9 116.59 100.95 94.5 99.8 139.87 123.48 79.84 70.93 121.35 86.64 91.02 89.29 110.76 72.45 92.93 77.66 112.82 105.32 76.62 104.86 115.74 113.07 104.43 154.5 130.24 122.18 96.84 128.75 108.51 106.35 143.46 103.43 108.92 122.16 118.56 115.42 96.06 98.23 116.03 82.15 101.43 108.16 97.41 118.52 112.42 88.3 105.97 103.43 97.11 113.22 79.96 93.25 109.21 89.3 88.53 75.97 90.6 120.47 80.97 92.49 84.54 123.03 102.85 59.24 67.88 57.67 74.1 74.83 74.9 83.49 52.9 76.01 57.7 78.24 60.73 86.53 204718_at EPHB6 69.95 26.3 98.87 51.51 34.99 27.58 35.27 24.92 28.62 89.32 40.42 30.46 29.01 30.83 39.33 50.5 97.9 209.21 49.34 29.46 47.23 28.21 150.62 60.03 36.4 33.17 54.28 31.17 34.18 128.54 19.73 20.33 23 41.59 104.19 62.02 60.03 77.75 52.09 174.56 79.31 649.73 56.73 46.81 86.34 63.55 50.87 44.14 83.48 51.29 70.36 25.32 34.44 50.96 34.05 64.79 38.69 74.06 37.47 93.27 21.96 22 34.24 28.91 68.96 42.22 105.05 29.76 39.51 31.61 55.24 54.39 23.7 27.82 19.61 43.44 154.17 30.67 27.93 33.28 21.64 28.71 59.25 24.02 39.18 37.22 33.93 49.38 30.35 39.65 42.89 31.76 22.8 23.48 37.3 42.51 27.42 21.54 25.63 28.94 49.7 68.47 25.82 28.29 22.71 44.76 29.95 43.03 34.25 40.54 111.97 45.67 46.1 41.59 50.59 55.46 21.23 66.37 36.39 81.66 34.4 29.27 42.21 61.08 45.82 31.83 26.04 30.7 23.54 77.38 33.87 32.61 90.21 84.11 22.03 22.84 212.63 83.96 27.02 26.38 20.69 26.61 15.61 20.96 23.57 28.09 22.48 42.34 47.79 22.85 81.67 53.08 29.53 60.21 30.35 90.85 23.94 53.07 21.44 58.1 24.27 42.92 21.2 26.03 35.94 33.83 20 51.05 27.59 47.5 32.63 41.92 34.03 69.94 171.96 109.38 48.58 21.12 36.96 33.43 36.57 29.97 26.99 27.88 59.76 20.11 31.62 22.44 25.68 26 24.82 37.86 34.09 78.3 54.67 36.62 81.71 38.32 57.55 60.6 36.47 32.38 43.23 53.34 29.46 32.43 36.32 37.89 45.29 24.85 58.53 37.05 41.93 36.18 22.03 29.43 39.94 59.17 32.49 28.27 44.67 96.35 25.79 34.82 64.65 27.17 70.69 204813_at MAPK10 35.3 49.96 51.17 82.37 164.45 59.74 49.6 54.43 74.08 75.55 27.34 52.24 30.96 53.41 42.65 43.08 49.35 30.13 28.07 23.9 26.65 53.06 80.25 44.63 35.97 45.68 142.96 95.33 55.3 43.16 111.88 172.34 120.37 39.86 107.95 41.23 52.38 46.29 54.66 42.63 78.53 72.46 44.96 36.02 95.2 87.26 41.54 34.59 29.92 123.26 38.65 44.78 35.68 31.56 41.88 42.49 36.41 35.13 84.19 31.61 69.07 36.24 66.03 34.64 34.31 57.39 187.34 35.52 38.28 22.21 33.13 95.21 64.78 67.88 71.72 159.27 32.13 35.25 36.22 52.39 49.42 47.89 52.11 46.85 68.38 73.63 108.4 52.62 37.03 44.97 63.81 35.42 80.66 42.29 49.71 45.5 41.36 98.11 43.91 68.76 127.24 275.17 159.25 54.05 152.42 45.55 129.92 52.85 140.02 124.4 33.69 36.81 33.11 32.01 53.26 87.23 31.24 99.74 35.45 220.99 68.26 33.85 62.48 58.72 47.22 38.65 31.26 43.13 54.01 67.36 142.95 48.23 81.6 94.59 41.31 54.26 32.88 71.63 37.76 132.64 60.37 31.95 30.92 81.81 32.84 74.06 64.81 41.12 43.43 23.28 33.96 67.4 29.22 100.7 100.88 35.99 57.69 33.35 55.15 39.22 39.62 44.02 43.82 60.03 37.75 64.79 44.33 86.02 76.19 38.29 29.29 40.36 48.49 44.98 35.4 87.96 30.28 24.56 40.77 33.08 30.6 265.54 38.51 32.51 56.21 35.23 111.59 29.39 293.86 108.03 82.25 32.49 48.65 100.13 45.27 39.8 39.15 42.35 89.68 111.24 54.71 29.96 36.72 42.02 38.89 37.63 34.66 43.85 45.57 46.78 44.88 38.22 127.15 248.51 42.01 111.21 98.84 51.33 62.35 63.7 42.34 37.74 337.52 46.42 54.83 45.03 144.01 204822_at TTK 201.26 109.16 17.53 200.34 186.69 6.86 327.95 48.5 79.57 136.03 123.1 18.47 29.03 132.67 7.67 137.33 293.87 161.61 720.95 671.13 69.2 78.68 361.83 87.97 255.57 123.96 71.95 219.84 193.17 181.54 74.91 76.22 97.16 71.64 249.23 109.59 109.11 60.35 12.13 58.25 277.84 131.46 118 102.31 94.34 13.45 144.6 184.54 87.34 79.95 78.82 77.12 67.75 104.87 78.64 33.12 119.97 123.51 47 58.38 49.86 269.96 205.58 67.39 65.32 339.45 297.22 296.82 212.32 138.54 257.15 145.88 241.93 121.74 185.56 103.99 657.06 43.71 17.4 24.07 170.74 64.25 88.24 33.52 9.97 25.29 32.75 32.96 42.91 34.78 12.75 14.94 28.11 41.43 12.87 97.67 17.62 120.31 47.19 162.71 22.51 38.21 46.92 15.2 106.2 127.99 11.4 20.88 14.49 9.46 256.11 12.49 73.41 173.25 111.59 610.2 204.99 40.52 80.61 49.99 36.4 228.83 84.79 331.19 9.39 37.64 34.58 17.58 110.55 270.79 15.12 21.09 11.54 26.39 53.36 48.08 200.99 151.41 41.7 85.59 48.33 220.49 372.93 55.22 71.1 65.9 259.57 27.6 37.46 33.21 244.02 12.21 169.48 30.81 27.09 158.5 44.85 187.24 18.78 143.63 19.28 27.98 48.04 13.34 39.27 36.44 67.86 63.12 96.9 78.79 55.59 30.85 41.86 502.95 74.33 76.82 79.37 14.99 14.87 151.07 15.42 125.7 59.15 15.42 49.44 13.63 60.21 41.11 250.47 128.99 45.75 9.18 28.7 189.12 24.94 67.62 184.66 25.03 130.83 73.29 37.53 37.67 49.9 127.16 102.29 9.95 171.18 319.33 10.93 30.87 264.65 39.21 18.32 21.1 39.5 25.42 36.17 243.16 70.19 27.1 170.89 184.53 23.67 206.99 243.75 425.21 18.65 204825_at MELK 486.13 224.27 83.15 287.21 300.81 18.62 555.57 231.01 115.08 256.01 266.7 44.89 59.57 395.47 33.52 166.23 584.06 600.24 866.21 739.5 120.09 342.5 575.4 340.46 556.34 181.18 420.13 343.4 628.76 819.66 184.45 231.02 310.02 188.84 409.17 891.04 208.54 297.44 45.84 282.28 434.51 767 371.08 384.82 334.67 73.25 152.67 461.53 523.99 285.44 100.4 236.33 105.64 301.94 188.11 114.64 333.82 370.93 263.14 203.05 133.67 460.57 613.75 166.03 128.57 690.76 526.33 604.55 412.68 449.84 246.63 333.81 186.94 346.68 365.46 188.66 584.93 92.76 59.54 238.14 179.32 123 118.26 70.46 20.25 71.45 74.92 112.56 151.4 169.66 36.76 90.68 93.45 196.24 67.57 278.86 49.75 369 201.82 284.29 102.41 119.27 211.43 18.15 217.66 338.95 46.33 111.73 211.24 46.6 432.09 13.6 185.44 174.07 228.63 441.15 155.32 74.1 176.61 121.71 78.19 183.65 222.85 320.34 26.15 389.36 160.65 80.38 193.87 206.66 47.81 174.7 24.53 50.34 163.98 172.64 226.49 121.61 134.67 348.71 115.93 222.68 970.89 163.78 140.72 236.09 202.99 84.19 188.67 74.71 475.15 107.09 356.11 296.95 76.27 523.33 138.53 365.01 45.8 383.17 123.96 59.98 232.98 44.05 63.6 162.38 151.09 142.76 124.1 322.42 171.36 139.55 130.63 611.45 124.44 175.48 165.95 46.84 97.12 220.49 66.57 540.59 139.82 172.9 111.52 182.51 157.76 108.64 694.73 233.67 266.22 17 133.54 144.62 58.67 176.78 450.03 74.72 143.9 148.59 122.56 165.76 164.76 139.58 315.12 17.36 419.01 473.58 67.09 108.45 295.38 104.03 78.38 58.39 185.21 72.7 88.08 327.82 172.5 73.41 279.63 232.21 47.8 192.44 330.97 314.74 32.44 204831_at CDK8 201.31 110.55 89.07 135.28 261.07 106.43 81.32 80.44 150.68 181.01 171.69 164.38 123.08 84.88 89.91 134.68 178.45 114.21 260.03 269.37 128.37 125.91 342.88 154.2 158.94 228.89 261.15 224.56 100.49 136.78 99.61 88.99 191.73 73.66 544.79 131.86 185.02 71.32 89.04 92.32 140.14 124.34 178.21 173.15 148.27 92.39 121.94 172.88 95.58 69.82 130.92 86.21 82.74 255.58 79.01 159.46 84.1 63.43 88.33 43.97 33.8 105.97 145.5 55.97 59.7 144.04 87.25 176.06 322.19 251.71 215.05 80.35 113.84 101.54 95.98 110.68 396.6 127.59 45.35 159.09 51.05 157.66 72.72 65.48 106.98 76.96 196.95 89.35 55 137.05 24.85 114.6 375.33 135.96 140.72 85.03 124.84 118.85 121.46 259.54 138.83 150.7 126.79 116.75 141.13 94.41 81.87 97.65 70.27 20.05 89.97 82.41 100.15 109.53 131.85 120.69 114.82 122.49 71.72 67.12 91.27 103.72 136.9 60.45 107.98 122.17 91.98 75.58 194.06 110.93 115.85 163.17 51.43 76.14 76.12 109.79 138.75 104.19 156.62 110.07 106.28 104.9 176.91 122.25 93.02 63.2 193.86 103.47 61.21 147.41 103.16 54.99 152.87 99.99 92.15 195.37 64.4 124.2 104.26 167.63 63.61 103.75 62.31 69.37 67.24 137.68 91.87 105.68 43.86 73.2 71.66 61.97 123.33 96.76 31.25 103.61 77.87 62.43 80.53 35.58 83.97 62.66 80.46 65 68.41 47.42 111.22 36.07 54.32 98.67 101.22 77.95 66.31 69.7 74.48 78.51 106.27 54.42 99.34 63.93 98.36 55.72 84.14 23.74 93.77 44.95 196.98 132.81 65.86 80.85 86.05 42.41 70.3 66.78 43.67 54.79 113.54 116.34 94.37 52.78 153.33 214.84 73.48 197.43 159.49 295.89 165.78 204878_s_at TAOK2 83.45 103.37 110.68 151.15 83.86 105.09 105.55 96.36 119.14 113.51 109.03 104.58 100.17 141.95 100.55 108.74 98.71 113.94 97.69 89.77 128.98 86.96 76.77 111.57 97.94 67.38 113.49 103.32 99.05 139.79 108.97 133.46 134.6 147.11 164.36 132.99 135.91 106.75 129.62 107.41 94.99 116.8 129.2 108.72 118.59 111.07 108.74 98.75 108.69 98.26 92.04 118.03 152.23 114.88 164.03 124.44 158.98 122.94 136.51 149.8 122.91 95.01 102.78 113 95.29 82.47 90.93 75.35 108.39 81.46 113.34 96.36 99.2 89.54 99.15 88.76 97.12 93.39 116.79 105.98 118.17 87.32 83.81 82.1 61.81 84.35 95.11 67.47 88.22 77.58 273.72 76.04 98.44 79.13 83.91 77.43 92.82 83.25 88.2 75.09 90.05 93.02 98.86 77.53 90.26 121.09 120 109.7 148.08 331.36 108.19 97.42 82.53 122.85 94.04 104.19 145.45 127.29 120.61 117.48 122.7 157.47 120.27 108.95 70.37 100.28 69 70.88 66.48 70.18 81.46 74.05 93.58 86.81 84.99 78.84 88.91 68.09 80.04 77.6 74.81 66.44 80.67 67.15 70.98 91.22 84.02 82.35 92.92 85.11 111.26 112.38 110.27 97.81 101.61 115.42 171.75 94.67 95.37 95.25 109.78 100 83.36 144.12 100.82 95.57 110.92 98.13 139.32 98.76 122.63 145.76 124.91 113.41 284.46 158.68 117.52 126.82 133.01 154.25 117.34 132.91 123.32 101.24 117.84 138.81 134.11 118.21 121.44 130.15 107.74 120.18 103.82 127.71 74.16 101.7 73.06 142.82 104.53 116.92 115.33 112.5 97.33 137.28 101.97 77.46 74.61 99.51 120.19 102.5 98.64 99.58 130.58 106.31 86.39 89.83 82.17 103.78 85.38 91.65 80.42 57.75 119.1 90.51 77.79 91.37 75.62 204886_at PLK4 26.97 28.66 6.71 37.55 29 6.19 104.07 45.27 22.75 14.95 23.96 9.11 10.35 43.78 5.13 39.73 32.55 30.91 41.27 44.39 34.39 34.53 42.8 19.1 24.96 44.54 17.6 29.34 30.85 42.46 21.78 23 27.73 26.25 30.97 33.48 27.07 33.9 5.46 14.93 34.5 30.83 15.07 25.47 14.94 9.13 27.49 20.82 15.62 17.44 44.69 22.66 28.17 22.9 23.68 13.75 19.9 24.76 18.08 18.89 24.49 32.67 60.24 12.28 13.22 21.84 16.2 24.44 27.14 21.11 21.12 20.21 13.91 28.87 19.48 12.81 23.71 8.86 6.42 10.89 14.5 12.37 18.71 10.89 6.07 11.28 8.69 10.63 12.35 15.26 7.09 6.51 10.31 13.99 19.21 17.58 13.6 21.9 24.15 28.62 19.57 13.6 15.53 11.43 18.96 14.1 10.15 8.01 12.12 5.66 11.3 4.95 6.05 12.15 11.82 27.83 18.45 7.35 6.85 7.49 5.93 11.22 8.31 11.56 7.57 22.04 10.62 5.81 12.76 15.87 9.09 10.55 5.2 8.69 10.9 12.46 10.93 9.64 11.03 12.03 9.84 12.82 30.95 11.09 8.48 9.09 16.18 11.21 7.92 8.7 29.13 6.78 14.48 13.43 9.96 16.85 11.19 22.47 8.95 17.74 10.61 6.27 9.19 8.73 6.37 10.53 9.01 10.78 7.66 21.16 11.48 8.97 21.1 13.61 8.85 9.53 8.32 6.65 8.51 21.33 7.79 9.23 11.69 8.82 7.6 9.41 6.52 12.07 13.21 9.24 20.43 5.39 8.5 19.06 8.63 15.23 18.84 8.44 12.1 16.37 9.72 14.91 10.11 7.81 19.73 7.21 49.68 14.89 6.43 8.1 10.13 7.51 9.21 6.44 14 9.03 6.06 15.76 10.34 8.01 17.48 18.61 9.31 22.54 17.88 25.11 8.06 204891_s_at LCK 92.67 42.7 166.3 216.79 148.16 131.98 68.31 56.25 69.62 125.96 33.9 25.26 97.83 32.32 92.88 38.02 64.63 500.85 159.76 90.83 74.99 312.8 49.86 299.64 110.94 746.8 99.93 112.09 265.33 37.57 91.93 78.86 56.31 131.59 124.35 259.04 150.72 91.06 345.97 457.05 380.73 322.08 100.9 61.86 106.1 166.6 107.43 173.61 323.34 68.35 84.85 213.79 89.72 281.07 27.5 45.15 86.31 206.16 512.33 63.79 32.2 85.5 51.39 120.32 259.96 112.89 134.31 277.8 91.22 101.72 58.21 236.55 54.63 98.87 25.08 27.93 48.39 409.23 34.63 44.99 30.43 98.05 36.79 144.52 142.83 183.77 82.25 494.38 432.07 330.37 39.88 199.24 91.02 44.61 71.46 200.65 24.76 40.47 176.6 108.29 40.96 145.07 134.77 61.83 26.41 301.12 53.92 193.8 73.4 38.46 182.31 86.76 421.9 165.29 403.97 122.12 20.87 283 620.73 116.92 186.68 191.66 246.23 464.62 111 281.07 60.53 130.26 40.05 77.41 478.11 53.38 69.52 321.54 35.3 60.11 109.35 89.39 61.53 33.81 38.85 31.09 61.35 52.97 160.85 153.61 38.37 99.82 311.12 79.9 132.5 609.99 29.99 229.2 386.92 75.92 15.84 590.29 33.14 388.39 119.93 99.35 36.72 75.15 23.41 331.58 58.57 611.74 34.94 34.58 220.33 84.91 97.68 327.57 42.82 29.69 340.59 44.01 51.72 44.85 575.9 65.16 60.59 48.64 172.95 51.93 54.27 28.32 75.33 21.88 34.52 62.33 26.46 131.41 76.05 109.9 91.65 264.28 311.62 75.22 105.53 214.59 428.61 58.49 69.59 36.92 65.97 126.37 89.63 106.51 129.01 317.82 132.11 40.04 21.82 175.44 202.09 119.39 71.44 70.43 416.39 77.91 31.16 35.23 79.71 79.63 202.06 204906_at RPS6KA2 174.39 117.41 362.33 179.65 297.51 170.92 142.04 89.26 180.33 261.15 202.9 140.21 152.41 209.41 239.51 1285.16 101.89 77.15 153.08 124.1 225.54 78.75 98.58 118.91 153.88 103.16 253.39 231.29 89.61 97.18 89.4 104.52 95.78 119.54 123.11 67.22 148.37 263.29 236.63 150.43 68.59 82.95 103.85 187.67 150.39 255.98 103.99 200.2 153.55 199.54 89.99 142.25 237.01 174.46 70.8 148.76 75.6 61.94 115.6 303.05 99.01 93.4 105.75 102.73 445.41 103.66 174.98 121.09 97.71 127.18 156.62 128.84 269.08 195.34 229.47 254.15 128.1 149.49 94.77 204.11 250.68 148.04 168.22 176.71 274.37 155.62 288.72 253.89 113.67 158.63 339.13 160.18 76.92 125.34 129.28 137.19 123.66 186.86 71.2 106.4 149.99 181.63 111.18 159.89 187.85 162.44 282.92 178.61 208.03 145.49 261.86 194.01 313.47 207.42 171.15 98.08 206.57 278.4 183.62 334.85 600.74 450.69 381.09 185.62 428.56 102.73 127.04 319.2 120.06 98.82 243.85 175.72 340.27 215.88 161.44 97.54 128.4 170.23 132.73 150.14 126.89 129.57 79.36 158.19 235.29 168.35 197.48 262.48 513.68 127.79 57.48 223.42 255.04 171.66 125.16 150.81 53.33 137.65 310.43 157.45 173.6 241.37 110.19 160.05 126.6 184.55 159.36 207.09 169.13 136.66 141.43 276.24 191.79 182.01 313.3 316.67 197.15 135.02 325.38 74.8 196.12 128.53 167.1 206.67 290.99 194.78 235.29 223.91 117.22 97.37 274.24 192.26 303.37 304.27 258.41 203.32 144.52 266.11 200.81 250.57 202.2 169.81 187.66 133.32 122.28 223.74 209.3 133.33 209.86 106.38 238.99 199.89 265.49 352.57 174.79 204.93 165.37 104.08 154.13 392.94 307.72 256.97 176.52 179.8 209.81 174.9 287.5 204936_at MAP4K2 44.25 36.96 57.5 44.17 27.41 31.36 46.58 32.08 41.79 67.78 43.74 36.33 42.7 25.89 29.06 34.08 84.48 45.94 55.33 36.13 39.31 49.9 99.48 50.12 26.25 35.19 48.23 57.33 57.47 38.87 52.95 64.12 51.02 54.16 46 49.65 31.31 40.15 54.39 37.63 62.36 60.28 62.95 48.72 44.14 47.52 45.22 51.84 55.7 69.24 35.45 60.3 51.84 94.22 82.5 66.65 72.31 43.42 58.92 48.76 37.24 86.11 46.3 35.49 56.37 46.17 50.89 41.87 49.39 49.51 72.48 44.86 47.42 35.27 32.9 22.62 34.14 31.11 47.58 51.98 36.12 42.36 35.74 34.65 32.56 43.2 32.79 31.92 33.16 37.05 66.86 33.19 43.82 35.48 32.46 40.41 46.62 113.48 44.5 33.81 30.28 41.8 40.13 42.45 32.07 72.84 35.68 47.51 123.92 82.59 45.75 37.19 47.71 39.57 66.09 24.32 34.61 52.22 32.59 40.26 31.61 52.89 56.95 65.46 31.8 47.3 34.18 23.54 26.75 48.54 37.21 66.27 51.56 42.29 32.43 30.13 43.21 35.94 23.52 25.94 25.09 28.44 40.08 26.51 33.5 33.75 32.5 54.34 51.93 51.54 108.3 74.88 73.1 44.29 40.24 70.05 37.03 55.7 42.56 49.15 65.77 43.38 44.42 29.38 46.26 47.89 41.27 61.4 69.94 35.26 65.74 57.61 37.48 60.14 63.62 82.52 59.91 59.39 52.32 45.63 45.53 66.62 33.11 55.68 52.21 69.04 62.74 56.56 73.97 71.07 45.4 36.42 81.87 68.33 48.35 106.27 67.66 66.74 44.71 56.14 55.76 59.2 57.85 65.62 73.16 55.49 70.96 58.34 66.66 51.92 91.79 40.94 46.87 37.45 46.24 42.55 38.16 45.28 51.37 26.94 91.41 77.91 49.64 47.01 63.32 56.68 48.2 205051_s_at KIT 76.99 288.82 104.29 148.58 254.07 248.13 79.38 174.31 74.92 60.46 69.11 64.71 370.64 44.02 265.8 257.26 134.68 298.88 177.95 120.26 80.87 343.71 267.63 508.73 297.77 196.87 74.31 76.73 245.75 225.65 148.11 106.35 117.53 76.71 1101.99 477.05 1457.74 266.6 388.38 131.3 111.55 67.02 165.74 91.42 320.43 315.09 185.97 183.3 98.86 99.38 1522.92 161.39 362.95 860.43 30.04 359.38 160.27 105.4 284.47 173.51 127.25 2037.05 517.91 129.33 25.89 112.65 425.37 108.45 25.33 151.65 280.48 357.86 178.99 60.23 12.99 42.8 1055.69 117.95 1640.32 55.32 25.36 340.33 109.48 81.39 218.4 262.32 132.79 158.31 45.19 158.27 21.73 252.29 60.58 252.34 335.2 73.06 54.01 95.98 145.22 77.55 1672.33 219.52 47.89 76.64 38.08 100.28 168.47 323.78 130.38 29.48 145.03 1278.42 106.99 307.31 129.62 226.58 21.17 178.95 220.25 38.3 70.69 271.8 1006.95 820.98 605.66 28.56 80.58 101.94 66.78 168.55 59.72 61.88 247.88 132.77 107.95 127.65 310.78 104.02 93.19 41.46 45.34 36.53 20.36 176.22 161.05 13.05 87.18 324.1 187.45 29.71 127.84 104.1 55.86 107.74 45.67 301.72 350.77 20.26 291.29 42.92 221.32 352.38 52.57 197.6 50.84 128.76 71.81 466.51 35.31 170.44 55.11 101.45 69.09 313.55 22.73 39.13 149.4 26.5 285.7 19.41 340.35 213.08 35.22 242.15 495.33 68.32 43.92 58.63 165.7 122.29 65.76 325.42 148.53 60.22 1262.64 33.45 508.66 140.59 118.61 198.05 85.98 126.26 258.71 48.15 197.94 118.66 188.89 95.56 182.79 78.76 168.32 1116.18 435.45 146.64 62.76 89.32 48.86 137.61 36 171.05 205.34 177.73 137.95 154.64 154.77 136.12 148.22 205126_at VRK2 336.21 340.22 101.99 204.18 152.77 119.47 151.78 86.92 131.24 245.68 504.48 203.48 143.5 116.43 102.17 108.8 276.79 272.47 471.81 228.49 198.19 390.59 565.39 135.34 179.23 417.77 281.5 226.66 354.19 162.1 176.43 152.38 1028.82 155.55 380.17 290.85 299.58 220.98 111.14 243.39 1091.47 291.11 353.84 318.48 240.73 140.26 160.23 303.23 318 112.14 283.85 195.98 80.17 507.32 159.77 158.99 222.03 274.49 240.99 205.05 168.26 101.22 267.4 126.69 202.69 418.77 313.85 837.39 377.23 160.81 223.41 353.83 411.97 283.63 170.67 215.1 421.97 270.05 179.14 285.59 99.71 313.82 139.28 146.47 98.89 218.75 93.74 192.75 164.06 139.56 53.63 197.53 148.15 122.88 166.42 330.64 148.38 285.03 333.18 400.29 161.39 190.35 145.45 180.03 159.44 207.63 182.54 148.72 131.99 58.65 207.42 136.7 232.44 250.48 259.23 430.39 72.78 102.59 167.99 118.81 127.52 499.24 185.03 154.25 80.13 144.29 89.34 115.81 154.23 325.77 140.49 112.74 74.72 149.04 156.88 130.89 272.75 207.01 117.15 198.31 153.66 137.62 187.24 137.33 100.49 135.15 168.84 105.6 114.73 308.73 222.04 117.14 321.89 204.25 172.1 209.83 117.46 259.73 135.45 229.28 131.97 140.63 204.6 139.92 97.87 94 46.87 37.45 46.24 42.55 38.16 45.28 51.37 26.94 91.41 77.91 161.64 139.99 155.13 140.8 120.23 136.22 311 216.24 376.89 74.9 235.6 256.59 256.29 74.54 420.24 181.26 110.01 137.12 89.88 170.41 89.22 141.23 90.66 87.89 132.64 104.88 134.79 141.84 149.9 131.99 197.63 252.07 108.24 178.04 239.07 110.13 147.29 283.01 161.18 123.63 377.25 484.25 437.66 189.95 340.58 191.04 147.81 81.35 155.59 1847.37 332.91 123.04 152.05 112.67 163.02 274.39 156.4 84.13 125.16 166.11 316.11 85.65 205130_at RAGE 35.44 82.84 77.4 47.13 63.94 189.9 168.53 111.97 39.74 56.06 135.02 164.89 154.41 186.79 114.62 180.58 123.23 57.82 89.79 127.62 152.83 153.49 83.95 130.46 100.86 41.87 82.77 64.62 90.24 64.01 59.38 147.21 62.21 128.72 103.71 87.96 124.05 75.95 74.68 57.21 49.7 47.78 152.74 394.37 61.52 64.13 79.91 134.32 54.5 75.45 70.9 72.75 101.27 150.19 102.94 77.35 51.1 79.19 88.54 60.67 162.96 186.69 85.24 40.86 24.87 42.74 137.42 51.13 35.43 44.53 60.41 79.38 144.46 80.04 65.66 59.6 92.39 120.42 67.73 47.99 60.99 113.78 75.76 115.42 96.16 112.63 81.33 109.7 42.72 59.68 206.04 79.93 90.87 48.5 121.79 75.04 91.5 94.09 76.1 50.76 127.13 79.11 101.8 132.27 75.35 107.37 84.69 62.99 69.79 212.95 39.38 68.86 55.76 98.96 139.22 58.92 228.54 148.46 264.55 55.02 70.15 30.18 60.88 71.36 49.28 48.16 46.65 59.1 92.41 122.95 60.69 114.4 111.66 68.67 48.05 58.92 63.38 58.18 67.06 56.8 63.33 71.99 125.27 64.71 75.82 211.43 53.21 89.48 53.8 144.53 45.14 54.5 113.59 75.51 105.72 81.89 59.73 64.74 103.56 68.37 97.74 114.92 57.58 88.92 101.03 62.3 151.89 48.76 80.01 63.05 89.13 73.6 92.99 73.3 94.54 112.38 33.57 87.79 63.17 91.34 74.36 69.98 47.8 98.75 59.62 45.73 89.85 51.35 61.86 89.87 119.98 64.49 70.48 68.43 67.9 68.08 120.21 59.7 88.56 201.12 57.38 68.99 89.73 76.98 38.26 77.2 111.34 57.1 61.56 40.63 138.6 95.32 52.11 92.34 105.01 60.57 54.83 145.67 37.23 91.53 76.89 68.86 132.39 51.71 61.57 1352.31 64.79 205168_at DDR2 103.94 170.74 447.33 213.23 254.89 204.53 74.63 111.23 133.28 163.85 128.81 147.75 157.18 96.73 280.6 459 141.98 117.75 160.87 75.91 183.92 87.25 395.31 176.62 100.12 252.23 184.42 157.33 220.02 287.37 191.75 211.43 134.11 104.62 212.75 86.7 215.62 149.71 481.84 459.06 51.34 176.24 292.67 194.61 185.9 394.56 297.81 101.53 412.48 225.12 253.33 211.12 245.14 449.79 80.31 190.68 161.62 221.61 240.42 613.63 204.54 173.01 244.34 119.84 197.85 228.9 203.59 170.75 139.43 332.78 347.98 285.33 341.44 240.75 99.36 174.43 93.46 221.11 104.93 287.17 322.09 226.06 111.31 154.54 426.08 279.65 266.93 470.46 118.29 147.51 250.3 75.66 28.48 57.68 93.11 60.81 51.3 16.2 68.32 45.08 511.95 1256.65 85.87 36.94 64.9 182.75 344.25 238.48 162.01 119.17 112.1 234.37 274.17 282.39 123.04 112.21 127.02 269.99 171.37 854.46 593.07 484.9 135.77 200.21 471.85 48.79 155.57 433.6 103.48 79.19 234.09 136.98 482.61 228.12 119.84 145.12 165.53 146.2 152.73 179.57 107.35 116.94 51.73 132.57 168.19 132.24 80.07 207.9 241.74 86.08 232.83 312.27 186.66 219.39 71.74 113.01 56.89 111.48 280.67 142.29 159.07 194.21 101.09 134.21 143.18 178.83 153.45 152.26 128.04 71.46 110.89 279.86 216.27 131.01 53.1 154.25 164.61 104.91 232.3 68.19 212.13 302.88 135.61 179.19 249.58 168.71 156.38 136.79 285.35 103.27 223.64 287.05 303.98 148.1 225.46 169.51 158.89 215.6 194.03 178.43 85.09 119.32 137 85.34 135.11 230.6 162.28 68.61 149.41 90.54 337.01 214.87 328.61 702.02 118.74 118.38 258.22 132.82 171.99 157.16 164.05 212.66 218.49 239.56 281.04 85.81 354.69 205192_at MAP3K14 39.81 52.63 79.01 69.33 62.78 60.26 39.82 48.58 41.87 60.34 31.41 38.03 51.19 39.69 55.74 34.86 77.82 127.46 75.71 63.2 45.16 107.71 59.7 66.8 46.21 79.5 64.51 59.84 79.07 44.86 43.21 64.87 40.32 36.54 44.72 81.36 94.44 42.66 88.49 94.14 107.74 142.68 58.67 62.34 57.25 72.65 82.11 67.87 90.7 57.48 49.66 63.35 54.31 61.73 50.05 56.31 63.62 114.67 94.71 55.58 65.3 109.26 42.17 67.06 110.16 77.89 62.18 86.44 47.25 41.53 39.32 124.2 50.77 40.54 36.75 15.76 39.27 51.61 23.09 35.12 22.48 41.77 43.46 42.71 52.91 61.63 51.35 82.86 70.61 50.2 106.72 60.91 45.08 33 51.26 61.74 48.9 42.48 61.05 58.58 53.44 83.6 62.35 67.66 41.34 93.28 62.65 67.11 77.67 133.47 90.79 79.75 87.45 68.55 82.32 28.93 70.9 93.06 92.73 75.86 72.68 176.59 47.29 100.18 54.32 90.7 49.31 62.27 30.96 91.2 98.2 46.89 93.63 98.86 50.84 48.29 44.8 84.62 39.25 46.55 45.99 43.3 42.61 44.2 54.78 49.18 33.84 57.44 94.85 56.66 224.78 146.92 47.17 69.56 67.08 60.08 69.62 98.03 55.59 149.6 69.35 64.22 47.39 54.91 45.43 75.13 55.07 93.36 51.19 50.53 55.26 58.02 49.16 65.71 35.74 126.63 83.1 54.01 72.86 48.03 104.09 44.34 37.11 56.14 69.35 50.23 48.61 43.09 58.9 43.97 59.78 65.71 71.63 57.92 64.88 63.07 44.12 109.72 81 51.63 50.72 89.59 121.57 78.49 55.99 56.41 64.3 48.75 59.4 47.4 64.59 108.56 65.34 51.98 37.62 49.87 67.63 52.53 50.48 61.03 110.38 50.38 87.64 59.02 95.24 70.76 101.54 205214_at STK17B 39.89 24.66 21.04 34.79 17.21 13.55 17.48 14.19 12.95 23.33 10.88 7.59 10.63 8.18 10.2 9.75 30.18 49.76 34.11 22.48 10.71 70.03 34.39 27.89 17.74 100.92 30.34 20.94 38.43 25.03 34.93 23.26 16.87 9.85 59.31 32.8 27.24 35.35 44.22 71.53 69.16 56.33 85.95 24.43 30.73 26.41 19.15 51.79 38.48 38.2 21.71 22.97 12.54 25.26 10.94 34.32 26.49 59.81 34.33 15.04 12.8 19.92 37.26 13.81 139.31 32.11 24.95 39.39 33.61 24.99 21.72 28 13 42.28 11.25 13.24 38.38 54.95 11.97 21.69 13.05 39.28 12.34 18.74 16.99 20.58 12.45 25.84 34.59 18.73 10.5 18.82 10.33 14.85 18.12 43.48 18.77 13.76 18.86 30.16 7.7 41.48 18.84 15.62 17.23 19.37 18.03 13.55 11.36 11.58 28.23 16.02 53.7 30.54 32.58 77.19 10.34 17.37 57.02 18.06 22.91 25.47 19.88 23.61 14.02 19.92 14.38 14.39 26.66 26.18 29.57 10.98 11.01 20.88 12.39 11.69 10.75 16.52 16.46 14.59 13.29 21.12 11.68 15.35 14.34 19.04 17.22 14.35 27.07 45.89 55.95 25.14 29.08 33.04 27.49 19.23 17.71 69.77 22.96 30.15 11.16 19.18 25.46 13.68 12.54 21.58 14.78 40.94 8.36 16.13 16.03 14.33 14.74 22.43 12.32 10.11 26.39 11.58 9.9 11.34 32.68 18.26 20.6 14.87 22.82 13.99 17.05 10.71 12.66 10.62 13.09 16.76 11.92 18.81 10.59 17.14 12.19 17.79 41.79 36.41 8.5 18.85 21.7 8.55 15.3 10.93 11.91 22.56 17.23 24.65 11.41 21.34 10.73 17.54 9.82 12.52 30.48 29.66 18.55 11.8 43.91 29.01 14.72 23.44 25.97 31.77 27.33 205271_s_at CCRK 77.52 69.18 91.1 105.08 82.19 122.3 107.21 136.92 94.42 88.42 111.91 125.23 98.23 118.22 98.32 115.08 65.96 95.86 98.72 117.21 128.66 110.03 102.3 88.99 160.46 86.62 127.48 72.74 96.61 161.83 75 108.08 88.55 164.37 70.95 104.48 117.38 115.2 87.68 106.43 152.49 95.93 93.35 77.97 93.37 83.07 120.34 85.85 101.66 133.21 100.77 95.92 116.89 75.06 152.06 88.87 161.91 130.2 77.6 92.92 120.91 79.66 96.02 73.82 63.11 96.24 104.23 91.3 66 72.05 75.18 134.08 55.43 97.18 84.26 80.09 68.7 78.12 84.7 175.92 85.93 69.56 95.55 97.57 59.85 78.47 86.41 69.48 67.06 69.1 113.63 79.8 74.74 114.47 82.55 70.14 101.34 109.09 83.67 83.49 111.9 75.15 102.52 115.52 103.08 114.41 95.27 75 125.61 133.64 74.44 101.78 76.37 78.03 63.83 69.66 109.03 86.35 80.54 65.15 87.56 94.43 79.87 59.94 48.3 75.05 74.96 62.16 78.39 68.99 77.72 75.52 68.87 78.65 81.83 75.84 87.58 57.78 66.79 86.06 91.62 54.83 98.08 81.45 55.28 89.48 53.05 99.79 81.37 97.24 102.1 69.29 80.02 94.12 97.05 93.7 163.99 81.51 69.24 77.6 86.47 80.96 103.71 101.26 66.79 88.44 108.87 75.17 83.02 98.4 119.42 86.23 89.37 76.04 84.79 82.8 75.62 110.9 87.54 87.22 85.69 151.58 91.41 92.65 111.38 103.96 80.43 113.92 136.01 116.97 120.15 87.64 98.94 88.66 91.11 102.02 89.7 119.94 82.06 80.39 103.68 80.95 86.47 83.38 76.84 91.71 77.8 97.31 77.59 100.77 72.97 82.8 105.86 89.86 102.61 80.55 70.32 61.59 80.03 92.14 60.72 71.94 85.18 64.16 69.93 85.37 54.86 205394_at CHEK1 68.06 61.72 19.08 79.86 58.5 10.66 77.26 60.93 59.97 80.73 71.28 14.73 24.46 32.89 12.48 50.62 266.7 72.48 101.1 156.86 37.37 153.73 134.73 53.91 131.05 95.28 73.62 221.09 272.81 91.2 76.36 183.77 78.94 78.82 149.41 134.35 114.14 305.84 31.68 58.53 66.69 160.2 146.5 85.06 72.41 26.14 106.16 101.81 124.59 67.06 55.07 36.67 21.52 69.04 34.12 41.98 122.47 111.67 45.76 49.15 80.23 204.61 83.96 43.92 53.37 116.47 196.92 135.79 173.84 154.79 77.63 87.97 130.11 59.81 270.07 66.51 145.49 26 24.08 59.88 39.87 36.69 20.86 13.93 10.29 15.51 22.89 18.8 19.15 29.4 14.97 14.82 23.72 33.19 18.42 67.93 34.16 58.51 29.62 148.09 43.82 18.94 52.85 12.6 56.64 73.15 16.9 23.29 18.89 16.13 79.57 12.85 31.62 82.85 109.33 174.02 36.25 17.8 15.52 28.83 23.19 81.16 60.62 27.61 13.41 71.9 16.42 21.86 56.14 286.96 16.15 29.97 12.17 29.5 66.4 23.78 37.09 35.49 59.84 20.09 24.14 38.24 150.49 24.06 29.41 20.14 79.19 32.75 11.92 43.08 121.1 19.55 149.06 52.57 20.83 110.76 32.82 119.84 26.07 109.36 24.13 27.4 65.33 21.53 29.97 24.31 27.1 27.29 18.8 70.88 44.04 27.61 16.7 95.86 8.65 28.15 60.46 14.36 15.97 26 16.61 42.04 22.86 13.43 24.23 23.31 28.25 16.8 70.15 40.79 24.31 10.54 16.99 91.3 20.18 49.94 97.26 11.73 34.21 33.96 13.84 21.45 33.09 15.51 319.6 8.84 130 33.24 12.03 20.18 28.54 19.22 11.33 13.46 17.23 19.43 18.21 98.15 23.56 9.21 88.84 88.1 9.62 44.77 71.53 274.32 11.42 205399_at DCAMKL1 41.14 78.03 446.73 499.79 349.94 1186.81 69.68 453.84 4984.71 49.67 352.98 325.27 392.68 1758.74 293.74 817.89 35.23 285.68 74.5 105.44 92.27 77.46 42.7 77.09 41.23 82.02 92.23 90.21 128.27 76.46 794.54 105.1 256.26 34.59 46.06 93.46 625.65 66.87 80.35 53.71 60.01 84.97 45.83 68.76 67.56 634.56 151.23 110.85 77.22 175.72 48.73 1679.66 440.99 94.89 1009.6 152.41 28.99 104.95 121.95 30.53 817.8 538.62 63.57 54.5 99.87 155.18 31.98 87.45 56.15 96.12 84.32 248.85 38.29 109.53 25.39 192.79 20.97 799.87 340.05 63.5 30.65 160.82 838.15 824.85 475.2 403.88 118.38 245.85 156.99 608.15 48.3 878.41 968.53 565.78 1170.92 87.81 176.85 104.17 581.73 190.09 1366.59 253.22 1211.45 601.87 108.57 114.05 194.13 151.77 89.88 52.28 97.86 422.78 156.87 68.34 62.91 26.32 1212.98 49.71 111.3 220.38 274.99 67.92 145.12 151.85 1143.35 70.95 119.88 479.44 109.66 44.33 177.78 58.84 537.16 280.73 54.5 261.5 182.93 166.45 108.51 132.89 536.66 30.53 78.16 136.35 218.32 30.95 368.79 716.93 222.9 581.66 50.09 222.84 233.48 178.29 124.31 91.94 63.63 58.68 284.6 51.41 135.47 831.91 633.33 95.76 150.34 117.47 230.72 320.7 51.56 204.39 1977.69 246.96 213.48 51.69 25.65 257.31 125.14 299.67 433.97 33.33 662.45 28.69 249.19 248.62 92.69 106.4 74.96 1256.61 37.26 184.83 686.19 403.23 237.23 99.26 799.1 129.63 24.45 128.3 256.75 85.9 235.71 304.14 245.55 46.52 37.79 909.09 68.07 26.63 41.25 240.91 46.95 267.88 270.55 83.48 166.09 460.39 25.41 113.05 52.57 234.44 61.01 212.77 212.4 80.34 83.42 24.09 535.64 205418_at FES 87.36 39.73 54.4 46.39 56.89 42.56 38.09 32.79 32.52 58.6 32.65 28.02 71.06 38.16 58.72 69.92 104.19 79.3 88.51 58.05 30.6 39.99 39.82 53.97 30.17 78.39 37.32 34.39 57.77 42.99 37.52 33.18 28.84 39.48 57.34 81.15 42.14 48.71 75.31 55.02 73.43 51.77 43.19 33.73 40.79 56.76 57.38 124.94 58.86 42.12 159.23 47.46 57.73 155.59 30.11 36.58 78.64 50.61 63.17 49.49 85.2 65.31 35.57 29.73 100.63 34.37 37.02 57.21 30.73 43.75 31.96 27.17 28.42 28.05 35.63 21.35 32.18 48.43 31.08 54.17 25.98 40.37 32.31 39.83 52.25 50.09 36.17 50 35.53 35.3 54.57 45.35 34.11 30.41 61.61 30.99 30.26 43.98 28.9 36.93 39.22 31.83 31.02 46.48 25.64 49.37 44.56 44.43 32.05 38.3 46 45.01 41.9 46.37 39.83 20.74 35.28 47.24 47.69 39.57 49.17 43.48 47.33 39.54 77.11 32.75 38.5 47.73 30.63 47.2 52.09 40.1 79.41 45.06 40.17 28.63 42.17 34.1 33.55 33.62 24.16 23.27 34.14 36.67 35.69 46.39 34.15 41.74 68.93 43.15 32.85 55.98 40.19 40.63 30.89 60 29.31 75.65 30.56 60.32 41.56 53.51 46.57 27.41 32.69 50.03 36.68 60.07 40.19 62.59 38.21 48.05 52.21 49.32 40.96 45.89 50.12 41.61 49.65 30.96 59.56 78.15 39.69 29.25 42.75 39.23 38.3 36.38 71.68 33.55 39.68 43.21 46.15 43.87 49.64 40.25 41.94 48.73 46.84 55.89 37.22 38.06 55.18 57.86 29.52 40.31 42.56 37.14 43.3 25.79 85.21 54.37 75.07 52.99 70.88 29 52.31 40.46 53.23 53.38 60.26 58.37 60.72 36.1 46.33 87.45 60.45 205448_s_at MAP3K12 55.12 79.41 85.57 82.74 83.69 214.19 104.34 175.34 161.5 55.02 100.95 52.02 82.93 101.74 101.29 83.92 55.77 61.06 60.48 44.14 66.11 57.74 41.12 67.23 46.55 68.19 70.8 46.6 39.13 43.27 52.43 55.98 67.12 81.04 58.71 49.47 80.99 52.92 115.66 72.97 55.41 62.72 54.26 84.26 76.78 104.66 52.51 48.57 78.21 91.32 57.08 72.9 111.37 57.2 106.56 121.55 63.08 48.2 117.34 74.28 93.81 55.77 68.96 88.61 50.5 48.52 55.21 52.05 66.37 54.77 61.79 67.13 46.59 60.55 116.63 94.59 63.08 69.23 88.71 85.55 83.05 115 97.01 131.57 154.18 114.8 147.8 99.78 92.26 99.98 160.6 115.96 82.47 103.31 141.28 62.74 140.48 113.73 122.89 56.02 103.9 80.77 116.66 125.37 78.3 105.69 215.8 127.11 184.82 585.55 37.83 86.43 58.42 48.54 50.52 39.11 194.48 115.13 54.63 73.12 110.01 51.37 60.46 65.15 139.23 169.43 178.54 132.1 88.54 52.16 96.95 87.02 121.77 99.83 77.6 151.38 76.46 66.05 111.05 69.79 119.44 81.48 85.24 83.11 100.16 89.62 63.35 101.59 126.48 171.82 55.31 121.14 60.55 85.42 121.86 64.48 125.01 68.16 181.61 47.76 229.76 103.08 80.09 124.22 134.22 94.29 93.02 105.25 141.32 90.03 128.05 99.86 174.05 66.86 168.76 66.79 84.49 210.03 152.12 116.65 115.85 75.44 93.79 97 77.9 111.32 86.3 106.91 67.51 74.22 115.53 123.87 129.17 118.02 142.38 76.9 55.73 162.74 98.21 63.67 125.04 123.73 124.2 160.52 76.53 129.98 51.5 77.92 123.67 101.74 89.04 135.13 148.14 93 161.75 161.62 110.11 79.99 98.78 125.65 71.13 76.7 107.72 100.66 59.22 70.07 100.94 205455_at MST1R 41.51 85.07 136.93 33.72 106.52 57.8 127.05 228.07 122.63 156.66 217.86 55.2 76.11 262.02 94.44 50.95 37.01 205.73 94.24 91.3 80.07 53.87 97.39 70.01 31.02 71.9 202.12 70.88 69.57 82.25 50.82 135.29 94.53 273.87 108.9 166.74 102.49 115.61 46.87 116.69 194.1 236.84 92.88 301.33 91.08 86.75 118.22 110.78 107.74 377.06 68.97 99.58 27.91 43.25 43.26 141.07 248.98 254.01 71.62 171.51 203.71 64.31 118.52 111.96 47.49 100.41 35.42 192.3 147.04 58.56 57.5 232.73 70.32 157.04 223.09 85.63 83.21 70.76 129.67 40.35 52.44 159.7 93.02 97.27 39.91 102.57 38.84 71.72 29.92 47.75 181.58 88.88 45.7 50.42 70.62 134.66 28.59 116.77 54.14 120.2 107.36 173.58 125.52 197.63 41.85 390.63 122.04 119.94 160.17 756.54 151.18 60.59 45.35 130.69 48.89 81.85 98.87 34.3 58.93 133.21 210.35 94.07 60.96 60.07 39.12 48.17 146.08 110.23 103.78 214.91 101.14 88.48 80.51 205.18 87.7 133.53 66.59 122.88 77.07 54.35 212.7 130.63 32.65 41.24 54.74 116.06 139 135.72 151.26 159.71 176.25 93.46 246.68 228.04 233.87 136.25 127.14 193.51 81.1 275.32 93.13 95.18 218.03 56.73 132.12 69.34 82.47 124.99 161.91 214.12 242.44 143.73 96.31 117.74 247.81 96.36 95.27 150.55 97.92 209.91 88.96 78.41 38.59 90.25 63.47 52.16 301.72 43.17 72.6 216.23 80.74 81.33 89.05 219.33 79.84 249.85 142.54 149 165.89 37.66 56.21 187.88 61.04 247.41 175.26 152.5 171.81 84.82 104.35 106.49 151.66 106.17 43.17 299.95 253.26 200.76 56.14 309.54 102.92 122.34 77.75 86.45 142.4 82.69 104.77 149.67 85.57 205486_at TESK2 60.74 308 67.52 165.25 121.69 87.77 124.45 87.36 192.57 139.16 116.7 239.44 90.46 272.64 93.7 95.84 111.97 79.37 89.23 148.39 43.79 110.67 142.44 56.47 94.44 79.23 68.82 59.93 108.48 64 170.52 281.59 57.72 274.11 49.42 51.51 94.67 134.46 92.63 115.9 174.53 73.09 79.6 61.63 71.46 152.46 96.21 92.98 70.07 89.07 144.69 101.29 105.81 77.44 98.87 205.25 43.67 133.36 306.06 76.83 165.58 81.41 138.97 148.6 76.79 56.98 95.3 107.62 57.3 102.97 56.07 114.75 58.92 68.26 146.62 131.98 131.14 108.36 92.16 195.36 45.53 192.12 148.13 290.75 66.53 122.17 87.66 81.92 109.91 220.99 35.38 107.11 336.88 122.42 230.01 112.7 190.98 161.93 196.63 69.17 111.52 169.43 68.54 201.61 175.41 242.21 133.61 209.54 229.06 72.02 96.88 178.1 91.72 87.35 124.99 134.57 30.74 52.27 80.76 187.33 72.49 63.67 65.39 100.95 72.38 70.39 44.95 63.04 145.83 70.23 65.42 139.36 86.02 104.81 92.74 66.95 92.48 60.82 161.18 162.67 64.87 52.6 129.37 148.67 79.92 99.92 131.87 186.43 125.29 137.97 77.25 128.57 69.48 168.79 105.75 103.35 223.06 60.52 167.97 101.57 248.21 119.42 125.67 155.65 67.92 75.41 67.33 161.39 71.37 183.29 103.23 91.99 113.43 82.03 93.8 85.97 82.3 48.21 107.49 200.42 64.96 85.18 57.71 79.04 43.39 61.43 70.56 82.75 127.23 131.75 54.52 141.13 86.98 142.99 118.14 52.66 149.89 62.93 162.04 103.94 126.5 173.55 190.11 27.13 78.86 160.49 77.17 86.15 90.17 99.06 79.2 73.21 126.42 101.31 107.13 105.88 225.95 56.23 88.62 149.73 129.65 116.48 158.36 57.2 120.77 58.8 153.22 205504_at BTK 102.07 85.36 115.55 232.94 112.42 144.56 87.94 66.03 142.4 115.94 61.79 77.69 84.27 65.78 92.26 58.08 79.41 286.6 104.89 57.36 71.6 133.92 66.09 174.93 93.05 406.85 94.95 89.58 182.78 96.28 94.55 78.89 75.19 75.97 106.52 118.49 127.8 101.01 167.46 174.78 186.37 145.23 97.81 90.08 80.41 92.95 63.11 110.8 138.99 70.69 67.55 120.17 88.63 159.92 61.45 77.26 117.37 115.55 221.87 86.77 70.6 66.22 62.58 92.68 825.73 87.8 75.57 193.85 81.89 70.37 58.48 108.21 65.56 93.59 60.38 36.82 57.86 163.88 59.34 59.76 51.06 44.61 34.49 81.54 58.92 89.42 62.55 78.67 98.39 112.96 73.24 102.55 58.37 74.72 92.26 83.36 72 28.4 60.15 76.67 52.28 84.1 90.35 71.13 61.38 97.01 105.2 78.1 72.85 74.26 104.75 42.11 165.5 78.52 157.64 62.04 44.57 131.43 109.81 101.15 92.23 86.96 120.84 125.52 68.01 125.16 65.3 70.94 62.11 90.79 154.17 69.99 61.61 79.91 73.73 71.97 74.65 64.09 64.75 79.05 55.91 55.67 60.3 71.19 85.57 83.18 54.05 49.01 81.03 112.99 41.29 186.31 57.18 123.15 108.58 72.85 92.7 262.76 67.18 85.98 83.48 63.99 72.31 47.18 6 121.04 61.91 157.9 49.15 107.13 106.1 92.6 90.83 131.81 89.21 104.43 155.26 96.15 104.99 53.5 121.82 100.49 68.69 60.48 107.52 90.52 77.26 73.35 59.79 51.92 70.68 65.71 73.53 83.07 37.04 105.93 51.65 85.64 101.42 91.77 58.49 86.17 144.85 81.69 48.63 56.54 50.69 69.76 118.77 61.3 63.77 96.97 99.45 79.79 65.95 66.21 122.51 91.25 86.79 60.74 100.61 63.03 35.07 38.94 41.51 52.02 66.05 205546_s_at TYK2 302.73 555.05 611.16 534.81 688.04 652.9 361.78 441.49 452.72 1066.94 570.87 514.96 557.95 877.44 661.79 616.23 370.76 869.97 659.45 374.34 612.13 494.62 598.24 755.71 369.9 596.27 536.78 520.68 513.51 317.94 324.21 623.24 411.63 931.49 555.32 652.44 859.48 598.53 867.95 738.64 801.65 775.13 647.15 633.96 717.59 526.86 1266.3 540.91 615.5 645.66 360.86 547.34 815.82 436.36 712.47 551.09 678.87 390.99 894.97 586.59 696.78 768.64 523.33 425.97 962.87 477.3 636.96 757.22 436.52 390.34 487.67 770.42 509.93 351.93 641.97 383.36 366.57 471.75 272.51 485.47 473.56 516.03 527.55 605.56 357.7 467.21 596.27 641.16 515.32 470 1035.13 486.11 347 242.06 269.63 402.1 403.17 607.06 307.17 414.38 756.89 566.62 584.22 399.47 513.26 875.75 629.13 760.95 640.43 1657.8 709.53 479.34 796.15 657.92 629.74 627.72 794.91 921.85 707.25 930.77 1070.48 924.04 928.45 765.01 461.05 318.97 518.72 478.28 275.51 390.26 719.57 471.35 663.16 470.83 467.94 441.91 499.68 352.17 462.59 466.68 328.86 384.64 527.59 467.93 418.33 620.19 522.2 496.52 707.33 486.15 834.03 897.3 518.47 556.76 666.92 599.81 407.09 725.73 676.59 792.22 804.14 544.92 592.3 672.2 538.19 703.65 622.25 710 583.78 1013.96 770.63 866.02 784.39 819.14 627.76 708.19 620.76 602.13 632.51 722.05 694.09 596.01 720.29 630.22 673.13 696.18 513.37 421.25 465.95 716.5 686.34 407.1 682.87 743.1 508.82 587.2 515.84 715.53 766.38 732.06 480.73 748.92 739.2 618.06 654.13 480.03 609.01 397.52 517.82 397.11 478.02 989.84 585.27 923.12 680.08 733.99 1540.58 357.85 462.16 710.09 448.82 308.52 416.9 317.92 320.78 389.05 361.67 205578_at ROR2 37.4 73.68 110.94 79.43 43.99 46.72 46.93 34.15 36.19 49.14 32 86.77 30.19 31.14 35.41 83.05 59.07 52.52 55.59 51.75 42.62 33.88 17.53 33.27 23.41 64.57 49.68 28.2 30.6 56.56 38.65 47.59 37.09 31.96 46.49 19.92 23.47 43.58 61.25 55.57 103.08 72.6 78.13 54.41 46.5 58.36 93.04 21.68 41.01 80.45 25.31 72.52 59.31 45.13 9.82 37.16 210.63 54.12 48.9 30.39 37.77 35.22 44.24 28.11 25.96 92.41 36.18 40.55 62.03 43.66 64.79 115.15 45.69 57.78 41.63 101.57 27.92 52.66 83.22 60.02 53.15 123.86 16.5 24.72 60.73 50.43 106.88 78.28 23.48 35.04 34.36 38.47 28.81 35.45 49.89 24.75 47.23 81.02 38.19 31.29 38.39 43.71 40.51 33.25 55.65 118.83 111.83 102 106.78 37.44 54.47 42.17 56.37 41.85 41.37 45.94 23.86 25.01 36.16 70.93 81.29 36.05 27.76 23.08 56.09 18.5 53.87 99.96 78.89 64.12 71.95 98.43 67.99 68.49 45.74 48.29 42.97 40.96 59.33 142.76 29.54 32.4 19.72 46.9 51.93 38.49 33.1 43.15 41.7 107.6 79.22 58.17 86.08 74.59 18.65 35.57 23.65 32.72 98.8 27.19 75.53 50.51 127.66 66.09 333.69 60.38 54.23 44.1 52.19 49.8 38.46 71.47 80.44 37.47 29.16 57.03 97.01 120.41 82.18 21.01 91.05 60.12 31.71 80.24 38.83 74.61 84.32 54.36 46.3 113.79 135.37 171.9 112.19 78.17 60.47 87.49 28.66 116.23 60.17 22.11 75.39 51.51 77.55 35.08 71.49 86.46 26.25 33.03 57.1 36 39.95 68.02 98.54 80.42 65.23 36.79 64.55 53.28 59.82 39.69 40.08 83.4 41.3 38.31 47.42 38.42 40.12 205698_s_at MAP2K6 48.74 40.9 61.98 59.92 38.26 23.36 44.95 29.44 76.21 71.33 124.39 83.96 54.08 32.78 64.24 120.5 285.12 65.66 68.82 27.98 50.99 43.11 211.81 64.42 30.7 84.45 37.33 130.63 61.03 71.86 34.91 72.36 111.84 32.66 293.34 179.29 102.65 63.65 70.1 72.43 75.64 148.56 78.59 39.3 126.37 46.82 56.52 84.85 46.01 49.11 824.91 43.98 42.48 79.21 26.37 39.19 58.76 70.1 59.09 196.51 33.13 43.46 35.46 40.79 76.16 53.68 62.88 69.38 72.56 54.53 60.07 76.32 54.47 33.66 35.41 42.83 63.29 67.12 38.33 48.46 37.87 37.54 45.77 46.43 37.38 68.73 36.55 54.24 40.84 30.28 67.12 38.24 44.44 35.44 33.24 81.03 31.04 51.27 71.6 36.86 27.84 140.51 38.06 36.68 45.64 54.33 35.39 56.07 125.38 46.84 95.83 102.2 65.03 184.27 69.05 86.81 37.79 44.02 104.52 204.27 46.55 46.62 85.07 69.53 41.3 44.41 27.2 43.63 82.32 59.37 62.24 42.31 70.86 50.7 39.46 63.45 61.9 37.48 31.64 45.83 33.12 40.35 81.02 46.8 41.29 40.52 43.76 65.04 32.58 38.7 74.36 59.38 33.66 35.65 62.43 72.47 32.99 46.43 56.26 56.7 45.24 113.18 33.61 62.13 35.86 48.71 62.99 55.05 49.09 63.12 57.89 49.05 44.61 97.4 26.56 75.88 70.45 38.31 31.65 52.25 52.03 36.03 44.14 60.12 52.9 49.8 38.75 28.59 36.96 61.74 28.13 58.74 49.93 33.69 128.79 88.02 43.02 35.61 50.86 30.58 52.16 44.48 48.37 51.6 36.37 34.55 37.97 76.23 39.5 167.27 45.57 42.02 39.12 149.89 24.78 24.29 32.53 26.38 27.71 26.4 53.93 39.87 21.56 38.29 49.16 127.16 89.07 205880_at PRKD1 24.63 66.76 119.95 58.12 44.8 37.33 93.14 36.83 28.45 33.29 19.49 36.68 24.95 20.45 43.93 71.79 15.19 9.54 33.01 42.46 28.58 28.04 8.64 18.69 24.76 60 51.87 26.53 23.95 28.67 87.53 17.93 21.91 15.99 38.88 20.32 18.61 47.48 63.84 19.52 15.31 24.07 14.93 43.95 39.8 68.32 58.85 41.49 31.79 44.95 18.63 31.32 32.03 41.54 11.19 23.18 24.14 12.92 22.45 27.35 22.42 14.18 48.26 16.45 12.32 54.28 19.66 25.83 12.09 36.18 36.13 26 19.64 47.4 27.64 22.15 22.9 40.48 20.26 41.42 23.68 18.16 17.65 18.57 63.55 64.66 64.67 62.76 14.86 35.51 19.97 48.29 16.17 79.77 116.01 52.01 96.36 78.88 60.35 75.87 35.79 82.09 128.87 60.51 115.05 42.71 97.93 45.72 64.82 13.01 25.12 93.35 44.52 44.48 33.48 114.69 43.12 18.87 41.79 47.58 76.09 11.16 18.13 28.33 122.58 15.13 32.78 104.72 54.91 44.82 53.2 57.1 106.2 34.89 76.77 43.71 43.69 65.34 123.33 91.69 26.07 44.27 24.98 80.25 46.8 20.31 52.75 39.5 44.41 56.97 30.31 37.85 35.11 79.82 17.95 24.46 15.5 15.3 90.06 13.08 27.73 50.42 23.6 22.84 28.84 41.86 29.89 36.96 13.84 16.16 29.72 46.15 54.66 28.91 28.84 60.52 57.92 28.69 79.75 29.88 58.2 22.29 28.62 54.09 68.91 26.95 62.8 30.49 16.8 17.31 99.59 138.6 70.48 40.93 73.91 76.96 21.12 63.39 49.63 24.31 19.49 52.79 34.84 18.95 23.39 79.69 24.07 21.46 86.82 49.76 27.73 51.99 59.17 63.21 29.28 32.47 49.26 34.28 71.67 33.82 24.4 76.44 55.28 62.99 55.05 16.17 75.76 205977_s_at EPHA1 273.95 100.72 99.79 166.05 168.83 130.69 127.92 125.83 76.88 129.19 147.43 119.02 110.32 85.29 74.39 69.72 100.47 104.91 79.91 64.42 105.32 88.95 374.69 112.58 110.3 115.44 131.38 46.81 73.41 123.68 64.73 91.69 78.55 123.69 68.02 161.43 89.65 144.19 51.8 188.98 247.99 152.63 92.45 113.64 105.85 100.06 91.66 117.45 168.28 101.32 74.09 83 97.94 97.75 151.43 95.55 67.73 105.09 256.19 131.75 362.92 73.9 393.76 63.51 38.67 70.12 56.7 54.25 86.5 76.64 117.85 92 103.47 92.2 127.96 116.96 94.91 103.88 94.28 173.13 91.09 97.28 134.53 125.36 86.32 106.21 107.56 128.71 138.86 62.75 104 113.4 85.17 52.86 48.09 116.82 91.17 170.21 65.75 55.98 163.45 165.11 155.64 88.06 108.7 172.39 97.78 174.5 185.69 307.87 77.21 106.18 135.65 94.9 125.09 105.93 108.37 121.78 169.15 158.82 156.9 160.8 124.81 192.69 44.87 69.72 87.91 113.16 58.25 84.77 106.32 148.54 101.82 171.17 107.07 96.46 127.94 120.23 107.05 124.55 95.7 115.9 74.98 144.98 83.28 159.22 120.57 154.07 130.46 100.2 138.47 138.25 246.53 160.44 242.64 114.22 109.16 117.8 108.03 124.62 173.81 135.35 90.32 65.74 181.4 139.23 110.41 213.65 63.62 113.03 108.52 109.03 105.36 90.87 123.77 345.49 100.27 123.2 94.56 86.85 130.2 247.72 110.06 81.34 87.05 75.2 185.37 90.97 286.5 232.58 85.37 89.83 84.48 137.8 123.19 151.42 249.85 113.92 220.94 168.31 172.8 108.02 123.42 132.41 95.54 79.49 76.2 119.23 52.21 82.58 117.43 115.3 113.84 99.83 103.9 119.29 172.62 74.19 96.19 126.31 187.04 174.83 276.33 142.43 110.35 103.9 89.26 205986_at AATK 57.73 55.72 69.04 98.1 52.62 68.53 62.76 92.44 77.45 66.45 49.59 54.97 51.71 82.34 56.85 54.46 63.48 56.48 45.91 47.98 45.19 50.07 48.9 79.14 62.2 61.77 51.65 65.24 61.9 107.46 65.77 78.32 56.17 72.34 82.54 72.34 55.16 62.94 73.51 76.18 49.71 59.65 60.06 60.44 75.21 60.15 61.08 62.61 62.51 79.79 46.11 55.57 103.03 51.82 62 61.43 77.26 83.64 52.95 71.37 60.33 55.23 90.96 63.5 41.82 53.35 56.79 40.88 46.16 36.81 72.04 53.24 50.38 47.55 56.8 63.8 99.17 97.03 162.51 100.7 128.4 42.27 64.66 53.65 77 81.42 71.68 59.32 66.83 57.5 190.48 48.78 71.96 72.71 54.64 62.48 85.65 71.8 71.45 65.67 72.07 63.6 88.64 57.49 53.38 90.65 70.85 74.75 91.05 195.04 92.69 69.36 63.36 72.15 66.94 85.02 79.48 78.04 65.57 90.5 74.7 102.18 101.68 63.92 86.85 89.48 66.41 63.45 73.67 72.13 67.69 86.57 113.07 97.32 88.46 95.66 120.84 88.07 85.42 91.96 100.36 79.35 67.23 87.7 81.55 82.68 87.88 81 61.09 69.17 68.99 72.87 87.91 85.79 71.94 71.01 106.21 56.42 53.21 74.46 72.86 74.78 74.59 91.63 81.3 73.02 57.62 63.03 159.25 87.98 97.98 97.42 84.77 64.16 122.65 100.34 72.58 78.82 64.65 105.35 62.57 108.46 50.62 73.2 65.63 79.34 73.18 80.61 80.29 89.15 65.34 69.19 120.25 90.26 66.61 94.46 62.4 88.99 79.6 88.03 108.01 70.74 68.74 145.28 94.43 98.24 91.65 88.47 127.3 75.18 77.54 86.98 81.31 90.82 95.04 111.94 82.81 91.3 94.05 103.52 44.17 64.86 49.43 49.67 38.67 49.17 49.37 206028_s_at MERTK 84.13 137.32 94.29 101.8 125.41 99.31 77.69 134 104.74 86.26 76.23 67.93 43.07 135.91 100.94 278.68 89.79 196.18 136.49 40.17 88.31 141.24 88.23 61.32 149.22 309.18 95.45 249.66 55.53 77.35 115.85 78.59 154.51 46.35 153.75 84.89 95.15 249.01 241.19 229.1 178.27 165.09 129.12 116.97 115.67 90.43 99.91 77.1 209.25 93.65 48.54 69.98 90.44 192.42 29.64 126.34 195.83 173.37 60.82 160.48 87 45.41 78.8 37.68 34.26 70.55 58.9 216.97 81.81 94.96 82.02 78.06 78.43 132.18 50.64 81.08 91.37 113.35 63.47 194.35 44.58 162.78 53.77 59.68 85.22 156.04 106.38 111.88 58.4 72.06 28.35 98.39 81.93 110.19 141.47 89.05 158.2 58.67 101.59 91.63 58.95 107.19 101.82 64.67 66.42 217.86 155.25 56.74 114.98 29.5 123.75 119.74 137.11 85.35 142.45 107.25 42.11 100.66 96.75 164.29 108.77 94.6 93.42 127.81 133.86 60.43 81.54 91.02 78.34 157.88 133.35 112.93 81.68 108.67 132.16 78.19 120.86 92.38 86.74 163.54 84.98 81.41 87.51 111.63 126.09 64.25 39.87 43.76 77.61 109.1 53.86 127.11 112.19 145.75 135.59 106.28 97.64 225.63 81.18 145.98 148.92 127.92 205.67 35.89 108.89 232.1 84.08 108.98 16.19 105.19 131.09 108.85 86.5 177.25 16.98 139.68 119.1 166.61 79.22 46 70.03 205.24 110.29 75.65 84.45 76.86 61.94 59.99 135.15 35.57 94.35 68.94 90.38 144.1 70.78 81.09 109.13 69.53 85.85 48.38 28.62 125.86 69.92 27.61 52.23 65.66 72.51 73.29 214.1 88.55 79.62 75.7 70.87 137.78 151.26 57.12 75.12 93.04 132.58 91.75 96.38 149.24 49.85 92.9 64.27 74.73 128.4 206071_s_at EPHA3 11.99 10.5 14.43 12.12 9.33 10.72 10.34 10.83 48.88 11.06 10.85 10.94 10.15 12.33 12.02 12.06 15.26 9.45 9.7 9.35 13.88 10.19 12.18 10.8 11.39 9.84 10.54 11.06 11 10.07 92.78 12.25 10.09 10.28 11.08 13.17 13.78 11.09 13.55 15.96 10.58 10.96 11.35 9.72 12.61 13.05 13.32 11.94 14.34 12.15 12.69 11.35 9.71 10.09 139.55 11.93 10.54 13.89 12.61 12.24 13.36 15.22 11.32 14.53 10.9 11.5 14.87 11.79 12.13 11.83 15.56 10.85 14.28 12.23 12.96 12.2 11.03 10.61 14.59 17.3 10.06 13.53 10.07 12.42 9.37 8.43 9.84 8.64 10.26 11.68 11.25 12.54 10.99 9.1 11.04 12.27 12.55 9.96 9.68 10.04 10.44 9.45 9.83 12.32 9.71 12.16 11.11 11.15 14.12 45.99 9.89 9.41 11.37 10.97 12.29 9.3 11.47 12.38 10.52 12.32 12.63 10.65 10.15 13.91 13.04 11.82 9.15 17.35 10.99 11.51 9.79 10.65 12.51 8.7 8.91 13.5 10.49 11.3 14.99 11.84 10.84 12.6 10.41 21.89 10.39 12.37 10.18 13.01 11.88 17.55 11.61 12.64 11.66 11.65 10.98 11.7 11.81 11.91 9.94 13.63 11.64 11.09 13.26 38.18 11.19 12.1 11.93 10.71 16.19 11.87 12.5 11.33 11.67 10.98 13.2 11.68 10.62 11.09 12.7 11.71 11.04 10.94 13.42 10.7 21.02 12.51 13.98 10.57 11.96 11.23 11.09 10.58 12.65 11.46 10.65 12.45 13.35 10.86 10.11 16.97 12.4 12.46 12.77 17.23 13.51 17.17 13.37 12.01 13.36 10.1 9.59 15.21 19.13 9.95 10.92 10.04 13.24 10.1 10.4 10.07 7.73 8.78 6.72 8.14 8.95 7.22 8.43 206114_at EPHA4 61.42 32.9 77.88 61.54 50.5 53.79 34.69 32.34 37.43 51.68 53.15 51.8 55.37 86.19 81.07 209.26 43.73 56.58 27.34 24.65 43.81 47.23 44.93 51.69 43.55 69.19 31.87 197.01 32.32 31.34 169.92 362.21 24.19 49.97 59.18 41.09 50.71 38.95 80.87 80.23 29.33 73.92 35.19 39.14 80.78 87.08 54.43 76.92 40.49 33.44 49.37 44.97 50.9 45.43 58.07 44.18 31.65 39.34 41.63 63.28 40.75 269.57 175.95 41.12 50.75 82.05 115.19 159.93 39.42 117.42 273.67 144.95 117.99 52.75 137.08 31 75.15 80.48 31.47 68.77 54.68 36.66 79.31 43.88 62.51 259.12 40.32 75.17 98.7 136.32 81.83 85.04 50.4 58.15 83.25 54.3 44.44 58.17 46.83 66.74 75.78 72.46 27.52 67.09 39.9 252.68 40.53 38.56 41.59 51.91 108.64 105.66 60.69 45.64 92.73 42.01 26.5 155.02 109.51 87.13 156.51 146 102.88 178.14 150.16 30.3 49.73 135.22 32.61 64.57 74.09 64.5 307 68.03 59.62 50.05 117.29 155.86 60.41 46.32 38.84 43.52 117.89 49.98 68.43 95.72 74.97 98.35 113.33 52.53 34.72 97 53.53 69.79 45.11 101.8 34.27 40.35 39.6 55.5 103.89 54.58 96.08 52.66 38.34 71.3 34.74 104.51 89.78 47.32 43.65 53.34 72.23 40.58 146.97 63.31 60.64 38.64 61.76 96.95 54.61 70.62 48.42 57.02 70.65 57.59 49.11 61.91 65.21 34.19 56.24 45.55 37.6 19.19 70.4 69.38 36.93 56.02 98.61 245.08 35.72 37.74 28.96 30.1 73.01 93.2 51.06 55.33 64.02 28.46 66.69 61.61 58.49 64.33 278.99 33.36 24.23 31.64 66.57 75.05 51.04 120.59 46.28 69.57 106.73 36.87 61.25 206216_at STK23 54.96 32.24 64 60.14 666.79 34.49 47.18 46.24 51.89 122.61 46.44 35.45 48.27 48.01 220.86 41.21 76.39 43.86 57.12 37.16 58.58 41.66 162.15 55.43 73.81 47.99 74.15 91.13 66.08 74.64 43.95 148.9 36.74 54.49 145.98 52.83 62.79 55.15 53.21 121.12 54.52 71.43 116.11 118.17 83.43 54.63 41.12 49.99 61.57 77.5 68.67 98.96 260.86 84.55 66.32 133.65 77.85 58.3 98.32 66.23 97 65.38 55.93 68.33 39.32 50.97 56.16 48.26 58.63 46.06 73.65 58.02 50.65 96.4 138.36 49.95 45.19 29.37 42.94 50.64 96.47 172.26 48.29 52.96 48.14 32.57 147.8 55.06 52.08 29.59 135.89 36.02 36.65 34.24 50.36 317.75 54.17 161.69 32.65 332.01 41.48 63.56 87.23 39.84 46.87 36.03 39.03 30.57 35.28 57.2 65.71 37.1 111.59 54.3 150.1 67.04 64.66 75.04 112.76 77.06 85.7 312.75 297.9 79.21 72.99 32.28 43.06 49.57 35.26 59.32 96.92 144.44 97.11 156.09 37.38 28.25 44.1 47.56 30.91 38.26 35.34 39.31 35.14 36.85 35.99 49.47 62.71 42.34 56.32 40.03 53.53 60.34 54.26 34.34 45.71 72.28 55.73 50.89 44.08 59.82 50.42 67.75 55.69 52.41 47.78 58.25 46.94 127.4 67.75 60.69 52.69 56.04 105.5 45.44 314.81 69.6 170.06 38.8 56.18 46.13 49.98 144.75 39.74 48.09 58.63 109.71 169.26 62.43 110.6 53.53 59.61 41.86 64.72 286.69 56.85 621.98 321.17 46.21 512.86 75.96 69.84 53.97 36.61 262.57 97.3 135.68 76.09 94.52 63.71 54.34 112.9 105.2 48.42 209.48 43.96 41.09 77.8 236.63 53.53 48.21 154.42 37.65 117.97 63.64 83.68 50.09 47.19 206267_s_at MATK 40.36 35.84 50.37 58.88 37.8 47.8 46.03 28.63 33.68 49.73 39.7 40.34 46.96 40.14 44.94 43.79 38.83 50.4 38.6 36.16 54.64 53.19 37.77 54.2 102.02 54.77 55.48 38.36 49.88 55.61 45.79 39.94 69.03 61.72 50.97 60.33 56.16 48.93 66.37 72.17 51.21 70.68 68.75 52.71 58.69 56.8 39.67 54.82 67.77 57.44 38.9 55.9 66.3 50.92 53.04 43.41 63.42 60.05 71.82 60.32 36.04 37.66 34.52 77.85 51.15 49.83 61.53 76.37 70.21 40.26 50.66 60.76 38.29 37.4 37.35 45.44 34.27 51.87 50.27 53.38 45.85 47.33 45.59 63.06 56.27 48.99 52.77 99.08 79.73 50.87 86.95 47.36 38.05 34.52 41.09 51.74 35.17 32.87 41.19 45.33 31.6 45.33 53.95 40.89 29.45 47.87 40.56 35.07 34.06 83.69 78.04 56.98 77.98 69.29 73.73 68.84 62.72 72.07 133.6 60.01 49.58 70.08 72.99 70.17 40.76 55.74 46.49 37.16 32.46 41.05 43.36 44.05 36.68 60.01 33.83 41.07 46.56 46.99 33.22 31.36 37.1 30.92 44.25 37.64 41.28 46.21 30.7 55.44 67.94 188.62 56.81 78.63 76.1 80.67 82.77 59.51 63.19 98.31 51.62 63.88 62 52.82 50.45 60.51 49.27 73.12 43.82 76.72 53.12 56.12 76.16 64.11 49.7 68.45 50.73 64.43 77.98 43.87 71.14 58.71 103.15 102.36 65.46 52.98 60.15 51.82 48.23 56.39 72.95 107.92 50.8 48.31 68.82 80.3 67.37 61.9 43.29 73.45 58.74 63.45 79.2 67.39 64.12 84.45 51.34 47.01 46.72 73.84 68.23 54.01 63.62 53.76 81.23 57.06 46.1 64.62 65.07 74.86 62.08 46.66 113.28 45.08 49.85 59.57 66.34 44.96 57.58 206412_at FER 78.55 106.78 111.97 66.91 59.4 67.6 59.81 110.19 105.48 42.47 70.61 64.96 56.88 63.52 55.57 59.98 94.43 64.13 115.5 184.62 69.67 69.85 79.95 77.2 65.24 70.18 61.71 82.54 87.56 99.67 91.58 60.94 61.05 74.9 73.12 82.98 56.14 76.88 74.35 59.56 59.53 46.2 77.49 92.67 60.9 94.96 93.63 84.09 86.42 71.98 86.81 72.97 76.79 86.02 87.87 77.57 61.44 73.5 58.43 84.94 49.02 86.63 121.08 79.23 48.67 88.07 110.37 80.95 67.98 92.22 63.59 76.27 77.48 142.38 70.87 77.73 102.03 124.52 163.45 101.81 92.12 142.4 111.4 101.58 127.07 118.52 104.11 109.82 85.3 67.44 84.63 72.98 46.27 83.49 56.63 62.99 63.09 54.34 56.96 53.78 75.23 71.73 65.16 62.46 80.54 61.82 78.38 71.26 68.91 44.82 54.74 113.51 94.77 93.24 66.31 62.07 64.28 54.96 65.64 70.03 75.76 60.47 59.15 57.62 100.37 62.03 70.46 103.1 69.32 54.18 66.38 58.67 68.11 70.75 65.14 77.98 67.67 96.98 67.79 67.79 62.19 68.78 55.66 72.24 88.47 59.8 58.17 98.84 73.2 54.49 65.49 67.9 92.22 88.32 83.58 99.41 82.01 76.56 87.91 75.45 73.66 82.87 86.42 54.5 78.75 81.79 79.47 66.14 54.48 68.8 75.89 62.83 70.97 73.15 53.01 51.61 61.76 65.25 77.72 46.96 68.4 65.39 66.26 61.76 83.28 69.09 69.49 72.18 53.82 71.97 76.32 104.52 45.36 47.42 57.45 47.7 50.19 62.86 51.09 50.54 45.32 45.49 44.49 40.32 46.36 55.09 36.93 57.2 66.55 49.1 50.3 46.51 63.1 63.79 58.96 83.8 71.38 63.85 75.33 62.63 51.68 78.12 55.9 95.65 80.86 79.85 78.89 206464_at BMX 9.85 8.59 11.92 13.37 14.55 14.44 13.18 9.88 10.01 9.58 12.11 9.22 11.16 15.78 10.54 12.39 12.18 8.5 9.31 10.28 11.59 10.5 8.19 13.35 9.87 9.93 10.44 9.45 11.04 9.72 11.31 10.38 11.09 9.68 10.76 9.88 8.76 9.31 19.47 11 10.96 9.76 9.69 9.46 10.67 34.87 11.56 9.35 11.19 11.28 8.77 9.23 14.73 8.65 10.39 11.96 9.39 9.3 10.87 11.83 12.42 212.17 10.33 16.69 13.74 8.51 8.92 8.35 10.17 10.69 113.29 9.16 9.01 11.56 9.74 9.14 9.51 11.86 9.41 11.1 10.92 13.38 12.33 10.96 27.73 18.12 13.12 15.31 11.26 12.64 14.32 12.5 9.53 10.72 13.81 11 9.96 11.44 11.89 9.12 16.65 10.68 9.57 11.99 13.02 13.36 11.79 12.84 12.09 12.94 12.1 14.67 13.98 12.93 11.41 20.22 14.41 11.84 12.88 14.63 26.91 13.91 13.61 14.78 34.32 21.55 12.39 16.82 10.57 9.92 9.28 10.61 22.25 13.02 9.95 10.99 13.23 16.49 11.9 10.89 12.85 13.34 11.27 10.69 14.2 13.18 12.88 16.24 28.02 16.95 11.4 16.53 11.32 15.56 11.15 16.7 12.76 10.88 12.78 12.34 11.39 12.89 14.36 15.35 13.12 14.57 13.13 16.27 13.24 11.82 12.11 12.95 11.7 11.19 15.51 13.1 11.26 10.87 14.05 12.43 11.01 9.44 13.08 13.14 10.24 16.75 12.94 16.34 12.8 11.11 10.12 16.4 9.27 12.41 16.3 10.3 10.2 17.7 13.29 13.51 20.55 13.08 12.72 12.15 262.55 11.47 12.03 12.69 12.05 11.47 12.96 18.17 22.45 17.33 11.69 10.9 10.51 8.98 13.15 14.04 9.46 7.58 13 9.42 7.1 8.77 20.2 206674_at FLT3 18.1 41.75 18.01 31.53 38.8 16.88 54.45 65.12 16.29 18.62 27.49 91.94 44.22 16.17 18.76 33.66 15.62 23.88 21.16 16.17 85.46 28.68 13.82 20.53 18.77 32.02 16.3 18.17 25.82 19.39 22.52 16.46 16.11 18.19 19.32 21.12 31.32 26.73 42.33 28.53 53.92 27.04 22.12 23.74 20.72 24.87 19.72 25.45 24.47 18.54 19.65 21.6 27.03 20.54 17.33 19.54 19.87 21.24 51.18 19.55 15.26 17.22 18.89 28.71 29.32 16.33 23.77 71.45 26 20.01 19.76 24.29 16.55 16.8 15.29 16.96 15.3 40.88 18.54 17.04 22.16 16.27 17.71 81.98 44.63 25.43 25.27 63.5 24.96 43.03 47.01 48.24 188.78 75.91 35.13 25.12 58.1 18.81 30.1 27.81 22.97 56.69 20.41 38.81 12.54 24.39 44.69 18.93 21.93 34.74 17.49 16.65 29.01 15.66 26.41 35.46 56.18 23.83 30.01 20.95 28.63 21.6 31.37 34.21 32.21 39.5 23.23 29.47 23.45 20.05 26.55 19.74 24.88 42.23 19.14 36.13 31.76 29.14 26.32 27.37 22.79 21.93 28.24 23.43 34.54 24.88 23.21 67.47 23.43 77.49 23.41 50.94 22.5 23.88 26.85 22.9 24.36 37.2 23.57 41.86 26.29 50.24 24.47 32.1 30.17 28.05 22.08 42.23 23.74 23.32 35.46 34.07 34.59 27.32 26.39 31.58 36.71 26.37 38.46 82.77 50.54 24.5 21.56 20.43 23.75 20.51 26.65 138.18 24.85 22.72 23.61 28.56 55.01 18.18 30.14 21.05 17.29 24.12 39.2 16.2 17.86 32.84 48.06 22.5 19.2 19.45 18.72 18.62 17.34 24.95 14.78 15.68 26.21 44.63 41.74 24.74 23.77 25.15 29.78 23.65 25.86 14.6 14.72 18.61 14.01 21.16 56.91 206702_at TEK 40.85 41.57 88.18 76.2 144.59 124.38 66.32 70.82 78.38 26.07 75.04 39.64 71.5 59.37 71.03 32.42 76.8 25.2 90.09 36.03 79.74 126.27 37.02 21.11 43.37 60.04 62.08 56.9 38.29 27.84 61.52 27.99 46.28 45.86 39.52 22.06 34.77 42.76 230.09 26.86 43.67 40.83 17.32 31.88 76.28 165.76 27.03 21.37 66.44 47.6 35.76 84.56 63.27 41.55 19.48 61.96 35.8 28.58 44.4 46.1 41.46 21.78 71.1 36.98 19.08 21.98 21.27 48.51 18.38 42.31 21.98 45.76 28.35 42.52 21.71 27.18 25.15 99.61 34.98 24.12 34.99 86.93 43.88 83.25 133.66 87.01 50.18 93.19 53.98 124.77 41.25 98.9 55.89 51.04 172.01 52 59.81 50.21 113.83 46.89 53.35 90.62 35.05 184.96 39.9 57.87 58.79 68.53 46.01 17.46 44.96 76.62 40.19 23.11 44.86 46.88 31.37 40.26 35.75 46.38 116.49 33.84 30.52 38.03 205.87 64.1 46.84 160.47 31.62 24.91 47.73 34.77 239.29 68.29 43.63 39.08 43.43 102.19 68.13 25.16 44.05 34.27 31.92 55.27 91.79 33.77 36.06 93.96 106.47 56.99 14.88 54.74 17.01 66.51 22.83 31.29 24 32.34 46.1 26.05 39.77 57.63 26.23 40.09 31.38 64.97 44.56 103.91 16.09 40.19 57.2 52.64 66.55 39.19 17.63 47.87 47.54 35.46 136.78 22.48 60.73 50.31 57.26 62.11 67.93 102.73 60.51 117.39 22.24 28.04 33.15 82.37 31.61 42.55 94.9 36.37 25.98 74.98 47.81 37.45 51.92 47.23 45.41 14.6 19.95 59.41 20.35 25.64 24.93 26.86 60.47 87.45 105.73 50.12 46.08 44.93 23.43 29.53 54.55 90.52 38.55 33.23 72.87 38.56 34.62 20.08 91.89 206854_s_at MAP3K7 228.23 220.99 171.32 219.68 325.57 240.59 170.77 139.35 208.85 202.43 289.22 225.95 239.25 209.53 159.9 249.46 199.87 239.96 529.24 720.02 216.04 158.46 307.35 179.63 243.45 293.54 251.85 287.57 263.04 243.52 197.37 192.71 222.73 172.51 200.23 210.1 228.1 125.79 205.95 157.96 274.13 173.92 184.88 321.17 209.54 154.14 330.93 148.2 140.87 203.5 204.02 190.6 212.41 238.85 176.52 191.49 271.11 402.76 170.04 155.15 176.81 397.05 311.91 216.37 353.05 389.73 364 379.26 163.47 417.83 309.84 259.96 322.54 228.73 282.73 286.25 596.04 324.39 181.36 114.69 138.26 300.09 361.3 279.85 265.08 284.96 397.24 218.99 169.36 198.09 31.21 164.47 118.2 213.57 246.76 207.22 152.19 244.43 105.46 214.59 362.97 222.75 164.82 116.98 234.17 240.47 262.33 235.35 109.56 18.25 227.57 257.79 345.18 362.02 158.25 259.75 554.98 286.21 222.51 152.78 269.67 436.28 148.91 245.3 175.65 107.15 223.75 235.57 199.16 239.19 226.41 163.15 116.4 164.08 286.99 147.12 346.99 334.63 178.72 188.73 295.63 219.24 312.19 241.93 759.14 189.62 238.74 200.1 199.29 186.53 185.27 145.62 130.03 153.47 228.25 231.21 102.98 213.86 239.31 250.08 134.24 215.73 110.17 113.26 172.13 200.92 188.97 188.12 105.09 114.98 153.51 124.15 168.86 283.02 27.74 137.65 157.87 143.52 205.3 145.91 177.7 130.14 162.82 129.76 138.06 100.07 205.37 183.92 206.68 212.24 159.22 200.6 174.9 179.74 177.16 115.84 171.24 109.2 182.34 266.06 152.73 137.97 191.73 29.64 125.96 162.96 227.88 229.74 70.14 148.18 247.94 206.45 115.77 113.82 41.74 24.74 23.77 25.15 29.78 23.65 25.86 14.6 14.72 18.61 116.88 142.33 274.2 292.97 136.31 176.22 312.93 249.8 218.49 296.43 274.1 205.22 192.23 206875_s_at SLK 354.58 442.79 179.08 215.52 266.46 263.87 205.45 443.36 296.47 331.07 383.06 470.06 283.17 411.84 180.19 191.42 193.48 152.38 241.62 527.05 319.26 346.35 327.61 150.09 211.68 297.6 264.44 384.09 219.01 506.88 369.99 177.2 350.17 330.13 182.7 135.98 168.67 182.41 209.37 117.3 297.2 148.03 219.92 195.66 213.96 245.55 146.99 140.06 177.48 296.86 352.51 225.14 118.84 288.54 247.84 395.76 132.44 181.31 232.91 116.7 282.77 155.1 308.49 160.65 82.65 206.17 141.83 187.11 176.99 229.1 264.66 190.29 177.13 371.99 202.23 195.49 486.43 352.72 488.67 233.22 152.87 670.12 395.73 364.1 277.18 305.58 223.85 333.42 157.72 344.78 28.56 430.71 413.69 591.47 400.24 321.17 198.32 375.8 351.47 234.71 185.71 459.19 322.41 261.04 347.84 154.48 253.32 232.08 239.35 18.82 179.17 268.34 181.01 205.25 229.93 158.01 194.88 90.26 138.67 97.07 110.71 100.83 58.17 90.32 220.2 229.92 207.01 261 324.2 181.82 197.62 244.66 70.12 173.82 365.79 204.61 127.83 334.93 229.68 476.66 386.45 245.24 357.54 321.85 213.23 142.91 253.13 229.04 201.52 487.36 252.48 127.89 362.49 277.5 262.86 198.73 293.13 206.35 300.09 240.26 190.62 354.17 361.41 165.99 109.85 204.39 362.41 165.8 48.04 317.67 92.25 101.92 237.21 165.55 24.91 118.08 194.01 191.01 192.87 181.71 266.48 130.99 281.41 254.9 114.51 128.47 144.75 235.13 145.34 158.63 201.21 300.96 182.04 169.02 186.03 123.61 220.12 116.83 100.52 69.7 166.59 225.98 231.91 12.68 117.62 151.44 224.3 101.59 133.49 172.81 111.61 141.06 152.28 138.07 139.25 211.25 173.62 254.04 231.71 204.5 155.64 223.27 206.21 300.22 292.57 143.07 223.96 207011_s_at PTK7 315.3 290.05 584.69 204.58 276.76 103.28 292.61 146.84 136.52 438.07 422.61 311.36 231.84 142.07 297.35 366.3 397.76 370.53 532.44 488.33 213.8 544.38 279.03 369.27 575.53 322.7 464.28 712.31 205.82 353.96 291.01 460.08 167.44 86.7 288.94 315.86 239.79 338.95 299.6 238.36 225.94 207.84 319.87 661.07 326.08 500.94 486.34 209.72 718.88 298.22 299.2 312.47 434.76 750.82 45.21 290.47 312.28 316.02 341.8 318 297.54 796.85 522.33 184.96 64.59 214.93 94.27 108.87 156.49 338.47 502.5 283.57 294.55 234.95 191.6 288.16 203.25 150.2 100.35 225.63 216.85 117.28 138.77 98.08 159.38 201.44 340.98 288.64 85.22 79.19 217.25 138.78 98.44 95.04 136.65 95.5 155.5 300.35 55.29 191.14 331.91 195.98 230.75 124.67 356.65 287.21 458.13 498.13 418.56 197.64 296.48 243.23 541.76 464.55 284.96 199.02 375.49 159.72 307.2 242.8 277.33 595.36 236.17 203.62 118.76 57.34 138.29 185.82 141.17 238.96 165.06 196.2 128.66 309.34 206.93 103.81 343.43 227.23 297.68 258.42 94.63 228.2 188.44 175.21 88.49 101.51 198.98 202.57 198.88 196.62 265.3 266.23 446.8 391.7 161.05 555.89 90.39 122.29 442.01 214.82 203.81 361.13 201.55 247.99 230.92 284.6 170.95 257.35 394 165.83 81.38 265.2 223.65 203.22 300.91 335.34 255.29 222.26 301.41 212.93 268.61 209.9 126.01 312.48 219.42 113.16 227.05 142.44 194.78 638.54 329.39 372.62 430.2 316.47 227.66 305.56 403.73 263.05 228.41 355.13 180.38 223.17 282.35 114.72 247.41 191.97 149.3 242.46 178.94 187.86 331.61 285.11 288.19 155.24 174.89 159.46 175.38 231.32 170.96 208.99 285.73 477.32 294.79 387.53 376.28 247.98 163.7 207106_s_at LTK 44.18 37.19 53.25 74.07 35.22 38.61 51.2 37.95 34.39 52.02 42.17 39.34 47.67 34.29 44.78 47.65 43.06 54.09 45.95 37.33 44.27 62.01 45.44 58.9 38.54 35.13 39.76 34.85 54.51 64.38 43.34 50.65 52.88 61.94 47.58 54.82 60.75 47.22 48.12 60.28 44.48 59.97 74.99 51.57 69.6 51.8 44.59 54.02 52.93 42.91 50.6 51.2 55.86 42.09 51.98 36.06 57.5 64.02 58.43 42.32 39.36 33.21 42.15 67.94 64.56 51.88 41.58 43.42 57.78 42.06 61.94 40.07 48.37 45.02 37.73 51.23 34.57 37.34 54.84 44.43 41.93 25.85 34.86 35.97 26.76 27.28 32.89 38.67 42.24 50.96 108.8 35.97 45.51 38.15 30.69 45.15 37.82 42.49 63.63 51.67 49.5 46.91 56.06 54.24 41.85 56.51 42.54 50.5 61.19 146.21 63.27 50.88 51.17 45.75 76.4 60.59 51.77 71.6 62.92 63.72 48.08 63.36 71.39 33.3 35.68 46.8 41.04 27.5 34.64 35.1 40.75 33.11 50.39 38.52 33.07 30.99 41.95 34.15 37.87 33.17 31.06 28.79 32.99 31.17 30.71 52.9 31.87 42.04 46.82 41.91 60.86 72.47 62.96 48.21 58.57 78.14 81.5 64.68 48.66 39 44.06 47.37 44.16 63.78 51.3 50.86 41.38 56.92 86.72 52.88 61.65 63.19 50.36 58.34 104.68 69.98 78.65 50.59 66.03 70.13 53.91 84.82 56.21 48.04 56.54 63.23 46.49 44.79 66.54 71.71 58.15 48.69 52 66.68 53.93 58.33 39.47 44.25 66.21 67.81 69 47.6 39.53 59.7 52.96 44.11 48.77 58.11 70.17 41.19 55.18 38.52 53.24 65.27 41.58 36.83 44.6 39.21 41.15 39.3 44.76 21.01 51.95 28.9 37.25 52.99 22.54 207121_s_at MAPK6 694.33 543.47 304.83 296.84 228.49 252.31 419.91 536.01 432.09 365.26 710.26 435.92 335.82 388.74 191.61 387.48 1023.7 541.13 738.42 794.09 449.01 683.74 239.78 378.78 348.58 498.36 345.72 545.94 729.36 621.03 492.3 512.29 488.9 251.55 788.04 462.8 381.12 716.46 265.84 446.47 477.83 545.28 576.63 479.48 360.7 472 467.56 651.35 378.29 294.59 755.25 624.99 283.84 490.61 329.63 588.09 399.91 1107.58 294.87 327.41 513 374.6 597.06 242.71 258.5 556.41 352.11 408.69 822.51 435.74 667.63 427.08 356.51 1029.97 500.06 437.6 787.62 428.25 324.1 527.27 411.32 705.31 600.03 391.81 383.87 390.41 387.4 612.32 234.04 372.24 43.89 439.83 342.13 507.79 593.28 759.41 615.27 416.45 506.79 522.43 785.46 879.56 596.66 310.77 415.97 420.76 399.9 297.67 446.25 34.76 315.48 308.99 321.53 575.71 445.46 581.5 425.19 176.73 208.66 486.56 300.22 494.87 233.51 276.53 247.29 402.93 466.98 416.13 843.18 492.62 634.64 440.45 140.82 351.74 922.43 245.68 451.61 572.61 301.43 587.2 382.86 332.17 565.76 254.64 252.91 221.01 570.73 370.31 245.87 769.64 1074.42 220.18 597.22 353.66 480.89 413.54 416.62 591.15 397.92 415.46 268.14 495.86 398.52 132.04 302.12 264.85 293.06 359.93 171.31 325.04 312.19 210.76 419.3 471.75 60.64 409.01 366.22 283.6 293.16 443.8 320.82 524.82 488.06 307.97 286.69 129.37 576.05 398.46 519.62 186.69 453.53 522.37 307.63 372.19 290.28 410.52 556.97 257.75 368.24 303.24 170.1 386.13 297.87 135.72 408.51 261.76 260.17 430.98 141.83 664.18 183.54 237.78 164.01 269.04 242.75 287.04 198.4 612.13 329.64 211.49 319.39 484.35 497.63 698.61 719.05 453.27 442.83 207667_s_at MAP2K3 168.85 110.82 205.35 197.88 161.39 118.74 102.31 151.42 139.15 354.07 185.13 117.15 181.75 158.66 139.32 264.69 270.87 191.14 135.63 110.88 129.73 142.79 235.45 211.21 191.54 166.25 299.8 102.67 184.46 227.95 127.58 130.21 206.18 188.22 224.98 168.78 175.96 193.88 244.77 213.47 174.3 297.36 166.75 285.53 304.95 216.37 166.5 141.3 258 183.26 101.78 221.1 220.03 221.41 220.19 176.32 201.4 246.07 184.23 249.47 241.92 135.97 186.28 177.43 231.71 150.99 307.15 156.89 194.36 181 197.37 235.89 210.05 324.24 111.16 143.67 139.95 126.09 163.94 168.51 203.86 146.03 119.34 168.38 142.6 137.6 245.03 178.39 156.79 109.78 190.44 140.09 116 103.66 123.94 182.37 89.11 111.68 103.44 120.03 160.76 159.8 136.33 79.3 105.56 175.94 140.31 166.91 155.39 200.96 212.47 142.89 168.83 145.71 137.54 134.64 120.62 196.78 144.64 162.66 159.24 225.75 331.59 233.53 118.18 131.68 157.25 135.06 84.02 108.87 124.84 172.23 141.67 134.45 77.79 113.71 110.82 115.6 118.29 94.96 96.89 108.95 94.67 105.14 116.67 173.45 123 180 259.34 247.99 203.21 247.78 309.05 277.84 152.99 168.16 162.47 232.49 120.71 208.95 200.88 203.04 162.35 154.81 210 194.47 150.64 234.42 267.57 231.05 134.27 236.9 144.32 164.52 232.23 198.51 150.29 92.35 93.88 172.39 139.57 176.39 132.79 133.21 149.04 103.33 125.24 141.92 219.06 160.47 110.66 135.26 152.71 163.44 144.78 169.48 242.17 153.71 135.26 193.25 176.99 137.95 150.1 216.93 183.84 123.76 127.27 188.76 130.41 119.82 136.31 160.06 203.06 190.46 119.53 101.53 120.4 463.52 138.79 171.87 312.79 180.56 107.34 219.47 255.65 169.65 167.99 207764_s_at HIPK3 29.92 19.03 17.43 21.27 12.99 14.16 11.91 29 23.57 12.01 15.46 13.67 9.41 12.35 9.72 8.16 34.76 15.26 13.7 59.19 12.49 41.97 88.75 13.32 19.2 30.78 21.37 25.02 34.95 22.54 89.4 53.52 28.4 11.41 26.47 27.99 29.64 35.85 26.88 38.82 26.49 27.15 48.94 50.47 26.89 27.4 24.06 100.63 23.41 17 54.39 23.5 14.06 19.19 16.44 53.67 37.4 27.54 12.08 23.95 20.92 65.15 64.13 14.37 10.45 11.23 18.17 11.09 26.31 15.01 17.39 19.62 19.05 18.82 21.32 13.18 63.15 20.81 20.22 17.29 13.2 19.92 16.32 16.53 20.12 18.46 13.67 12.28 13.9 14.29 15.74 10.68 9.35 13.71 9.46 10.79 9.53 10.33 12.55 13.52 11.03 14.31 10.93 8.29 12.54 11.87 13.59 12.56 12.12 14.71 8.34 18.76 13.49 42.06 82.53 15.52 19.35 21.59 18.24 18.72 33.67 20.18 17.25 24.01 11.87 16.53 30.73 15.68 17.91 16.47 18.59 12.67 14.01 21.64 9.24 9.28 9.36 15.56 9.47 9.62 8.39 10.88 8.77 13.48 11.5 9.41 13.02 9.89 9.7 11.11 11.21 9.85 11.62 10.09 9.16 11.75 11.33 11.28 9.65 10.3 11.44 10.76 10.45 13.12 10.57 10.35 10.11 12.78 10.29 10.3 10.21 14.71 11.58 12.9 9.9 10.42 10.19 11.41 8.64 9.02 9.26 10.43 9.55 8.4 11.07 9.03 9.28 8.34 10.55 11.4 9.6 208018_s_at HCK 248.01 151.21 158.07 378.67 144.46 130.87 150.4 140.68 233.77 176.5 93.2 75.75 71.9 58.84 62.79 135.25 173.63 446.58 291.72 155.77 222.43 295.47 109.21 256.01 110.56 578.14 161.53 129.6 491.87 93.06 182.43 141.52 155.62 85.14 275.07 379.59 287.3 267.46 535.78 762.03 594.22 391.91 146.74 183.8 141.08 140.93 290.71 262.59 348.36 147.62 90.94 213.74 166.8 454.72 42.16 91.04 678.07 412.76 410.57 204.34 100.86 2036.93 125.15 124.16 757.11 261.38 168.99 686.66 431.63 189.27 788.3 198.68 155.9 179.98 99.33 76.52 142.8 216.96 77.44 129.43 50.42 157.98 69.93 140.78 195.55 175.19 123.98 278.12 266.28 67.32 66.25 184.74 46.79 91.95 55.15 156.27 113.89 53.55 62.57 168.51 248.34 170.33 192.32 40.86 118.67 211.03 161.43 135.35 123.84 33.87 225.03 63.89 282.26 309.69 456.12 109.33 54.16 196.62 274.65 207.53 198.72 91.34 518.03 283.9 174.88 132.69 53.43 102.58 82.27 305.77 232.64 86.67 90.97 194.36 94.14 61.75 97.96 82.83 83.06 184.13 59.11 80.76 61.13 76.34 133.51 86.17 56.27 91.12 162.34 167.16 205.93 266.19 146.99 239.52 191.45 169.72 89.65 752.89 106.22 331.94 147.21 89.75 171.96 73.04 86.87 268.86 92.22 294.17 34.44 97.94 230.24 146.92 147.22 183.97 41.67 128.05 331.87 170.56 67.03 53.64 249.93 201.06 62.13 48.21 129.41 87.32 86.6 93.41 209.76 56.33 106.59 85.39 119.87 277.3 43.12 191.49 110.74 76.41 148.48 65.63 89.15 179.21 260.05 72.18 182.5 53.01 226 113.24 143.72 160.56 154.81 159.48 108.17 98.23 100.64 110.39 189.01 174.01 210.28 64.46 434.76 243.12 76.55 157.75 187.95 179.7 208.14 208079_s_at AURKA 464.14 236.6 62.8 808.07 486.41 15.16 1289.77 287.78 167.67 264.86 150.71 59.19 79.77 340.51 34.63 137.36 653.41 325.35 558.95 563.18 218.23 301.21 456.94 487.53 299.82 171.88 125.69 134.29 184.11 459.59 396.44 269.09 327.27 218.56 449.35 417.99 162.02 316.79 25.05 249.32 309.14 399.24 222.44 444.78 174.49 64.03 214.96 217.47 296.33 147.12 63.65 160.72 2961.48 281.66 232.27 206.4 334.93 214.09 226.35 174.22 124.71 278.94 168.97 256.87 235.3 261.17 395.9 296.84 483.66 238.66 128.04 307.01 251.99 233.16 240.14 195.71 648.61 67.45 108.73 173.77 288.45 83.69 108.9 85.27 63.16 116.02 268.39 114.6 397.51 205.74 96.78 72.02 223.34 207.2 126.12 386.62 101.41 174.28 360.04 405.66 47.54 72.62 121.09 61.51 242.15 292.15 40.81 55.5 271.78 71.87 268.17 18.89 151.88 291.2 328.26 578.52 281.03 89.23 186.62 193.84 89.42 166.86 574.63 434.04 22.34 330.73 149.45 57.34 227.53 201.69 54.9 253.02 30.04 80.86 93.66 220.36 406.6 127.08 120.45 170.54 66.79 242.93 382.33 86.48 141.32 155.81 494.82 152.66 144.21 86.93 276.16 113.13 242.07 85.98 106.96 674.73 104.41 513.48 35.58 139.76 133.6 48.91 118.03 74.26 70.99 103.54 116.25 151.39 111.78 199.03 707.61 104.86 231.67 703.18 164.78 325.48 131.12 47.05 47.8 387.42 40.63 423.28 102.5 160.55 98.64 121.72 114.95 238.89 491.87 121.86 118.24 18.44 53.74 262.02 42.27 140.8 305.51 124.47 336.58 77.46 232.91 137.84 92.74 412.83 246.74 22.72 77.28 262.39 89.53 112.76 196.08 70.17 115.43 42.65 222.25 69.98 70.93 255.93 198.21 55.52 217.17 201.85 52.97 208.14 301.57 292.14 53.84 208212_s_at ALK 34.87 18.12 18.08 24.43 18.76 22.49 18.31 22.85 40.04 19.52 20.31 17.57 20.1 17.48 38.17 27.91 20.72 16.47 24.52 16.34 19.43 20.55 21.87 14.33 67.46 20.44 25.38 17.68 19.61 26.33 17.45 21.6 17.13 22.05 18.34 22.67 19.63 25.49 22.83 25.41 17.82 20.93 21.34 22.94 20 22.05 20.35 21.68 24.83 20.88 24.61 28.46 19.88 20.9 22.8 17.27 18.03 16.89 18.76 16.46 18.84 51.44 20 21.23 23.4 23.51 42.7 19.38 23.94 18.09 25.12 20.32 25.04 22.35 18.4 21.58 25.96 21.55 27.38 39.08 24.97 17.65 15.52 17.88 31.65 22.83 22.66 22.66 22.95 21.05 26.09 21.79 26.32 25.88 26.28 25.12 28.04 21.36 32.82 85.9 20.57 24.35 23.61 21.14 23.73 38.07 19.41 23.87 25.68 42.42 33.52 29.59 33.55 30.9 33.69 43.34 31.37 28.2 35.91 61.01 32.55 83.89 35.78 23.61 23.05 19.49 20.3 17.42 20.19 20.59 16.92 22.98 20.33 18.44 22.69 19.28 36.77 24.57 22.15 21.52 19.66 22.17 20.35 23.06 26.75 24.07 25.07 20.74 22.22 19.76 25.22 25.3 24.5 26.17 24.9 28.76 23.01 35.64 20.89 19.58 29.62 28.74 25.41 22.93 24.72 27.17 24.24 27.63 23.21 21.86 18.92 25.93 22.64 36.83 28.23 41.1 18.62 19.54 26.5 18.83 17.91 25.56 20.65 24.74 17.25 15.37 18.28 19.66 22.57 21.16 23.09 17.43 24.62 34.81 18.48 20.05 19.69 23.99 21.07 23.19 21.33 20.32 19.16 17.92 22.36 18.45 24 21.86 25.62 21.8 23.34 21.67 18.81 19.7 24.66 23.57 27.06 24.06 32.04 23.67 16.23 16.93 14.81 17.48 15.87 179.14 10.99 208438_s_at FGR 96.62 52.28 88.93 117.08 65.57 55.98 49.95 57.61 68.78 79.26 37.23 29.88 43.05 46.64 52.46 46.14 68.64 157.38 60 49.01 43.7 77.04 61.67 76.5 72.59 169.05 60.32 54.76 195.26 50.45 52.37 50.87 36.88 55.56 69.83 153.42 139.97 135.54 227.02 167.87 89.33 148.86 56.9 110.31 48.46 81.51 423.34 83.63 105 53 33.87 90.06 64.33 156.3 26.03 66.61 160.68 77.26 159.8 61.2 45.74 55.76 68.19 45.37 137.04 73.36 84.65 123.14 87.88 50.27 52.2 69.44 55 59.48 35.93 43.29 49.73 82.82 43.73 61.76 41.02 45.27 43.96 38.83 89.83 79.67 61.06 111.72 78 108.37 55.22 75.76 52.56 47.43 54.27 114.61 43.27 38.57 54.15 88.68 57.76 142.08 81.31 54.97 50.32 97.26 79.24 65.45 56.34 40.05 70.69 31.61 96.21 103.43 240.99 50.43 40.15 162.77 127.96 106.51 92.81 83.95 124.71 99.3 90.32 62.24 37.03 72.57 59.91 69.52 111.13 55.84 103.71 128.78 46.17 41.16 64.65 47.57 47.1 88.32 50.05 62.9 35.42 50.37 80.72 67.78 36.74 53.89 98.87 47.57 49.65 116.49 146.08 104.86 67.07 59.25 49.21 254.56 35.79 141.88 60.66 75.67 40.13 38.75 44.98 97 47.79 102.49 52.33 44.58 84.51 69.23 69.1 91.14 37.96 94.42 118.93 47.38 46.85 30.61 154.45 55.09 41.28 35.77 140.03 47.57 48.46 41.78 52.9 35.26 65.25 49.6 116.44 144.18 42.21 102.45 67.25 101.8 122.93 35.66 41.43 85.86 138.03 54.33 64.16 57.11 61.76 70.62 113.1 63.57 63.45 134.04 82.82 127.57 48 57.21 114.16 115.73 100.05 88.11 164.93 77.13 33.34 84.63 73.63 89.84 108.23 208694_at PRKDC 1946.8 868.28 399.24 501.4 607.76 657 688.06 964.3 353.36 753.71 554.31 830.81 632.88 496.64 341.11 717.98 948.11 508.11 3664.18 2074.34 668.84 877.78 1224.15 928.19 1573.86 446.1 241.79 720.19 791.95 1134.59 507.97 661.45 1065.86 703.64 600.82 1137.98 650.46 830.53 345.2 224.92 644.22 1407.31 1443.41 558.91 500.55 340.71 631.2 530.3 1091.78 997.94 231.75 515.33 921.06 593.64 1048.9 664.34 482.73 416.73 707.83 807.1 815.2 1372.5 458.35 538.28 363.27 690.04 575.69 1066.46 963.22 1514.01 347.96 410.3 704.3 1150.69 631.48 851.79 895.33 407.72 709.82 610.22 136.78 485.93 473.78 777.37 350.84 362.09 272.19 288.22 795.25 385.49 52.4 342.66 1032.73 463.73 488.36 694.12 696.69 1264.13 1118.45 337.94 532.02 421.84 428.76 332.55 486.15 394.55 273.73 691.36 496.55 63.17 647.41 300.41 323.64 375.4 513.73 1077.98 481.11 154.62 156.55 296.08 164.23 389.31 435.18 512.82 239.63 332.61 659.78 541.31 746.25 683.93 253.83 208.85 192.72 254.83 518.75 820.77 729.6 339.48 359.83 687.59 281.69 707 990.6 401.16 322.38 146.63 339.78 407.03 279.81 437.14 518.95 441.89 490.84 558.88 327.67 690.01 1453.98 454.7 441.19 652.11 657.35 437.39 586.22 262.15 489.13 344.82 304.78 394.28 147.58 918.4 284.65 157.68 252.45 1173.23 47.53 479.77 140.86 316.74 282.59 391.28 275.76 755.55 403.17 323.63 492.05 530.72 274.01 461.48 1137.62 438.71 301.4 250.43 294.73 234.54 290.41 295.44 525.29 295.9 234.32 174.51 433.02 528.33 382.37 97.18 476.34 311.5 558.93 728.6 315.92 252.97 263.25 327.57 515.94 189.8 667.01 343.68 188.03 240.12 473.22 216.13 262.31 701.27 429.59 1633.27 1020.2 569.2 350.27 208820_at PTK2 1797.6 986.11 554.53 704.92 1114.95 1143.82 739.82 1826.2 669.43 964.1 587.34 867.3 582.65 793.13 477.57 735.34 479.21 534.21 859.18 1160.04 429.27 671.08 818.18 765.38 773.47 535.25 662.94 1007.34 824.21 742.53 609.9 732.3 988.16 767.15 442.27 780.21 1364.71 996.39 410.73 350.98 829.44 909.96 1131.79 624.96 525.73 508.37 550.8 369.41 674.09 711.15 324.45 795.22 847.3 889.28 1137.86 601.36 1269.42 848.24 715.64 734.43 694.37 645.31 691.09 641.2 325.04 722.49 337.47 559.9 971.05 684 748.3 334.25 470.32 460.19 1034.63 921.86 506.73 461.29 963.87 1486.64 727.49 400.49 576.62 742.58 609.59 766.01 583.06 403.01 677.73 631.41 250.72 502.69 1365.09 873.99 681.81 842.4 756.1 1069.65 1701.32 1131.06 713.61 502.36 671.64 765.85 722.67 400.28 483.51 1376.86 550.96 232.32 676.37 565.49 687.44 376.97 680.47 831.92 1211.1 437.05 495.45 495.71 664.55 723.91 401.01 965.52 544.19 1097.64 790.72 782.26 1035.14 457.87 378.35 915.26 577.87 330.72 646.36 1404.01 704.35 570.39 552.6 697.72 520.94 678.9 1194.06 1421.05 590.92 421.35 494.65 580.04 418.66 604.06 815.45 575.65 1046.6 583.1 517.38 1732.78 1349.2 318.88 503.71 525.39 826.12 503.38 487.11 743.38 489.88 987.83 482.96 440.28 716.97 1184.93 541.72 473.09 589.25 888.1 236.78 366.15 467.13 687.31 454.63 677.02 377.96 525.58 615.38 476.4 855.99 912.09 440.4 730.8 581.53 316.64 465.9 462.29 512.56 443.13 376.19 402.86 637.76 423.18 477.26 823.09 846 680.37 481.92 124.19 432.52 491.24 590.72 904.63 446.1 657.3 588.07 439.69 796.46 495.65 556.7 493.5 289.75 269.16 318.95 454.07 406.97 639.2 558.28 1467.91 608.81 725.69 457.56 208823_s_at PCTK1 204.98 144.41 133.81 226.39 142.17 117.58 200.88 147.99 158.15 241.22 183.82 128.16 154.44 212.13 137.4 154.98 196.76 199.38 166.09 181.56 202.15 90.47 202.67 238.88 136.23 79.13 107.39 188.15 117.43 138.75 105.73 162.55 187.83 164.76 184.33 308.71 185.54 177.54 92.67 160.54 232.74 229.52 193.73 322.02 147.45 138.24 136.2 171.43 282.99 105.98 178.33 153.87 238.11 175.76 160.59 161.89 351.4 209.42 192.85 236.73 212.52 194.09 258.98 173.66 90.28 143.68 84.33 89.53 101.13 191.18 169.51 260.73 126.74 152.4 125.57 144.29 188.8 101.41 83.73 131.44 147.4 103.14 183.72 119.88 119.4 88.4 152.48 104.34 118.73 133.6 173.79 122.18 142.23 149.66 118.05 110.72 136.03 173.67 122.81 154.49 189.67 107.63 173.64 121.6 157.63 238.31 135.26 173.95 252.78 262.92 205.01 102.47 115.38 222.81 172.6 110.71 256.03 222.16 119.59 197.9 126.52 192.79 253.86 101.85 107.24 125.8 132.84 115.05 90.45 115.16 114.67 140.4 138.31 128.39 105.1 118.54 132.81 108.22 108 138.12 83.38 113.89 175.3 127.82 106.72 113.53 118.56 190.52 160.03 122.07 357.61 156.12 174.64 128.08 165.16 250.55 171.19 110.66 138.89 186.46 162.01 131.1 125.91 122 140.64 131.19 142.31 154.98 246.13 178.81 164.26 177.62 231.67 183.65 300.62 232.31 154.44 140.61 191.93 237.3 132.32 271.97 196.75 217.16 160.95 177.27 193.35 162.67 335.83 157.82 116.82 150.42 159.15 237.14 155.7 342.09 186.65 196.48 143.79 260.69 629.03 163.22 142.27 219.61 346.46 160.15 231.01 175.45 120.48 136.84 245.27 184.21 176.23 122.36 174.06 168.18 204.72 304.48 171.72 179.52 137.03 182.28 163.16 163.98 162.16 173.16 119.27 208865_at CSNK1A1 1175.29 1513.22 984.89 1462.38 1673.29 1606.31 903.82 2213.22 2314.64 863.47 1532.02 1616.34 1455.88 1775.23 1232.66 1161.46 1011.22 907.92 999.83 1015.74 1266.67 1356.25 479.76 775.8 1219.44 1024.33 1219.71 861.7 1171.73 1015.77 1594.3 1008.33 1229.01 1711.08 1158.19 761.89 931.9 712.96 870.35 847.87 774.08 651.06 765.74 993.65 539.89 983.92 937 678.87 746.76 1531.62 1022.76 1564.95 1219.7 1044.83 2705.26 1477.16 739.32 719.82 1212.98 1365.9 1023.51 853.62 1010.31 1137.84 525.34 860.65 511.28 568.98 527.25 682.8 781.03 667.77 620.57 927.68 694.27 1218.83 907.9 1246.65 1839.57 954.79 1308.92 1932.19 1407.61 1846.21 1166.4 1131.05 1425.17 971.11 1098.24 1362.1 594.63 945.95 1316.72 1411.9 1538.65 638.31 1817.61 742.26 1812.06 1113.59 694.01 1401.73 1545.58 1019.02 1153.86 1339.2 1405.26 1197.3 1336.16 310.49 889.78 1088.12 766.09 973.65 799.09 1060.62 961.64 478.78 876.62 1000.13 962.91 618.05 612.91 768.83 785.49 1213.36 1511.46 1087.27 1125.7 706.09 951.4 1127.05 831.76 759.38 1425.83 894.62 1314.55 1484.85 967.76 1445.55 1379.04 1218.22 1028.17 1216.26 990.51 1242.62 1241.15 1359.53 778 1542.91 876.52 766.87 852.7 1156.06 1340.61 510.54 1970.28 886.57 1319.64 1032.74 1000.64 1390.6 1070.67 1133.57 1302.5 1047.05 1469.75 887.48 918.72 1088.99 1033.99 753.02 1239.32 776.84 694.14 966.22 717.08 969.51 1093.52 1165.2 824.17 651.57 1505.61 918.36 938.26 988.32 1305.57 1202.42 713.38 1268.17 1389.42 1125.49 697.07 1070.97 964.29 851.94 915.28 1644.52 783.56 824.75 849.11 1235.41 1290.07 887.32 693.74 724.57 693.24 673.38 790.82 1096.15 578.21 847.67 701.77 1211.41 845.38 1097.05 859.75 759.12 837.92 1053.13 707.55 771.77 1524.52 964.44 958.37 789.02 900.33 208877_at PAK2 323.45 407.2 285.58 501.4 295.2 318.5 231.32 521.86 469.52 418.99 418.54 461.72 343.74 422.09 334.72 257.01 296.21 382.86 568.05 536.99 322.16 386.57 450.86 205.58 372.67 322.14 376.16 681.74 392.3 356.25 369.18 325.57 295.25 302.03 319.27 449.06 375.91 330.52 286.08 383.34 341.71 374.39 268.97 242.39 179.42 258.32 408.41 300.09 359.27 305.34 336.53 330.94 239.28 372.6 500.13 285.09 166.78 185.05 206.22 189.84 404.34 239.75 428.77 166.71 350.6 348.39 243.45 310.02 240.28 344.46 280.73 178.9 363.19 191.67 282.74 266.51 375.14 268.07 242.5 163.68 230.87 373.04 358.99 351.64 324.1 211.76 294.32 273.15 297.5 288.84 55.78 219.5 186.96 381.48 379.69 191.17 242.76 248.05 248.88 167.09 297.62 271.64 178.61 191.22 126.09 270.54 295.21 166.36 259.87 26.35 202.28 253.43 237.09 196.94 261.94 166.94 173.14 87.09 180.66 204.01 205.72 205.41 128.18 279 199.01 234.44 166.27 228.51 171.03 176.04 194.76 156.67 121.86 179 170.76 208.26 143.97 179.88 103.2 141.23 200.68 172.81 133.14 222.51 171.33 145.72 196.39 268.52 269.5 377.32 289.59 229.52 158.82 240.42 292.07 140.97 344.41 243.75 327.11 283.69 194.27 201.53 324.39 142.24 267.4 227.58 437.81 215.73 121.54 118.85 186.4 120.43 150.64 112.48 80.35 215.02 204.73 164.57 179.57 200.59 183.96 87.42 194.52 137.87 106.26 138.81 154.94 132.53 71.05 128.57 134.33 203.76 128.21 145.9 152.62 143.64 274.92 258.97 120.63 91.99 116.14 235.42 218.52 102.92 209.8 157.12 118.1 135.98 211.38 179.69 94.31 186.28 285.98 143.5 137.08 161.02 185.92 153.89 317.43 203.14 247.24 273.68 251.99 313.48 321.91 483.71 264.19 208944_at TGFBR2 431.45 749.03 1002.8 843.88 684.89 965.68 448.7 456.48 508.62 393.72 416.11 275.96 725.87 208.49 790.23 432.04 339.75 644.17 683.04 518.04 792.14 963.34 388.52 722.36 324.43 1293.35 762.72 1019.91 758.6 429.43 957.87 348.65 457.37 269.49 683.67 637.99 547.74 636.28 1337.63 944.55 669.35 634.57 397.94 550.9 777.56 1811.33 595.04 505.79 1101.7 568.52 551.12 1117.26 877.28 885.12 136.09 1172.87 517.96 599.73 1086.29 565.21 394.77 233.72 1128.44 344.05 1186.54 453.89 388.27 623.52 182.08 778.73 448.75 531.58 290.59 696.78 227.43 280.77 313.59 1016.18 237.68 357.74 185.91 766.35 401.23 569.74 1573.37 1198.43 596.25 1457.47 508.77 1118.24 126.86 861.2 307.48 565.97 1233.92 559.35 493.72 178.1 498.98 559.14 632.88 1174.29 486.09 440.04 407.85 696.88 978.15 631.08 723.56 46.26 633.15 1268.06 1107.53 647.02 694.23 263.5 153.94 396.37 839.29 520.22 1227.76 388.33 308.99 568.61 2395.74 576.67 414.78 1675.53 353.33 203.31 765.16 379.38 1214.21 893.84 362.06 380.12 590.2 591.9 355.01 608.45 309.64 283.91 187.72 416.98 642.26 103.82 249.61 785.67 1521.14 608.13 311.21 1180.22 593.91 1069.19 678.39 568.2 225.47 881.91 898.47 954.34 458.44 806.81 437.13 308.5 330.56 955.5 410.56 1030.82 26.87 257.65 460.16 486.15 569.68 400.92 15.78 328.76 715.24 467.96 1068.71 62.8 1046.81 329.13 480.82 434.74 425.75 495.39 376.31 382.58 159.68 157.93 592.86 1248.81 508.06 558.53 839.23 546.4 182.87 578.01 567 299.46 319.73 418.53 808.85 21.38 133.34 530.6 182.89 297.98 402.63 389.72 399.75 685.48 897.81 402.76 272.9 368.68 427.32 367.52 618.22 464.06 893.48 528.49 629.36 278.73 498.26 268.42 1284.08 209019_s_at PINK1 237.71 342.1 347.8 283.29 341.58 450.12 299.32 490.8 287.67 250.5 276.44 305.71 293.39 299.1 327.91 326.89 222.31 182.57 205.74 338.88 270.83 184.98 401.33 255.96 235.43 229.64 301.99 170.54 187.69 336.47 290.17 188.79 237.94 378.12 298.43 202.98 189.81 224.47 358.25 250 310.62 204.75 236.74 194.03 292.46 286.79 250.43 187.91 398.46 271.37 399.55 346.78 416.51 358.61 471.08 292.86 151.75 307.79 232.59 305.88 217.47 155.82 222.67 164.51 144.2 169.62 193.75 129.08 146.16 224.67 262.69 269.22 186.38 244.18 198.44 178.7 260.88 206.78 206.01 206.65 230.09 385.32 278.51 280.81 324.72 294.75 288.07 277.85 201.11 238.17 235.48 372.19 335.66 286.11 271.41 225.51 313.27 351.88 309.83 259.83 285.63 322.61 295.55 406.69 229.32 264.69 350.42 335.3 401.56 237.11 143.22 366.25 263.74 242.22 165.53 166.42 362.72 172.44 242.07 247.76 322.25 202.51 319.88 174.71 319.24 244.2 226.24 272.65 182.25 111.26 234.32 239 330.16 229.51 253.09 217.06 233.69 248.21 228.97 202.59 158.17 221.86 227.76 226.18 233.2 169.52 161.56 381.2 295.34 300.48 126.43 282.29 247.03 279.02 274.86 250.69 222.17 221.07 296.2 172.61 264.63 260.54 204.15 272.35 216.38 261.2 238.12 236.96 201.03 183.8 309.26 250.63 281.02 147.41 112.71 238.06 235.53 263.77 276.68 211.09 247.84 187.57 193.04 281.1 225.13 224.41 151.83 267.86 178.86 188.77 202.84 294.5 244.75 186.19 264.45 254.8 108.2 210.3 198.57 196.38 214.32 248.06 225.7 118.66 163.41 290.92 117.98 163.92 217.52 142.19 188.64 169.97 381.06 203.82 238.24 288.78 301.93 232.49 229.51 390.93 299.83 227.82 418.69 239.76 335.89 160.16 433.66 209033_s_at DYRK1A 603.44 792.15 575.02 533.12 601.95 813.91 689.3 1039.36 709.14 602.9 711.94 641.53 559.23 852.61 544.24 518.24 536.98 700.96 775.23 1148.79 1251.61 684.7 718.45 688.59 605.3 907.41 738.72 691.64 555.73 599.44 929.6 771.87 1237.49 862.06 600.08 550.24 557.01 652.23 478.4 397.78 803.55 714.84 365.84 551.6 381.25 831.75 532.59 624.36 498.02 847.08 847.82 641.5 926.73 873 413.88 537.61 512.61 621.85 534.07 410.68 912.41 995.43 751.16 322.41 522.87 518.12 371.8 344.37 670.84 726.63 503.79 728.92 429.18 474.83 537.76 481.39 944.68 756.45 407.51 320.65 353.11 792 591.28 515.77 744.66 872.76 617.15 665.19 585.06 984.81 73.6 772.48 878.29 751.79 996.08 566.29 764.81 860.76 883.87 971.12 844.43 595.27 927.43 709.48 1024.55 784.41 714.86 759.01 851.16 30.47 488.45 840.43 654.29 711.23 682.56 831.99 412.69 357.54 610.93 358.47 523.95 737.67 367.08 541.09 502.46 469.5 503.25 524.28 637.06 595.57 465.81 388.35 388.86 444.97 539.13 492.24 621.31 361.79 516.81 476.42 463.57 487.81 1001.23 378.76 394.57 229.68 141.48 770.3 480.28 678.85 639.65 420.4 513.71 729.02 677.26 730.56 345.24 347.24 550.01 629.28 542.06 578.43 590.75 347.84 464.22 540.49 645.9 564.4 138.43 440.47 602.7 342.43 553.62 506.91 117.25 539.1 392.87 593.31 599.4 464.68 669.06 293.22 541.84 512.89 433.54 462.34 496.16 546.71 334.65 341.31 755.12 706.79 289.81 476 506.6 533.53 491.07 373.24 375.39 236.82 347.75 393.11 518.86 245.22 298.72 486.6 539.22 383.84 317.3 430.1 352.71 397.68 347.6 367.57 403.4 463.14 438.11 397.61 357.74 420.71 594.36 632.19 587.66 578.17 709.74 737.82 524.69 209193_at PIM1 189.02 114.51 152.08 155.76 107.13 125.19 186.57 100.19 92.75 152.45 77.1 100.01 129.72 212.04 167.61 142.41 98.11 196.66 295.51 225.36 102.05 108.59 126.42 214.45 243.24 246.38 105.07 114.68 172.33 70.63 99.19 86.22 76.19 130.36 108.85 166.49 141.61 176.39 252.3 160.6 164.98 156.72 219.48 125.15 128.63 145.92 132.51 132.28 302.24 143.97 117.65 151.92 109.52 178.14 61.39 141.23 135 153.95 164.1 112.62 63.67 122.48 133.42 109.3 161.48 135.67 192.72 156.96 127.39 210 133.17 136.64 103.63 118.91 99.33 87.94 361.7 136.17 96 98.4 120.51 103.26 69.73 114.62 99.49 98.69 94.74 178.18 125.63 175.79 129.76 132.73 87.61 81.62 148.38 109.42 71.32 71.47 110.32 203.51 91.27 106.03 104.72 148.07 107.53 127.93 129.83 120.28 93.25 94.79 152.4 69.16 181.64 149.02 123.27 185.45 139.14 171.41 176.26 133.33 162.19 193.69 132.25 132.62 119.44 105.18 81.15 148.49 105.37 124.45 144.26 82.28 145.38 128.37 87.57 78.84 89.14 111.9 89.22 80.42 101.01 80.03 72.46 89.05 110.15 153.37 77.59 93.86 114.57 95.27 157.73 149.06 200.55 116.7 91.24 137.84 73.56 143.93 123.63 145 82.11 100.08 102.72 82.7 68.1 96.67 81.82 170.52 102.42 94.21 125.23 117.94 102.5 125.97 107.06 103.61 159.2 95.34 110.34 74.32 159.43 161.54 74.27 76.39 119.01 113.07 91.18 71.52 214.63 95.39 95.27 161.31 116.35 105.5 66.37 100.31 111.11 83.84 131.58 143.23 85.07 103.51 127.89 94.16 110.46 114.53 106.22 108.5 107.62 73.41 123.78 141.02 119.91 108.79 76.94 80.83 153.4 98.93 87.11 69.27 137.42 155.74 71.12 137.46 246 126.88 95.4 209333_at ULK1 126.44 154.63 255.9 130.45 205.26 171.18 204.36 661.3 169.59 211.39 408.12 325.7 254.12 266.92 231.59 190.3 217.85 266.6 231.38 166.55 183.78 158.46 170.38 179.07 148.76 108.22 337.34 158.08 214.2 378.04 102.79 172.58 513.94 253.15 205.08 252.33 165.99 244.25 223.46 208.58 172.15 223.82 414.27 221.01 157.51 224.72 304.39 100.86 273.15 298.64 157.51 199.87 352.62 154.84 265.35 289.96 127.73 170.92 440.07 215.78 539.66 240.51 185.14 209.59 109.29 126.04 187.55 115.58 138.88 147.43 192.79 178.61 160.78 173.44 263.36 247.85 112.96 96.12 169.38 143.85 295.99 168.43 181.88 175.57 119.56 148.48 197.67 123.99 162.14 149.58 213.73 166.11 266.2 108.5 154.96 184.6 300.61 329.3 377.22 116.86 280.07 133.98 212.69 185.1 227.15 321.76 258.26 266.06 580.83 311.81 163.24 234.44 273.76 252.46 225.4 202.91 320.38 376.86 153.04 238.74 196.83 217.53 305.73 217.53 116.76 204.89 190.76 138.93 109.92 130.29 182.34 186.94 143.06 212.37 167.88 297.58 97.31 229.84 170.79 260.11 134.86 236.49 207.04 152.66 165.59 156.7 190.08 297.78 181.85 213.42 302.28 194.73 206.63 220.42 253.77 272.89 150.8 118.24 233.62 139.35 393.85 193 177.99 149.17 214.16 181.06 259.89 285.96 267.44 261.81 267.17 276.02 183.29 146.2 407.36 765.47 286.98 248.04 275.44 219.08 218.29 317.49 323.91 233.63 189.67 291.38 200.65 257.04 329.98 257.12 284.24 158.69 192.45 384.3 185.88 466.39 189.46 652.29 271.84 163.1 262.4 348.56 325.14 145.56 252.03 225.86 281.01 215.7 254.69 165.6 195.07 185.13 334.71 157.86 183.43 212.19 210.42 167.28 168.2 288.55 115.82 148.57 285.04 146.54 185.03 117.65 121.87 209341_s_at IKBKB 148.49 1460.5 795.68 239.34 1288.99 1271.27 608.94 4300.94 416.82 356.03 1954.52 624.72 1131.59 2227.88 1014.93 314.74 197.2 535.16 816.92 601.11 627.69 420.73 418.95 353.41 594.3 464.45 228.26 327.12 289.04 594.04 535.77 367.41 829.72 884.9 330.33 286.66 417.63 417.22 535.57 413.79 580.69 496.45 208.9 680.96 381.71 515.02 435.28 291.02 446.91 2202.18 250.68 467.39 934.07 392.53 1026.67 652.66 344.32 224.12 605.66 1050.95 1847.02 323.36 339.6 151.42 507.41 209.96 269.47 347.5 298.55 301 128.67 353.83 353.82 197.69 253.18 1321.89 273.39 394.05 229.95 1306.43 639.74 507.37 573.63 1498.57 465.77 525.84 530.58 462.76 759.51 487.19 296.88 633.18 362.26 615.68 598.79 296.05 1288.06 291.82 781.76 381.29 739.57 476.03 1066.81 909.31 708.89 1299.79 1346.14 697.32 468.13 2482.46 349.3 629.82 337.87 264.11 313.09 959.26 2008.43 745.52 370.26 391.17 862.23 420.59 370.5 284.42 552.78 455.94 1487.56 568.45 202.84 648.61 579.39 514.54 567.32 327.46 434.43 449.2 410.85 275.57 619.22 223.23 1052.75 264.49 668.53 432.38 304.87 344.98 326.07 558.14 1014.56 797.67 307.31 1141.6 192.3 505.37 1154.19 383.55 572.24 354.53 541.77 380.82 396.43 702.62 1245.14 496.36 1171.19 556.14 760.32 1235.75 32.24 457.19 328.32 481.61 841.27 546.31 385.44 306.94 373.64 1321.92 892.72 853.87 600.57 769.71 501.2 638.18 239.47 540.83 511.21 912.4 687.35 422.07 1161.41 698.75 324.34 428.75 526.76 224.54 287.29 789.15 389.24 351.51 499.94 1038.99 716.7 200.36 348.96 494.66 470.49 349.58 814.35 510.24 381.66 671.04 489.13 392.02 1110.86 570.23 352.08 205.06 309.79 934.38 269.66 221.71 1387.95 167.14 467.96 182.49 377.06 209464_at AURKB 89.76 55.26 38.75 71.61 82.33 11.64 85.95 58.69 51.22 77.03 55.41 14.89 39.83 144.01 15.89 85.74 157.66 102.36 249.6 311.97 80.43 68.66 284.65 123.64 109.39 48.78 37.57 60.09 134.68 78.91 75.63 64.5 100.08 98.61 137.18 192.7 64.47 97.61 28.48 84.62 196.98 187.92 81.62 115.13 39.82 39.23 98.31 115.51 240.6 54.06 44.26 59.28 77.27 74.26 80.61 109.53 79.78 123.11 101.69 43.94 202.3 128.87 74.6 89.1 74.15 126.89 156.23 166.95 83.03 84.87 89.02 94.32 138.05 81.85 98.52 52.49 235.03 30.43 27.03 71.06 129.61 25.37 31.91 30.47 11.85 22.93 18.86 31.47 76.66 16.41 39.94 17.5 34.23 24.71 12.28 28.67 20.08 51.5 24.2 56.68 33.92 17.57 37.82 11.18 39.78 47.08 20.49 25.03 50.32 73.67 88.23 14.83 49.97 114.89 65.52 30.87 101.15 45.63 48.9 38.35 30.4 94.16 61.34 97.64 15.19 208.95 52.91 23.3 42.2 63.58 31.49 49.81 23.03 58.15 23.69 62.71 88.14 40.38 37.86 50.4 32.22 42.06 83.59 30.89 41.32 38.88 104.36 34.07 41.65 35.43 115.54 33.29 119.28 43.85 51.01 183.59 95.65 111.71 16.95 92.89 5 31.79 52.72 23.84 43.29 46.22 32.94 47.72 71.54 93.31 40.01 38.15 81.13 151.69 84.79 32.44 54.56 26.94 24.77 155.86 27.34 94.45 62.55 38.24 31.41 34.95 37.6 39.75 129.79 52.74 27.16 14.62 56.83 116.75 40.67 77.65 139.74 42.73 52.46 56.78 56.89 56.78 35.03 71.67 108.21 16.4 69.19 153.65 42.03 39.56 97 37.1 47 20.65 91.21 45.48 26.93 78.68 57.66 31.14 96.66 83.67 22.47 92.75 121.5 154.44 23.11 209481_at SNRK 306.07 374.58 363.23 289.19 447.58 438.9 321.46 674.2 518.27 190.08 249.5 210.41 395.3 361.47 274.62 232.08 259.21 372.2 421.34 443.84 458.75 425.66 268.31 319.82 243.94 513.6 357.55 372.59 297.49 324.54 487.7 322.21 350.57 234.95 262.14 175.79 218.39 289.4 466.96 196.75 284.5 304.2 172.81 431.46 307.94 609.63 225.31 241.15 303.94 453.46 338.69 417.01 380.97 356.97 996.09 380.02 227.71 219.39 292.4 234.23 420.45 262.69 362.87 342.04 344.96 287.52 314.62 278.6 228.42 409.38 256.57 333.81 170.06 318.22 433.54 340.27 301.5 656.99 369.54 237.36 227.78 563.18 471.29 488.82 485.79 471.71 408.32 565.07 306.6 480.14 131.43 592.98 235.05 413.01 600.33 338.36 456.41 242.69 533.06 313.79 489.66 416.65 345.09 402.68 352.39 346.07 454.36 381.34 220.75 19.11 239.62 445.52 380.39 325.7 364.62 252.31 476.96 191.97 425.3 238.32 460.1 246.76 192.45 320.96 532.14 405.45 341.17 514.95 354.89 193.25 390.04 209.72 502.33 342.5 297.67 359.15 369.81 381.07 307.2 305.62 486.04 226.91 392.66 236.47 414.13 180.94 232.09 447.64 554.92 547.02 228.63 454.18 345.08 414.81 445.12 324.45 394.14 328.44 356.39 433.67 195.46 342.82 384.22 429.95 300.03 327.63 270.53 447.08 57.07 192.35 252.66 212.37 324.69 373.91 74.87 265.12 326.82 214.05 441.1 216.9 520.29 278.02 379.94 287.89 286.15 285.44 251.34 414.64 159.1 344.9 294.08 409.79 262.08 292.61 358.48 393.66 194.08 335.31 272.03 221.09 232.26 346.23 312.89 78.32 180.65 282.28 134.96 243.63 201.05 332.8 233.21 353.5 356.45 232.05 248.78 247.66 216.06 207.42 267.15 326.24 413.85 265.58 366.06 278.31 303.86 368.14 325.57 209544_at RIPK2 77.04 55.58 26.66 67.27 53.61 35.36 41 53 35.81 72.05 43.87 38.86 29.98 46.43 18.68 46.51 42.01 47.23 94.3 137.86 29.07 53.17 123.79 43.28 51.19 50.91 75.05 42.42 47.01 38.52 86.14 33.36 38.03 52.44 48.38 54.94 75.36 58.16 19.83 37.81 106.75 45.15 71.88 32.12 46.27 21.55 27.04 33.01 36.87 32.14 33.17 31.88 30.97 78.8 46.79 36.71 80.34 63.62 34.07 106.61 36.5 18.8 49.11 41.71 36.35 154.68 36.57 38.66 189.42 34.84 36.35 88.11 76.05 40.45 21.86 59 51.15 26.8 47.86 45.81 31.14 39.74 29.19 34.68 26.46 30.97 30.42 29.88 35.12 30.14 26.27 33.32 35.33 30.14 26.75 40.72 34.27 43.08 74.24 53.79 32.75 37.61 31.96 23.62 33.39 30.06 25.24 36.87 35.61 23.18 41.38 23.67 42.75 79.38 64.47 66.25 19.73 26.53 44.76 31.56 42.79 99.54 30.82 43 20.59 73.31 32.8 33.7 50.57 38.23 28.62 32.99 28.08 47.3 36.19 30.66 35.83 34.29 30.16 28.32 33.68 34.97 75.88 50.87 25.62 26.69 34.7 27.25 26.71 50.98 44.28 29.98 105.6 37.87 38.64 19.58 76.63 57.57 20.14 51.96 32.6 34.22 37.78 64.55 25.31 30.66 24.68 23.85 28.47 46.08 30.87 24.3 31.94 57.36 32.31 42.55 40.26 19.56 14.64 49.5 27.88 23.38 29.34 26.15 44.34 39.54 25.95 21.96 23.8 32.22 25.96 27.7 23.81 24.71 18.24 26.83 29.14 20.9 21.99 22.69 26.42 35.12 24.85 37.04 31.88 25.04 25.52 59.89 31.26 26.62 18.63 23.31 22.77 23.47 28.6 22.6 29.76 25.4 39.63 20.72 37.72 33.98 11.88 59.8 30.74 46.22 17.99 209588_at EPHB2 60.17 57.6 131.4 106.45 60.17 57.08 85.01 67.41 69.52 87.31 59.1 53.77 45.69 58.77 51.4 100.52 119.48 59.39 60.95 53.67 55.78 62.04 148.81 99.4 51.36 84.82 66.53 118.65 60.08 57.37 58.75 62.52 64.79 51 94.83 65.55 98.34 103.62 69.34 113.42 83.72 83.17 69.35 71.38 91.98 66.49 145.5 74.37 67.56 68.01 45.63 67.7 80.11 93.25 61.84 52.54 94.81 77.27 96.54 104.74 67.96 91.44 104.8 111.7 67.08 63.97 66.12 89.83 92.07 61.59 87.23 77.99 58.19 76.66 78.56 72.02 44.34 53.82 42.99 64.04 41.84 71.87 51.63 63.67 57.34 55.24 77.24 69.67 66.77 69.85 127.09 61.26 45.54 48.03 72.88 78.34 62.65 64.01 72.86 77.62 60.22 48.83 54.1 52.97 53.3 95.79 73.84 68.67 80.1 73.13 78.85 46.12 91.85 92.78 99.97 84.26 85.81 49.93 59.26 88.52 90 196.45 137.18 105.42 64.48 58.61 50.9 71.86 56.81 55.62 92.79 68.47 79.88 105.71 48.15 81.33 100.06 69.44 79.07 63.02 48.25 74.47 54.44 64.06 68.18 82.69 58.83 80.54 73.36 68.18 60.75 75.8 120.79 79.06 58.33 69.28 68.32 104.78 68.4 73.23 66.47 46.97 47.14 74.93 66.93 93.43 59.23 76.15 77.29 85.58 86.15 82.9 72.75 73.07 89.43 90.24 86.78 73.31 64.18 59.51 64.18 85.81 61.21 59.18 74.88 79.94 81.66 74.6 129.9 62.52 64.18 58.06 79.07 83.35 51.84 78.17 50.11 71.96 72.5 60.48 55.18 75.99 66.52 107.96 54.08 57.11 51.43 65.47 88.39 58.9 56.22 78.37 94.68 80.34 59.62 66.64 71.75 80.65 83.27 58.3 72.22 102.06 60.72 52.65 64.09 50.84 61.16 209622_at STK16 63.51 64.12 39.04 48.65 78.93 89.28 38.09 51.06 85.61 51.37 67.09 110.79 91.29 89.63 62.23 57.45 71.9 52.55 43.74 37.6 45.23 64.22 67.76 52.77 39.49 61.74 94.51 58.08 54.88 58.61 85.3 65.6 52.5 99.03 54.26 64.4 69.31 56.06 55.06 60.79 62.79 51.08 46.85 71.97 50.41 45.73 76.9 61.58 64.01 56.58 53.37 70.1 78.76 59 101.1 70.46 32.53 62.24 73.76 54.77 63.57 55.59 52.74 38.62 53.76 24.94 55.07 40.23 43.9 27.39 30.56 59.63 43.07 45.55 36.69 50.29 31.38 37.81 47.85 48.26 45.95 58.2 53.83 79.11 61.2 53.75 60.44 26.57 37.21 23.28 17.13 24.08 49.14 31.85 20.76 22.82 45.05 53.8 24.09 26.62 50.38 84.9 46.43 24.73 46.99 48.46 40.73 60.95 42.54 56.4 44.73 39.58 42.98 55.85 39.2 27.61 39.29 39.94 40.82 77.66 75.47 52.38 46.48 49.77 25.67 63.34 53.69 38.88 49.4 43.18 46.07 45.09 51.65 44.68 51.91 42.17 37.42 43.76 37.92 40.15 34.89 43.64 45.08 64.86 29.63 54.36 34.81 50 53.43 116.79 40.94 25.26 52.36 53.63 50.84 42.26 77.42 70.9 61.53 43.78 79.52 60.21 50.56 57.66 77.06 62.65 71.77 38.22 53.84 74.34 86.71 52.66 84.21 51.37 37.79 62.57 61.2 67.85 41.09 87.12 60.87 69.42 77.43 63.6 60.48 81.63 76.79 72.83 64.13 71.74 63.43 69.02 73.15 46.06 51.65 94.04 62.01 73.33 72.94 61.63 106.38 120.18 64.34 66.01 70.11 60.72 22.73 45.46 38.69 80.32 46.88 61.83 66.71 61.19 60.22 54.88 58.84 71.2 67.19 41.5 67.29 68.01 39.14 52.45 51.97 40.35 52.29 209642_at BUB1 262.01 74.53 15.21 95.81 60.2 8.3 109.9 95.67 74.27 109.22 53.23 15.77 19.54 111.66 10.07 40.76 512.64 102.11 268.57 266.63 53.75 120.43 414.89 107.76 131.57 120.52 52.33 119.99 149.27 149.41 120.97 98.85 146.43 42.01 222.27 289.81 54.19 233.26 12.45 94.12 151.34 218.06 90.37 77.03 29.8 48.3 86.82 133.06 135.14 48.11 38.74 62.73 62.25 100.4 96.35 36.35 86.94 90.75 54.89 36.31 16.61 149.5 176.54 43.06 49.59 220.48 148.15 204.84 296.14 115.8 130.33 110.73 76.54 229.55 96.12 77.97 278.54 36.16 16.86 51.77 24.1 54.3 44.7 17.68 9.74 35.52 13.6 31.97 79.94 51.32 12.67 29.2 47.12 41.17 19.89 77.29 10.84 61.74 55.16 132.22 22.92 23.58 25.99 9.43 51.51 91.09 10.84 17.29 72.51 9.83 59.85 8.02 46.72 62.96 39.62 76.28 48.99 29.31 27.79 14.69 13.84 62.96 24.43 102.63 9.04 77.57 35.95 16.54 49.87 136.01 14.33 28.75 8.15 23.47 28.78 51.45 77.33 25.02 32.36 48.18 16.81 68.88 152.45 30.65 24.3 24.63 63.62 27.78 19.11 27.3 156.44 23.54 96.16 42.34 37.4 116.01 44.63 129.66 13.41 57.6 30.44 11.3 38.04 12.34 29.79 44.24 41.21 46.19 13.75 103.01 25.85 10.26 38.8 179.78 23.19 17.46 35.4 13.21 14.13 51.92 13.83 70.75 46.84 24.61 23.29 23.66 24.65 20.36 96.94 31.4 21.17 9.52 14.84 76.3 12.7 50.92 133.63 29.15 43.55 29.18 21.63 37.21 52.8 14.36 184.19 9.13 129.7 91.78 13.69 23.98 66.95 18.23 21.19 9.43 57.36 24.06 19.56 104.92 52.19 12.84 64.55 116.89 16.59 79.39 75.59 66.41 12.46 209666_s_at CHUK 236.55 203.82 61.08 183.29 168.05 98.39 135.36 140.91 172.85 171.21 169.12 153.56 76.89 120.07 36.82 82.76 169.09 95.45 214.94 182.15 65.31 195.11 134.95 67.25 100.1 152.1 110.63 86.91 101.1 99.04 159.79 112.78 140.35 122.98 95.03 94.31 133.86 59.38 68.66 100.15 176.9 115.45 105.23 175.9 118.38 61.74 40.94 62.59 86.08 81.79 93.67 77.67 78.24 142.23 154.24 110.32 81.08 75.92 72.49 38.65 58.77 72.22 90.21 90.83 94.43 81.06 83.18 97 130.9 101.51 63.01 72.76 125.31 105.25 61.59 102.21 216.19 102.91 94.73 67.72 66.87 131.17 86.71 123.38 95.41 108.75 170.01 80.18 94.14 122.29 23 102.87 164.76 225.8 145 157.32 86.63 111.03 186.14 116.12 124.83 146.06 121.82 99.59 97.58 77.62 85.62 99.35 80.53 21.33 140.02 67.86 113.31 125.07 134.96 144.94 122.03 78.38 77.22 68.67 85.15 59.7 106.4 126.19 66.31 130.24 85.1 91 108.97 121.19 90.7 109.36 71.61 73.73 109.15 120.41 88.74 106.59 94.3 117.06 116.48 127.04 168.15 154.28 99.43 79.42 107.74 102.41 106.89 175.91 84.65 51.06 117.56 99.78 108.99 94.35 102.28 89.43 101.2 79.81 86.18 88.08 117.99 51.9 75.76 72.83 177.83 83.42 41.31 104.17 69.03 48.58 121.89 102 36.97 88.56 97.01 82.76 55.59 111.18 87.13 60.53 97.05 82.44 62.51 77.88 55.66 113.04 68.75 95.88 153.85 122.83 87.25 70.6 54.39 62.72 96.35 55.32 76.86 42.01 54.46 102.06 112.15 14.66 87.52 73.63 67.94 96.38 62.64 103.43 51.63 60.75 58.01 79.81 46.58 89.32 73.52 98.86 106.45 58.67 92.88 79.08 63.01 120.21 67.51 80.82 75.92 209902_at ATR 116.33 117.41 74.74 79.4 84.14 115.84 54.2 125.25 94.33 64.37 89.45 140.48 102.04 146.21 58.5 79.82 108.77 198.89 438.58 129.77 121.48 152.41 183.55 77.69 157.31 134.61 138.28 68.81 100.75 64.07 84.37 103.73 138.11 153.48 111.19 128.6 106.35 127.63 75.37 81.61 237.46 147.51 123.95 154.31 70.97 80.91 73.35 48.77 82.34 98.89 119.48 100.65 75.06 96.28 120.6 94.66 132.75 79.8 94.71 147.27 93.26 124.39 65.42 48.9 81.94 174.07 147.7 117.31 108.22 90.15 60.09 115.35 124.99 90.03 87.48 85.16 110.51 87.4 71.82 61.82 47.39 88.7 69.06 66.93 76.34 86.64 76.42 80.62 65.73 58.29 32.7 47.27 61.28 91.25 60.77 51.68 82.05 40.57 85.04 110.89 143.49 114.59 96.46 49.45 85.22 92.12 98.08 101.6 106.83 32.4 94.85 74.4 66.05 72.52 84.09 101.1 46.5 77.11 82.95 67.47 70.95 63.29 105.21 72.08 68.34 82.17 86.17 87.54 109.19 184.86 100.08 82.99 55.71 95.11 112.44 77.42 103.4 95.5 43.89 67.55 122.96 84.12 79.48 77.97 70.07 66.39 50.74 87.14 83.9 127.42 201.84 57.01 44.76 90.55 75.16 102.37 135.3 93.19 84.19 101.84 88.73 93.85 126.6 62.27 92.66 63.83 144.09 67.32 44.12 84.33 74.91 55.08 49.58 166.94 39.2 102.88 91.2 76.05 78.02 90.5 76.35 93.41 84.68 55.1 66.61 88.08 63.1 89.86 99.88 100.72 62.26 76.3 59.1 101.69 84.43 74.05 127.56 90.93 114.91 95.09 94.53 86.85 106.39 50.09 49.11 61.42 157.15 51.37 58.71 78.36 85.96 69.27 56.36 56.64 61.02 65.36 72.88 54.81 72.21 71.37 99.89 67.66 58.35 63.49 90.79 79.95 62.72 209945_s_at GSK3B 212.49 126.95 379.12 250.99 207 161.8 55.97 256.96 207.59 293.95 274.38 248.18 170.38 191.82 153 217.64 358.96 171.38 155.99 230.5 235.61 191.75 233.53 210.21 209.84 187.2 181.36 475.99 132.75 170.57 123.02 390.25 248.52 300.09 225.62 247.41 144.31 181.06 167.65 154.46 189.97 244.28 282.23 372.55 156.67 263.29 194.84 159.45 219.96 350.63 297.27 290.67 234.16 298.11 367.11 221.65 172.18 109.22 246.36 216.73 307.32 530.98 379.7 448.39 375.69 534.8 544.34 493.9 449.37 410.71 420.71 724.88 568.67 454.77 428.23 508.6 556.09 247.19 255.13 271.18 558.73 646.81 542.58 587.57 333.99 219.84 375.02 354.11 354.33 310.41 421.95 322.02 843.85 198.78 537.61 372.1 198.72 173.03 270.86 279.53 233.76 289.55 382.23 181.1 324.23 322.06 304.39 227.71 483.81 261.05 317 249.35 341.3 384.66 377.22 103.87 312.07 534.27 345.08 786.52 424.02 735.46 460.5 326.44 303.44 221.39 366.69 439.83 362.01 245.78 526.72 503.74 627.86 326.21 499.55 677.48 435.17 460.07 315.54 358.22 308.42 345.94 565.1 311.53 315.7 638.74 363 428.12 329.93 268.34 412.73 313.39 253.64 299.76 246.11 520.4 201.83 311.76 249.47 246.37 283.5 222.74 164.3 252.38 307.46 318.78 233.56 216.47 539.64 262.9 429.19 726.56 294.4 568.72 754.98 709.72 367.74 300.83 417.94 750.48 313.36 739.13 395.31 445.48 487.02 278.58 616.29 526.95 957.75 551.88 384.56 303.81 232.3 312.93 247.85 575.8 201 478.99 485.83 541.23 340.73 443.83 241.84 1279.27 336.12 477.31 315.85 308.62 401.07 494.95 543.73 342.33 346.81 543.36 302.77 313.37 521.41 496.04 294.06 375.91 298.96 359.37 460.49 447.47 586.89 369.24 257.32 210038_at PRKCQ 28.98 15.21 45.6 56.73 48.29 44.55 15.44 25.55 31.38 34.33 15.3 16.59 35.27 13.62 33.31 16.36 39.52 106.65 106.76 114.63 25.23 99.82 15.53 123.15 26.55 181.45 26.24 33.17 72.14 24.45 32.84 22.99 19.6 60.53 38.11 115.17 47.07 33.47 68.66 89.07 69.26 82.48 98.6 27.15 54.46 58.13 56.99 39.95 200.1 26.37 21.29 74.61 35.98 58.85 17 21.16 37.01 71.61 124.24 26.78 20.28 33.48 27.37 49.2 86.44 44.22 76.96 93.77 45.95 36.56 25.38 71.82 16.77 37.44 13.93 16.64 22.53 116.81 25.04 26.91 18.95 33.36 23.65 57.98 55.44 58.84 44.99 109.74 138.69 119.41 25.37 66.61 25.34 19.9 43.99 67.26 20.2 14.8 86.28 76.65 22.89 74.53 42.75 22.21 15.68 98.53 31.44 63.36 36.54 38.75 52 38.96 137.11 35.29 160.8 65.19 15.39 142.77 181.97 53.75 76.7 57.97 66.8 109.6 56.14 112.09 27.16 55.6 25.84 33.93 168.05 33.94 74.03 99.75 22.71 29.88 60.2 49.87 39.49 24.11 27.08 30.83 29.61 33.75 76.71 83.48 29.68 39.5 73.82 24 72.86 155.76 23.18 57.3 79.18 36.9 16.5 132.84 18.64 95.92 30.71 36.91 20.22 26.13 20.32 114.9 19.8 93.36 33.43 18.91 70.89 37.79 41.18 107.36 19.44 22.49 106.07 13.31 29.04 20.73 139.48 64.55 38.39 27.54 49.63 23.77 30.49 22.36 81.22 19.64 27.12 42.85 15.69 40.47 47.73 45.08 19.38 89.1 93.74 33.09 36.66 55.23 87.11 29.37 43.38 18.86 29.46 47.94 43.73 36.52 94.84 80.59 41.07 29.77 15.66 48.31 49.94 37.14 35.2 40.43 102.04 28.99 18.45 21.73 34.33 32.77 60.34 210058_at MAPK13 234.67 153.67 149.19 491.96 250.03 222.75 238.92 218.59 260.01 294.52 283.58 226.97 214.02 182.84 111.62 101.08 623.09 236.52 251.47 268.32 228.97 313.45 130.25 272.46 173.37 233.56 227.19 322.18 186.85 211.07 202.3 364.18 247.67 467.11 232.32 393.97 306.67 191.83 127.74 170.46 198.94 203.55 328.98 107.7 216.09 87.89 134.97 257.41 424.41 261.88 262.4 234.03 176.64 234.82 712.69 263.89 158.84 253.98 276.58 92.26 192.41 235.47 482.72 237.82 22.89 273.3 308.43 218.5 109.08 145.04 314.76 186.99 204.98 311.78 327.01 187.35 773.33 262.39 331.78 202.46 272.47 407.51 322.75 362.85 315.03 185.57 274.57 130.73 273.3 148.06 61.19 140.13 190.94 113.72 115.25 239.59 269.88 444.71 104.11 204.37 80.46 214.42 318.07 147.68 437.63 305.57 158.4 276.28 284.89 89.1 300.33 175.84 290.25 234.76 492.51 175.09 366.65 224.49 181.51 273.51 99.26 210.44 267.36 290.86 74.32 341.03 197.71 119.97 121.59 276.38 166.49 393.37 76.8 295.71 239.55 201.28 313.9 156.09 302.25 346.81 194.35 326.92 372.62 357.24 167.88 254.74 255.94 359.31 261.37 551.39 249.57 172.97 387.67 177.61 340.66 393 81.17 410.09 161.82 136.4 183.85 245.46 128.69 228.57 188.05 509.13 233.35 185.33 138.35 397.94 257.16 296.35 252.28 267.76 150.73 256.06 189.74 180.14 164.07 501.1 101.35 249.38 265.47 116.16 117.69 276.32 152.89 601.88 318.08 580.12 353.16 141.07 189.01 351.94 194.66 280.64 338.89 123.05 351.68 193.92 514.78 225.67 272.14 269.91 426.48 225.44 171.33 289.93 228.27 362.22 303.04 157.21 234.34 133.3 151.36 269.08 396.58 278.74 282.37 123.34 494 592.72 487.46 220.98 348.5 259.43 185.44 210316_at FLT4 25.62 22.91 30.66 23.52 31.26 25.74 26.1 20.15 25.16 24.23 25.29 24.17 27.93 32.54 33 158.07 23.42 22.44 21.4 32.76 20.36 25.43 19.71 24.82 18.05 23.2 28.06 20.93 24.9 18.88 26.94 23.94 20.63 28.56 26.61 19.81 27.74 25.7 23.14 23.55 21.93 25.03 27.3 27.58 23.29 30.27 29.34 22.64 23.66 24.76 26.66 24.66 31.61 24.93 20.88 29.57 24.05 44.95 25.35 22.38 23.85 20.13 23.77 25.17 27.33 21.68 27.24 23.47 17.38 25.03 27.17 21.13 20.77 23.87 25.91 17.34 25.8 25.13 27.18 17.04 20.86 20.71 19.33 19.81 22.58 21.75 16.62 20.53 19.83 21.86 34.43 18.66 19 17.44 24.32 20.33 19.24 23.48 23.5 20.25 25.76 20.52 19.75 26.88 20.4 24.96 20.79 21.96 24.17 35.42 28.16 26.92 27.83 22.92 22.14 28.26 34.54 36.66 23.96 25.81 26.26 28.28 22.71 21.73 29.55 24.64 23.22 28.15 23.56 20.12 21.82 22.51 37.91 27.92 31.31 26.6 27.7 23.03 22.83 21.21 23.29 20.04 20.68 20.02 23.52 26.01 24.18 27.91 30.05 20.53 19.37 24.76 24.63 20.87 18.11 24.07 18.09 20.28 26.51 21.83 25 20.86 16 23.09 23.12 18.15 21.09 19.69 19.31 21.01 27.71 19.9 26.16 25.72 46.77 24.21 26.02 26.46 38.46 50.43 30.43 33.05 24.1 33.11 23.07 30.73 27.54 27.39 25.61 32.54 31.33 33.43 24.4 22.74 27.56 18.44 18.38 36.07 17.7 23.86 20.27 18.14 19.37 16.97 27.97 17.96 26.68 23.03 18.1 21.2 35.6 21.52 24.56 23.8 24.66 22.03 17.87 23.31 26.5 24.15 20.1 19.47 21.94 20.64 19.92 15.43 20.88 210416_s_at CHEK2 185.39 99.35 45.83 44.9 60.99 29.45 46.1 76.59 47.69 96.23 37.36 40.02 61.51 43.61 40.47 78.17 152.5 141.74 134.8 128.4 63.95 87.31 113.15 65.34 168.14 77.39 65.84 57.67 119.75 68.98 132.97 89.18 49.07 137.47 156.45 99.94 94.95 65.23 51.09 90.62 139.03 172.42 155.38 64.07 77.9 56.11 31.4 84.69 62.79 63.91 73.11 33.68 62.79 95.86 65.06 69.82 83.49 136.91 78.73 50.99 114.44 70.59 64.95 55.98 57.02 77.88 39.83 102.72 98.45 61.01 98.3 68.85 119.97 46.41 212.55 80.47 112.37 43.06 38.09 44.77 52.69 67.63 59.86 38.77 27.6 40.74 35.82 32.74 55.12 48.29 42.62 26.16 36.12 37.58 41.83 41.73 43.14 44.37 45.35 51.27 45.97 37.86 37.9 22.93 30.26 53.72 37.53 44.57 45.15 99.96 57.91 42.53 53.18 75.24 55 98.36 43.99 127.41 36.92 44.23 40.45 72.43 127.43 81.97 34.84 47.99 32.79 44.14 100.26 96.82 35.5 45.69 39.49 68.47 64.41 66.63 42.42 47.93 41.02 57.9 54.13 63.93 50.67 42.53 54.26 48.12 78.48 40.69 56.36 81.89 140.01 48.55 95.18 66.11 48.01 117.42 105.11 62.75 66.37 81.74 41.21 46.05 74.44 34.53 54.75 32.38 54.68 45.31 82.86 55 40.69 37.54 49.67 85.73 44.62 54.78 56.08 31.85 27.39 88.99 44.77 106.58 33.55 28.87 52.07 39.76 37.98 47.33 99.77 63.04 35.51 35.01 45.4 93.01 56.17 59.85 125.6 53.49 53.71 68.75 40.28 62.5 58.23 61.88 59.48 32.83 95.11 77.36 97.82 51.12 60.18 82.64 44.54 43.14 56.07 67.52 55.82 75.5 46.51 47.5 69.19 73.38 47.71 72.69 66.75 102.32 49.35 211107_s_at AURKC 22.94 24.64 24.82 32.87 31.79 26.77 32.5 29.04 23.45 21.2 23.82 21.45 22.17 33.35 33.64 42.07 29.13 37.7 31.52 20.17 29.76 23.2 27.57 27.63 23.14 27.83 30.33 189.6 27.68 23.75 21.57 19.51 28.14 27.87 26.55 24.89 97.32 25.65 30.31 31.36 35.72 28.97 45.55 22.86 19.59 29.07 31.28 18.12 21.99 27.37 26.13 87.22 36.07 24.96 33.91 30.86 31.27 27.25 27.13 46.07 29.63 36.51 30.89 25.02 32.75 16.96 22.41 26.08 22.45 27.42 22.1 23.76 24.57 25.21 24.52 21.71 32.47 24.93 25.32 21.98 26.99 21.78 16.89 20.85 31.73 32.92 61.56 36.08 24.47 30.81 52.05 22.16 27.23 27.04 25.34 29.85 28.16 26.5 25.11 22.58 20.58 24.74 27.78 36.04 21.47 31.86 28.47 41.83 28.5 33.28 31.06 20.12 33.18 29.97 25.87 40.41 33.02 30.87 28.07 36.85 32.21 31.82 37.33 30.18 28.54 29.06 29.1 25.78 27.66 27.55 31.03 29.48 46.22 31.97 28.13 36.69 28.88 24.49 25.68 34.32 29.95 35.18 29.35 28.59 26.31 35.58 26.17 26.72 26.52 24.42 22.5 29.67 25.31 23.81 26.95 23.5 25.25 29.14 21.64 29.53 22.25 27.57 22.55 23.19 26.25 29.17 26.39 28.68 34.02 24.16 40.93 37.28 33.15 19.71 37.79 36.65 35.41 32.88 35.98 26.35 28.17 30.18 27.72 32.91 38.82 27.9 38.59 26.8 43.65 29.37 29.25 24.88 40.44 31.93 29.5 34.53 40.51 39.29 31.41 22.8 25.44 26.17 28.54 33.51 27.67 22.75 27.7 26.41 43.47 29.56 31.13 27.78 19.74 32.69 16.83 19.88 17.55 20.03 20.72 21.43 20.64 20.35 20.23 26.63 27.65 20.04 29.65 211208_s_at CASK 283.88 487.55 328.53 416.98 453.8 218.55 366.16 458.28 261.57 386.47 251.74 535.7 342.65 634.83 281.82 303.2 411.96 288.23 405.33 440.84 290.03 245.85 745.55 441.03 290.97 288.42 401.15 315.3 379.13 434.33 236.95 417.51 445.16 407.94 496.98 55.48 415.65 392.51 148.26 255.69 274.21 336.21 605.81 682 429.39 332.86 741.12 289.65 299.9 174.33 351.15 362.19 727.3 373.9 278.41 468.62 171.55 381.04 385.8 500.69 1046.92 216.06 595.25 323.15 264.31 847.96 290.06 238.27 341.98 224.05 471.19 463.91 648.73 455.38 479.68 294.11 297.73 387.4 208.32 365.35 418.1 606.34 333.4 380.66 476.64 434.48 572.36 250.91 336.69 283.55 82.16 251.32 193.92 611.12 326.06 347.3 369.15 437.57 201.07 272.48 195.82 314.28 289.02 155.76 493.59 182.68 250.56 170.36 271.04 73.58 234.55 260.87 270.69 648.04 380.76 256.83 526.12 261.12 232.49 344.17 229.3 624.03 238.34 204.41 214.58 338.87 414.53 593.13 332.25 391.97 353 367.35 266.06 334.8 395.79 535.09 398.78 362.52 420.04 407.09 483.96 297.57 419.16 1678.95 370.98 173.57 429.3 323.25 232.56 371.61 314.11 575.93 324.94 188.24 466.45 285.68 226.36 183.56 324.63 221.39 215.4 383.26 310.14 171.09 183.31 222.94 176.95 250.41 214.36 184.78 274.45 395.29 230.95 282.46 342.22 610.61 231.52 196.61 309.7 486.52 382.41 511.89 291.66 244.97 388.81 239.49 306.72 327.76 521.38 324.97 287.83 356.69 249.18 205.05 224.39 444.56 206.51 233.53 213.51 398.63 142.68 190.53 223.5 90.22 252.25 351.3 269.16 383.47 176.24 410.81 227.54 174.53 248.25 230.12 326.19 297.1 211.67 288.36 388.69 194.57 344.51 530.28 573.62 379.05 531.69 258.16 239.47 211297_s_at CDK7 187.6 372.76 96.7 166.67 215.26 149.47 146.95 177.07 219.73 226.6 268.41 322.27 144.32 209 111.22 161.24 241.85 178.43 232.37 262.89 216.3 231.7 161.24 94.49 307.54 166.45 153.18 109.43 77.34 299.13 173.17 218.36 222.52 166.89 272.36 122.49 90.68 107.09 110 213.48 244.57 201.8 323.42 127.76 147.33 118.51 150.01 89.3 124.1 154.66 131.85 255.06 159.24 166.76 244.73 272.52 142.31 215.82 170.64 163.38 93.3 89.6 107.73 105.49 93.41 122.91 116.58 192.4 216.01 128.77 94.34 98.73 249.05 154.31 167.18 511.39 252 143.37 428.43 155.36 127.44 261.31 213.66 188.72 127.46 173.01 94.93 145.73 166.36 85.35 41.31 89.77 91.41 212.7 96.71 108.13 214.6 174.04 138.4 108.6 118.63 138.3 267.37 105.11 218.36 194.07 226.29 166.48 118.7 117.79 214.58 189.57 131.95 168.09 111.02 141.94 140.7 93.57 140.85 152.08 174.06 137.11 153.89 175.79 85.56 185.28 97.11 179.56 314.63 190.68 133.42 140.91 79.95 135.98 260.4 127.01 68.01 143.41 100.38 159.08 142.66 109.08 114.44 159.75 151.63 121.56 119.8 169.35 129.24 329.64 132.19 82.47 448.08 176.4 187.53 222.74 330.71 160.79 203.07 144.21 137.89 114.38 362.34 70.25 239.52 123.65 239.5 123.03 109.05 172.25 217.46 160.21 192.38 87.3 92.17 120.36 158.36 172.82 92.22 247.92 155.23 142.16 275.79 198.07 212.37 125.47 198.79 239.72 136.43 310.2 193.65 189.68 198.75 209.31 193.4 150.33 269.04 121.78 147.58 89.62 115.46 213.19 182.09 109.62 142.95 177.86 92.14 219.98 105.4 210.22 86.55 94.17 75.09 115.09 118.44 107.01 128.72 164.6 125.81 133.06 135.82 157.31 153.54 168.62 186.05 93.82 154.98 211339_s_at ITK 49.67 31.27 91.63 124.82 103.01 139.67 17.3 45.05 25.99 44.97 14.17 18.34 38.3 15 39.62 16.09 34.48 395.8 151.58 62.81 40.36 293.58 25.31 145.51 60.88 612.76 51.41 78.15 184.46 20.46 50.24 30.63 36.55 97.41 46.44 112.67 104.99 65.7 159.13 287.69 203.03 252.19 46.17 36.83 69.8 103.25 41.85 90.96 168.85 49.17 21.75 118.53 32.97 134.38 14.91 35.46 44.58 84.88 282.76 30.35 19.37 36.78 22.76 65.77 143.73 79.12 57.5 129.61 54.66 53.47 25.45 131.94 21.28 80.03 14.81 14.54 24.8 180.89 20.42 17.49 17.49 25.52 11.78 47.61 74.11 87.45 60.43 216.88 226.03 138.02 15.32 77.49 31.61 23.65 40.21 80.09 15.02 22.96 55.07 40.57 17.87 93.34 55.51 16.83 14.13 120.24 25.38 62.54 28.95 17.43 76.25 31.97 202.49 64.78 220.11 58.92 16.48 148.46 331.42 50.93 87.1 61.09 98.15 169.01 60.11 179.68 21.18 63.87 21.78 34.06 202.14 21.57 19.8 106.51 15.56 23.32 40.33 31.44 22.75 14.7 17.62 15.35 23 22.93 77.01 64.88 19.37 34.63 148.91 32.15 55.1 334.45 16.46 128.87 187.19 27.31 17.3 267.79 20.12 160.48 37.52 44.42 29.13 27.96 18.49 255.46 22.31 256.01 15.54 18.53 63.07 36.04 36.91 73.25 15.17 14.96 103.47 20.67 23.36 16.41 210.22 23.64 27.37 16.34 39.35 17.56 26.59 15.97 20.54 14.27 19.4 33.08 15.37 64.25 21.87 47.47 26.96 75.34 131.47 27.35 34.06 96.25 262.59 17.08 20.15 22.22 28.56 73.23 45.76 41.99 39.41 134.6 33.41 23.45 12.96 70.38 94.26 44.39 34.09 24.49 174.18 33.1 13.12 11.24 19.62 29.51 85.05 211421_s_at RET 21.61 134.67 15.6 505.83 788.25 340.48 364.32 160.8 356.48 55.39 162.95 86.68 312.94 633.38 30.8 20.99 11.59 25.32 38.19 59.22 261.22 187.72 15.07 16.2 11.98 17.97 629.09 11.3 37.23 17.12 25.51 59.1 297.62 939.16 13.73 15.1 10.25 11.86 15.89 16.46 245.56 279.94 10.98 18.97 15.03 41.17 10.78 12.79 15.59 67.01 13.64 18 474.02 14.04 342.01 8.04 20.82 10.16 426.98 13.74 33.38 13.25 12.23 226.62 16.6 24.4 29.74 22.75 17.69 10.07 12.55 47.15 25.92 29 95.87 46.83 8.86 95.27 161.24 17.04 22.6 344.75 400.18 145.95 156.14 133.49 363.89 19.76 13.63 135.95 22.1 15.05 143.01 31.02 233.55 17.21 24.95 44.53 153.79 16.87 9.53 16.15 85.91 10.51 10.58 101.56 17.23 26.47 147.6 17.64 21.97 14.37 7.83 12.58 10.89 14.18 76.86 12.44 25.4 13.34 10.89 13.46 15.51 12.61 24.7 20.38 26.08 176.18 150.34 10.36 71.17 29.07 23.11 21.65 16.51 824.08 8.96 71.91 116.43 37.1 24.22 23.86 432.47 84.29 381.94 19.34 56.15 309.54 32.54 326.68 19.74 32.79 13.48 11.54 246.76 20.81 174.38 12.13 17.28 13.28 199.65 71.27 128 30.98 21.34 14.42 167.17 24.61 67.36 15.17 3113.45 15.73 49.07 20.07 63.98 15.28 19.21 760.03 152.01 349.22 16.33 13.62 85.17 48.01 11.58 33.44 244.02 192.4 17.08 328.61 18.18 10.42 17.07 22.39 99.77 197.85 12.41 22.69 97.29 12.34 137.17 100.7 56.35 352.88 16.46 285.8 19.63 9.71 22.57 25.6 13.43 138.83 26.6 19.53 457.29 1140.31 10.27 75.9 426.2 435.48 8.03 11.02 14.93 11.14 8.5 8.12 13.21 211432_s_at TYRO3 102.05 73.84 110.14 125.37 82.21 65.29 93.34 78.01 76.97 99.21 98.11 68.22 69.83 83.81 77.69 89.5 143.83 110.14 91.71 84.22 73.43 80.66 88.83 94.73 88.31 60.99 83.85 110.15 94.39 123.85 61.45 65.46 78.59 80.32 104.73 114.47 82.54 78.05 63.8 75.44 96.22 79.91 130.09 81.26 90.27 76.56 86.18 95.38 74.9 102.72 104.11 62.09 105.46 82.76 107.33 102.35 275.8 110.37 82.14 129.42 89.67 90.9 122.7 91.13 63.8 67.99 87.12 81.54 111.39 80.57 97.32 96.94 60.5 58.02 67.36 63.9 50.79 60.33 45.57 66.15 106.81 46.28 76.96 63.13 69.05 70.26 81.56 77.48 59.07 59.22 177.37 58.47 64.91 49.55 57.73 83.27 60.63 59.28 79.95 88.69 89.99 83.93 79.85 45.1 58.57 157.72 77.76 65.57 191.58 152.82 133.39 71.63 78.3 104.97 81.39 101.78 96.74 75.89 77.11 161.37 141.06 174.38 118.81 87.16 88.86 111.27 68.25 104.13 58.19 104.02 58.15 83.48 101.06 97.66 76.28 58.56 104.56 90.19 82.6 60.02 64.06 56.54 85.8 73.99 63.79 82.4 122.34 87.01 74.12 58.81 106.06 72.47 122.07 79.59 72.13 114.29 70.47 101.89 66.69 76.15 77.45 80.2 90.45 64.27 98.41 78.6 50.5 80.39 89.19 72.73 86.85 104.24 114.07 113.78 123.82 107.55 112.11 77.38 91.76 105.14 76.93 151.08 116.07 116.64 112.09 111.03 102.76 80.61 156.97 95.48 59.61 89.26 114.53 125.02 100.11 88.88 133.82 116.99 111.43 182.08 87.08 75.42 69.77 127.63 99.38 64.86 93.04 118.68 128.1 77.84 117.09 93.38 130.64 92.03 83.64 67.74 79.46 93.74 90.54 72.05 67.26 60.24 53.52 85.6 84.82 118.79 92.35 211500_at MAPK11 73.59 49.47 80.04 85.13 72.64 113.08 74.14 102.64 86.43 84.09 66.91 65.55 72.73 69.27 89.16 79.08 63.26 67.08 50.71 50.84 67.83 60.91 56.41 92.92 64.13 73.2 73.95 49.75 81.21 84.24 60.78 57.96 60.36 65.07 60.01 73.58 55.63 59.51 105.94 80 75.87 73.65 55.39 55.35 76.54 78.02 91.96 63.66 80.68 107.88 56.38 82.9 96.38 64.51 126.68 66.93 110.97 57.18 99.31 100.79 70.6 53.62 57.46 57.92 59.75 45.15 54.45 38.81 59.37 39.78 68.6 59.82 48.13 48.82 50.08 37.35 33.89 50.83 56.84 43.61 51.1 61.95 46.59 48.93 51.08 41.51 64.9 59.49 44.42 57.51 90.55 47.65 48.79 48.09 49.87 43.33 58.45 42.94 41.92 61.81 53.2 56.65 76.21 56.36 61.46 47.97 52.84 54.61 48.5 105.77 43.73 41.71 56.86 55.11 52.66 47.17 62.25 70.89 55.45 76.04 69.38 58.38 61.39 50.44 51 49.75 45.16 40.15 41.97 47.49 45.78 46.19 57.69 50.65 43.33 42.55 50.57 40.42 46.28 42.88 46.66 45.19 43.41 37.15 40.78 47.04 38.89 59.37 74.7 70.57 59.57 115.1 70.53 66.37 59.36 75.92 59.25 66.41 50.79 63.27 64.24 66.33 57.18 55.65 66.59 52.79 60.33 59.59 81.98 49.19 61.01 52.69 62.68 53.71 92.04 67.68 59.54 65.79 50.55 51.75 56.89 55.81 49.98 52.84 65.34 54.41 52.67 52.48 49.86 45.22 56.08 50.58 68.99 51.83 60.39 69.22 44.33 83.57 59.43 66.23 71.83 56.32 55.98 55.99 60.21 50.23 53.48 60.23 69.46 49.65 60.42 72.42 67.64 73.99 58.75 72.19 58.4 46.16 58.06 66.54 59.19 67.1 103.6 69.29 69.39 58.11 72.78 211593_s_at MAST2 214.42 164.71 199.03 116.9 290.3 325.68 169.65 374.41 186.27 253.15 130.98 340.29 168.43 307.8 203.59 450.7 118.73 122.35 254.81 234.76 187.84 105.71 181.45 163.51 278.85 132.52 167.12 252.97 136.38 110.29 89.29 124.27 179.49 752.99 171.98 223.81 116.88 284.75 189.75 151.9 146.18 164.66 214.79 371.21 170.52 205 155.29 214.02 336.45 166.75 194.06 188.3 286.5 206.82 711.17 216.89 194.56 230.71 209.04 262.41 351.85 776.6 442.1 139.33 100.57 121.43 160.09 108.77 183.24 171.64 181.38 169.02 136.3 138.21 131.31 199.45 117.37 98.45 108.17 168.79 159.88 145.63 122.93 130.99 141.97 138.85 157.75 158.77 148.99 291.09 330.97 219.61 243.33 133.09 210.12 132.32 206.58 385.14 220.87 239.95 173.13 152.68 324.95 169.02 164.16 280.8 199.07 226.85 354.44 516.43 140.55 158.19 169.15 284.89 232.39 130.34 578.31 212.77 220.8 274.28 194.48 467.22 359.47 159.78 147.04 123.15 97.54 159.43 112.59 117.35 168.52 119.84 202.7 210.54 122.25 167.01 193.37 105.28 145.61 142.59 97.19 104.64 153.78 173.96 110.32 125.79 187.73 240.23 245.24 238.81 252.18 251.49 187.66 214.85 194.25 246.03 277.86 153.08 201.85 139.55 328.7 215.85 127 320.18 175.05 174.65 186 225.23 311.8 113.38 208.28 187.58 200.63 205.56 323.09 173.43 204.6 135.28 292.01 283.87 121.63 193.97 114.18 188.65 202.48 162.18 213.97 184.28 150.98 452.92 187.59 143.04 248.18 246.32 155.69 251.93 181.09 242.34 367.73 288.11 239.79 308.98 203.26 392.54 178.3 240.38 234.35 166.1 191.85 147.6 192.06 222.53 317.76 355.15 365.48 307.53 233.29 176.27 160.61 341.83 218.45 195.63 244.54 175.28 176.92 278.26 108.21 211992_at WNK1 542.9 1201.46 1567.17 1539.96 1553.85 1263.24 241.42 1070.86 1072.34 1193.46 1320.53 1225.53 832.85 921.78 1083.86 663.96 1952.72 1003.13 1429.45 673.39 1371.47 593.12 1010.05 1263.96 887.3 1045.48 792.9 1331.93 775.11 686.02 802.91 1254.47 1404.93 1679.49 1407.94 913.1 1086.01 933.23 886.83 827.61 671.51 721.01 1343.71 729.07 2051.86 766.06 791.56 553.84 1404.32 781.78 1206.27 811.27 2045.05 1052.51 1318.86 1241.44 968.71 1435.58 776.67 1165.83 1767.96 1688.73 1092.91 1436.73 1953.59 1217.69 2150.32 1697.96 2161.7 2001.82 1145.18 1777.58 713.2 995.97 1692.46 1052.25 1023.3 1170.05 1211.8 1918.69 2437.76 1919.72 1420.45 1995 1449.42 1213.51 1302.59 1514.97 1811.65 555.97 2298.3 1045.64 887.26 476.74 616.63 749.27 1028.12 825.12 468.7 500.81 1179.89 891.23 774.6 437.89 1275.19 1754.64 1020.67 1004.01 1339.88 1029.87 1707.09 837.15 955.01 1251.39 969.07 1003.26 1185.62 946.94 1170.42 1453.54 1760.83 2868.58 1400.59 1635.24 1233.6 1013.75 1241.89 1504.64 717.09 1620.14 1257.3 1200.68 1516.69 1140.83 906.64 1553.21 715.67 914.99 1300.27 936.41 932.04 993.08 1309.38 1115.55 1234.59 1453.16 626.97 1122.65 1095.17 582.31 1674.06 1225.49 818.54 1020.74 966.13 1093.98 599.2 903.19 981.16 835.95 755.62 848.1 554.79 1256.46 1073.99 1414.05 655.73 754.66 2187.21 885.15 1344.17 1470.05 1308.89 1319.04 1193.17 956.23 1248.14 1010.68 957.19 1089.89 1304.5 1140.76 925.28 1158.23 1236.07 1097.44 1168.68 1617.58 1209.68 1351.02 1319.86 946.68 914.45 719.98 738.74 814.3 789.38 1138.84 1105.06 740.22 1262.18 797.8 880.96 3294.88 1317.36 1146.87 2014.24 764.56 1252.34 1041.63 2581.61 1676.41 924.46 1176.08 1160.68 1234.59 1180.65 1068.44 1151.71 1305.48 1362.83 921.94 1114.24 1108.54 2388.59 1062.03 1036.8 212073_at CSNK2A1 105.67 168.29 124.93 106.18 302.31 172.04 34.64 283.58 216.42 159.69 86.82 115.44 103.59 179.65 103.72 171.73 174.05 115.06 229.83 194.87 220.92 155.26 129.61 104.73 188.03 155.77 123.34 222 110.08 112.53 106.27 148.47 147.11 160.19 107.95 114.6 129.45 189.03 90.09 147.42 93.49 111.76 157.26 153.54 141.02 121.46 151.77 101.94 95.64 101.29 99.86 181.26 255.32 133.95 137.26 179.82 103.49 65.81 124.2 83.38 90.39 160.73 116.52 125.17 151.03 118.82 122.98 110.62 153.27 192.27 181.38 159.63 126.57 210.96 120.35 167.13 78.47 106.71 74.98 123.91 143.43 169.34 212.21 98.27 569.74 132.42 124.83 103.55 148.75 200.77 81.6 158.58 252.06 200.4 256.52 136.18 146.75 92.4 133.07 89.53 177.39 125.94 145.29 128.31 124.19 129.08 131.95 120.62 120.42 105.64 165.44 211.36 153.24 152.91 234.08 35.5 223.82 177.26 112.99 332.89 188.32 161.45 173.41 289.04 170.67 86.47 196.78 213.21 142.75 94.14 142.9 167.34 179.41 136.18 152.34 303.71 193.81 179.47 129.19 146.13 133.32 78.28 167.59 124.9 138.45 178.76 160.39 230.56 115.14 169.64 113.88 130.62 170.22 94.04 146.76 140.21 200.42 136.84 135.02 141.05 133.52 94.12 162.28 122.02 118.35 240.39 154.71 140.07 123.05 136.25 159.56 136.54 198.68 125.97 179.19 98.13 117.03 107.55 134.45 210.34 119.31 112.6 88.24 121.97 131.19 159.85 139.04 260.84 132 184.27 150.44 118.08 109.15 126.1 137.13 162.56 101.67 161.11 210.95 154.64 166.72 112.56 134.11 93.82 180.94 117.01 122.55 130.51 131.8 131.06 132.44 207.07 204.63 108.05 139.72 193.25 151.74 144.14 133.45 154.53 130.07 149.56 186.49 198.33 152.67 72.64 117.44 212252_at CAMKK2 153.08 215.85 217.07 169.96 185.55 211.73 163.82 247.66 178.03 186.15 233.16 255.74 227.19 205.62 194.13 171.27 155.59 234.39 348.86 270.32 263.69 125.71 165.72 158.66 212.66 144.08 181.48 134.24 124.96 198.28 528.25 220.19 227.97 240.13 192.26 171.97 171.83 191.83 189.01 238.97 270.4 220.91 434.66 211.22 139.65 199.68 167.97 134.86 190.34 230.62 165.62 211.8 293.21 174.97 396.69 231.01 221.37 175.78 247.92 189.92 289.14 184.92 228.29 238.18 154.45 177.56 176.83 182.21 220.01 160.65 141.07 186.43 207.34 137.76 192.05 155.95 173.43 128.66 139.52 140.05 271.21 191.92 212.64 198.94 167.26 194.13 181.63 174.32 165.53 89.79 118.64 148.82 127.66 119.43 148.74 89.79 189.19 185.02 153.33 103.2 206.22 119.47 172.01 128.35 160.87 205.04 158.45 196.29 231.96 157.23 107.11 176.95 178.95 206.61 139.24 115.45 198.3 149.98 134.22 128.03 163.72 231.56 168.32 211.74 157.78 169.73 184.45 225.02 108.55 136.42 182.87 171.08 221.93 184.34 142.02 165.07 123.04 102.11 133.98 111.68 138.02 114.03 150.38 132.82 134.95 136.34 108.34 203.98 192.75 206.95 288.74 183.42 156.12 191.15 188.7 175.78 103.27 133.66 188.03 241.65 309.46 157.11 134.24 131.46 215.19 138.72 244.33 175.75 140.37 180.42 188.59 188.01 219.93 131.56 220.6 133.56 170.91 169.37 210.55 265.26 185.11 163.91 230.03 146.18 175.18 200.45 117.86 292 165.01 194.14 217.1 190.76 215.68 171.09 239.76 238.79 188.24 246.04 217.95 173.66 209.29 224.44 213.28 176.71 221.07 175.57 248.78 143.31 158.6 161.43 158.43 219.7 243.77 121.56 170.58 165.34 182.04 164.82 127.04 163.44 186.36 157.93 216.03 180.36 180.2 161.48 117.92 212293_at HIPK1 543.13 681.92 353.06 519.17 440.66 472.96 463.41 801.04 747.91 346.16 536.31 449.12 358.89 455.27 310.3 408.99 336.71 504.06 638.85 734.58 427.8 506.42 472.5 258.19 468.52 622.89 557.46 1118.77 493.65 367.61 564.91 384.7 421.72 562.72 427.35 259.67 266.52 454.26 341.66 204.36 333.04 401.25 239.56 339.32 195.61 407.55 306.47 416.53 341.15 280.04 416.01 411.31 297.76 506.37 472.23 429.39 279.94 183.91 255.51 265.6 405.36 417.52 316.47 250.71 171.89 255.85 374.26 323.88 483.87 427.66 261.16 212.21 359.2 323.63 465.71 304.4 375.53 465.83 494.97 261.79 165.52 873.65 645.09 514.22 521.73 442.99 232.72 417.07 325.72 494.62 38.91 461.32 515.84 492.98 609.73 368.71 318.11 344.05 532.99 439.69 360.99 482.08 418.55 325.95 272.93 286.44 298.55 305.62 326.98 15.65 172.56 394.45 338.77 283.62 386.47 348.59 129.22 124.27 240.48 275.52 216.58 189.3 199.99 305.88 501.79 307.32 236.83 448.95 420.51 430.96 363.6 198.02 221.06 437.27 438.91 371.45 339.9 344.82 391.54 367.04 563.65 477.53 435.2 351.61 365.37 147.43 264.14 409.78 367.39 332.88 260.84 303.38 222.94 325.25 358.76 361.06 314.1 297.28 370.17 386.94 404.06 331.35 282.87 261.11 249.05 340.48 338.39 357.62 49.44 220.99 314.3 177.2 281.53 280.18 17.71 166.08 262.66 317.42 369.97 241.45 368 225.14 243.41 473.39 239.99 493.17 337.79 363.43 262.07 398.09 286.3 519.27 250.13 347.82 298.41 226.82 264.23 261.95 253.31 325.29 183.54 336.95 494.02 45.26 371.2 284.08 395.6 349.1 272.35 457.98 214.14 308.08 296.41 201.48 253.51 327.97 333.65 349.59 335.57 295.7 339.15 334 281.4 345.98 416.23 515.64 326.4 212299_at NEK9 234.15 470.59 494.74 296.76 448.6 784.06 672.62 668.1 341.59 240.19 421.12 706.73 493 705.18 610.67 606.52 158.78 523.52 432.02 343.38 448.51 350.87 214.08 297.51 296.67 444.3 342.19 226.62 268.82 263.94 316.14 328.58 297.78 601.62 270.25 245.42 298.34 259.72 452 274.68 298.59 265.92 156.26 355.54 275.92 427.87 253.03 230.25 304.87 298.86 194.95 417.41 532.51 331.6 269.67 327.91 210 206.33 412.86 491.37 587 397.94 289.17 427.82 503.26 237.83 450.73 326.46 129.06 274.45 254.62 318.19 129.62 262.8 209.47 225.6 218.94 489.75 400.67 287.91 293.58 382.24 363.77 410 398.57 448.92 295.63 371.91 344.48 370.06 452.33 327.46 375.73 296.11 391.85 231.77 240.91 224.11 354.27 207.01 336.51 274.7 282.35 374.34 370.32 411.39 406.39 409.2 323.55 290.07 133.6 404.32 285.46 299.75 275.92 220.37 529.96 263.55 303.22 237 351.42 230.9 213.73 228.43 392.78 219.65 359.15 387.24 235.47 171.14 341 247 367.52 273.95 290.44 244.46 334.11 194.42 324.01 227.84 257.98 248.36 234.71 258.36 194.32 387.21 184.63 280.5 317.75 311.49 193.14 348.56 216.04 351.58 333.06 276.96 245.21 242.3 361.97 258.22 420.31 314.58 274.11 424.22 359.93 339.82 266.81 340.4 218.8 330.29 256.5 308.14 404.54 202.97 226.22 179.5 242.66 297.25 428.29 367.88 311.87 150.78 285.93 291.35 204.21 282.35 320.01 234.07 85.01 352.3 269.51 317.68 294.06 276.11 326.93 236.29 224.16 294.69 365.14 206.93 223.8 344.76 305.96 235.26 179.07 362.94 133.57 135.88 234.47 198.69 227.14 396.96 336.01 394.93 384.27 267.65 261.04 250.92 223.66 399.75 302.92 176.28 356.32 178.65 193.04 237.26 269.18 212401_s_at CDC2L1 365.38 279.09 496.6 281.77 439.55 368.03 508.53 448.58 256.17 385.13 285.79 430.69 446.68 297.97 479.5 349.24 393.82 669.71 638.35 482.36 419.79 420.99 357.03 421.7 449.08 427.4 339.62 306.96 379.74 211.64 291.53 348 509.91 347.44 476.98 449.41 428.83 261.93 417.09 334.09 341.26 346.32 335.24 502.11 210.55 310.22 393.4 178.05 299.5 258.43 152.96 374.27 434.09 476.2 326.89 395.33 416.68 356.83 384.65 305.23 378.18 770.34 444.1 214.96 364.06 203.69 233.88 322.55 261.16 383.99 431.58 480.14 244.84 183.72 297.1 171.66 202.74 229.02 184.22 229.59 447.45 326.19 297.05 326.96 301.68 322.61 228.31 319.57 288.92 546.39 640.72 428.45 536.57 392.79 514.28 406.45 393.55 722.79 632.44 412.33 676.68 512.81 471.03 881.26 397.06 473.94 447.49 410.76 614.09 642.14 444.43 361.58 406.47 406.08 337.63 246.41 487.45 962.51 419.35 328.87 401.01 501.21 519.76 405 369.33 302 386.69 303.7 156.35 237.49 398.77 264.25 481.98 350.84 396.69 297.62 255.43 287.84 231.39 275.86 186.1 246.57 349.97 306.67 319.16 336.36 197.98 390.61 487.8 347.69 478.35 455.92 240.59 368.55 329.78 385.98 155.65 414.48 345.36 507.07 503.53 355.25 261.83 354.28 235.86 311.2 360.3 450.56 825.98 179.64 376.76 399.19 388.49 266.98 502.12 461.96 378.77 305.03 348.53 426.87 317.6 344.07 159.25 275.57 282.17 241.21 232.94 283.31 397.56 193.17 251.95 264.01 380.77 363.8 259.67 290.83 209.93 240.06 279.69 239.74 208.5 264.1 327.7 367.07 324.87 248.24 233.91 260.27 270.58 181.89 316.1 440.54 328.76 413.54 335.52 245.95 291.46 278.59 281.96 357.44 311.11 280.92 352.37 298.72 428.97 253.49 320.8 212435_at TRIM33 405.93 418.33 169.3 281.7 302.81 374.13 285.07 555.25 386.65 198.98 227.17 369.71 245.54 351.77 204.68 272.35 223.91 447 474.23 467.08 286.97 270.3 351.39 204.58 319.59 321.03 188.14 440.04 276.74 213.54 202.45 234.11 340.78 417.79 198.57 296.04 270.87 240.13 221.14 178.52 312.4 275.65 242.48 327.48 225.13 229.78 266.32 228.85 197.65 129.07 273 237.94 259.13 345.71 469.65 225.27 161.38 129.11 200.49 165.67 303.55 321.84 139.28 207.36 111.78 156.86 174.45 200.89 373.56 256.71 227.6 127.24 218.72 205.72 234.02 221.37 221.7 202.37 274.59 125.63 107.95 273.19 282.32 242.61 201.84 245.51 125.56 212.35 170.15 297.65 68.02 216.6 277.59 296.36 333.01 158.3 209.5 172.29 311.42 219.01 437.75 282.16 223.48 214.31 174.67 234.68 295.97 301.32 399.56 22.06 208.75 281.74 239.49 231.33 290.97 314.86 95.63 155.06 199.76 218.1 164.96 315.52 176.97 244.42 222.58 313.6 186.87 217.46 251.76 363.04 230.96 115.82 177.44 318.96 229.19 265 173.33 166.64 275.75 204.45 340.06 299.76 293.59 286.61 248.43 121.87 172.97 263.35 204.17 243.78 133.55 174.69 149.58 229.7 276.58 293.31 193.5 176.15 224.5 278.21 256.75 221.11 192.12 229.02 157.47 177.76 230.63 211.54 136.16 132.02 232.51 191.5 296.53 240.73 163.74 96.31 201.91 254.8 206.95 290.66 246.05 221.48 133.81 338.82 184.29 348.74 199.51 259.09 236.32 285.43 162.88 281.7 221.48 275.66 311.1 205.58 219.21 199.65 275.96 301.21 215.15 282.8 329.66 243.3 290.12 259.74 296.07 411.31 237.55 404.93 151.27 252.03 191.48 212.23 237.18 203.22 194.79 176.62 189.54 265.31 208.66 254.04 241.58 268.5 243.21 340.79 217.43 212530_at NEK7 579.17 604.84 301.86 673.35 291.3 314.35 473.54 368.69 344.35 603.51 686.3 238.98 324.77 346.35 120.44 277.89 448.35 294.6 546.19 1188.99 348.59 710.44 375.52 330.38 335.83 502.93 467.98 510.5 494.82 509.88 902.38 432.91 832.07 282.97 493.62 257.14 363.29 286.04 499.68 228.65 859.01 427.59 692.83 290.06 446.95 380.05 341.59 599.53 270.81 340.7 485.83 316.45 258.11 542.64 221.4 583.56 490.13 312.79 306.7 168.1 466.9 188.43 369.2 231.59 225.2 592.43 323.07 250.6 464.18 569.33 436.44 254.36 595.9 425.07 459.17 423.94 995.91 873.33 557.06 389.57 268.68 470.17 334.05 455.97 706.49 604.05 383.61 540.61 480.39 538.23 52.44 422.3 828.98 1076.03 586.41 785.13 565.3 620.3 632.02 903.09 332.36 563.2 644.84 397.18 514.41 452.94 462.15 328.94 334.83 26.86 574.2 377.73 637.82 508.41 636.91 516.36 181.62 254.19 349.59 186 311.86 379.98 136.9 392.41 472.47 427.24 436.41 526.83 1059.45 556.37 408.7 398 81.01 301.26 388.32 438.25 361.75 471.17 526.5 932.33 835.13 333.97 416.93 431.86 400.55 223.2 260.52 281.06 326.86 779.46 362.05 240.29 559.93 386.01 481.09 269.69 731.38 343.24 380.7 633.66 486.61 530.93 1069.41 266.61 580.59 488.47 533.84 319.86 108.57 417.89 255.91 164.7 433.24 585.76 113.61 196.74 376.39 459.14 347.65 648.36 520.58 287.53 541.09 501.15 431.66 319.01 556.98 687.98 256.29 290.73 555.7 854.74 440.28 305.43 405.24 305.33 412.31 402.03 223.38 341.4 398.24 473.65 357.51 67.62 249.97 382.26 341.8 555.89 311.31 609.01 264.08 325.38 357.56 194.6 235.69 306.11 353.52 289.27 337.86 191.65 440.32 436.74 212.18 676.83 514.54 547.19 306.67 212533_at WEE1 468.55 724.44 292.88 432.23 873.01 320.8 644.04 904.4 571.59 289.34 615.6 520.22 683.44 718.74 438.12 448.95 329.94 398.63 1002.67 1193.11 622.56 623.46 588.62 476.91 556.03 305.62 458.96 456.89 656.51 420.63 625.09 305.73 596.53 496.1 435.86 417.13 405.07 774.3 221.9 428.1 625.95 800.77 869.25 1715.99 324.16 378.17 668.14 612.55 449.06 1183.01 496.29 497.15 754.95 811.5 551.9 709.09 456.02 270.98 441.83 328.73 778.05 1671.11 927.97 360.24 572.07 412.35 1272.92 555.71 591.76 1106 480.96 758.62 643.32 352.69 325.43 264.02 773.7 595.05 457.49 322.88 452.48 922.3 722.24 883.7 438.66 528.97 304.59 449.37 520.17 600.08 70.4 438.82 415.04 277 558.42 443.32 389.7 713.82 442.96 543.41 1190.78 1030.13 740.14 669.75 393.57 534.28 475.87 371.03 355.94 38.29 540.45 409.61 300.61 640.13 585.4 1118.58 1379.46 262.77 374.8 318.93 464.43 307.29 328.64 650.35 365.18 615.79 830.27 285.21 463.1 346.45 248.06 188.95 231.19 508.49 484.85 224.46 179.04 450.19 316.75 1412.36 717.16 489.56 533.58 395.67 218.78 103.57 479.16 467.68 305.97 606.16 495.91 287.21 410.89 473.69 449.46 444.88 459.2 186.24 316.83 481.39 264.42 407.18 670.36 188.98 309.18 186.3 320.7 476.51 117.29 677.31 300.5 250.39 385.17 510.96 115.09 271.17 298.31 301.54 292.54 663.19 306.89 404.25 536.2 467.43 161.26 337.31 229.65 467.95 557.8 396.35 358.96 392.52 159.54 245.16 332.9 817.81 505.57 174.11 324.91 218 155.92 278.54 205.02 93.47 278.94 777 942.48 553.55 110.51 305.28 595.95 105.7 216.61 322.28 368.05 384.2 231.28 379.72 547.48 329.14 297.1 239.97 282.9 369.99 663.36 720.42 320.88 212572_at STK38L 321.17 433.32 509.86 245.6 210.86 163.04 154.29 307.4 267.91 365.62 285.23 355.71 234.26 222.15 145.02 156.08 1045.36 251.6 369.7 244.92 320.52 344.12 294.16 242.23 766.04 524.81 473.37 502.71 550.22 215.67 651.81 365 265.4 342.54 326.49 187.61 167.07 1021.32 288.36 248.71 817.49 334.3 1062.71 392.86 239.5 339.39 549.9 347.77 347.65 244.83 2294.79 347.87 143.52 1115.52 132.22 329.96 305.32 384.38 103.28 219.94 473.89 325.42 419.63 175.36 149.19 500.02 430.7 239.2 785.17 548.1 476.42 227.39 449.76 489.62 158.81 297.14 238.46 261.39 125.55 235.06 184.01 391.83 301.27 204.24 184.6 262.77 406.29 196.77 138.92 249.04 41.5 284.8 167.48 531.56 407.68 365.88 282 340.6 282.05 1017.73 286.75 260.77 364.23 178.05 267.11 224.12 283.32 204.9 152.7 16.23 1858.85 187.46 281.36 551.9 458.61 251.27 122.62 165.71 193.79 352.46 277.53 784.21 254.94 413.5 398.72 226.79 222.93 361.29 392.43 901.83 215.6 271.41 78.24 224.25 407.67 167.01 119.59 327.58 343.6 424.2 318.47 381.99 144.08 225.79 182.53 117.85 280.21 194.94 153.06 551.2 273.71 156.63 468.85 377.55 213.13 195.98 134.59 191.39 266.41 265.48 138.37 976.24 408.9 142.34 328.07 191.98 371.92 208.1 44.43 156.02 183.08 134.64 336.14 369.31 31.29 182.91 260.79 202.48 247.91 125.6 306.89 254.42 185.98 180.49 315.18 155.85 164.76 203.66 267.54 185.82 202.23 379.15 187.54 291.43 173.51 370.88 1019.67 235.56 271.54 272.03 131 269.74 199.31 23.52 137.26 219.09 485.16 469.46 318.6 420.58 395.48 265.98 153.18 240.78 337.83 202.77 164.67 234.01 354.02 269.56 797.08 336.67 168.54 775.96 667 141.37 251.82 212607_at AKT3 113.86 127.39 263.25 149.36 112.43 114.56 96.63 138.95 111.28 86.74 415.98 160.74 71.25 61.44 106.88 200.47 79.99 95.38 355.96 184.86 107.03 103.57 33.6 246.08 200.94 165.06 144.41 96.43 90.8 88.66 365.14 95.55 75.61 55.9 68.03 47.15 519.27 306.92 182.35 166.35 744.12 302.87 605.79 131.59 153.76 244.35 261.26 341.02 161.08 153.56 94.78 152.73 123.89 134.47 414.98 145.69 408.74 310.01 87.09 98.56 63.04 204.37 135.46 70.66 60.86 260.49 176.58 616.35 1407.24 258.98 121.27 703.8 50.39 132.43 109.29 100.42 563.61 140.24 115.73 360.42 52.92 103.76 52.76 95.99 148.4 154.19 129.37 171.82 80.72 159.44 52.45 142.17 90.84 155.46 241.53 102.91 162.95 79.82 158.86 449.02 80.25 158.17 98.78 146.42 126.39 614.12 321.51 201.22 189.14 23.03 156.43 166.36 254.16 89.32 127.18 85.96 49.03 51.72 81.01 133.12 217.31 75.93 54.84 299.99 230.41 62.83 95.61 178.4 131.91 62.84 130.06 89.84 135.4 117.13 120.21 74.62 91.03 121.36 159.89 131.7 76.51 85.25 56.14 127.61 145.7 49.32 53.6 128.05 143.74 82.3 207.22 179.97 105.93 193.2 78.66 378.22 47.2 79.47 282.51 73.1 71.18 123.84 83.65 85.68 93.77 142.88 96.46 113.62 92.85 59.97 84.7 107.2 105.58 152.04 18.45 115.08 131.43 68.4 170.36 35.9 183.76 70.64 112.17 129.49 176.03 111.37 84.78 487.53 60.18 68.39 112.56 205.27 201.17 115.46 121.23 133.67 60.8 131.96 112.44 101.03 78.04 99.1 129.51 56.76 96.49 161.53 26.06 97.54 152.64 95.22 174.17 128.78 114.79 112.1 53.51 77.23 95.88 85.01 125.11 85.56 108.25 121.93 108.82 1021.62 129.18 953.45 144.74 212628_at PKN2 293.37 363.34 137.21 181.02 221.85 301.53 170.09 297.36 328.55 153.96 333.56 399.11 217.56 379.92 124.33 289.56 339.61 292.65 436.23 638.38 335.02 244.15 253.09 153.88 348.11 203.41 196.38 582.05 289.87 160.57 274.5 268.35 399.51 282.7 241.94 95.64 199.97 240.98 158.66 79.41 325.54 256.48 152.74 273.87 111.3 189.53 197.42 248.31 100.7 76.48 663.42 216.76 670.05 284.82 206.41 206.66 169.46 271.52 131.06 169.29 153.59 353.37 343.77 93.96 139.11 148.6 203.72 159.23 189.09 241.9 273.07 102.23 181.97 124.99 184.9 139.33 196.55 182.63 69.84 43.77 54.26 338.94 238.59 248.98 233.23 258.49 105.47 170.19 147.79 205.74 13 199.98 159.66 157.97 211.6 129.34 229.99 316.7 210.43 209.77 186.61 238.08 246.74 189.2 194.62 204.85 193.79 179.6 152.49 21.4 138.58 179.53 175.2 174.01 227.17 93.5 167.02 45.88 82.89 51.97 70.79 176.5 100.8 150.92 117.74 126.61 92.37 147.84 146.43 225.19 116.38 72.31 60.7 102.47 198.42 123.42 176.48 142.95 144.66 155.98 118.79 223.63 234.71 125.55 146.61 72.61 154.77 281.97 177.7 331.91 238.32 120.31 179.48 171.55 173.04 195.41 151.95 191.8 241.87 269.9 196.89 228.81 239.68 243.39 131.56 228.43 222.19 164.34 15.62 115.25 221.81 86.13 186.38 227.45 21.48 47.72 127.1 208.19 100.66 178.1 179.17 151.76 102.57 131.93 88.87 63.53 148.37 165.76 154.93 165.55 90.98 217.36 95.11 140.55 159.08 111.74 181.18 88.99 115.78 172.2 93.14 158.81 188.09 36.98 152.29 101.45 245.9 162.46 75.28 145.9 94.38 139.23 150.23 121.74 180.4 139.36 124.66 171.99 149.26 170.23 223.97 329.75 218.2 241.92 245.75 369.9 199.68 212669_at CAMK2G 86.12 129.7 175.88 192.17 180.87 202.32 75.22 174.83 159.79 191.87 86.3 115.75 83.72 121.24 121.51 100.2 281.01 134.74 154.42 114.48 108.1 77.97 86.42 127.28 154.92 112.32 220.72 128.39 114.15 94.86 107.66 140.66 147.63 205.53 141.98 116.24 186.52 131.6 101.25 188.36 171.55 124.74 270.35 176.67 230.02 140.53 116.77 88.65 150.81 127.12 51.85 160.96 158.75 117.7 260.45 93.98 107.6 130.92 169.11 110.91 133.42 147.24 157.74 96.24 115.9 118.22 146.14 84.37 135.8 90.08 184.08 181.36 174.92 169.14 126.4 161.52 210.92 128.14 97.49 167.31 215.5 115.1 116.25 105.29 179.26 111.76 127.2 165.78 119.5 46.6 178.65 83.56 125.84 54.52 43.7 98.95 95.02 131.11 49.42 63.54 208.46 185.79 190.8 67.39 69.5 183.96 113.34 165 200.72 240.61 168.37 141.07 127.76 255.98 90.73 103.49 182.63 264.97 124.91 161.01 165.87 190.51 76.23 115.83 246.86 125.97 93.3 133 104.43 343.65 124.44 176.96 163.17 202.28 104.27 207.54 123.73 127.85 115.57 148.8 85.82 233.95 151.85 120.33 83.84 266.83 104.05 166.24 151.82 93.72 239.41 162.15 120.61 148.9 87.42 174.53 76.6 120.62 116.87 152.07 153.1 109.02 119.42 107.26 95.87 157.52 136.16 143.07 118.79 110.88 170.19 154.1 161.86 152.4 297.85 130.38 221.68 122.36 150.01 171.97 127.25 149.07 146.76 162.59 319.19 130.72 216.7 172.38 138.73 147.34 114.61 118.92 205.21 128.68 174.6 203.35 188.21 198.93 163.81 148.6 159.13 181.53 143.24 233.05 193.76 134.91 114.73 132.81 96.41 99.32 165.9 144.2 208.83 219.17 98.35 191.76 282.63 218.68 174.8 222.7 161.35 221.2 160.59 115.99 152.11 157.31 166.66 212672_at ATM 151.47 152.16 252.37 169.64 177.82 284.72 106.52 182.78 166.23 157.84 144.42 252.08 205.1 117.29 205.43 204.36 139.23 446.78 191.43 155.77 159 388.73 70.71 212.06 182.83 406.16 192.65 349.08 181.5 149.86 209.81 118.78 183.77 200.23 164.89 105.88 158.88 202.76 287.03 149.77 187.04 246.36 104 133.17 153.62 261.8 159.85 161.95 196.42 169.78 191.79 191.04 150.96 204.11 91.27 114.64 68.84 121.87 233.95 167.6 145.05 83.62 130.98 130.05 503.98 144.13 100.07 218.46 115.47 149.65 141.36 185.8 169.53 149.59 200.02 117.64 88.61 291.84 138.48 100.5 75.35 167.26 127.83 159.81 185.46 203.51 133.38 246.6 251.04 395.43 60.26 297.36 124.82 190.04 313.04 204.47 205.51 115.35 295.33 208.2 324.02 245 267.49 314.19 230.38 291.43 234.69 250.67 191.92 101.5 170.14 244.45 359.2 184.72 334.63 127.79 161.51 260.55 417 140.49 211.28 186.69 196.25 267.45 270.96 311.22 174.32 285.45 195.56 204.98 357.38 126.04 157.55 278.08 281.26 157.7 157.54 190.08 182.72 167.71 177.85 128.89 150.5 180.81 255.7 134.62 150.43 145.61 184.27 219.86 171.06 338.21 135.61 296.32 291.1 124.93 113.92 291.89 229.31 311.86 165.47 218.8 332.52 115.89 168.49 280.16 126.94 311.2 71.74 123.21 182.99 145.25 142.09 187.76 72.06 88.07 288.74 272.02 225.67 108.33 349.27 122.87 133.82 166.31 244.61 208.01 117.83 117.01 94.76 191.8 147.52 304.74 151.91 246.5 207.48 144.11 151.74 184.45 208.81 131.51 97.21 167.8 313.61 72.23 113.91 163.28 219.24 136.99 214.62 147.46 123.61 262.26 140.83 146.55 130.43 162.32 176.15 156.62 145.22 155.59 262.81 144.34 123.87 118.24 118.43 144.28 226.61 212740_at PIK3R4 172.6 249.82 196.86 224.62 162.18 183.78 212.3 227.27 267.25 161.55 187.91 247.14 152.26 213.62 89.42 175.59 386.34 228.62 211.81 279.59 191.92 274.7 259.78 177.02 144.96 240.02 219.44 188.1 262.23 118.58 269.62 288.36 217.85 240.25 232.73 232.52 186.22 251.15 140.1 218.12 304.44 275.22 226.95 300.75 162.63 172.17 231.34 114.4 121.57 299.71 198.8 260.22 123.47 226.16 188.97 190.51 192.42 130.14 190.8 203.12 234.87 292.07 273.87 169.22 104.66 223.87 324.56 187.26 251.14 164.29 183.11 191.11 199.19 241.32 243.57 189.25 392.34 226.45 302.12 200.39 124.6 223.25 153.54 202.29 196.18 195.15 249.31 165.78 119.96 230.03 40.01 184.61 269.87 369.11 284.59 216.67 310.54 219.6 445.09 298.05 269.09 233.79 180.9 341.31 219.5 221.35 234.24 178.91 256.75 29.76 220.91 216.44 198.95 175.97 233.42 362.68 59.11 233.16 152.74 166.44 209.18 221.6 139.81 221.27 166.27 236.47 180.16 180.26 216.3 134.26 149.47 177.58 160.27 184.07 238.03 154.67 211.46 242.6 186.81 191.42 192.25 185.58 190.22 123.29 147.1 165.35 137.39 221.03 149.59 269.48 281.88 191.67 89.62 216.29 188.08 239.48 363.92 183.77 216.21 235.43 151.04 172.55 332.41 75.61 310.72 190.46 222.36 200.71 77.22 260.69 171.82 147.84 166.18 305.04 41.38 120.58 192.4 178.94 206.18 215.27 208.91 215.4 272.87 134.04 174.43 213.27 191.75 296.81 266.17 203.48 139.81 231.24 175.86 318.3 184.66 183.62 242.74 229.69 193.25 127.75 175.45 244.18 213.55 55.59 145.45 212.87 188.33 201.87 121.69 258.78 167.7 141.98 172.11 146.48 192 196.65 207.57 302.88 227.6 104.8 206.69 143.21 194.29 147.87 236.21 108.79 128.31 212801_at CIT 113.5 137.75 104.83 164.38 198.14 47.07 226.63 242.75 161.57 115.66 80.95 43.83 144.15 311.74 62.09 220.97 203.22 239.56 313.08 276.63 151.44 81.56 167.84 170.24 133.32 104.55 90.04 117.77 244.03 186.99 186.08 147.94 250.91 274.36 233.33 217.71 151.73 344.15 83.61 210.08 177.19 187.92 202.97 148.73 80.66 95.78 409.19 157.49 189.26 220.68 183.07 131.38 221.45 114.84 216.13 121.77 160 153.91 175.02 138.18 140.84 623.36 225.92 307.41 138.17 259.13 248.65 203.07 290.62 229.04 173.72 218.83 172.79 122.05 189.92 139.3 235.84 56.91 95 83.78 372.96 88.42 312.47 74.07 63.93 122.11 106.22 164.45 155.87 116.96 131.27 150.38 102.63 93.74 81.18 114.25 75.23 100.07 149.66 125.47 214.11 82.22 189.17 54.29 162.24 173.94 74.86 83.22 303.8 151.53 128.79 42.91 162.56 221.77 112.76 165.72 543.9 175.28 130.97 141.38 158.36 272.63 159.65 203.99 103.27 210.95 218.22 126.09 91.15 137.14 81.64 95.19 94.3 94.49 79.04 221.48 208.29 98.55 111.45 116.01 96.8 143.27 288.27 101.76 102.98 200.71 260.52 152.86 106.65 117.69 449.74 92.49 152.69 101.95 146.35 192.18 79.05 233.14 69.45 157.05 262.66 80.38 142.15 53.09 159.24 75.72 152.9 106.69 136.13 228.41 129.85 140.18 180.61 243.38 306.87 189.89 134.56 80.1 99.19 462.65 135.74 256.87 148.36 85.4 129.71 135.56 92.35 219.2 283.64 125.74 174.83 50.7 102.27 242.56 60.47 125.94 158.03 171.84 142.32 166.83 84.14 106.82 103.14 291.64 470.57 47.25 270.64 170.65 80.93 70.71 138.04 94.86 151.19 73.38 330.19 116.63 104 152.64 131.57 147.06 148.38 148.82 111.18 179.26 229.11 187.43 55.13 212871_at MAPKAPK5 90.17 143.64 63.21 94.94 163.94 88.85 204.85 91.57 122.36 81.62 119.42 115.55 92.59 96.37 47.05 94.14 208.91 103.58 207.27 164.33 103.75 123.62 155.65 96.09 115.09 100.23 104.85 74.96 141.73 156.15 86.03 110.33 200.79 129 104.28 154.41 175.99 116.73 48.89 150.23 175.99 157.01 181.64 87.63 93.93 65.08 93.51 70.01 83.73 91.23 118.09 103.16 139.91 84.67 147.26 159.65 102.11 179.4 138.32 65.75 114.53 206.69 72.96 128.89 56.74 104.39 143.47 88.52 307.85 103.47 86.8 99.01 106.72 98.62 169.84 75.67 157.13 55.23 65.34 76.09 154.53 102.6 77.65 101.68 86.67 119.24 77.09 98.43 95.64 32.07 17.84 51.58 56.68 45.31 43.93 57.39 99.44 108.16 44.64 60.87 94.79 90.12 75.67 61.02 96.43 58.88 68.14 75.17 51.55 39.22 70.68 76.76 101.94 125.33 64.6 158.34 73.41 61.64 65.29 67.36 56.95 76.2 63.62 151.2 48.56 132.04 86.67 67.06 100.17 86.05 61.04 144.28 43.34 59.64 91.24 88.07 59.06 61.89 66.26 73.37 54.55 85.82 125.23 104.33 59.65 66.01 49.01 110.34 73.21 146.56 83.78 72.36 100.42 73.58 71.62 100.43 64.34 86.34 81.8 67.21 119.16 70.32 73.04 27.74 95.1 95.9 97.67 69.65 27 80.26 67.26 38.52 105.5 87.71 26.84 39.79 57.45 51.23 32.78 57.83 52.48 104.26 48.34 29.2 60.25 34.27 34.15 72.95 115.9 70.41 76.68 59.74 65.33 71.08 63.66 82.02 51.82 83.49 72.02 40.69 72.9 85.56 67.9 19.34 133.25 45.02 66.27 94.73 52.56 82.24 46.95 42.37 87.54 53.9 55.62 59.75 79.03 118.94 76.74 47.71 77.54 104.4 73.17 102.35 90.03 103.04 80.19 212897_at CDC2L6 459 187.63 199.49 286.97 258.96 187.93 114.34 112.67 129.81 344.75 229.86 156.07 234.26 356.68 150.67 160.86 193.65 253.43 155.19 290.17 220.59 145.06 163.13 175.09 358.53 231.95 146 367.47 187.8 259.44 198.69 404.78 175.14 343.75 133.69 106.53 199.88 158.53 169.07 176.74 265.74 175.95 181.62 539.62 254.29 195.26 259.35 269.42 311.46 183.04 360.6 119.24 196.54 346.73 441.81 98.89 255.43 319.73 112.35 105.16 272.28 363.55 281.67 280.28 357.24 472.76 268.98 475.05 268.74 554.09 330.42 355.38 507.59 159.59 379.14 480.06 537.65 348.44 220.94 256.02 198.23 254.41 324.01 291.19 248.8 233.77 229.59 188.57 216.35 409.97 254.55 307.38 185.77 376.08 394.84 234.17 120.73 336.78 166.93 396.41 302.88 210.66 170.02 231.88 288.96 313.23 243.96 223.07 147.78 86.62 220.81 299.51 342.67 326.34 268.37 205.77 361.33 1500.02 358.4 312.38 328.28 801.42 254.22 467.19 280.73 182.99 304.61 324.91 138.17 425.8 257.85 159.48 363.33 179.1 283.32 209.18 371.61 308.37 668.46 363.98 372.07 324.88 216.18 456.65 296.71 296.91 206.88 246.92 215.96 130.67 258.09 199.54 241.23 134.4 162.62 227.11 162.58 177.07 185.39 213.33 154.09 205.97 107.7 204.74 165.65 139.57 210.4 165.97 499.4 163.85 253.87 256.37 274.63 778.51 269.14 812.38 232.71 211.76 301.39 258.52 227.43 433.86 430.97 267.97 418.49 189.03 314.86 236.4 662.11 717.54 283.23 257.56 173.65 200.6 191.74 217.06 268.61 215.92 244.38 744.33 224.01 167.51 200.66 281.04 213.64 477.51 318.45 347.26 158.88 150.02 490.82 232.77 256.83 209.89 179.69 146.4 587.91 142.81 148.48 240.86 473.55 220.88 326.79 710.04 441.06 367.15 226.1 212954_at DYRK4 116.29 111.63 79.29 108.94 86.15 77.2 119.17 74.94 108.14 161.82 137.59 105.04 75.81 138.55 68.3 66.05 87.76 66.34 104.52 75.92 71.01 67.47 189.3 104.23 94.93 92.72 92.84 31.8 66.08 93.08 54.51 77.09 86.01 154.66 126.39 149.62 82.04 111.2 100.53 96.02 70.51 120.27 173.99 39.65 241.69 65.77 66.93 79.34 100.08 59.69 178.37 78.01 85.44 71.16 101.03 56.66 50.19 170.56 90.1 195.68 79.09 266.37 52.77 82 99.91 105.79 124.56 127.36 78.88 82.51 66.28 52.56 75.35 49.98 192.12 84.11 103.14 106.17 63.22 133.39 126.85 99.57 75.11 71.21 66.42 80.94 55.5 83.41 48.95 23.73 75.35 45.67 31.49 29.11 23.03 45 72.04 68.06 33.89 58.62 80.21 108.23 82.18 49.11 67.17 95.27 86.45 81.08 64.74 124.76 103.36 107.56 89.24 118.56 73.23 224.97 68.99 67.96 78.22 77.12 101.13 181.19 111.78 153.83 66.72 108.25 69.19 49.37 83.42 149.01 71.97 90.99 81.72 72.1 74.34 68.45 56.16 47.57 72.62 67.69 56.37 60.87 51.84 67.89 63.87 103.72 47.23 81.1 87.39 111.45 134.88 76.13 96.81 85.38 101.23 67.53 74.65 91.22 95.43 73.01 44.86 95.01 79.27 91.26 71.06 72.38 61.88 68.7 153.29 93.21 92.14 91.32 87.36 117.6 107.24 47.37 88.44 67.18 52.84 57.55 82.36 106.55 63.53 58.17 98.72 47.54 47.9 42.01 115.26 96.78 85.86 77.6 85.24 90.12 102.87 56.38 153.59 83.66 89.38 84.37 41.39 91.39 74.91 135.25 88.27 67 121.68 97 78.32 74.99 94.1 53.39 46.7 71.7 58.11 71.4 61.44 71.44 64.95 51.57 97.78 84.15 62.56 92.1 116.18 103.02 82.39 213034_at KIAA0999 111.02 184.71 197.56 197.57 236.59 365.2 92.48 210.28 126.7 131.31 126.72 163.14 323.94 198.68 215.41 140.49 201.66 228.74 110.46 121.69 153.22 219.9 70.87 157.28 160.46 173.63 116.24 204.74 174.43 112.15 108.3 149.24 67.57 314.23 115.5 111.19 118.82 171.57 204.08 132.7 93.99 184.17 168.57 103.24 142.86 187.45 117.6 134.56 129.7 131.57 101.28 150.55 115.74 124.21 82.03 78.3 170.14 142.04 159.18 120.06 525.51 101.27 117.33 125 182.12 130.83 141.27 110.84 173.07 235.84 115.74 184.73 144.79 91.49 176.21 71.88 80.46 145.86 27.32 130.85 99.95 331.83 69.03 184.13 235.81 153.75 81.94 151.67 141.23 252.45 98.31 273.05 227.11 307.17 330.47 103.29 190.76 130.29 284.89 284.29 215.96 170.46 459.72 428.43 72.3 139.85 241.07 116.37 144.62 149.44 188.76 235.12 196.57 144.92 143.46 263.8 92.17 216.98 158.82 97.18 144.68 234.5 143.59 135.33 208.29 122.8 120.83 144.95 73.54 167.3 161.88 95.97 209.69 88.22 184.46 130.9 87.26 112.17 91.27 147.88 60.67 71.49 148.9 99.44 156.74 89.34 93.02 153.25 142.15 281.25 83.39 173.74 94.17 109.12 132.95 118.34 106.38 133.73 248.44 171.51 134.79 230.09 257.49 105.28 250.74 137.78 188.92 165.73 36.26 131.28 247.52 208.8 124.82 164.75 116.51 99.37 139.7 141.5 185.88 189.51 223.18 112.2 83.97 117.85 140.41 80.2 60.39 112.17 115.59 21.37 163.96 231.46 117.2 83.15 170.36 83 87.28 111.76 111.39 120.04 119.57 115.55 216.14 110.22 257.49 111.1 62.8 76.68 105.7 64.65 79.27 169.07 126.55 109.15 154.1 115.97 115.67 70.63 76.79 160.84 143.64 158.66 155.02 89.71 98.01 115.37 130.48 213045_at MAST3 81.79 68.91 159.51 114.32 101.79 186.69 124.03 132.68 129.41 88.36 120.19 106.63 132.93 98.4 102.39 100.84 66.1 149.3 115.88 86.49 125.05 124.48 101.53 155.69 84.82 147.85 95.29 83.75 116.76 66.65 77.88 83.3 340.05 124.84 121.05 117.26 75.21 101.77 201.72 161.55 111.25 201.68 111.5 38.26 92.97 132.06 93.13 95.3 121.76 103.88 54.53 127.61 153.91 96.27 158.58 148.06 150.66 120.62 133.94 108.01 100.69 93.06 118.51 80.71 290.77 71.4 105.1 94.25 89.1 77.45 125.71 117.46 92.75 76.71 93.03 73.42 44.67 89.58 69.16 81.21 68.79 119.83 76.99 128.1 90.75 95.65 78.83 101.98 109.1 197.38 174.35 124.93 137.38 99.4 147.13 112.21 99.57 162.28 170.8 91.48 128.67 118.24 115.26 122.61 83.55 144.76 108.75 131.61 186.31 118.07 95.37 94.44 126.33 82.62 127.21 67.12 72.95 92.8 132.49 145.56 135.69 108.04 151.42 120.01 89.2 99.14 74.18 138.2 60.18 75.49 122.45 116.58 184.16 99.71 62.97 90.59 78.8 85.03 63.19 90.25 67.65 85.82 120.79 88.91 101.91 99.5 101.61 125.38 125.08 106.12 140.68 201.14 72.2 92.66 152.39 115.71 77.54 118.14 131.81 147.81 148.49 103.05 111.1 77 127.23 118.72 108.01 199.61 101.57 322.55 136.85 105.65 113.1 113.04 176.56 141.69 121.36 111.57 124.62 83.98 131.88 101.92 71.05 111.33 118.17 158.65 92.01 121.82 86.31 148.96 100.82 103.79 97.36 81.37 83.03 113.52 70.91 140.37 106.64 73.06 132.33 119.25 141.21 134.79 106.62 87.91 75.96 66.68 91.88 82.29 99.98 130.38 122.31 116.85 126.64 110.74 98.97 84.52 96.76 107.98 143.94 73.03 111.45 59.47 75.13 86.77 119.66 213093_at PRKCA 49.29 22.19 41.48 43.94 55.9 34.55 33.41 18.86 32.97 17.1 28.55 10.72 28.51 13.19 30.26 278.76 40.01 123.28 82.43 55.72 157.56 22.82 14.1 40.58 139.82 48.29 27.65 20.04 155.36 38.07 36.25 7.91 26.89 14.5 26.19 39.3 72.45 244.63 73.76 30.52 204.4 155.73 46.17 59.24 29.1 54.59 65.35 133.56 40.74 57.31 92.44 48.83 59.7 48.07 13.12 28.9 262.02 264.77 37.55 31.45 19.48 98.79 64 12.38 37.21 75.5 33.68 99.69 173.72 243.07 26.52 41.32 173.17 24.54 10.93 67.48 19.78 22.64 11.91 17.86 11.57 29.95 30.49 18.14 67.75 33.96 33.04 54.88 19.31 57.44 14.79 39.46 20.91 56.37 103.74 19.65 56.12 21.32 58.29 161.63 18.68 81.26 32.06 38.11 27.67 38.42 46.16 29.32 30.37 12.75 57.82 69.86 47.56 14.18 26.57 96.24 13.96 17.74 58.38 38.65 47.94 21.05 15.47 31.19 96.53 34.14 20.66 80.76 15.39 41.06 41.09 22.8 44.69 31.3 14.5 19.2 48.78 41.77 24.28 23.64 14.51 23.99 18.9 45.13 32.82 8.36 23.43 46.3 55.49 15.23 97.19 55.48 71.24 47.87 20.03 18.77 17.27 67.53 39.4 132.19 30.49 25.93 12.53 16.46 21.55 41.52 10.5 39.38 13.23 15.61 26.05 22.42 17.35 63.23 6.69 22.09 34.96 19.57 75.51 9.96 66.65 30.37 24.06 32.33 26.32 23.15 21.74 22.14 20.8 12.18 26.1 90.67 32.76 24.51 37.36 45.6 11.38 31.31 37.86 18.34 15.91 19.41 52.58 9.19 89.45 55.56 74.81 27.37 29.01 22.53 75.59 32.86 42.83 27.83 15.9 25.14 15.59 15.55 52.26 19.71 62.35 30.18 26.71 168.07 52.11 261.27 94.77 213107_at TNIK 13.98 56.57 62.53 18.06 21.8 18.33 28.22 16.8 26.1 28.67 277.04 67.55 120.8 19.6 64.54 64.53 69.29 29.29 44.64 15.73 85.6 115.14 37.01 17.4 12.13 34.29 95.73 82.79 27.81 95.64 51.56 20.2 39.39 24.76 48.29 16.43 113.98 39.33 33.07 25.99 17.57 15.41 19.9 31.48 66.13 54.75 24.82 17.12 17.84 37.8 14.01 33.4 44.15 32.16 14.34 86.4 16.75 17.67 29.06 43.67 228.19 207.6 43.94 16.4 17.75 41.87 31.95 20.31 15.54 21.03 30.7 14.6 85.32 21.98 55.47 443.83 14.41 86.02 17.76 24.8 17.07 253.14 26.83 92.2 27.37 56.54 47.03 72.27 67.06 24.01 23.21 53.53 46.15 91.71 55.04 15.14 15.96 54.4 29.69 14.18 27.51 276.37 129.22 18.4 51.76 106.12 77.56 68.98 128.83 23.72 34.28 35.68 18.5 14.06 22 151.14 14.92 13.31 20.07 33.63 90.41 21.91 15.11 19.75 28.9 34.89 62.72 68.59 77.1 42.58 187.29 31.08 42.81 161.4 95.99 25.97 65.74 37.99 30.25 78.53 111.66 111.87 45.2 23.71 20.36 35.46 74.99 74.42 21.86 98.66 67.38 21.38 49.97 74.16 14.6 15.59 57.59 22.93 45.24 19.58 29.75 83.62 132.72 48.1 103.48 19.22 515.87 24.07 41.12 229.03 41.87 95.33 256.2 22.6 31.84 94.66 50.26 65.64 79.87 31.06 41.03 56.25 39.34 82.48 139.94 21.95 54.04 56.75 51.51 84.88 56.06 119.79 39.07 41.08 59.54 60.68 36.77 26.41 132.16 89.87 13.23 32.95 175.65 26.63 29.44 48.64 24.73 28.8 40.64 25.49 21.47 14.28 22.12 42.14 51.04 15.75 19.55 44.2 33.32 57.14 16.2 37.38 180.52 13.23 103.8 15.78 30.8 213116_at NEK3 88.07 97 71.28 48.38 292.1 153.16 114.6 43.9 121.3 93.7 121.69 86.23 162.57 163.79 179.16 150.93 73.09 77.21 167.36 245.31 163.91 81.5 242.72 54.52 82.48 94.42 106.22 75.44 56.17 96.75 98.1 54.59 83.63 140.13 125.87 85.54 71.11 89.34 124.28 59.81 72.79 79.28 75.79 75.83 88.32 117.66 78.65 135.86 101.45 67.68 48.35 72.74 103.09 72.59 110.19 107.72 79.97 57.77 101.5 34.49 84.04 70.92 102.84 77.04 63.11 67.53 34.73 72.64 37.1 94.29 130 45.1 54.09 65.63 49.49 87.19 130.97 74.25 24.82 234.4 97.94 57.96 78.11 51.92 85.2 84.69 59.22 61.66 73.63 91.99 110.37 84.2 58.84 110.49 66.31 62.48 110.22 60.91 77.39 63.18 212.87 130.16 151.43 169.81 87.17 100.71 93.4 143.38 78.19 73.55 120.12 80.31 101.15 126.17 72.92 217.15 44.25 430.19 66.68 69.66 188.55 85.99 63.71 89.18 180.32 60.05 159.77 130 72.22 109.96 133.89 66.71 152 66.83 70 140.58 71.47 73.1 68.31 101.51 130.74 45.76 124.18 91.13 56 77.43 114.07 102.11 106.47 104.09 58.71 89.69 71.18 105.13 133.13 89.74 24.28 99.93 133.22 188.49 83.63 102.62 106.92 56.73 60.46 93.02 117.56 83.58 36.26 119.33 84.31 134.11 107.02 64.25 67.53 59.08 114.44 128.75 139.94 88.78 115.34 65.27 124.09 99.23 65.93 143.99 76.83 79.2 40.97 92.59 132.94 113.69 131.11 56.45 119.73 77.07 32.36 88.94 93.9 75.98 93.36 76.46 95.57 41.88 56.75 87.5 90.86 60.16 77.59 86.5 148.6 51.28 96.43 78.79 58.61 72.98 83.64 48.14 55.8 143.23 61.74 89.45 143.82 71.55 66.78 79.68 73.29 213141_at PSKH1 94.39 94.74 165.64 180.71 142.4 114.72 121 109.15 134.5 125.5 108.59 98.16 86.89 109.56 117.86 147.74 111.01 95.25 105.69 75.52 140.74 106.42 80.33 128.94 129.53 100.67 162.13 154.74 133 123.63 213.07 104.52 123.79 135.58 114.37 136.9 142.89 101.21 126.36 117.62 109.88 125.05 107.25 145.29 140.21 144.93 120.01 114.01 140.87 131.83 89.17 142.51 179.85 181.58 139.72 140.44 192.84 132.08 126.95 139.66 120.1 121.18 203.3 139.58 86.73 89.37 137.45 97.26 111.44 108.71 129.23 94.74 122.79 98.16 109.36 106.44 110.01 94.6 120.8 116.32 135.81 106.3 73.9 114.68 143.09 100.44 123.89 130.57 128.52 70.93 155.34 137.06 134.15 99.9 102.35 108.31 130.51 126.43 102.79 118.48 195.12 148.6 115.79 97.74 125.09 112.77 96.09 93.39 120.68 148.44 135.03 153.48 115.23 101.46 129.5 113.7 126 87.26 118.55 228.86 123.53 117.57 99.41 132.91 113.13 88.85 93.38 97.42 88.58 82.72 103.14 86.25 111.8 125.58 89.87 103.92 106.85 82.14 84.65 86.48 72.03 93.94 91 87.84 93.02 89.69 95.87 182.84 143.76 138.43 128.43 141 133.67 129.32 98.96 143.38 141.29 142.66 140.57 87 140.81 130.32 100.77 126.33 144.15 130.5 93.64 113.46 112.2 126.1 127.98 115.33 101.41 79.85 125.09 149.52 120.13 103.99 139.81 130.73 107.17 127.56 103.66 112.96 119.54 153.66 150.58 126.23 128.43 94.72 128.45 112.85 115.49 105.91 124.07 153.78 131.51 149.74 102.06 103.63 137.84 96.47 126.5 102.76 165.38 113.74 61.19 96.67 100.51 108.64 104.94 91.24 162.06 157.66 106.49 106.56 132.5 103.15 119.7 107.37 164.88 171.59 147.41 177.61 163.41 107.99 191.43 213198_at ACVR1B 197.19 436.6 451.2 359.19 632.82 725.67 288.6 648.68 434.8 256.65 578.46 563.61 320.55 448.1 530.01 238.23 460.97 248.38 287.03 341.45 664.49 368.12 204.81 232.16 611.81 235.87 483.82 366.97 228.51 238 333.76 335.98 330.78 263.69 248.18 191.31 254.93 183.1 192.25 234.93 251.9 257.63 314.89 224.59 275.75 346.8 304.47 145.77 178.93 333.9 280.95 452.41 359.23 305.97 546.2 335.94 254.06 237.14 493.62 198.08 229.72 388.05 178.95 380.4 77.22 220.76 236.77 231.51 220.41 312.61 432.89 226.26 138.13 207.6 289.78 376.2 371.35 373.99 354.92 415.56 373.49 368.43 495.96 367.5 448.86 405.08 367.21 377.85 343.28 399.08 544.28 245 312.19 431.23 605.39 315.6 497.96 326.1 622.2 229.35 657.07 336.67 483.98 668.49 341.28 390.79 464.33 511.17 451.78 236.32 286.36 406.92 201.47 220.27 329.27 396.34 867.97 197.14 276.41 408.28 389.68 463.49 729.82 329.22 260.97 312.06 313.43 358.12 273.36 385.02 305.07 223.45 414.15 225.2 364.76 299.27 155.79 331.05 346.67 217.53 215.94 257.23 391.04 302.43 262.93 363.67 229.61 430.15 277.52 364.04 144.61 336.99 198.52 233.59 263.07 231.74 231.11 171.29 283.04 155.97 405.77 237.7 214.18 474.43 238.24 262.96 458.18 327.03 310.55 385.82 449.56 465.18 409.5 188.31 465.8 285.96 217.66 323.03 336.94 82.58 286.95 231.33 342.93 325.1 222.5 179.93 407.11 322.24 261.06 265.75 309.61 313.68 198.37 362.97 299.09 282.33 147.66 273.87 430.9 233.38 313.75 208.66 284.7 648.97 152.97 347.53 155.28 278.23 208.57 217.03 278.46 280.18 302.08 419.95 242.24 390.89 292.98 209.65 220.32 351.35 230.05 198.85 556.81 177.9 230.86 231.54 233.06 213324_at SRC 168.92 182.33 203.74 246.33 234.33 167.32 124.5 265.08 202.22 148.77 212.65 160.55 123.1 95.89 94.19 85.7 310.28 197.23 179.87 136.37 143.42 158.39 114.12 368.96 163.07 173.35 136.58 161.65 175.9 194.19 183.97 192.63 223.48 157.62 161.69 285.45 163.28 113.23 135.73 180.49 127.19 167.21 290.31 225.54 137.85 150.62 197.51 164.88 247.61 147.1 77.09 151.53 227.23 311.36 291.22 172.63 324.79 211.29 247.24 191.67 219.17 351.09 178.66 259.08 82.44 100.68 103.58 82.86 141.56 142.41 177.33 192.67 156.17 138.03 97.89 98.19 56.04 108.8 101 214.23 172.95 88.03 104.83 161 135.93 145.14 178.63 122.44 262.88 83.61 136.76 88.47 145.51 74.19 83.51 76.27 107.22 121.56 49.93 83 163.86 175.86 81.53 52.35 92.15 147.69 135.32 245.91 241.36 107.86 91.21 136.05 169.15 269.24 199.07 63.74 175.04 106.88 146.55 335.79 168.13 347.71 259.85 196.72 104.4 105.31 144.29 135.61 65.2 89.67 91.19 98.72 144.05 137.73 92.73 131.2 77.33 71.02 130.19 105.43 53.52 59.87 136.77 133.75 93.13 112.56 165.06 194.99 156.44 185.68 216.97 190.83 221.49 116.48 92.94 251.26 138.22 155.05 139.46 100.71 156.92 147.71 114.44 170.21 125.56 159.56 166.4 139.02 225.11 137.9 117.21 249.68 173.61 82.14 138.63 123.61 115.54 69.73 77.79 175.6 86.58 155.03 124.52 132.65 127.57 162.94 131.02 214.62 149.34 168.44 116.28 100.8 138.23 92.81 89.41 142.99 149.47 115.94 181.65 131.01 175.59 121.61 155.65 131.53 123.3 100.86 83.98 128.4 136.26 138.83 227.05 117.07 175.93 195.28 129.42 95.22 210.27 173.77 402.9 104.73 215.46 347.08 117.3 331.41 213.54 197.76 160.89 213328_at NEK1 44.42 58.02 70.9 82.94 81.11 76.62 26.14 73.46 60.18 241.24 208.98 397.26 200.4 328.02 222.48 363.91 34.87 54.46 58.15 59.76 51.26 80.56 76.3 51.45 52.57 62.65 37.61 46.21 92.08 54.58 50.4 55.55 56.71 128.61 54.79 41.04 50.49 71.22 64.16 36.67 54.03 53.09 27.66 45.56 58.55 73.98 63.23 40.04 43.41 81.35 112.45 60.61 73.6 40.64 56.67 47.32 52.41 26.65 56.54 47.65 96.43 59.69 62.25 79.08 253.39 121.58 95.21 206.17 123.62 140.17 95.51 143.54 119.18 114.32 93.48 77.78 370.32 164.34 197.2 142.46 242.27 300.6 218.61 239.36 146.97 179.68 123.09 105.91 104.71 184.72 184.15 164.41 210.28 121.01 194.69 89.19 73.98 120.7 66.73 90.34 127.19 66.06 58.57 106.66 137.43 84.95 121.32 86.75 102.07 139.52 111.51 262.82 193.42 170.87 115.61 62.19 206.81 186.27 142.53 135.47 231.76 178.73 157.88 131.59 195.11 77.99 142.15 211.87 92.2 103.08 198.02 142.23 286.94 76.92 245.31 203.4 185.11 129.53 148.66 206.93 134.28 106.83 186.24 277.75 126.56 201.7 85.97 127.89 101.47 77.72 64.99 95.05 54.32 97.68 106.51 80.32 60.68 94.13 127.8 63.5 121.99 105.75 67.55 95.47 62.66 117.69 117.26 100.23 155.12 119.69 140.3 157.39 200.63 146.26 199.09 168.96 104.61 136.7 157.97 141.29 148.27 102.36 144.97 155.83 289.41 160.96 92.28 168.25 89.8 195.17 164.68 145.56 83.51 60.37 100.06 75.03 64.32 81.63 91.94 218.28 63.55 84.46 77.28 128.38 99.28 165.16 125.85 63.06 134.91 74.08 67.78 173.38 96.48 126.94 119.97 111.05 108.54 63.32 80.74 142.21 68.14 49.61 91.45 84.64 65.44 71.34 75.81 213518_at PRKCI 265.09 353.25 148.75 217.12 237.05 189.01 266.05 294.95 211.6 340.24 546.09 310.94 247.54 323.91 178.35 291.88 383.02 268.34 308.51 542.63 248.06 306.07 375.05 165.09 270.04 210.69 238.12 1460.53 235.68 374.25 240.94 328.43 265.02 284.12 243.35 374.19 409.49 300.86 141.11 307.64 402.52 250.05 302.66 216.83 216.43 205.26 177.16 369.45 202.31 220.04 405.62 196.47 270.89 314.87 298.2 250.09 265.64 280.09 161.96 124.38 175.36 300.48 319.08 156.4 45.87 414.74 302.43 111.69 256.01 149.86 261.16 106.3 249.24 198.78 138.44 225.17 228.61 205.55 138.47 130.2 135.58 206.51 192.85 165.85 149.42 162.81 144.35 143.81 150.68 125.73 33.46 63.46 101.06 151.19 94.14 74.08 122.73 79.58 101.93 136.04 224.51 177.29 116.75 55.13 88.02 163.17 138.11 118.32 120.34 25.94 243.38 189.59 211.4 286.13 236.62 592.38 118.57 80.55 257.22 93.29 168.96 259.69 227.26 197.03 83.7 146.7 93.32 101.21 97.16 133.87 101.51 82.44 78.95 145.79 110.53 130.05 104.67 116.37 82.31 88.79 101.46 99.18 126 119.19 82.35 83.06 129.29 151.89 106.29 196.69 333.22 75.25 254.64 161.63 179.63 142.2 149.48 161.7 151.41 317.31 152.47 171.81 217.66 71.4 170.1 89.78 373.44 111.01 121.68 131.05 108.68 87.09 108.8 128.25 80.15 69.48 102.26 92.73 112.08 136.58 113.94 73.08 124.59 145.33 122.97 94.6 122.75 178.91 88.83 118.01 100.83 148.9 161.59 111.48 135.89 91.16 203.34 105.53 135 118.54 111.12 107.01 105.17 19.51 121.18 109.9 147.26 121.63 66.82 97.54 97.28 81.4 91.25 91.9 116.3 94.21 104.56 108.49 157.2 101.22 156.33 145.18 104.37 121.62 256.9 157.95 104.6 213534_s_at PASK 56.82 94.22 74.12 50.97 147.46 115.34 108.32 162.15 70.97 76.1 59.41 69.09 78.15 147.37 73.35 34.41 58.77 176.58 104.63 77.25 117.16 164.77 69.16 73.75 57.33 117.79 86.74 80.77 72.82 57.91 55.06 63.49 265.68 163.32 66.55 43.21 62.54 75.65 117.8 42.44 56.6 137.98 69.31 57.38 66.19 44.27 98.78 134.22 68.97 93.11 40.65 101.21 85.76 99.07 95.07 53.5 61.76 43.02 132.06 35.85 100.28 207.67 71.02 53.26 194.31 114.04 133.3 107.62 40.44 66.96 43.95 83.04 58.48 53.11 66.98 35.27 56.51 84.41 36.72 59.83 100.08 68.7 55.81 62.37 41.51 60.14 39.38 92.91 81.54 148.09 112.37 86.24 42.11 73.99 66.92 61.85 78.89 69.96 119.31 58.82 79.33 46.24 78.18 67.53 45.92 151.11 51.02 53.19 59.41 178.7 100.4 29.41 93.48 51.43 77.36 82.93 116.51 115.93 72.06 53.86 63.87 89.03 87.61 83.41 32.69 165.13 48.31 47.26 36.78 106.57 76.56 34.88 47.05 76.6 42.22 100.24 79.65 46.71 66.28 127.48 86.15 34.42 142.68 53.13 81.95 70.26 82.83 82.09 90.97 91.66 118.41 141.49 29.5 55.34 63.63 102.55 39.48 68.21 39.3 137.36 123.33 45.93 49.05 36.58 59.25 59.74 91.04 130.9 65.55 72.11 80.57 62.74 98.68 83.43 73.23 29.27 79.67 65.21 55.96 135.35 96.73 42.23 70.28 56.91 74.66 94.79 51.43 80.33 41.75 104.14 70.05 33.35 65.26 64.82 42.89 64.91 65.35 72.97 80.76 127.38 59.33 41.03 65.76 285.63 49.58 51.02 83.4 62.53 34.14 41.86 59.4 98.96 60.18 64.34 104.21 78.21 72.5 51.21 59.27 82.8 68.63 59.08 57.78 56.14 51.91 55.57 50.43 213578_at BMPR1A 559.35 405.35 183.97 229.43 257.28 159.58 293.81 302.51 257.56 422.94 398.56 251.7 153 340.23 87.56 402.51 718.7 216.68 172.67 304.73 181.53 269.92 505.54 266.15 167.76 281.87 318.5 244.17 271.06 212.01 257.12 166.97 215.84 235.46 258.38 205.73 243.3 201.39 81.43 129.29 382.27 190.13 453.1 321.8 378.46 184.91 136.86 108.54 119.85 117.07 330.07 129.39 108.95 198.94 230.13 157.22 216.22 194.76 183.3 80.73 234.41 311.64 120.7 143.61 52.15 287.8 127.86 80.32 331.77 227.04 210.04 148.06 316.86 227.98 248.4 200.42 104.41 153.3 259.27 114.68 172.96 274.42 214.68 150.54 225.88 215.68 190.02 149.3 100.89 190.27 27.72 213.32 261.31 297.31 403.08 310.91 187.32 261 300.62 238 811.02 289.8 178.19 211.07 190.06 111.28 155.6 174.55 142.98 27.04 279.63 212.73 130.76 181.4 157.64 289.15 140.77 127.44 124.13 121.29 125.35 297.19 68.85 121.45 180.55 152.67 146.87 111.63 230.26 355.7 98.48 182.25 103.38 75.69 159.06 182.79 112.56 193.49 205.84 189.48 236.15 182.12 296.41 224.93 123.73 44.11 176.27 209.9 86.49 93.3 403.39 71.38 216.57 155.03 136.6 222.59 210.05 50.67 177.38 90.97 135.34 191.3 284.11 133.5 164.87 150.55 186.61 115.52 62.22 271.42 664.77 63.91 205.47 59.8 93.27 207.85 204.55 121.39 133 234.48 124.7 77 156.05 177.86 94.62 78.12 180.36 200.39 113.94 231 179.33 329.02 186.79 126.54 217.28 149.24 213.13 122.57 150.14 186.7 287.96 172.13 145.22 69.25 446.17 182.12 148.05 178.58 139.33 242.86 196.95 98.07 167.97 184.4 53.48 123.73 111.19 174.86 158.08 173.47 107.81 174.51 94.8 217.09 234.04 155.19 177.27 213927_at MAP3K9 68.83 58.5 54.65 35.19 91.59 117.17 73.01 122.3 63.19 27.82 173.74 120.34 64.81 95.75 52.79 45.46 48.78 54.23 130.68 46.36 64.9 17.42 64.61 14.97 54.71 17.34 48.06 105.83 19.47 60.43 33.94 43.89 41.05 60.75 53.11 21.81 18.54 35.4 17.77 21.72 17.24 52.87 28.47 40.77 39.27 27.05 25.58 23.76 34.33 30.58 23.95 37.95 81.07 66.98 72.61 42.24 34.35 16.15 24.56 67.92 62.49 110.21 80.12 34.22 31.05 29.61 37.51 20.65 56.12 25.72 108.4 25.88 36.66 44.29 34.99 49.63 48.5 23.72 31.58 46.13 41.84 122.53 62.63 84.71 43.44 25.47 50.65 23.99 75.67 27.93 36.82 32.82 38.44 21.61 64.61 52.39 37.6 69.01 49.81 44.34 52.62 68.25 144.49 31.6 64.18 67.47 28.56 82.92 56.37 28.31 33.58 33.61 25.19 61.59 36.06 85.28 77.17 64.72 24.14 42.11 54.6 100.29 101.34 52.3 19.09 29.71 48.83 36.84 80.01 58.78 52.89 67.23 46.22 52.66 57.88 48.89 65.81 38.67 31.78 38.46 41.04 59.11 76.08 24.24 28.14 50.36 70.56 61.05 51.52 60.95 38.27 31.32 39.13 28.42 38.8 69.3 62.82 71.82 52.56 33.1 62.48 29.79 34.31 38.7 134.12 34.03 28.66 30.25 90.81 39.71 40.06 34.66 23.27 42.88 52.3 90.44 21.3 34.54 36.72 32.67 34.48 40.3 28.86 33.06 27.83 28.02 103.8 42.11 54.84 62.74 43.15 29.18 23.88 46.15 30.9 49.9 69.42 23.2 88.71 55.29 52.53 35.28 59.63 86 67.33 32.33 35.98 42.28 23.06 33.92 52.31 28.83 27.98 118.66 40.61 78.52 43.71 77.29 19.96 77.28 48.19 57.75 59.33 78.76 70.78 49.14 52.9 214032_at ZAP70 57.84 35.75 93.13 69.04 73.74 93.97 53.12 42.23 44.84 73.98 41.17 32.93 78.13 32.49 88.85 39.27 36.14 207.9 73.48 50.23 80.09 133.13 43.4 158.37 52.35 233.72 46.56 54.15 104.39 32.5 48.14 109.81 41.92 116.35 67.35 88.5 56.61 42.66 146.98 97.85 117.63 111.98 49.28 35.38 55.96 86.09 65.8 73.1 129.05 41.8 40.01 95.49 66.14 93.24 27.35 36.39 53.18 89.79 186.74 57.89 55.81 47.59 48.33 71.38 100.04 50.53 62.87 103.8 41.41 41.99 46.07 119.51 29.74 43.95 36.82 22.32 54.16 134.24 30.09 34.55 34.16 34.64 29.36 53.43 35.27 52.68 46.05 88.99 104.73 120.24 52.91 58.86 49.9 24.96 30.22 86.03 32.03 29.9 80.58 54.47 34.7 60.57 62.03 57.99 28.95 111.82 31.12 59.05 43.05 69.81 69.77 49.26 163.48 66.69 121.94 67.08 38.16 250.63 315.53 71.98 76.92 84.58 121.61 144.86 50.54 75.84 36.53 57.14 25.47 32 172.07 33.99 47.15 64.2 30.17 29.55 36.3 38.72 30.91 19.53 24.72 27.36 32.18 30.16 46.42 91.3 26.1 33.73 110.67 29.86 34.44 141.47 32.5 62.13 94.03 34.66 25.92 101.13 30.32 95.86 59.23 49.4 24.87 39.2 21.49 74.5 23.42 135.06 44.48 27.92 70.76 70.51 60.22 81.36 92.46 41.74 114.97 40.58 50.15 43.93 180.57 56.4 42.74 47.82 73.94 43.86 51.86 41.46 45.52 36.66 46.37 43.52 43.02 71.48 42.67 55.63 45 119.81 110.96 57.86 68.23 109.2 109.21 100.58 42.87 43.12 51.23 57.16 60.49 48.04 82.19 148.97 49.9 35.09 34.69 69.94 78.09 41.43 36.83 47.58 193.14 58.37 46.64 46.08 57.23 59.66 86.24 214339_s_at MAP4K1 64.75 55.17 139.27 147.02 105.83 166.9 88.64 51.37 67.28 131.42 48.07 41.85 89.45 97.12 93.08 57.65 54.85 287.82 106.47 58.32 71.78 120.74 51.3 235.4 61.32 252.19 88.16 183.57 149.55 53.76 69.05 53.32 47.84 136.73 87.7 175.14 122.96 78.08 219.36 219.18 112.92 198.26 71.84 82.51 83.29 116.3 81.46 119.61 157.89 67.59 64.16 139.04 93.86 128.48 40.11 69.57 96.9 139.69 341.65 95.25 55.98 201.55 62.28 96.66 905.1 62.12 87.68 106.51 54.72 53.23 70.04 120.55 61.06 59.87 95.26 40.62 50.13 118.31 57.42 40.44 56.13 43.87 42.2 67.06 73.76 86.2 73.69 176.85 210.54 223.66 103.84 111.48 80.27 51.04 85.91 124.57 56.41 63.17 106.05 97.06 65.2 133.15 108.56 91.65 56.64 154.5 60.98 97.07 58.45 121.42 66.81 33.11 140.21 67.78 180.29 142.03 31.78 246.23 203.71 93.23 108.59 116.53 181.35 159.54 76.92 104.06 53.62 73.3 40.29 60.98 223.46 56.49 74.4 132.59 40.08 44.82 64.85 61.76 51.23 51.7 46.36 51.08 55.86 42.31 64.46 112.55 35.93 53.82 191.9 54.45 73.55 296.39 38.16 121.58 148.07 64.09 58.17 231.14 50.51 155.92 92.89 79.17 47.18 75.3 41.2 148.7 56.65 292.01 84.68 48.01 112.08 103.15 100.83 204.36 88.28 83.01 196.14 73.86 96.86 68.41 207.81 70.18 83.26 67.88 111.49 69.01 57.45 56.69 74.1 42.24 52.68 59.74 38.28 82.87 35.43 73.09 55.54 147.18 129.29 81.4 63.04 98.29 167.61 124.13 66.36 36.19 52.02 74.38 84.55 49.19 82.25 126.17 115.38 82.45 51.04 95.07 110.49 69.22 64.09 130.48 115.05 44.71 30.01 30.54 27.06 54.76 97.32 214607_at PAK3 48.99 34.97 47.21 54.77 55.78 42.2 32.97 50.82 40 32.41 40.56 28.3 43.67 93.48 58.65 97.3 124.12 119.33 51.14 52.57 33.3 46.33 85.08 62 32.35 45.01 33.18 33.69 67.15 31.55 30.92 28.82 29.72 37.88 30.16 105.2 123.74 75.29 51.09 102.93 84.13 358.58 59.97 26.21 105.13 55.18 143.93 404.68 37.89 37.51 54.82 4 47.02 53.54 25.02 25.63 81.65 85.03 45.69 44.87 39.47 37.55 31.7 41.79 47.54 180.1 343.35 202 152.06 49.56 32.08 113.12 69.01 39.35 30.31 51.96 31.05 54.8 21.73 32.71 39.86 43.51 33.45 36.54 97.67 63.99 127 62.25 27.38 48.5 79.54 98.23 55.39 138.64 101.05 34.55 43.17 52.03 110.15 202.9 64.29 106.33 51.36 85.36 45.7 43.78 53.08 60.03 45.45 54.28 603.07 238.5 87.81 49.48 47.6 83.5 65.73 62.36 95.62 45.7 80.88 285.74 50.99 97.71 128.44 45.13 58.05 104.39 86.79 168.06 50.37 63.22 143.98 66.11 48.83 89.85 48.16 73.43 49.03 59.36 45.69 40.41 50.73 58.79 71.38 50.52 55.66 54.34 78.22 34.52 53.98 54.19 35.07 31.88 37.64 64.44 38.29 27.76 44.87 92.42 31.82 100.38 28.76 42.29 34.93 61.57 40.13 60.77 46.15 38.29 42.3 54.98 48.58 54.83 52.54 49.59 33.25 36.89 83.61 43.64 88.02 85.4 46.65 66.16 50.18 42.69 42.02 46.32 101.82 36.64 33.24 54.48 31.57 35.4 139.02 50.21 54.09 39.94 37.17 113.9 38.79 28.11 52.34 44.96 89.8 34.05 53.37 97.2 43.89 32.23 45.2 35.17 53.63 60.07 31.65 66.58 31.74 27.19 32.68 51.13 117.27 38.83 42.35 49.44 44.36 51.16 111.01 214625_s_at MINK1 91.9 56.4 103.78 79.06 78.26 72.36 54.55 62.17 81.39 71.87 80.2 45.62 73.56 63.08 72.15 63.12 85.7 75.5 69.11 94.2 73.76 54.95 58.23 101.11 69.49 112.25 86.12 64.62 78.73 68.03 63.03 71.82 82.8 115.77 104.49 108.08 97.94 86 78.72 121.87 136.04 86.15 153.81 110.94 103.3 102.46 100.96 94.52 117.82 65.91 64.27 69.33 128.63 83.06 84.49 92.66 127.96 154.57 112.32 78.83 150.03 107.48 80.74 58.7 69.28 64.88 80.08 64.54 74.44 62.21 72.11 98.81 75.16 75.53 70.93 70.67 79.76 60.19 80.7 93.63 80.13 53.86 50.19 69.65 63.16 70.74 72.14 85.34 94.37 50.75 65.16 75.97 73.75 48.09 30.57 64.44 65.54 54.32 34.76 56.95 72.04 64.84 76.83 46.36 50.17 104.91 99.9 80.13 113.01 184.65 76.51 69.49 70.93 66.88 89.72 53.65 78.5 77.82 81.05 83.66 67.47 78.93 103.97 120.99 58.9 80.48 54.37 76.49 62.73 58.95 63.98 55.57 65 77.69 52.48 65.68 65.14 71.08 66.88 61.4 61.66 58.38 60.75 55.64 62.4 48.63 69.19 85.66 103.39 76.02 98.68 116.59 104.02 66.26 82.46 106.86 121.24 80.71 61.23 70.76 94.84 86.33 80.5 82.31 102.35 99.33 70.27 83.59 67.13 77.04 84.04 71.4 69.61 75.58 93.41 99.88 68.56 84.59 57.34 70.34 59.49 83.33 89.25 82.76 66.31 67.51 61.89 66.18 72.68 71.3 73.9 60.67 85.95 75.28 69.27 62.53 66.91 68.62 81 93.29 50.95 60.56 70.66 69.91 76.96 70.68 51.17 65.93 52.62 54.91 57.86 77.82 97.85 105.58 103.83 101.55 100.26 73.09 87.81 101.75 81.61 53.78 66.21 72.6 85.4 48.4 48.78 214663_at RIPK5 141.91 143.77 116.6 177.06 82.28 136.82 214.88 114.12 131.67 112.83 152.01 165.47 185.63 102.85 119.92 113.37 109.63 115.84 297.01 224.33 145.05 81.17 117.5 142.64 326.05 78.56 81.7 151.92 65.19 111.72 87.5 91.67 119.73 98.62 148.14 184.7 119.98 64.94 93.36 101.88 126.03 156.19 198.11 74.86 133.4 109.39 83.22 131.77 128.32 74.24 98.87 96.86 139.35 154.42 102.11 151.29 110.01 64.33 92.69 87.61 83.83 74.87 175.72 103.56 127.58 190.78 64.31 110.46 220.22 202.86 148.94 101.96 194.44 134.58 231.67 143.87 113.57 85.25 128.28 162.32 334.2 73.78 138.51 110.14 183.39 118.52 114.89 125.71 181.97 113.36 22.33 132.89 194.26 158.54 162.84 194.79 187.74 356.1 108.98 222.14 88.28 76.33 168.17 170.8 131.15 153.09 145.94 188.98 157.33 40.03 152.83 149.31 106.92 110.68 91.43 49.61 254.14 62.49 154.9 61.2 91.09 105.64 67.97 232.99 126.64 76.55 173.75 100.4 172.45 254.39 136.73 144.7 54.68 49.64 81.53 194.82 104.39 87.19 114.24 110.79 143.43 98.4 95.28 161.5 85.57 52.68 108.56 130.14 127.84 94.71 142.1 89.19 93.39 57.53 106.98 128.26 101.68 63.35 107.14 76.34 208.89 111.79 111.64 94.68 125.17 100.93 114.6 103.12 173.92 243.13 124.83 67.87 92.25 114.57 229.18 64.2 94.82 149.05 152.16 302.14 106.11 113.77 105.9 207.68 244.06 185.43 133.63 215.41 136.98 175.2 144.29 93.35 126.49 82.43 108.13 88.56 200.74 302.18 58.74 145.68 151.48 135.35 91.33 129.29 87.46 103.36 129.47 172.82 57.8 101.07 134.76 107.61 183.77 46.18 232.5 133.63 104.11 84.3 95.19 138.61 101.84 288.46 120.75 387.05 143.51 264.17 130.13 214683_s_at CLK1 377.77 516.01 398.47 298.45 629.34 708.85 417.05 325.34 427.72 313.54 376.82 677.94 456.92 1361.53 550.62 373.76 128.93 556.34 417.89 321.87 333.16 342.54 497.07 207.94 534.81 548.3 436.64 275.37 356.72 256.95 266.24 223.68 318.62 871.39 327.2 258.52 450.04 171.11 502.37 137.12 188.62 204 236.75 326.78 215.45 432.57 266.6 188.89 153.49 455.11 189.89 279.12 171.38 223.82 507.18 306.62 325.79 163.1 349.03 355.75 207.06 249.76 178.86 135.56 335.68 254.18 315.75 239.27 156.41 317.54 229.01 217.63 257.35 190.86 244.25 230.48 320.97 670.59 429.13 281.12 340.28 416.44 495.08 526.38 346.37 259 278.15 324.64 322.12 315.96 362.83 491.18 580.19 235.14 498.63 237.18 155.15 250.23 204.19 181.37 480.2 337.77 315.09 383.05 207.06 242.87 177.15 313.14 82.21 180.13 203.12 357.46 344.27 267.67 169.05 317.86 366.26 301.69 371.47 148.46 336.11 340.44 209.23 211.1 345.63 215.49 319.18 302.71 185.6 243.37 429.79 177.57 303.62 240.41 277.68 345.09 203.8 287.6 359.29 327.37 584.17 389.01 332.01 298.78 286.7 328.62 266.35 371.03 253.78 221.59 117.21 256.4 232.12 264.06 413.42 227.29 227.48 264.42 425.04 255.67 310.1 422.29 218.64 265.96 235.3 346.35 360.57 326.87 239.72 236.1 164.08 240.31 371.51 231.69 92.46 217.87 205.15 423.32 258.69 401.59 317.21 213.39 177.56 323.43 264.83 214.37 345.53 347.05 186.9 144.85 122.42 263.81 211.49 236.75 397.47 315.24 234.05 279.1 385.54 348.91 213.4 229.4 298.05 444.18 236.5 413.13 531.98 207.42 471.24 244.56 275.49 424.1 219.62 666.5 359.3 373.77 311.54 188.64 225.25 589.63 216.21 196.41 404.43 234.58 226.26 218.17 361.29 214797_s_at PCTK3 100.81 88.53 71.51 146.46 45.86 83.64 59.97 85.12 100.31 69.91 45.56 29.93 52.33 98.04 55.31 42.63 171.9 125.1 79.25 81.33 76.31 46.65 68.01 133.69 106.58 74.68 42.39 38.71 86.18 130.44 65.18 127.28 71.06 88.34 204.48 161.86 253.28 94.75 94.03 170.79 233.68 322.84 349.6 88.11 124.6 66.91 185.84 200.47 79.57 140.18 83.01 53.5 65.51 164.04 73.11 50.57 314.29 169.71 59.53 171.53 89.79 130.2 107.67 36.39 63.4 113.23 93.75 190.42 184.93 68.16 109.26 188.13 63.43 38.2 65.34 62.66 62.4 51.38 56.77 65.77 104.38 60.53 72.53 38.22 51.72 76.3 45.09 39.05 111.78 86.62 114.68 75.44 97.36 36.38 73.77 53.36 45.06 72.37 96.19 98.07 65.84 45.25 55.51 64.33 35.19 108.79 49.71 67.72 113.72 173.75 229 64.86 81.76 62.89 71.81 77.68 38.23 113.62 92.17 47.59 62.16 97.76 80.78 120.16 43.28 68.31 66.69 47.73 61.11 210.81 60.28 56.19 79.15 64.63 50.68 71.11 77.44 42.17 41.18 66.42 77.23 43.15 57.37 60.19 50.92 61.31 37.51 90.07 62.17 137.54 166.57 82.36 106.76 45.56 73.66 96.68 111.23 58.38 44.67 97.74 106.19 53.96 51.43 70.01 121.08 60.79 62.03 96.28 59.49 120.39 170.16 71.47 59.23 300.68 143.29 74.74 133.23 107.8 48.3 137.91 55.11 93.49 52 102.4 113.86 115.14 88.32 50.51 104.63 96.83 58.27 55.85 120.01 85.88 90.96 67.48 123.44 67.56 79.03 132.26 134.04 86.75 41.67 109.27 178.46 71.39 131.76 177.52 94.21 78.39 111.82 102.76 86.58 77.35 238.57 162.19 44.68 59.63 63.4 95.17 91.41 141.37 42.08 96.78 81.84 164.97 54.23 214846_s_at ALPK3 26.2 27.98 60.1 34.86 38.52 46.07 32.72 132.67 36.01 26.48 39.49 50.05 26.79 47.65 42.91 22.99 27.08 23.56 31.37 20.64 38.45 30.44 27.43 32.81 32.34 28.58 48.45 36.2 28.71 39.47 33.2 28.66 38.37 39.96 30.63 22.81 28.55 27.51 57.85 33.4 25.68 53.56 36.69 50.84 38.95 47.4 31.48 32.46 42.58 35.84 31.84 39.93 34.05 31.61 35.32 38.74 26.73 37.13 27.92 58.31 30.84 34.36 37.69 47.12 31.28 33.23 36.86 50.25 25.53 36.59 36.03 32.43 34.04 57.16 34.3 27.86 24.07 27.13 22.23 39.45 43.19 29.86 43.05 39.1 35.45 35.28 41.51 41.06 28.11 35.43 85.46 37.48 36.32 46.33 33.04 40.75 60.48 40.2 31.95 37.74 37.88 314.16 39.58 31.78 38.53 44.98 47.04 39.93 54.62 79.62 51.21 38.98 39.99 47.02 41.44 58.55 49.35 107.94 37.39 83.43 108.6 44.22 46.03 37.18 69.22 45.54 48.13 59.5 44.8 38.93 65.96 71.48 51.14 51.98 59.16 35.63 44.1 56.29 55.17 55.84 38.3 105.01 39.92 33.32 45.9 43.74 89.9 42.65 80.49 35.57 42.97 65.07 45.7 52.89 40.22 46.75 48.69 53.82 47.86 351.91 35.44 45.33 38.95 44.5 55.84 43.49 47.09 46.73 45.16 40.76 54.59 71.47 53.57 42.14 51.12 59.4 51.41 54.38 56.33 35.25 45.49 62.44 47.65 49.18 55.43 50.15 180.08 57.85 45.13 61.71 53.73 47.31 47.4 57.61 41.13 163.15 41.64 51.14 44.69 57.55 36.44 41.19 32.43 36.34 43.44 34.46 68.07 25.95 39.55 28.8 57.81 50.28 41.76 72.42 39.06 37.31 41.08 67.86 45.6 51.33 36.45 32.76 53.2 32.37 44.85 36.22 73.8 214911_s_at BRD2 481.54 726.24 907.59 862.91 1250.36 862.56 449.35 698.03 597.39 1841.71 1806.44 1657.96 1508.83 1906.49 1546.18 1171.19 665.51 909.26 881.5 1168.22 961.83 495.57 653.93 842.86 818.33 681.18 314.12 615.21 720.95 390.31 483.82 735.65 750.5 1532.95 332.55 890.42 662.31 618.05 709.06 525.82 505.98 975.82 578.63 700.36 415.7 611.7 662.47 500.71 960.21 948.8 629.54 911.87 828.56 519.69 842.74 544.95 549.53 325.98 984.33 798.95 645 1625.92 750.74 1221.24 1428.27 947.14 1189.79 912.72 961.56 1157.21 895.04 1577.6 913.68 698.09 1017.21 695.82 1072.22 830.07 899.82 665.34 1386.22 1031.52 799.79 889.57 816.62 619.32 702.19 711.78 660.24 708.7 2751.52 956.07 875.67 351.36 485.36 547.09 1007.96 1129 593.12 730.92 1157.84 745.26 831.98 768.88 1011.21 999.57 663.38 815.11 1216.72 1484.18 854.78 1012.39 975.2 982.37 873.21 412.36 2488.69 1482.01 1245.74 1916.38 1526.3 1643.26 1167.15 1103.86 910.47 727.14 1361.07 896.09 480.51 604.3 997.11 1065.95 2082.8 672.02 658.11 1074.41 1303.39 470.55 878.44 847.39 655.14 636.08 882.62 840.87 760.46 1921.04 661.85 794.11 1084.52 536.95 666.3 1028.52 450.38 567.67 801.9 1019.52 364.81 682.69 738.03 699.25 1195.51 788.73 346.33 687.42 650.53 1341.1 852.97 790.64 1985.21 881.72 1549.73 991.83 1238.81 938.4 1780.15 1174.24 802 1030.64 1220.42 1818.03 900.94 718.02 1139.85 1243.77 997.49 1692.72 1225.25 1205.17 813.95 980.2 1540.05 644.86 695.91 796.71 833.2 1298.12 625.68 1285.54 933.63 740.01 1067.19 778.62 819.58 2409.32 1269.32 896.94 967.7 1045.29 1067.35 630.89 1029.38 1137.98 1434.93 1233.96 840.47 847.58 697.7 1266.38 564.48 1630.92 1014.88 1258.96 1420.26 1032.38 867.87 1015.8 667.45 215184_at DAPK2 53.31 124.42 69.6 68.86 58.4 118.07 89.12 63.89 87.2 66.39 71.48 71.83 61.67 170.22 73.57 50.46 39.97 71.38 144.86 56.98 73.22 61.47 333.74 86.6 208.82 63.85 42.36 73.28 80.71 57.67 38.18 78.16 47.81 99.22 77.82 71.99 54.36 124.24 89.19 104.47 130.37 93.12 77.56 292.63 62.92 63.46 64.78 163.32 83.81 73.67 96.27 59.06 1147.33 123.91 104.37 63.67 57.93 56.34 65.18 144.95 102.87 113.86 43.43 75.75 74.26 104.23 268.15 61.13 49.88 152.28 52.33 80.29 111.04 39.32 37.17 53.54 52.99 57.58 65.79 35.67 71.33 80.73 94.38 48 63.75 64.28 70.82 84.6 52.49 71.26 212.18 64.05 83.2 62.5 78.45 80.5 58.68 63.06 93.52 95.27 293.66 59.03 62.57 80.76 50.63 90.76 53.71 77.08 73.7 212.89 111.51 143.29 62.99 190.03 87.17 133.2 67.52 130.3 91.19 57.48 89.23 227.62 59.61 51.98 71.3 43.61 57.72 50.94 56.49 52.3 84.87 54.98 87.08 42.59 57.78 66.8 63.66 52 50.56 45.39 47.14 40.5 41.55 43.81 59.23 55.87 46.2 87.88 94.32 53.57 106.63 70.55 52.69 40.47 82.17 92.25 112.16 60.1 39.23 88.56 103.41 70.64 75.45 89.31 46.54 55.98 50.99 77.62 115.6 62.1 80.88 97.14 77.08 84.32 99.73 86.68 50.64 95.42 92.38 117.22 79.44 144.4 65.46 59.07 59.38 88.25 64.01 80.36 111.23 92.73 74.36 48.86 49.61 42.02 74.46 39.07 380.19 60.58 59.56 114.06 64.7 57.36 47.28 126.7 63.52 59.1 69.07 98.44 55.71 42.21 95.19 63.4 65.72 46.61 105.99 62.91 45.61 49.06 56.12 161.74 100.72 65.36 33.48 58.54 218.16 94.56 49.13 215462_at PLK3 107.93 106.21 214.51 80.53 73.59 183.33 171.03 68.47 106.03 154.81 118.18 98.64 167.74 77.94 430.17 91.14 81.22 120.61 103.88 76.48 131.16 97.26 142.51 140.68 120.45 124.61 149.74 88.51 85.16 124.74 108.79 104.96 158.62 51.43 130.87 111.52 116.72 151.04 212.86 131.41 70.81 104.58 96.61 138.34 137.07 243.73 133.98 121.31 139.02 177.62 77.53 123.61 121.78 134.46 73.25 240.7 105.86 122.25 171.84 248.93 119.98 71.78 98.99 114.89 158.85 119.86 279.21 128.66 105.04 247.28 162.95 78.85 146.47 240.61 111.23 205.73 97.56 119.52 108.41 212.76 273.44 95.57 98.75 192.79 121 88.67 285.36 180.54 111.04 115.09 158.33 179.01 116.69 119.22 84.88 94.07 140.68 156.97 95.38 110.78 48.92 78.49 114.76 143.48 195.32 89.18 178.38 224.94 138.58 201.14 115.32 131.1 133.04 201.66 106.63 75.37 158.55 278.21 147.8 134.3 271.01 61.73 81.33 108.97 162.87 75.06 153.44 122.23 86.1 104.67 225.23 203.31 51.69 109.87 138.69 99.07 95.78 97.26 212.06 154.1 124.61 112.12 75.03 90.26 124.12 234.44 97.47 67.36 269.28 96.85 69.68 204.47 170.01 173.2 91.77 113.33 74.28 134.6 156.33 110.43 214.32 177 86.88 134.21 119.21 117.96 152.42 149.13 227.68 101.27 101.79 148.12 135.03 81.37 133.67 111.53 189.79 171.72 185.07 80.55 135.03 176.1 118.82 177.11 182.75 166.91 210.67 150.85 125.57 123.63 264.14 85.93 188.57 115.25 110.1 142.53 92.71 218.24 66.17 140.33 106.04 122.93 108.44 92.48 121.78 144.36 163.02 123.06 248.09 88.15 169.65 232.48 187.78 121.51 131.1 108.04 211.94 108.38 136.18 157.18 75.3 151.82 132.93 107.59 127.55 74.18 75.04 216836_s_at ERBB2 644.48 616.77 8994.38 707.99 1713.95 1215.9 787.1 1034.77 931.93 981.38 1324.22 1648.59 1063.43 1339.86 1803.7 643.38 366.24 555.59 471.47 380.19 517.49 1796.12 2055.27 397.41 704.3 210.56 12104.85 3068.01 452.73 644.86 1851.49 1849.77 518.7 1204.85 506.21 1005.19 446.46 695.16 260.25 762.15 935.51 771.19 279.86 9140.12 528.37 6155.6 628.06 461.71 1190.86 1129.57 842.43 1565.42 1842.16 736.26 2296.62 1571.89 396.18 1200.31 6667.17 14330.58 836.47 99.2 659.91 822.03 49.16 394.56 724.52 203.53 283.3 873.49 639.18 395.69 577.77 8488.64 346.95 4827.25 443.8 396.48 721.94 645.15 822.26 649.87 1110.13 1019.34 622.88 769.64 11198.63 346.49 645.25 490.99 1264.47 854.63 1405.73 629.62 602.36 5023.3 1343.91 600.08 825.91 483.28 661.81 930.73 606.82 1237.5 538.24 1370.48 987.28 892.53 2325.28 1455.36 570.11 898.36 366.96 471.02 421.2 338.67 659.16 380.4 324.23 520.51 729.85 708.76 357.57 489.88 505.99 469.08 1115.46 486.7 7265.79 411.83 4997.9 783.8 3053.43 5484.58 7369.67 362.32 372.39 4005.61 5692.71 443.78 468.38 3983.07 201.58 401.52 347.46 396.45 12895.29 741.91 9538.45 674.72 483.79 393.78 480.9 8514.5 1048.47 852.63 1066.67 330.72 1087.07 558.17 1260.44 910.74 669.11 1230.35 424.75 648.7 1320.13 4927.62 4901.05 874.23 291.07 999.08 648.98 372.19 7560.58 1184.61 307.14 651.1 561.43 590.82 494.01 343.94 632.81 490.02 746.94 1169.54 9071.77 935.58 408.99 1654.9 421.23 684.27 754.29 10923.22 620.86 6039.1 618.09 830.79 4727.02 438.94 719.87 4764.71 353.57 372.82 221.19 891.24 290.75 451.46 262.3 584.52 609.23 716.02 3951.46 564.71 386.3 639.73 1638.16 5538.18 2990.5 923.75 800.24 425.17 2072.33 742.86 580.54 238.1 1362.57 217270_s_at DYRK1B 35.53 44.09 57.42 57.19 37.82 51.36 48.78 64.81 55.63 42.18 49.09 54.39 54.75 42.84 40.22 39.16 49.65 44.56 43.43 51.54 56.98 45.06 49.17 54.29 39.75 42.73 45.37 85.99 47.74 51.82 56.12 60.57 46.78 52.9 120.54 60.42 62.73 81.85 65.53 346.03 66.5 57.54 82.95 59.69 64.98 58.48 73.37 62.59 77.28 67.22 58.69 69.33 83.58 56.44 272.76 71.78 67.11 93.2 64.64 63.63 85.23 72.45 66.92 48.69 46.63 45.42 66.66 49.07 46.3 36.96 48.03 44.25 40.28 45.81 52.4 45.6 29.73 28.29 46.17 32.08 32.83 33.64 33.44 36.18 40.97 34.17 47.29 35.97 47.31 32.06 43.85 41.04 38.65 28.93 33.27 39.27 49.45 39.79 49.39 27.22 33.19 29.88 46.67 38.1 30.73 48.69 61.88 50.9 70.77 107.19 48.62 43.18 46.94 36.18 47.81 42.35 49.24 71.06 47.8 42.64 46.21 61.5 62.68 38.58 29.65 42.24 32.27 25.47 31.12 30.98 27.39 32.93 33.24 33.94 33.1 31.24 27.16 29.27 27.5 36.22 27.69 30.88 28.88 28.39 28.1 28.2 33.17 174.38 75.49 127.19 85.02 87.05 75.6 75.99 95.07 88.13 134.15 74.15 105.25 75.85 83.59 75.57 79.77 95.62 73.59 85.3 97.2 71.68 80.86 69.24 57.5 60.54 55.09 51.01 82.94 39.17 53.85 64.34 48.45 62.32 47.68 46.96 43.18 46.64 42.07 57.46 48.03 50.92 56.05 52.86 49.94 45.81 70.89 66.26 65.04 64.89 93.48 64.54 68.06 76.89 81.16 69.56 99.8 71.75 55.33 57.05 51.7 59.44 66.48 85.74 62.47 61.89 70.92 50.56 55.61 61.39 65.56 49.64 52.8 63.31 80.77 35.05 108.78 68.79 75.85 58.39 47.14 217736_s_at EIF2AK1 807.55 1210.52 1222.8 758.76 1527 1548.37 602.37 2038.09 1674.49 1104.85 552.96 1348.63 909.02 1343.35 544.69 590.28 785.4 1093.68 966.53 1003.33 1196.26 1784.01 668.24 745.59 877.24 549.13 831.29 1472.42 658.08 1184.83 1093.58 1091.3 874.36 1720.6 2443.64 1167.89 1026.08 849.35 526.42 830.83 1395.67 1229.53 1189.2 1191.82 1210.93 678.13 810.47 956.84 886.09 899.66 368.25 968.37 1309.98 638.29 3093.9 1270.68 1572.2 1052.45 796.99 909.21 866.62 870.79 1533.05 982.24 699.8 1623.5 1504.35 1118.96 1365.34 919.68 909.62 1237.17 1100.21 680.79 651.51 797.74 694.71 712.47 1108 1097.61 1207.84 1431.46 1135.89 1109.4 536.98 877.85 942.53 579.86 1272.07 361.95 915.97 445.64 642.07 391.07 260.69 424.65 912.59 903.2 322.82 513.38 866.89 945.85 945.83 325.57 673.26 1373.05 962.28 1269.96 1964.27 1298.61 1156.87 562.61 773.66 586.15 920.81 826.94 1171.59 712.05 685.01 1687.1 877.47 1987.89 1322.98 814.97 456.31 1153.89 947.59 547.5 834 1339.62 530.14 1955.92 603.04 764.82 696.17 723.59 353.2 643.86 899.58 1194.09 468.42 795.25 703.48 846.31 698.7 780.89 662.95 952.77 1223.2 946.29 792.33 933.84 1200.74 725.64 739.48 726.27 1726.8 739.74 803.1 767.36 1259.39 695.15 624.95 1458.59 1094.06 813.68 826.27 715.53 987.46 908.2 961.4 1108.39 800.93 880.25 980.31 589.52 574.51 684.7 671.27 1236.95 433.95 672.96 901.53 981.54 692.93 1042.21 1048.68 1288.55 802.58 1001 758.06 561.07 699.3 661.96 561.61 799.92 608.61 846.78 848.23 972.26 968.84 760.85 646.02 579.04 807.12 738.92 431.89 537.29 460.35 817.69 626.41 657.18 809.43 1631.49 662.68 867.85 936.2 1496 576.9 810.11 1015.3 958.36 973.5 496.71 907.56 2025.7 1157.36 217765_at NRBP1 238.75 177.1 315.28 204.98 161.22 133.77 94.81 214.96 222.81 184.91 218.8 192.04 167.08 187.88 192.4 202.68 324.21 174.52 298.18 138.91 246.04 286.36 188.85 247.64 163.64 237.86 241.99 243.32 159.63 260.63 198.23 318.9 259.43 181.69 246.26 283.23 296.1 333.31 198.77 269.04 325.42 262.1 286.65 251.23 234.7 223.23 216.85 183.39 452.7 181.46 175.39 270.16 253.92 405.04 189.91 301.87 247.6 214.2 196.42 260.87 244.29 190.12 359.61 271.53 99.8 194.98 152.07 138.47 212.83 176.24 221.76 177.81 211.28 296.64 178.31 199.8 185.07 184.42 159.94 232.12 155.87 205 142.79 207.74 155.88 152.73 142.66 197.42 143.81 68.65 95.01 139.07 97.56 107.5 79.92 127.41 180.6 212.84 88.64 84.85 174.95 159.54 160.79 82.98 116.15 192.55 205.64 175.8 229.17 115.91 197.28 199.75 209.56 386.64 202.6 40.24 169.06 127.81 188.69 215.01 185.23 362.11 249.38 165.4 162.26 173.94 169.51 158.43 135.77 151.07 175.5 205.11 148.31 208.14 168.96 127.08 254.33 171.52 141.66 171.65 111.56 153.46 130.66 140.34 151.31 133.31 201.6 242.24 220.46 211.4 284.79 179.17 284.76 195.99 186.93 286.91 197.82 240 240.39 192.34 273.37 189.13 156.96 184.06 167.11 330 203.82 217.41 91.22 170.24 199.48 253.95 233.78 228.25 119.96 308.62 238.45 138.01 181.1 200.93 195.72 163.1 183.01 213.25 222.75 187.39 250.73 206.51 187.94 181.85 155.38 228.69 200.21 249.17 159.28 199.86 182 181.37 178.46 153.23 185.05 221.67 237.64 76.07 165.17 183.04 142.28 184.41 126.06 194.63 179.7 169.07 287.05 166.21 153.04 142.56 174.34 329.95 210.93 165.74 214.63 243.05 173.51 210.35 260.97 286.74 170.85 217849_s_at CDC42BPB 108.8 166.54 212.12 209.27 373.37 291 635.6 216.65 124.86 199.16 275.97 272.71 175.42 502.19 216.66 295.44 175.66 78.41 177.94 152.5 158.01 77.23 122.42 101.69 117.1 159.79 158.55 136.4 42.62 141.04 85.66 162.58 122.44 305.26 143.69 164.41 248.89 171.96 231.48 156.29 106.13 265.43 106.58 266.13 191.21 248.79 163.84 139.55 207.43 150.79 75.48 166.97 550.15 199.89 221.92 206.07 281.83 274.71 329.2 334.34 463.16 686.27 214.28 90.06 75.69 84.42 244.29 85.88 93.58 103.76 128.44 122.08 70.28 90.76 106.06 147.61 113.24 165.69 84.59 96.29 136.25 193.67 151.94 187.84 166.66 107.95 154.1 139.33 124.69 104.13 211.59 241.43 197.81 123.96 140.17 161.46 209.51 259.68 120.81 131.76 405.95 290.44 209.33 277.13 338.39 643.71 287.74 361.11 361.24 236.12 139.17 152.14 71.93 186.62 155.41 113.68 479.02 223.35 154.4 332.35 368.68 170.17 173.08 135.07 145.76 81.93 131.18 154.13 104.65 99.44 144.23 160.9 322.56 206.07 132.08 88.59 125.17 88.5 105.91 128.55 100.71 124.05 119.5 127.42 157.59 202.25 151.91 241.52 271.96 141.95 199.93 171.17 191.15 116.21 120.12 150.83 73.49 97.62 182.29 98.76 245.81 185.64 70.36 158.98 198 172.06 131.18 188.63 331.57 214.49 451.13 227.73 196.46 107.16 174.79 302.42 109.61 148.38 175.42 219.62 133.79 113.07 194.14 251.43 136.33 108.27 298.6 157.12 140.47 96.03 256.4 183.52 326.68 328.17 342.63 512.79 308.96 374.72 403.22 295.97 263.29 409.29 342.73 183.06 285.1 409.07 254.07 183.14 212.07 251.03 339.97 406.59 415.84 627.98 247.73 160.35 140.11 283.22 167.39 302.85 211.47 182.52 314.75 226.19 150.77 119.7 183.51 218022_at VRK3 284.35 304.73 232.57 346.32 355.51 303.19 289.12 286.52 272.08 265.77 411.96 344.92 418.34 396.45 210.03 321.04 397.42 258.69 242.04 319.23 259.36 234.94 255.49 321.85 237.94 254.83 385.53 211.15 285 236.42 199.06 243.7 244.58 395.52 336.64 321.36 187.24 237.43 243.71 511.3 365.64 281.09 222.47 388.98 202.57 296.41 284.88 210.76 259.57 323.68 289.98 307.91 330.59 384.22 312.47 283.44 160.83 307.44 343.37 307.13 394.12 270.8 158.49 410.25 478.59 406.57 344.2 290.81 189.75 159.61 169.38 347.58 276.83 192.07 239.08 145.09 163.45 254.14 196.18 190.66 227.01 314.44 363.74 315.4 263.86 304.6 229.32 279.22 310.73 327.81 297.25 263.08 266.47 208.62 214.79 212.79 363.46 358.39 248.74 211.76 274.8 282.95 345.37 242.46 295.26 475.23 377.2 312.74 336.93 359.96 308.84 326.69 365.67 307.06 275.44 238.14 252.82 229.46 354.91 367.21 277.88 210.88 468.39 360.5 175.79 360.29 180.25 187 319.84 198.9 236.01 310.41 352.65 248.5 637.46 186.47 250.36 161.3 141.71 199.12 172.1 253.57 222.18 233.59 197.35 201.72 209.34 339.81 446.76 442.32 209.57 323.14 195.69 233.67 351.2 372.52 370.46 289.9 251.56 237.17 357.8 230.69 312.95 318.21 404.97 218.82 495.89 343.43 386.03 350.19 348.08 283.72 266.9 244.23 180.85 253.49 221.64 227.92 173.47 422.11 218.65 237.05 290 232.57 226.22 245.07 272.3 251.36 287.02 397.07 252.02 214.99 227.98 298.3 279.62 201.1 315.8 236.86 277.4 268.74 300.6 307.21 353.22 433.72 208.07 294.9 207.15 275.29 206.63 223.88 204.62 290.83 246.47 186.55 265.52 238.98 238.52 292.23 191.06 254.26 385.12 203.76 374.32 254.8 291.24 203.65 320.88 218145_at TRIB3 103.82 316.9 235.4 132.23 330.55 126.91 472.25 181 169.52 346.04 192.83 71.62 195.6 341.92 141.95 636.53 233.12 68.16 258.07 168.11 118.17 434.53 212.65 105.97 366.01 54.36 188.76 49.73 179.62 465.2 192.03 352.77 326.02 262.45 229.17 159.84 234.49 154.61 61.41 104.82 45.69 96.61 359.02 338.12 67.94 168.78 87.15 77.65 191.51 228.13 47 301.37 360.28 138.11 170.43 253.11 223.63 152.07 409.82 245.09 190.29 237.17 278.59 384.63 142.5 91.83 79.31 88.18 91.85 97.73 71.31 142.35 167.14 1435.61 237.09 373.99 119.44 90.07 136.84 234.46 221.7 211.33 138.61 101.31 229.7 120.1 282.59 128.89 155.32 197.68 62.9 149.97 128.32 351.64 181.26 529.88 679.91 159.66 366.12 165.61 62.05 167.65 151.07 322.08 160.28 235.41 189.83 205.52 211.07 102.87 65.15 136.26 59.86 188.58 259.17 147.53 138.84 124.94 89.73 434 346.36 141.12 221.08 229.77 60 234.81 215.3 125.98 233.65 42.89 151.19 416.35 161.01 244.62 179.19 59.7 65.92 264.89 337.47 361.86 109.31 431.63 327.77 100.49 157 171.82 555.35 250.47 312.61 149.37 114.49 177.72 395.51 194.47 181.28 110.77 408.64 132.71 209.48 87.35 235.97 148.56 338.12 169.32 167.25 611.08 170.46 239.74 201.47 281.38 110.43 219.92 193.4 173.62 54.33 139.32 187.62 111.61 101.23 441.04 101.54 126.18 114.18 248.64 184.37 271.16 187.84 274.87 174 109.55 204.34 103.89 230.97 625.34 126.74 552.08 251.67 357.63 423.89 69.94 263.47 215.58 277.33 74.88 391.74 230.72 116.85 240.97 223.52 268.44 274.04 171.51 198.38 140.65 206.14 87.38 163.87 371.42 176.98 103.96 201.34 211.48 379.63 323.71 146.22 125.54 228.54 218168_s_at CABC1 285.64 220.93 236.62 250.76 222.07 248.67 276.22 325.31 210.78 259.17 285.85 161.36 352.18 203.45 227.39 200.63 227.87 243.49 875.52 614.4 218.54 384.59 381.54 255.34 735.12 151.99 105.12 225.17 318.67 177.19 216.49 337.34 163.93 353.33 161.17 217.99 308.67 186.32 221.33 248.31 478.31 367.2 646.43 229.76 214.2 203.74 240.95 202.59 329.23 289.54 210.25 297.6 203.64 284.22 301.28 218.54 522.27 250.5 198.14 191.81 223.68 229.76 83.53 184.56 194.8 318.6 123.53 256.49 298.78 224.51 100.49 412.42 308.64 197.42 313.03 245.34 226.73 146.47 174.33 96.63 163.68 131.44 196.07 205.86 201.33 248.2 129.66 238.89 256.03 220.27 215.94 235.08 543.83 261.54 323.97 199.98 245.4 280.88 350.5 396.94 308.65 234.26 258.13 387.01 170.73 335.4 273.81 356.83 302.56 199.09 247.53 375.16 247.77 126.68 184.81 145.09 180.12 189.23 197 156.56 183.07 213.16 236 294.86 250.29 180.92 260.37 192.67 157.48 355.82 166.27 103.64 412.49 150.12 95.39 174.39 250.34 161.49 150.78 99.41 179.31 93.62 166.91 159.36 147.74 117.4 127.45 235.21 245.8 252.45 405.18 258.86 145.71 174.81 142.2 376.93 379.66 104.28 162.85 158.9 329.18 203.89 383.5 158.59 201.95 160 130.04 210.44 234.05 374.41 113.25 129.69 174.34 304.56 149.21 161.24 183.27 273.89 239.84 554.4 192.22 238.39 213.02 214.33 134.14 276.6 175.31 311.07 357.75 582.39 122.57 155.07 172.91 145.34 305.09 121.06 356.18 172.88 172.25 548.86 413.32 193.66 141.24 140 124.49 412.73 164.63 240.02 126.61 151.37 310.7 175.55 283.63 155.34 181.38 159.02 157.88 138.38 157.66 155.65 306.78 263.11 171.16 229.53 176.55 519.98 221.86 218311_at MAP4K3 145.92 228.6 84.47 83.27 201.07 165.9 128.21 229.4 191.43 83.37 226.41 250.95 101.42 187.95 91.9 101.6 218.85 74.18 152.01 135 186.67 151.77 204.35 73.99 157.77 139.95 133.73 362.97 47.72 200.64 199.89 104.18 209.79 111.85 216.82 118.48 119.34 127.39 98.04 61.5 190.97 151.09 98.22 161.28 145.04 121.04 92.55 132.13 55.23 109.34 132.24 132.81 87.78 222.14 302.74 167.1 117.57 74.89 54.24 56.85 84.79 119.87 121.08 38.17 27.73 137.52 166.35 52.51 123.81 98.04 127.44 89.92 92.93 125.08 142.83 166.69 256.59 116.81 160.81 61.03 61.13 188.96 231.17 205.62 234.62 168.45 116.54 101.68 99.27 112.8 13.06 101.22 123.38 128.15 225.04 74.65 183.82 109.95 94.25 53.21 140.35 187.06 111.03 119.4 82.42 104.72 94.18 113.41 86.56 11.37 74.27 111.5 53.93 108.86 122.04 117.73 84.82 29.65 38.78 49.8 49.7 158.96 40.24 68.07 75.16 95.46 131.52 121.42 106.1 111.08 95.41 92.71 36.79 62.92 114.96 144.16 159.97 108.1 106.24 150.62 213.14 95.71 122.94 103.52 345.06 96.92 173.58 130.74 125.46 201.77 80.04 59.48 123.73 102.19 125.73 90.07 195.54 58.1 134.73 79.92 86.41 129.37 195.71 88.08 175.85 99.95 128.87 101.51 19.19 159.4 147.65 73.47 106.35 83.73 11.87 71.79 55.08 131.79 68.92 106.7 71.18 62.66 100.83 99.4 62.75 97.36 136.18 147.98 42.95 114.82 94.28 199.17 87.93 105.68 115.23 128.12 110.41 74.7 78.56 68.57 161.11 147.62 129.85 19.1 61.99 143.9 190.76 70.32 63.52 236.76 47.73 79.85 74.59 117.38 149.41 118.56 152.53 190.27 96.07 130.71 49.74 104.11 106.97 107.5 125.87 186.86 103.91 218499_at RP6-213H19.1 524.26 238.28 56.32 345.84 113.27 65.49 307.53 42.18 156.6 163.77 255.95 57.45 80.78 165.89 25.86 131.94 727.98 433.37 783.43 679.67 236.14 98.69 646.99 230.08 396.39 240.83 172.54 298.22 616.34 359.11 640.11 385.85 333.53 2560.14 418.65 176.47 138.99 596.01 106.69 121.96 75.11 298.16 730.48 141.43 136.41 73.8 42.81 192.11 136.65 104.44 1298.34 72.54 120.1 338.42 277.52 206.86 164.64 245.79 177.06 161.59 18.47 273.36 313.57 247.64 317.35 491.54 149.16 175.75 212.59 243.14 334.4 54.26 226.32 244.45 420.5 100.83 426.55 125.48 184.94 211.04 138.03 335.09 236.85 78.19 66.21 213.42 111.64 199.72 143.22 605.29 17.46 119.39 453.14 327.59 286.65 231.75 124.7 171.26 516.16 331.6 393.83 141.14 140.13 40.86 114.32 86.37 62.36 67.52 126.85 20.47 150.08 130.87 116.89 251.93 249.92 295.57 28.15 55.54 154.23 27.33 60.19 367.14 186.23 356.09 46.02 159.11 45.2 100.91 190.05 426.24 98.95 58.8 21.61 72.63 102.97 79.38 198.64 195.91 61.7 124.68 135.56 224.84 194.06 80.6 141.18 26.88 372.29 76.69 91.66 32.56 650.88 92.04 436.73 128.3 92.52 70.37 282.18 127.02 101.67 215.19 431.23 257.4 530.42 68.77 228.71 167.28 232.81 160.42 86.53 1064.88 140.92 39.26 518.37 347.06 88.67 28.72 187.51 62.59 44.4 178.07 81.72 240.05 97.64 112.51 136.29 78.26 176.76 77.98 323.66 132.65 47.02 107.6 43.96 85.97 93.65 113.38 474.17 212.22 118.63 326.29 53.44 102.23 167.93 84.89 361.17 47.41 378.44 306.95 110.97 116.83 96.54 98.83 49.92 32.65 1005.69 136.66 74.51 272.18 128.51 117.46 355.45 199.98 103.12 48.13 194.42 447.55 95.27 218520_at TBK1 215.21 393.98 235.08 349.4 349.43 240.29 191.79 289.8 294.15 317.94 330.33 307.5 229.94 282.69 197.16 153.32 357.58 313.16 367.88 391.73 359.14 386.25 533.67 241.85 213.85 380.6 304.96 242.44 261.22 332.78 797.64 323.52 446.54 327.12 282.64 164.16 278.4 216.76 252.7 175.52 411.88 269.7 263.74 241.88 236.88 261.77 242.68 188.35 293.68 259.4 213.32 329.83 219.63 238.18 296.58 254.65 225.58 208.38 387.99 289.74 347.4 263.74 299.37 244.07 262.64 231.98 155.24 228.88 279.55 221.33 146.18 347.48 140.29 284.39 285.57 230.01 245.39 273.11 206.29 322.46 247.96 242.77 277.14 238.1 189.48 332.43 204.16 210.25 268.34 201.5 62.89 156.92 221.67 275.04 172.16 236.12 235.44 186.89 228.23 139.73 249.68 266.58 212.98 132.82 193.49 285.44 230.42 232.44 330.17 77.4 154.81 182.58 225.86 259.54 240.54 317.62 196.5 226.04 168.95 215.16 212.82 141.45 193.84 259.2 126 245.13 183.33 181.02 236.17 139.19 233.1 183.75 123.05 181.57 182.64 168.43 139.65 271.09 138.59 167.91 175.77 185.41 210.48 176.72 176.84 175.31 120.46 183.95 174.88 326.66 152.83 176.7 177.24 207.19 195.97 189.21 274.07 231.74 204.42 199.8 217.24 189.11 234.62 169.16 229 203.97 257.36 195.52 199.38 209.56 226.97 164.93 223.22 192.15 110.76 182.24 219.53 206.61 141.79 160.73 189.34 292.47 213.18 162.94 166.18 144.64 163.92 215.45 303.26 188.91 196.49 217.6 164.76 232.36 156.9 185.36 190.11 187.9 198.82 114.76 198.57 206.21 206.96 141.04 174.41 166.58 185.06 187.27 243.91 244.54 215.1 201.06 158.39 196.97 144.16 197.46 178.52 200.1 243.62 212.19 190.38 154.85 122.09 155.09 171.1 170.51 154.55 218535_s_at RIOK2 156.92 254.36 78.12 197.51 198.88 109.53 281.29 206.29 176.24 138.32 154.54 192.66 134.04 160.27 63.51 154.91 171.99 128.62 317.2 194.25 128.68 172.72 89.76 84.48 144.18 184.33 118.18 95.76 109.51 271.11 145.03 94.68 155.76 125.76 79.6 133.3 108.24 127.28 98.25 140.28 139.94 221.52 128.64 93 135.45 120.6 127.55 120.78 110.29 166.68 117.31 210.72 122.32 164.77 150.57 171.19 161.77 174.08 165.92 99.81 77.88 88.7 102.82 124.64 137.78 97.71 83.56 143.18 192.92 97.21 95.85 66.95 111.83 114.33 132.75 150.33 134.13 215.55 181.65 111.15 83.56 248.5 109.58 106.15 144.24 181.27 91.26 136.8 110.75 136.28 22.27 124 148.6 297.54 183.72 172.59 148.58 81.68 322.28 234.28 82.75 207.09 160.13 129.63 228.61 171.22 179.2 103.43 119.5 26.43 120.35 132.38 121.92 118.62 141.73 180.61 100.46 43.5 129.22 150.92 136.34 76.8 98.71 126.59 93.12 117.38 73.42 73.75 284.21 146.75 84.9 81.94 77.38 100.09 175.56 101.77 71.84 96.27 84.83 96.63 114.78 182.92 100.23 123.38 138.41 75.87 98.31 131.68 67 225.67 114.28 77.06 181.67 139.43 153.1 221.35 185.69 134.48 176.72 91.58 80.72 87.51 181.81 61.1 133.36 127.59 163.42 102.31 49.68 181.75 99.13 82.37 123.79 88.9 38.77 76.83 110.57 89.9 66.15 127.72 123.82 75.8 154.67 98.59 123.89 86.87 138.71 102.82 83.73 185.25 134.61 129.64 96.76 123.9 105.66 95.85 105.23 101.82 116.02 81.46 97.63 113.85 145.1 93.22 91.45 109.9 58.08 105.55 55.35 110.86 53.72 54.54 61.88 109.25 102.78 115.49 113.95 182.02 117.51 64.45 169.07 124.29 98.91 154.96 112.32 221.75 149.78 218696_at EIF2AK3 267.43 313.17 327.07 317.21 347.11 318.12 389.49 380.11 431.34 426.03 609.2 458.21 341.64 575.75 327.47 425.88 480.51 319.47 430.98 325.94 321.7 577.47 511.2 222.7 251.16 383.9 487.85 749.94 331.2 230.5 561.04 521.74 344.79 261.93 300.8 242.95 408.31 400.13 307.34 234.83 328.01 527.13 318.97 316.28 530.01 281.17 224.33 355.55 235.05 497.87 326.8 610.42 301.8 394.84 377.51 400.26 299.38 207.82 365.98 223.4 260.37 145.14 508.9 225.67 444.98 385.67 319.14 139.55 341.34 255.87 230.76 201.38 316.65 315.59 649.98 438.45 184.91 437.95 219.24 297.79 239.48 567.59 535.77 386.89 396.78 304 335.47 359.08 407.94 315.51 30.15 380.97 208.36 339.41 527.07 371.45 308.66 311.09 406.95 402.78 244.14 376.48 354.29 447.45 253.29 390.82 247.55 272.43 223.02 25.21 279.05 266.92 385.66 295.11 511.61 373.3 64.49 179.03 217.61 185.42 167.28 289.65 370.03 395.65 186.65 326.21 324.22 236.67 300.45 290.63 281.91 361.02 203.45 261.32 323.18 204.8 177.8 270 283.03 555.56 366.57 281.97 231.61 267.84 268.1 187.59 578.77 343.19 343.56 501.34 422.57 289.33 275.12 331.9 446.26 194.35 482.14 436.54 381.55 311.36 322.36 346.42 514.92 159.4 668.03 269.25 390.33 422.96 133.61 241.59 503.7 166.06 280.11 260.21 24.37 279.83 258.32 235.85 283.57 385.09 293.64 275.07 308.38 297.51 225.7 260.66 269.66 290.28 284.62 288.2 250.67 322.18 193.14 290.56 263.24 295.93 350.23 157.87 334.93 216.09 211.1 328.51 268.25 101.52 237.11 359.53 414.87 401.9 221.85 428.9 163.65 186.97 225.15 244.95 198.58 312.06 316.74 548.1 302.85 211.63 350.82 263.77 346.36 230.78 352.76 251.08 306.24 218764_at PRKCH 174.46 141.04 190.09 218.83 379.39 398.9 125.01 175.76 170.76 161.96 357.15 134.92 419.86 164.15 203.98 75 329.88 350.21 194.38 133.98 238.95 365.51 121.18 310.65 141.89 454.33 262.11 342.59 260.35 261.38 195.33 90.08 201.54 296.45 198.95 212.89 215.62 213.54 319.48 328.05 329.97 330.13 140.78 201.93 235.7 330.12 147.49 278.67 359.78 257.24 136.47 336.09 223.11 253.76 122.33 233.23 183.15 289.34 446.19 159.08 50.24 90.83 241.29 244.53 223.86 179.51 160.11 395.32 158.29 142.85 97.86 269.01 124.28 236.55 65.55 190.63 295.14 355.11 157.97 70.84 95.69 317.95 152.84 263.49 367.92 296.38 200.92 383.85 347.63 441.28 72.06 319.78 294.94 163.59 299.25 315.97 160.01 145.84 350.87 208.24 121.96 595.3 261.77 211.59 248.67 465.63 224.14 357.29 198.58 96.08 279.34 270.48 408.94 279.49 609.74 242.71 69.04 238.65 612 337.98 348.07 192.03 316.29 342.02 425.42 361.06 310.63 371.52 209.22 185.93 510.69 241.64 356.98 467.48 204.05 210.99 259.54 302.86 171.8 186.26 128.32 417.36 136.11 163 261.2 199.94 276.41 295.03 282.57 173.45 120.5 350.88 155.58 288.61 276.2 155.46 207.01 437.84 131.23 257.21 192.14 154.61 147.87 122.9 179.78 336.82 260.02 332.33 41.43 175.21 315.77 191.55 226.01 198 41.39 219.06 326.19 144.92 189.14 123.96 461.75 224.2 202.22 149.98 182.8 156.94 380.95 224.93 190.4 159.43 259.79 277.9 213.08 333.19 272.03 223.12 147.92 255.31 365.12 177.91 270.02 231.2 331.84 42.61 79.87 122.43 53.84 188.13 153.03 194.92 182.62 227.23 170.7 294.26 81.36 121.89 278.87 220.34 103.86 161.91 377.88 144.14 371.89 119.14 195.45 150 392.66 218909_at RPS6KC1 182.85 226.54 199.97 312.57 236.36 152.8 225.74 192.36 191.99 199.79 393.28 232.64 133.79 201.83 108.67 175.36 295.46 148.54 194.6 379.57 128.26 265.65 220.4 183.86 97.46 241.85 169.7 277.38 141.83 184.21 181.67 435.47 297.4 214.81 288.81 228.38 323.46 120.68 98.21 129.88 320.15 205.52 349.93 223.37 231.15 155.7 223.1 176.19 156.5 183.71 154.66 168.57 141.12 195.25 180.76 253.07 215.65 94.84 195.14 137.13 182.29 246.77 229.69 163.41 87.03 233.01 167.88 126.36 244.44 205.17 143.98 178.48 259.43 185.56 210.1 103.52 197.37 163.19 161.47 202.42 255.68 161.46 165.5 167.59 131.57 222.21 166.91 174.84 189.85 86.21 29.83 102.46 119.21 155.4 107.4 73.59 113.71 264.98 146.66 117.8 172.81 144.25 196.07 127.63 154.68 440.4 305.54 224.17 312.75 63.11 195.14 140.46 219.86 168.83 148.61 504.33 123.03 156.32 136.93 184.39 207.32 215.51 109.92 166.09 145.24 94.53 121.96 134.19 156.77 326.32 142.04 234.64 132.2 131.08 174.3 80.87 142 123.02 117.21 436.36 115.14 97.76 166.91 179.32 154.35 78.48 131.89 152.95 114.63 166.15 298.16 109.03 147.75 109.53 206.51 145.63 262.36 179.79 153.79 100.96 194.89 116.82 183.71 92.19 166.27 152.61 220.4 117.31 99.85 244.15 99.13 126.31 125.67 202.02 53.37 105.99 217.14 168.07 145.85 276.95 121.75 150.64 157.7 224.14 202.85 196.97 122.29 300.75 160.11 380.46 236.91 162.52 198.35 199.01 113.91 176.09 224.08 199.33 173.79 156.22 117.35 231.94 167.79 77.68 136.95 154.88 96.62 257.6 84.14 292.14 125.87 145.41 129.86 97.36 141.94 189.44 116.21 136.04 109.1 73.62 117.1 243.14 117.41 213.57 129.87 174.75 119.65 219148_at PBK 116.01 63.89 14.65 147.49 114.73 5.47 398.28 124.58 66.43 30.47 65.79 15.52 27.85 167.53 11.36 120.44 193.76 159.67 226.52 1117.52 85.4 68.42 142.67 244.18 151.63 159.96 32.96 64.39 166.07 494.03 68.19 153.91 82.58 98.43 254.34 392.87 110.42 567.19 13.28 137.45 156.65 111.97 175.9 59.96 142.86 24.49 88.43 252.97 50.68 68.6 173.02 43.65 89.72 69.64 194.71 129.04 125.92 126.8 47.3 23.75 114.92 125.15 181.6 106.26 48.06 176.99 94.87 167.71 176.99 48.27 115.73 101.07 276.71 44.63 311.96 57.53 272.93 31.32 44.61 102.91 112.1 98.72 152.86 20.09 7.74 26.11 16.87 27.64 47.91 75.25 8.62 37.96 104.03 117.14 16.66 156.73 26.22 128 92.68 182.89 29.6 49.64 131.82 8.86 223.02 84.24 15.38 17.45 29.71 13.01 94.05 9.95 31.91 114.82 75.12 156.93 85.91 13.33 26.89 19.85 21.76 109.68 64.22 35.81 8.88 309.07 46.27 26.84 179.01 61.94 11.65 62.85 10.39 24.5 48.47 241.83 315.67 33.13 65.24 47.4 103.06 72.88 380.82 217.9 27.94 48.91 222.96 49.63 34.4 45.97 414.28 68.51 146.41 41.54 38.08 233.45 148.75 132.36 20.01 117.57 26.16 15.27 272.8 22.6 146.35 31.61 80.98 25 21.4 298.36 54.08 20.48 85.05 384.48 21.87 19.7 56.72 21.75 26.08 196.2 10.74 241.04 44.54 46.82 23.03 44.42 36.7 57.6 251.36 113.89 27.02 8.89 14.19 87.77 36.11 24.98 210.06 34.51 42.76 39.28 41.18 75.16 29.53 20.24 278.5 9.77 85.31 139.32 16.45 57.06 44.86 38.79 46.76 9.01 176.4 24.16 46.33 41.32 54.91 35.89 82.58 103 18.01 84.93 211.75 118.43 8.73 219278_at MAP3K6 258.64 73.17 200.98 98.69 123.86 102.15 120.86 164.63 60.05 97.26 49.22 121.56 179.66 59.89 263.81 115.57 135.03 122.42 399.56 292.68 135.52 78.94 92.17 124.53 744.88 107.44 130.67 57.09 188.01 160.97 98.91 122.23 101.15 93.35 122.76 173.81 96.1 217.05 155.06 169.01 215.14 223.33 229.29 197.62 137.41 210.94 225.77 145.53 236.65 152.92 120.27 120.33 174.75 120.48 137.84 203.43 376.87 171.33 116.93 127.88 265.64 541.33 319.41 71.12 71.11 154.76 359.4 276.41 183.65 176.92 392.41 274.55 132.98 128.79 156.8 109.02 133.72 106.19 50.29 104.84 185.38 129.08 122.62 77.28 101.09 110.15 104.84 134.66 61.53 143.24 350.32 128.57 95.2 59.78 133.02 132.97 114.67 547.66 78.08 267.7 121.93 225.41 190.01 232.98 181.93 242.24 233.14 157.48 227.49 413.02 169.67 172.75 87.3 254.25 77.98 111.14 100.16 191.67 139.56 205.29 148.22 274.99 101.77 101.31 221.44 62.92 72.83 198.01 48.78 117.19 89.11 80.96 198.77 168.79 98.79 71.88 142.67 124.07 115.32 121.96 46.18 92.41 131.61 66.9 81.78 82.53 68.39 127.87 225.09 159.96 255.7 209.21 188.22 143.74 177.2 165.13 64.47 138.53 121.31 352.4 115.07 168.59 82.32 82.44 72.8 103.3 121 159.33 161.22 69.31 136.5 164.36 134.68 125.68 160.59 152.07 195.66 127.04 290.96 183.55 140.4 79.78 81.68 197.87 147.1 85.36 125.93 73.92 56.77 48.97 134.01 168.38 213.85 174.02 217.77 333.82 362.28 197.71 151.52 145.51 87.98 138.01 137.54 142.4 146.6 114.28 234.05 123.93 76.33 114.12 218.74 119.34 169 221.01 159.46 106.69 113.84 146.38 123.92 210.12 159.17 182.09 423.98 304.97 401.5 157.02 197.48 219542_at NEK11 50.56 96.78 34.11 45.93 64.89 112.54 91.48 104.31 79.89 30.32 74.66 131.89 87.71 124.52 65.18 41.54 55.36 34.48 36.74 109.57 27.75 57.02 89.62 30.34 38.98 38.08 58.68 22.37 40.16 54.05 37.03 48 37.33 89.78 27.52 79.37 38.57 53.74 30.5 44.81 44.06 27.4 21.24 32.61 22.53 49.55 81.73 31.61 30.29 118.81 53.96 96.94 49.31 42.35 102.5 68.8 30.89 35.79 48.3 51.04 118.64 31.65 23.2 43.43 27.78 37.37 77.53 30.93 27.46 40.03 30.56 32.21 74.34 49.53 47.23 61.95 35.87 74.04 75.41 55.05 31.17 123.78 95.32 122.29 67.89 76.11 49.94 65.79 47.35 66 49.86 117.44 93.29 95.39 173.86 73.26 110.85 81.06 99.8 90.65 66.75 58.19 83.64 203.12 116.92 65.42 82.68 63.13 59.32 107.88 61.85 67.3 71.11 45.45 48.37 114.56 45.52 27.85 45.36 45.94 60.86 33.58 33.19 43.53 61.47 34.46 48.84 47.62 81.19 32.59 90.96 79.65 57.48 46.78 62.38 54.11 63.68 48.32 76.33 50.06 86.34 53.55 54.97 26.64 37.87 55.8 47.22 77.77 48.58 120.62 33.9 45.13 36.38 57.86 84.24 53.78 91.87 46.68 49.97 47.77 58.29 54.23 81.2 41.17 88.61 44.49 106.59 53.64 58.18 36.99 38.44 36.55 42.99 45.29 60.36 52.25 47.61 66.67 69.12 94.84 54.77 32.72 73.73 48.41 36.78 36.23 73.58 82.21 29.48 41.93 64.73 91.68 45.67 63.97 49.53 37.43 54.16 44.91 48.94 28.37 43.32 79.96 73.62 75.47 25.06 99.13 30.22 27.37 36.35 40.56 42.21 42.76 33.89 35.94 92.74 61.3 54.07 36.43 47.26 54.27 47.48 37.77 94.79 57.25 44.91 52.66 52.25 219618_at IRAK4 115.69 92.59 73.05 74.44 60.51 94.83 87.27 70.15 88.07 48.88 55.01 76.01 93.04 41.61 76.09 29.16 93.25 118 90.21 88.24 75.14 219.39 56.71 60.52 69.83 121.47 93.68 72.46 61.91 88.21 81.87 59.69 74.3 66.2 73.4 91.69 87.24 53.78 119.03 124.12 142.07 123.1 104.28 65.55 59.65 76.25 79.97 153.9 138.98 71.11 59.83 114.7 65.72 81.96 139.55 100.49 66.47 65.38 157.66 66.82 49.92 63.7 50.93 64.5 91.98 108.23 64.63 112.52 61.7 78.62 69.31 53.08 42.86 80.91 62.16 69.38 127.46 132.55 75.17 77.42 86.14 72.25 81.72 81.35 69.58 96.4 50.3 99.99 94.45 38.17 21.78 47.05 41.87 38.15 35.38 55.91 64.14 72.94 54.51 38.32 33.28 58.25 77.67 45.1 83.35 36.63 107.04 68.01 45.08 31.5 41.72 51.12 75.15 74.44 82.03 18.38 57.26 34.15 100.52 46.34 37.21 33.06 37.16 54.24 75.45 72.53 108.02 61.41 58.69 198.5 75.95 80.22 45.25 59.36 87.87 67.38 68.53 69.16 63.18 56.19 77.16 80.25 64.88 76.25 68.37 48.83 38.66 54.77 100.11 135.52 56.68 82.93 61.02 78.83 76.76 103.35 102.86 113.98 66.56 104 93.61 69.66 80.47 57.8 113.29 109.17 66.4 89.67 44.09 75.18 82.79 49.58 86.94 89.8 53.96 48.2 116.41 76.08 57.94 69.72 93.94 50.85 64.45 78.51 93.29 52.18 69.93 60.91 61.09 65.68 81.86 74.08 68.7 60.3 55.05 63.78 76.01 68.31 41.62 54.77 70.28 80.43 87.85 58.98 60.17 51.11 75.74 84.34 46.04 80.57 61.96 81.42 61.92 45.62 64.01 50.12 71.42 46.48 60.69 59.93 75.28 57.13 52.07 53.42 77.36 86.16 103.14 219686_at STK32B 18.04 229.58 34.13 333.5 345.15 331.03 39.87 199.53 314.29 23.14 67.58 169.57 200.53 15.86 31.34 263.2 22.1 17.03 18.34 23.01 22.64 20.04 19.16 25.02 20.5 23.57 24.16 18.73 22.74 21.77 85.45 17.59 26.76 642.56 20.52 19.32 19.54 20.54 24.91 20.09 19.97 17.72 17.26 20.39 21.95 39.87 24.58 22.02 28.28 20.81 17.46 27.9 25.98 26.44 231.71 38.58 18.87 22.36 27.81 20.44 151.16 20.15 18.83 17.58 25.21 17.62 18.66 15.15 16.53 21.29 22.69 18.64 17.6 18.91 17.4 23.21 18.84 128.32 158.96 41.19 29.61 126.78 87.37 88.44 51.85 62.99 61.79 50.28 30.92 51.43 34.03 134.03 26.22 187.78 82.49 17.14 46.85 113.36 72.26 23.06 123.84 55.35 52.11 212.26 60.31 44.18 244.15 117.64 40.18 38.24 24.76 85.02 36.18 41.26 30.19 35.01 433.28 42.45 39.21 48.65 81.11 30.29 36.68 35.83 62.67 388.58 29.64 48.37 86.33 29.49 59.26 30.32 67.86 38.07 68.44 39.9 33.32 29.81 64.86 57.57 83.22 30.1 37.62 146.53 41.69 36.27 31.42 28.81 47.75 427.43 19.88 45.47 30.34 35.87 35.92 32.76 23.69 27.45 153.43 24.83 36.51 96.67 114.96 54.06 302.08 31.42 131.99 31.97 32.47 57.64 305.69 298.97 84.24 50.31 40.69 42.41 38.89 70.02 72.9 148.02 72.66 33.65 90.54 45.02 45.29 199.54 35.23 47.24 33.31 395.31 85.38 138.5 257.11 34.22 65.63 31.92 28.62 53.21 36.74 42.28 43.42 32.3 150.24 31.93 30.41 256.78 27.45 25.07 26.67 116.24 34.85 42.89 198.53 30.03 239.53 40.85 34.79 38.16 63.58 45.88 25.95 29.32 322.85 31.86 35.28 27.15 43.28 220028_at ACVR2B 42.96 60.91 33.74 67.91 61.05 61.5 44.7 77.49 47.71 47.02 83.45 51.77 118.55 120.2 97.81 104.01 42.55 47.5 72.68 50.03 59.49 48.6 44.09 40 86.8 33.58 23.73 51.02 34.1 42.69 34.37 41.32 57.19 103.73 24.53 58.49 71.9 34.63 48.12 47.23 33.75 58.17 58.03 63.76 31.83 49 50.24 66.02 45.79 78.2 44.72 31.4 63.84 29.19 93.51 46.24 70.03 50.23 45.25 50.12 104.6 79.79 67.14 163.49 29.63 68.5 114.81 59.28 63.92 161.64 59.48 69.59 43.41 45.71 107.84 76.72 84.52 72.99 96.06 60.62 92.4 87.31 119.06 109.82 88.38 52.13 73.55 64.83 52.52 53.9 145.42 80.04 61.22 46.97 65.5 47.75 46.39 44.9 34.51 41.45 95.43 43.94 37.63 55.28 52.62 69.63 70.3 82.78 81.51 121.73 62.88 95.76 56.23 51.5 43.43 24.12 125.33 85.32 47.83 105.2 57.76 71.08 72.59 86.95 50.58 60.89 159.82 53.02 66.04 37.31 45.42 39.46 115.45 43.56 34.21 136.28 44.27 30.7 58.07 91.27 76.45 63.16 69.72 83.36 53.78 65.2 65.04 123.7 59.15 34.38 54.47 38.61 44.09 29.43 51.96 50.35 71.55 24.25 61.55 35.11 55.43 47.3 73.6 74.13 51.86 56.14 41.38 37.14 137.1 89.88 95.38 92.77 126.1 67.46 94.8 78.72 59.66 105.24 90.3 234.13 76.92 58.07 83.63 108.42 61.71 113.99 81.03 127.48 54.33 97.08 49.7 65.04 43.94 41.32 72.31 69.04 49.2 69.81 52.98 58.1 129.76 53.26 32.97 171.54 43.01 113.78 71.86 62.44 62.1 96.04 74.23 63.01 108.75 115.68 92.96 68.06 61.4 46.4 62.58 151.62 49.31 64.61 153.05 77.19 40.19 156.38 51.19 220030_at STYK1 40.06 25.3 7.35 21.36 17.72 9.09 75.56 24.77 67.99 115.74 20.09 15.84 21.13 7.38 18.33 39.47 181.93 42.09 24.94 79.59 17.06 12.29 32.25 18.42 9.96 65.69 37.53 21.51 39.36 13.94 18.94 36.18 12.1 16.1 15.95 34.61 18.41 40.28 10.58 19.21 70.82 55.84 49 15.7 15.97 14.79 9.11 11.04 20.15 18.61 18.89 12.06 18.57 11.87 17.71 10.77 50.62 126.02 15.03 7.79 9.59 21.08 42.72 9.07 8.64 128.08 30.27 30.61 94.81 29.74 12.46 23.29 14.39 14.29 11.14 24.8 18.56 24.65 8.23 131.58 23.53 9.14 22.51 17.2 12.01 7.58 7.64 10.76 9.83 12.02 8.29 37.7 21.09 29.19 9.8 84.02 27.33 21.36 57.51 74.52 11.61 128.91 16.59 36.87 13.82 26.29 18.95 18.63 38.97 14.9 48.8 12.14 11.87 17.01 10.32 32.52 11.67 14.04 16.11 51.87 9.03 30.62 9.17 7.81 7.63 16.11 9.27 12.25 7.79 69.12 22.32 11.75 13.19 13.74 25.58 42.15 7.74 11.28 10.57 79.02 12.74 18.96 24.68 9.11 10.12 15.14 13.34 20.09 10.48 8 38.12 20.07 14.67 14.89 16.49 11.98 9.73 75.19 12.58 14.19 10.53 27.82 9.04 10.2 22.64 16.72 11.68 11.69 12.87 14.18 33.35 12.98 11.4 39.92 7.99 20.08 9.87 11.92 11.57 12.98 9.87 28.64 20.74 16 9.11 8.36 7.97 10.06 44.33 7.04 9.91 9.57 10.37 13.34 18.51 22.29 24.81 12.36 50.61 9.21 9.54 13.03 14.84 13.83 50.85 9 57.93 14.44 8.38 10.75 27.58 9.4 10.97 70.12 8.57 16.08 14.9 9.39 7.65 8.35 25.5 22.28 11.72 5.45 32.48 47.12 90.65 220357_s_at SGK2 39.05 49.28 40.8 32.2 43.08 44.57 47.35 36.79 46.33 33.98 69.42 37.5 33.97 45.51 47.99 48.72 50.23 33.78 30.8 46.07 42.28 42.65 54.73 50.7 36.13 41.89 46.51 35.11 48.78 93.09 39.18 39.33 39.16 35.76 45.47 50.18 39.73 39.08 43.92 47.59 32.25 35.46 40.96 40.04 40.93 38.26 59.11 37.71 48.88 44.11 34.94 49.61 38.3 32.23 36.86 36.88 51.78 43.37 38.04 69.99 58.16 44.58 42.66 39.57 37.21 35.18 36.34 30.34 41.15 36.49 46.31 38.28 37.71 37.39 49.73 37.04 30.25 33.2 29.8 49.62 42.97 44.62 43.59 47.57 40.89 35.99 36.24 26.27 32.44 40.33 84.79 35.08 40.34 39.97 39.74 38.17 46.55 50.79 46.33 47.47 54.34 46.37 49.64 37.65 41.98 64.28 39.34 48 56.79 127.86 34.62 28.9 47.49 46.48 41.54 41.87 44.74 48.67 34.32 39.28 48.09 58.39 54.44 69.59 38.37 38.54 37.83 81.36 46.42 45.65 46.88 43.12 78.1 45.91 40.96 52.2 53.16 45.66 50.06 38.63 51.63 47.29 45.62 40.41 47.38 44.18 48.1 56.63 54.72 52.92 45.65 62.63 61.21 46.21 47.82 46.54 62.44 45.46 49.04 53.58 56.01 52.61 55.59 53.68 75.06 55.32 63.06 53.1 78.74 56.48 45.84 60.06 45.08 43.98 52.44 46.92 39.78 40.73 41.71 47.92 39.44 49.69 52.63 45.78 45.9 54.93 60.81 49.3 59.14 41.63 42.58 47.01 58.88 51.4 49.71 43.51 60.95 48.08 41.05 63.11 43.66 40.14 41.74 63.99 48.48 61.44 50.11 44.24 53.89 39.71 48.78 52.72 54.82 68.57 48.31 41.47 47.23 44.14 58.87 48.49 33.47 35.82 34.56 36.98 36.64 34.38 42.56 221035_s_at TEX14 18.11 17.63 10.72 15.01 16.71 13.58 73.12 10.79 14.22 13.71 30.1 18.65 14.08 45.52 17.01 27.63 12.17 11.7 14.95 12.22 11.51 9.93 23.46 13.98 12.43 14.09 10.54 13.28 20.86 9.16 100.75 59.33 16.67 281.95 13.24 11.41 9.76 14 12.64 11.27 11.45 13.07 15.2 11.63 10.94 10.25 12.29 20.98 8.93 22.14 23.87 15.35 16.24 9.62 14.76 13.59 15.51 13.03 12.98 11.54 10.56 19.82 11.68 15.31 16.63 13.06 10.91 12.58 11.41 11.65 9.78 10.27 8.91 11.03 14.13 14.87 10.01 11.5 30.9 9.36 34.18 12.5 26.97 36.88 10.44 14.47 13.73 39.99 30.35 19.96 18.07 9.97 13.01 16.03 12 10.15 17.83 16.63 15.81 27.59 33.07 11.85 9.74 14.49 118.4 14.16 9.47 21.34 63.02 1226.09 12.07 10.92 11.03 11.85 10.45 24.11 10.7 10.28 10.03 10.14 10.35 11.51 15.5 11.47 9.09 7.11 8.4 7.69 11.09 8.12 11.29 8.21 9.1 8.71 9.67 9.34 9.04 9.85 14.89 10.16 34.52 10.79 9.71 40.59 10.74 11.37 11.48 11.42 11.51 9.2 10.83 11.43 9.58 10.7 10.66 9.49 16.41 9.54 8.88 11.72 10.46 10.42 11.26 15.1 8.71 14.6 9.67 8.74 17.39 89.54 11.66 26.2 10.11 9.73 10.43 8.45 8.52 11.63 9.44 10.79 10.85 8.47 68.01 14.27 12.38 9.27 9.79 12.63 11.1 8.79 8.83 8.61 12.46 8.9 8.77 9.4 32.38 10.48 10.26 12.43 10.25 12.42 10.95 18.07 11.71 20.61 20.66 9.21 14.01 8.44 11.95 10.49 112.87 7.39 12.13 13.28 9.55 12.93 73.59 19.32 6.39 7.83 10.78 8.66 6.84 42.2 7.5 221180_at YSK4 50.2 43.5 42.67 71.5 54.32 42.58 62.9 49.89 50.71 50.02 53.43 42.38 39.67 49.19 44.87 42.36 49.41 45.2 43.53 39.64 45.44 42.01 36.82 48.13 52.17 41.96 57.44 44.85 54.43 42.51 41.35 47.72 38.23 56.44 65.44 56.74 54.95 42.65 38.53 55.22 34.43 46.53 49.51 56.21 60.48 37.08 47.13 54.41 53.84 53.86 46.08 53.93 39.25 44.97 56.13 41.9 56.26 48.74 48.15 61.85 61.7 46.26 60.06 55.43 57.22 48.41 49.81 42.42 58.41 42.09 59.83 51.9 44.24 45 47.89 51.42 36.29 48.59 46.47 41.55 45.66 47.78 52.71 42.79 42.18 50.84 68.46 53.74 64.91 45.3 102.53 40.39 50.67 43.75 47.73 52.8 40.81 50.09 49.84 48.64 45.2 50.91 46.18 34.23 44.18 59.78 43.64 43.64 51.65 67.53 39.97 31.05 42.02 41.88 42.89 77.45 44.63 50.43 42.76 50.75 51.36 57.56 52.08 59.01 45.19 59.1 43.74 45.73 52.29 61.71 41.98 54.24 66.57 67.24 53.17 55.82 56.66 54.65 65.14 57.49 54.03 66.15 53.1 58.85 57.44 67.56 68.24 50.4 47.35 43.09 49.2 56.65 56.42 47.96 46.78 48.79 50.97 52.19 44.43 43.57 45.84 46.81 53.8 60.4 53.16 55.59 46.29 42.51 78.77 49.59 48.14 55.75 50.44 49.87 47.6 56.56 44.46 45.51 42.07 69.11 49.38 52.09 64.45 53.11 55.21 58.09 48.12 51.25 71.68 59.03 55.23 47.73 51.09 46.28 43.51 52.47 39.3 54.33 44.49 53.37 59.08 42.82 41.35 71.24 56.52 48.93 58.68 49.03 51.86 51.36 47.19 51.26 44.31 44.54 59.69 58.47 58.07 59.44 62.87 55.6 55.37 49.26 59.3 65.87 56.83 64.12 65.17 221215_s_at RIPK4 396.99 65.1 192.93 52.11 70.82 67.55 141.65 103.16 85.26 107.58 148.84 197.11 132.62 74.75 180.74 125.45 394.62 156.33 180.57 288.9 66.04 99.36 133 244.51 284.5 155.49 186.01 142.99 71.42 184.3 110.01 122.03 69.29 42.77 135.17 163.03 236.36 241.14 135.42 124.22 172.21 109.05 128.69 121.51 98.14 167.4 185.04 93.09 240 125.06 122.99 97.11 70.54 197.77 97.64 151.59 143.35 156.29 86.92 344.95 71.64 174.7 461.93 111.78 38.2 161.48 288.08 130.67 132.02 253.24 258.89 358.85 250.2 163.46 110.83 114.71 481.01 115.28 53.43 94.99 103.02 277.48 87.54 111.42 161.03 77.14 131.81 95.19 79.68 127.97 88.49 176.94 192.02 131.69 295.56 141.88 124.52 386.73 158.78 271.55 401.21 246.82 227.29 137.28 213.68 177.06 94.15 277.72 181.47 113.54 123.7 529.11 223.4 468.78 175.14 418.18 103.95 367.24 237.08 188.43 159.72 222 449.88 332.34 114.73 69.85 145.71 164.44 110.4 267.09 86.77 294.44 210.43 82.81 165.44 115.4 170.09 199.6 212.43 180.09 66.28 232.88 190.25 110.88 101.27 96.26 111.41 178.28 171.79 50.45 176.83 89.72 393.17 310.95 97.13 190.31 76.01 127.34 164.06 366.27 109.35 182.93 108.68 103.46 151.03 106.07 64.12 65.17 1215_s_at RIPK4 396.99 65.1 192.93 52.11 70.82 67.55 141.65 103.16 85.26 107.58 148.84 197.11 132.62 74.75 180.74 125.45 394.62 156.33 180.57 288.9 66.04 99.36 133 244.51 284.5 155.49 186.01 142.99 71.42 184.3 110.01 122.03 69.29 42.77 135.17 163.03 236.36 241.14 135.42 124.22 172.21 109.05 128.69 121.51 98.14 167.4 185.04 93.09 240 125.06 122.99 97.11 70.54 197.77 97.64 151.59 143.35 156.29 86.92 344.95 71.64 174.7 461.93 111.78 38.2 161.48 288.08 130.67 132.02 253.24 258.89 358.85 250.2 163.46 110.83 114.71 481.01 115.28 53.43 94.99 103.02 277.48 87.54 111.42 161.03 77.14 131.81 95.19 79.68 127.97 88.49 176.94 192.02 131.69 295.56 141.88 124.52 386.73 158.78 271.55 401.21 246.82 227.29 137.28 213.68 177.06 94.15 277.72 181.47 113.54 123.7 529.11 223.4 468.78 175.14 418.18 103.95 367.24 237.08 188.43 159.72 222 449.88 332.34 114.73 69.85 145.71 164.44 110.4 267.09 86.77 294.44 210.43 82.81 165.44 115.4 170.09 199.6 212.43 180.09 66.28 232.88 190.25 110.88 101.27 96.26 111.41 178.28 171.79 50.45 176.83 89.72 393.17 310.95 97.13 190.31 76.01 127.34 164.06 366.27 109.35 182.93 108.68 103.46 151.03 106.07 111.76 159.53 167.95 101.87 123.22 56.6 89.31 134.36 300.01 288.35 173.31 46.13 93.8 46.9 85.76 147.04 55.65 142.39 189.61 87.42 265.89 78.94 208.3 127.74 91.28 167.5 76.19 188.23 285.78 261.69 236.71 100.75 280.3 186.34 114.1 201.81 110.69 97.02 143.63 99.82 624.18 180.99 71.33 113.01 97.04 89.46 97.37 170.15 60.28 96.43 153.61 156.45 143.99 197.63 145.97 299.63 53.05 300.23 417.01 356.41 209.79 221508_at TAOK3 78.86 92.6 82.45 99.67 85.19 86.83 97.74 86.04 92.89 96.11 97.41 90.26 72.44 93.34 78.59 82 83.68 71.96 100.7 60.95 78.53 88.76 71.46 80.06 65.84 109.93 87.53 85.39 72.14 73.43 106.43 86.6 74.98 78.08 74.17 65.22 72.62 68.94 89.44 79.63 95.37 79.4 94.76 77.41 70.08 77.33 55.34 68.61 100.7 77.15 75.81 82.52 77.76 117.05 89.43 73.82 81.54 89.78 97.85 65.36 64.67 61.1 75.35 100.39 85.6 88.29 65.95 78.05 74.72 86.4 76.42 73.56 78.23 63.57 53.67 83.2 77.24 70.6 64.61 70.6 93.11 70.96 62.05 71.45 61.01 67.61 64.48 76.07 57.81 86.09 57.52 88.61 77.04 106.09 121.13 77.69 97.83 74.53 77.79 75.12 69.2 95.63 77.97 105.51 71.56 62.78 63.7 61.63 54.55 52.17 54.96 86.23 85.79 82.52 76.04 92.54 99.85 59.45 77.21 74.5 90.12 67.24 63.71 76.12 59.26 56.21 51.31 56.24 59.96 58.15 62.24 56.92 56.8 68.25 54.71 54.15 59.99 61.56 57.97 47.44 54.91 53.81 49.47 61.43 56.17 56.19 50.37 67.93 62.53 63.92 43.98 62.21 80.72 71.4 73.62 72.83 61.57 80.82 74.21 68.12 81.29 61.43 63.9 62.98 63.89 68.9 82.09 61.65 66.71 62.67 61.36 70.15 78.51 63.13 62.17 66.48 88.1 54.36 90.58 57.95 80.02 82.12 66.71 57.86 82.35 80.78 58.07 72.37 71.83 73.78 88.15 91.05 74.57 88.78 66.23 65.8 56.6 68.48 65.34 61.56 64.73 79.75 70.25 66.42 69.56 75.35 69.71 57.58 74.04 73.13 80.85 61.48 77.38 71.85 47.54 58.13 75.41 60.78 47.35 44.75 83.43 60.67 55.36 62.65 61.21 62.52 60.55 221795_at NTRK2 26.71 49.62 243.2 61.07 324.05 133.27 15.07 91 159.65 45.79 60.79 183.44 300.35 53.79 962.33 440.59 32.97 235.53 39.55 16.08 85.77 33.32 31.19 263.3 24.89 122.34 24.46 30.61 1338.19 34.74 43.5 29.82 32.12 724.02 25.28 46.41 479.37 493.83 133.46 53.46 39.9 211.37 24.49 49.99 40.97 399.84 2490.2 43.52 48.49 136.04 1196.32 100.8 218.64 169.25 25.62 195.88 317.79 450.6 46.88 337.1 50.59 24.87 28.37 65.83 72 369.33 1982.28 195.42 26.53 189.71 334.64 445.8 531.31 48.23 31.54 45.72 31.29 208.31 1201.89 38.68 66.91 143.36 2032.64 102.81 432.45 231.37 63.78 219.57 75.09 229.44 134 641.59 83.3 272.49 506.76 48.99 87.23 154.72 240.49 343.18 298.74 639.31 121.19 254.29 58.18 114.99 204.49 180.74 147.15 1758.16 316.68 1549.72 114.74 98.91 26.53 49.21 242.59 194.15 124.29 192.27 646.24 151.3 53.1 499.85 1873.55 43.36 175.99 1627.01 57.74 1217.5 176.05 30.36 1621.84 134.95 123.39 169.49 64.73 492.72 120.81 41.15 122.93 75.47 55.58 141.32 343.2 144.21 32.07 689.06 549.41 1075.16 861.64 219.68 11.46 114.85 32.68 140.43 43.64 15.32 191.77 255 116.83 1658.18 57.48 345.24 2321.54 134.53 89.65 216.63 48.48 158.83 160.95 137.78 181.74 113.3 18.27 86.31 76.87 191.69 702.35 91.55 704.76 17.99 137.44 594.2 1723.04 142.85 37.73 163.7 16.63 1632 58.13 298.7 46.89 25.23 2224.43 50.92 40.96 217.42 169.03 599.45 102.9 126.82 89.18 52.23 51.09 1171.45 148.52 12.02 58.59 129.52 129.13 586.2 403.99 477.5 1031.03 154.21 72.32 30.73 135.67 416.44 100.62 106.64 192.18 337.15 435.44 44.34 660.41 221918_at PCTK2 375.1 1017.05 347.83 482.01 721.76 655.02 473.52 1051.37 760.59 463.68 577.85 817.6 484.9 824.14 438.08 395.26 510.48 447.55 597.67 541.87 603.99 791.56 400.06 331.49 274.49 587.46 556.45 275.12 395.98 481.41 635.37 417.4 767.29 923.98 322.69 193.9 482.9 403.22 448.44 223.84 510.9 457.14 287.71 323.28 393.54 437.2 313.18 276.98 275.09 557.08 272.12 394.7 402.22 342.68 1115.59 498.3 239.24 163.69 476.78 212 571.32 273.75 236.51 439.11 386.37 374.95 341.94 353.93 280.27 284.21 240.66 390.82 234.14 432.71 366.95 469.2 230.64 552.86 505.14 306.22 421.67 1026.3 586.83 592.44 541.99 569.99 360.65 415.03 396.27 714.4 58.83 730.52 947.37 1015.05 708.96 542.29 1132.13 380.31 998.66 367.42 359.22 400.98 671.97 658.01 384.41 390.02 448.24 447.47 453.32 50.48 225.59 409.4 439.31 390.19 391.64 461.39 246.41 197.32 407.11 240.36 693.72 199.3 302.24 364.75 369.66 495.65 452.36 383.95 414.18 215.37 422.38 320.53 246 247.6 491.07 462.19 182.69 451.91 379.88 418.3 514.9 447.48 653.77 421.98 322.07 473.22 308.78 446.86 276.47 608.51 261.66 350.08 348.93 447.52 485.92 182.48 591.53 374.56 359.28 231.66 467.23 386.46 572.68 322.24 578.41 427.63 619.76 443.96 127.36 426.76 567.12 322.86 617.9 275.17 170.07 223.81 310.23 366.38 289.01 447.66 386.78 226.2 475.91 243 296.8 257.07 315.43 694.55 218.42 336.42 435.39 427.37 227.47 378.02 321.22 289.94 179.56 292.31 338.69 189.5 313.75 431.94 377.26 116.52 208.53 323.9 242.53 362.98 416.63 302.64 154.65 302.87 363.31 360.48 439.3 502.33 389.09 296.97 347.04 517.01 210.72 269.01 380.26 241.19 258.73 166.32 249.2 221957_at PDK3 77.82 104.52 101.3 144.34 72.19 83.4 81.23 86.45 162.02 90.16 150.45 121.21 69.56 123.08 56.25 108.82 292.25 167.78 55.7 87.5 128.19 149.34 276.86 147.91 62.06 243.44 99.19 132.62 149.63 111.04 159.03 259.62 162.1 162.64 510.59 327.88 202.46 480.7 85.45 95.4 160.7 87.59 370.94 67.98 211.48 72.43 91.29 167.64 85.29 70.43 145.29 130.29 85.32 727.49 157.58 108.37 142.67 118.89 160.8 562.17 186.36 392.47 492.75 110.23 70.13 655.75 108.57 208.85 143.76 95.29 86.5 598.49 365.19 118.01 185.81 138.44 121.85 168.83 148.74 238.84 149.4 249.73 155.48 104.03 91.43 124.74 112.73 152.84 135.75 128.01 35.81 100.93 107.68 117.34 114.36 141.17 107.31 186.05 102.32 396.23 181.78 71.85 140.31 87.98 193.12 99.87 137.89 73.07 143.27 49.22 104.99 185.44 186.48 416.7 317.77 81.59 72.25 149.45 85.11 81.27 88.07 744.72 188.57 102.94 116.78 124.82 143.92 105.46 203.34 131.78 77.99 84.11 60.32 99.11 109.73 136.21 61.6 100.45 102.94 150.84 109.07 38.08 130.05 205.18 76.75 116.89 136.71 145.97 129.6 216.75 851.96 99.1 87.6 115.7 102.64 434.7 129.75 88.29 105.1 83.68 84.07 295.92 145.49 86.25 193.02 117.38 116.14 69.4 121.86 86.6 227.6 105.57 110.88 190.46 17.29 102.6 101.41 112.3 79.22 61.78 90.27 171.29 125.24 86.32 133.86 51.08 111.9 115.5 224.44 102.29 57.84 124.42 78.97 93.14 101.67 83.65 114.2 68.48 87.92 174 47.14 87.74 167.84 56.85 140.61 136.78 139.41 295.08 69.65 138.76 88.54 90.66 100.95 69.11 73.55 108.18 87.6 96.33 81.91 89.41 211.09 188.22 107.53 27.09 205.58 429.38 77.56 222589_at NLK 154.23 408.77 79.22 171.92 173.36 240.63 226.11 434.07 308.43 173.2 216.01 315.1 198.1 213.81 142.66 149.42 137.24 151.11 190.83 201.42 151.97 269.86 261.57 50.16 95.94 129.38 305.78 532.19 75.24 125.17 108.49 211.72 790.05 202.55 113.9 51.32 64.76 87.4 76.89 71.23 196.82 94.8 56.86 100.59 8 129.47 136 67.27 126.98 106.85 78.32 165.14 178.49 109.74 245.34 152.26 71.39 176.56 278.11 88.29 66.34 121.79 81.99 103.32 111.83 92.06 103.44 140.28 109.64 137.15 78.14 75.16 118.32 141.48 54.64 669.48 152 190 146.47 114.6 72.7 156 411.27 245.39 201.57 154.06 256.44 84.85 268.05 242.65 40.13 155.77 250.67 388.36 286.43 131.1 530.5 143.59 476.02 157.84 173.2 123 116.79 190.16 269.57 188.79 132.31 211.63 214.59 33.96 96.32 136.4 137.61 93.12 113.41 265.99 168.65 69.37 82.1 73.88 83.23 103.87 189.55 93.88 120.25 160.77 137.8 125.34 130.01 242.49 149.42 86.24 128.43 139.01 273.69 255 114.56 176.12 821.15 235.61 174.52 562.87 470.21 178.24 101.91 195.58 400.13 95.67 341.31 199.46 94.98 134.32 77.69 194.85 212.48 90.69 315.28 114.51 183.93 99.7 137.89 104.16 141.34 168.76 162.21 137.8 283.97 440.25 393.51 139.01 138.4 109.95 123.85 122.13 193.16 67.91 92.5 167.61 134.98 199.23 131.65 157.36 385.48 174.27 89.61 195.55 149.99 219.75 186.61 291.43 105.57 185.87 140.79 319.13 127.45 78.01 320.41 112.65 157.12 95.55 128.55 134.18 251.96 58.39 95.1 175.99 167.5 127.52 79.09 115.54 91.03 72.39 287.95 56.77 125.56 197.95 85.02 177.21 137.33 101.57 104.87 109.28 126.95 140.91 155.12 94.69 143.06 223033_s_at SCYL1 119.13 176.13 188.08 176.92 128.83 140.91 222.12 137.31 123.51 271.43 205.26 164.66 170.2 135.68 160.41 227.8 177.24 190.42 135.49 113.94 155.81 138.52 525.35 168.85 329.45 152.99 162.93 163.64 140.56 103.69 178.66 138.1 136.38 173.36 166.7 176.54 122.83 155.28 119.56 116.72 147.47 154.53 189.26 168.37 147.38 136.83 87.18 99.54 219.06 135.59 216.24 193.75 224.69 179.62 200.37 181.56 132.64 137.52 148.9 127.14 127.97 193.76 164.38 126.25 116.32 83.71 118.1 129.83 179.75 128.26 239.48 128.17 167.72 131.87 129.76 137.68 105.96 108.67 104.21 217.86 136.25 134.34 122.43 91.33 137.65 133.36 160.4 137.96 95.86 151.81 203.36 199.48 135.26 157.46 174.52 185.13 224.54 269.37 276.51 230.32 154.93 158.99 169.23 196.61 177.93 280.06 134.77 142.87 182.63 160.86 142.34 111 121.5 132.4 146.23 48.96 140.24 142.88 110.54 235.37 118.44 172.65 162.65 207.93 100.31 172.19 94.97 95.13 88.46 124.35 98.95 167.34 122.56 118.93 123.57 73.92 105.36 74.53 103.2 104.92 39.71 98.62 107.34 86.44 80.9 86.88 100.3 151.55 166.37 184.29 136.53 161.49 177.02 133.52 171.89 147.76 111.12 163.72 144.11 112.04 206.04 133.13 149.41 117.34 129.07 136.54 128.41 129.31 238.19 97.52 152.71 190.53 237.85 192.4 208 159.26 165.32 113.24 132.55 303.71 113.78 160.51 95.22 174.74 151.21 124.56 119.49 163.53 153.34 157.61 158.79 125.13 169.33 163.7 127.71 177.71 154.99 149.76 169.01 190.24 175.27 147.32 108.25 215.58 220.03 175.66 100.31 87.29 88.46 132.22 142.44 99.58 160.87 153.04 127.96 108.53 137.11 200.31 128.67 131.18 212.25 203.09 182.56 120.65 186.72 144.33 194.64 223158_s_at NEK6 290.72 179.64 269.14 196.75 148.61 87.19 176.22 126.55 119.11 211.12 204.21 269.05 225 99.59 107.66 365.77 221.48 253.92 202.98 176 316.02 327.83 448.21 155.3 245.16 285.51 327.51 85.95 243.49 493.34 262.55 270.96 253.38 47.82 255.54 268.79 199.74 210.15 248.11 302.44 202.15 190.23 237.13 206.6 224.04 180.85 136.28 170.81 265.81 187.73 130.62 234.25 191.22 357.58 28.17 182.16 186.91 256.81 137.78 269.42 186.89 118.74 204.3 109.42 240.19 209.2 276.57 337.55 258.39 313.99 206.45 358.32 157.32 286.55 240.42 273.82 202.97 342.81 222.25 172.15 183.31 242.5 233.48 206.23 118.78 118.85 334.46 210.61 172.4 189.46 45.64 129.48 140.43 197.45 208.33 284.5 155.49 313.03 157.43 156.59 58.05 190.24 238.75 138.62 213.7 211.58 190.96 246.55 144.73 57.2 170.63 93.77 272.6 212.94 196.12 145.02 39.33 153.19 229.83 179.93 182.29 121.37 165.77 113.87 119.82 88.6 116.08 178.64 191.93 220.74 250.4 231.85 79.8 219.99 289.54 79.99 176.07 242.48 140.13 622.32 104.61 309.76 137.31 160.42 193.77 133.79 212.58 144.01 174.98 266.28 140.96 183.53 209.5 394.42 209.93 243.72 137.7 321.85 163.23 172.42 131.42 203.27 187.82 90.88 267.5 153.41 152.76 178.34 86.27 147.61 189.45 163.7 144.52 107.65 55.69 648.75 225.81 142.27 93.37 124.16 172.37 138.87 99.06 158 161.01 122.13 220.06 111.54 136.24 189.6 160.55 136.51 165.38 163.81 86.9 446.35 152.12 157.28 279.18 123.11 103.62 186.49 199.02 40.56 139.44 94.43 65.26 138.9 122.85 218.02 138.95 118.88 122.54 191.18 114.47 89.3 188.52 150.89 161.9 113.47 176.13 209.51 145.62 211.11 210.54 135.56 220.23 223199_at MKNK2 317.63 548.34 371.99 489.28 650.77 1017.91 349.46 400.84 331.18 535.35 519.53 624.14 741.06 671.93 712.28 582.9 562.9 441.01 304.6 201.22 511.5 371.26 558.71 417.36 469.63 268.07 634.51 265.02 238.87 552.64 186.27 339.83 327.27 670.23 588.12 425.91 424.28 314.76 439.87 382.69 474.86 1078.13 254.13 526.32 329.69 537.25 402.38 240.45 438.63 773.41 86.24 881.68 671.75 447.7 680.26 656.14 624.5 259.31 1008.91 1018.11 1314.46 550.86 280.98 288.3 865.81 202.88 254.1 393.91 277.45 166.01 248.84 314.62 308.72 272.76 155.65 407.44 372.09 484.92 243.65 163.49 226.4 904.89 357.28 600.31 303.55 293.46 193.52 240.91 270.5 410.19 537.21 402.97 320.68 224.41 302.71 283.64 211.09 229.13 287.14 188.17 285.94 909.83 264.89 398.81 319.65 802.28 534.57 466.71 649.18 907.25 409.06 404.4 213.78 310.51 343.2 113.39 359.84 194.1 254.86 772.87 453.01 341.83 348.88 376.87 232.64 227.32 249.94 193.7 190.14 118.39 190.71 287.57 285.25 379.35 250.4 166.01 290.53 242.83 113.01 114.09 200.87 162.52 98.66 225.6 150.64 344.48 386.32 419.92 799.05 645.31 279.56 724.15 488.73 387.62 718.86 437.42 340.02 423.38 281.84 454.98 590.1 415.99 386.79 701.52 435.93 307.32 778.87 417.13 442.63 269.99 596.42 440.03 435.36 390.63 754.72 948.77 243.79 332.5 612.54 392.7 327.55 308.79 548 391.33 208.45 294.81 259.53 243.97 389.05 142.81 372.67 235.65 303.5 354.5 369.71 353.98 205.53 294.59 348.55 259.4 496.34 392.99 351.24 233.91 197.99 306.46 111.58 291.71 308.81 370.86 170.93 316.79 506.24 998.12 410.54 302.44 213.39 225.93 237.76 762.28 386.36 275.54 821.28 343.07 406.82 438.4 848 223266_at ALS2CR2 150.26 216.76 119.81 464.86 160.46 136.43 179.47 136.36 224.6 288.43 381.03 255 365.35 252.66 219.16 196.38 115.16 152.38 223.43 134.81 198.19 208.8 241.45 117.94 79.93 205.4 212.4 255.77 192.56 184.93 141.49 231.06 113.25 333.12 160.09 261.71 164.74 69.22 99.18 144.71 112.41 146.31 141.17 127.85 142.26 175.7 91.1 101.57 119.78 122.79 89.47 176.44 102.71 214.03 156.12 176.01 155.95 104.55 191.21 75.27 207.64 141.55 105.22 151.71 120.17 142.54 110.17 73.56 123.5 76.94 198.99 137.44 201.02 145.21 208.4 93.5 87.94 346.41 224.53 149.47 194.37 342.22 186.13 186.95 145.31 143.04 119.87 116.35 104.69 276.08 114.13 156.49 230.19 115.95 178.1 135.65 168.18 181.29 173.1 133.85 248.88 388.16 217.78 295.34 234.53 166.61 172.09 173.38 91.3 49.14 111.76 177.11 114.48 98.8 294.99 282.99 75.62 81.94 115.48 226.17 216.02 110.73 169.61 117.94 110.46 225.46 114.49 91.31 145.52 119.89 115.86 319.91 107.62 144.4 153.2 99.75 126.07 99.39 127.04 145.3 239.35 133.49 95.25 160.8 103.59 56.18 100.78 166.51 194.68 488.54 117.06 84.31 149.59 139.13 203.85 203.86 543.6 240.9 238.91 148.41 128.44 187.86 162.03 136.37 189.25 130.04 265.73 104.28 133.97 605.84 223.01 130.48 171.4 96.42 48.68 224.73 125.86 161.1 150.24 296.83 159.25 199.44 248.08 155.39 77.62 199.74 262.08 176.62 183.26 215.14 136.54 181.11 105.36 171.15 169.4 209.73 96.18 152.22 217.18 174.06 219.42 190.62 135.68 149 212.86 175.41 87.47 82.92 110.77 165.96 78.06 118.15 161.17 194.96 164.55 145.15 241.69 130.39 90.57 185.63 147.71 150.07 123.23 78.32 91.61 86.49 199.07 223324_s_at TRPM7 84.14 169.47 94.32 87.95 112.06 131.76 137.08 132.46 155.14 93.31 157.19 153.96 106.65 124.82 87.28 101.33 113.61 150.28 165.14 181.99 106.17 202.1 79.07 85.67 111.91 124.82 62.32 152.9 147.32 77.04 121.19 51.72 147.73 141.1 127.35 33.83 104.76 185.69 104.21 65.42 89.75 115.74 108.8 136.13 110.73 113.5 78.28 139.15 74.58 81.52 156.55 95.74 88.52 111.9 158.48 95.79 91.41 86.42 97.24 113.41 93.02 142.36 113.61 60.03 80.44 79.46 81.53 115.62 90.05 87.35 74.99 84.99 53.48 112.27 99.53 46.54 135.26 95.85 53.05 107.05 73.9 128.4 87.4 100.02 88.71 94.93 55.17 180.37 67.12 48.52 22.27 68.79 50.21 56.37 55.36 54.39 67.08 49.84 52.38 38.76 123.87 164.85 52.09 45.56 43.39 92.23 78.38 64.22 69.38 12.04 44.73 86.12 90.54 90.2 73.16 125.34 112.51 41.79 66.68 64.5 84.07 84.78 36.72 69.31 71.83 99.33 97.12 98.63 98.34 98.48 83.85 67.62 41.93 106.19 104.8 74.05 74.48 85.24 74.46 88.11 92.57 85.28 87.09 86.61 76.39 64.17 58.74 83.01 59.95 109.17 127.06 67.14 66.15 71.77 92.77 57.85 82.66 72.52 98.6 70.62 67.09 84.77 121.45 60.02 69.41 79.71 76.58 61.93 39.53 83.84 83 70.61 81.7 72.83 17.18 57.07 88.86 79.13 82.05 79.21 82.42 86.4 115.15 87.37 60.09 108.86 81.8 105.98 102.47 55.69 83.09 132.5 97.03 99.5 90.75 92.06 92.45 68.71 95.41 59.47 64.54 78.39 116.23 14.42 102.81 84.3 76.94 60.62 68.98 154.04 50.91 107.52 77.12 102.35 61.78 68.69 69.6 56.47 72.49 77.28 61.25 73.55 69.24 74.12 76.95 75.17 102.7 223430_at SNF1LK2 68.3 50.08 107.3 76.91 62.78 113.71 137.02 117.7 54.29 114.96 124.4 102.89 94.44 98.17 100.68 103.88 188.66 75.06 73.69 110.11 106.85 68.02 77.68 77.94 174.24 66.26 115.22 93.82 86.17 69.67 77.99 99.13 70.07 33.08 98.65 86.65 38.19 135.89 83.92 68 68.96 131.95 166.06 71.36 110.96 101.15 158.73 92.28 187.6 118.64 229.83 118.12 82.39 103.74 49.41 90.62 139.82 132.56 86.01 75.11 108.14 51.44 85.71 149.67 56.39 143.25 181 105.69 97.91 206.72 90.12 115.29 92.49 77.22 159.37 66.97 102.47 58.45 65.24 66.24 92.99 135.38 92.56 53.57 156.14 100.95 107.02 108.28 50.73 93.99 90.86 63.13 53.34 56.35 115.33 80.73 60.38 74.43 82.53 94.2 136.86 76.47 95.78 100.69 66.84 109.29 81.7 63.74 83.8 60.3 91.83 143.12 64.21 100.92 103.84 80.33 75.23 87.01 111.39 48.45 93.25 92.35 79.51 45.65 125.11 79.95 76.59 127.25 62.38 103.16 125.45 73.3 203.53 93.08 116.09 60.16 40.32 67.47 69.78 70.44 49.97 81.84 92.99 40.5 80.55 63.62 79.34 68.83 99.46 149.1 142.43 81 100.37 65.03 59.65 94.73 35.24 83.39 95.92 101.37 76.23 90.02 93.15 35.84 54.53 67.42 32.19 85.54 21.68 20.69 74.93 78.71 64.63 62.59 53.07 80.96 84.83 37.06 98.71 105.68 82.52 98.63 56.58 65.81 88.35 69 76.9 82.56 98.92 57.69 82.29 89.94 51.35 102.59 110.1 89.58 88.01 81.48 80.81 75.72 52.17 63.75 57.33 48.77 71.94 60.21 179.76 64.08 60.28 63.51 89.25 81.25 97.03 50.5 53.5 61.19 45.19 55.64 40.43 49.44 136.77 96.69 66.38 73.97 73.54 142.7 90.18 223460_at CAMKK1 115.92 92.48 148.91 97.18 89.28 129.67 115.68 111.91 93.2 138.38 177.14 96.1 181.57 141.28 131.93 169.98 120.25 85.12 88.65 125.28 169.59 108.93 159 154.63 244.17 86.03 98.17 141.97 127.69 98.02 119.1 114.21 189.3 137.53 144.83 125.15 106.86 86.51 118.45 139.93 126.27 117.27 204.96 159.9 95.11 124.3 127.67 140.48 177.55 132.36 141.44 139.96 201.25 131.02 106.42 143.27 180.92 93.58 137.08 141.95 192.02 119.08 145.4 121.79 111.38 83.78 81.8 82.26 99.38 99.19 124.79 88.95 169.63 112.06 138.23 78.02 76.09 91.41 105.12 102.1 83.74 92.11 93.02 84.73 90.54 82.98 78.86 103.06 86.56 89.58 115.39 109.8 98.41 65.73 140.45 115.87 129.66 109.71 106.73 119.6 98.74 136.83 129.96 124.67 105.49 126.51 122.04 122.82 147.33 231.79 80.3 123.84 131.03 121.44 141.68 91.17 117.19 147.82 103.53 134.4 152.57 102.17 166.13 101.48 103.52 92.4 100.68 87.98 76.67 61.63 92.16 107.88 97.99 98.38 84.29 76.27 101.37 102.83 116.13 117.68 87.43 70.98 74.1 62.62 83.14 104.24 127.59 102.78 105.37 109.45 98.03 102.8 128.33 111.39 101.12 103.53 102.63 114.79 87.62 105.53 116.35 108.69 206.16 108.66 153.48 103.71 118.1 145.11 147.85 115.08 139.5 138.76 112.49 104.16 111.64 203.22 129.61 160.79 137.94 141.44 105.91 135.16 133.69 105.25 115.53 119.67 182.31 120.89 135.15 159.67 136.68 130.92 126.46 88.08 105.74 113.44 130.5 113.65 104.53 179.3 149.68 106.16 83.04 110.59 100.85 108.59 128.43 143.3 106.55 86.57 141.94 139.2 116.5 115.47 132.41 94.66 154.19 129.67 109.7 125.88 82.7 74.9 114.77 77.34 67.13 71.74 82.88 223534_s_at RPS6KL1 42.2 25.62 59.58 47.39 58.69 71.47 72.28 103.54 64.32 48.52 44.66 38.73 69.63 38.98 34.87 32.34 28.57 46.53 59.73 23.02 47.69 33.38 30.5 37.5 34.52 20.72 40.1 20.21 41.57 81.89 38.24 20.89 50.41 44.66 31.06 18.25 27.55 21.69 19.7 27.59 37.55 62.95 24.48 19.43 17.99 24.73 38.48 20.02 41.28 27.02 34.06 47.08 95.17 54.08 48.69 48.05 45.24 38.02 40.99 26.16 41.69 93.9 19.09 48.5 49.16 24.46 20.48 20.83 29.91 13.86 46.81 25.79 18.99 26.16 24.93 32.96 31.65 23.12 33.24 33.29 54.06 47.63 54.64 40.18 30.2 40.75 66.64 35.29 36.42 35.92 110.13 28.48 33.81 26.76 37.03 32.46 53.31 42.93 54.37 34.75 29.33 41.76 74.22 62.23 151.52 64.91 43.44 37.04 55.8 119.94 24.04 40.52 40.99 43.26 64.61 27.82 183.07 97.02 29.42 91.9 51.59 56.22 241.23 48.58 31.03 50.21 42.96 23.29 39.3 42.1 37.86 117.23 47.76 28.82 23.51 49.2 85.07 29.35 30.74 22.18 29.33 33.34 22.97 25.16 21.78 87.94 42.42 36.16 52.71 45.97 24.97 41.17 30.61 39.05 38.18 65.61 46.34 38.07 42.82 49.41 53.06 43.79 41.92 62.84 128.66 31.49 44.61 29.77 83.12 54.05 71.62 35.79 53.89 46.04 66.55 140.47 40.9 57.41 67.44 31.1 49.42 114.68 60.25 49.71 31.3 41.88 61.18 51.09 119.02 69.14 104.37 33.14 41.68 82.74 47.72 46.4 35.89 40.98 50.06 50.15 85.43 42.38 27.84 81.26 36.65 46.47 40.22 37.49 38.96 33.25 42.73 35.24 38.68 98.12 52.96 45.44 31.52 36.6 46.7 54.08 54.79 28.52 42.92 58.47 33.51 75.64 70.58 223715_at BRSK2 25.43 20.1 24.21 37.93 45.07 98.42 52.06 78.59 67.74 37.99 28.88 27.89 27.32 28.08 19.82 35 29.03 23.83 23.1 105.25 28.05 19.52 19.75 35.43 24.93 21.06 23.05 19.94 25.68 35.83 25.44 23.87 23.09 27.6 31.4 30.24 25.09 23.82 27.33 33.45 22.18 31.23 25.28 23.55 31 17.28 31.91 22.28 20.09 25.19 33.32 20.9 27.8 29.39 34.69 29.34 31.22 26.17 25.16 28.87 32.4 28.66 23.91 26.16 27.05 19.33 20.66 22.23 25.95 19.02 23.9 23.27 23.9 19.38 24.76 24.24 25.92 17.98 19.7 27.34 20.5 18.81 16.63 17.73 21.01 20.27 22.72 19.83 29.19 20.19 38.81 18.25 25.1 19.3 35.92 20.25 23.71 25.63 27.61 21.29 23.36 17.79 35.46 21.64 21.78 21.88 17.76 16 21.34 38.86 22.78 18.95 20.68 20.97 21.82 29.71 103.12 20.47 21.18 25.25 18.08 25.04 32.45 25.66 16.45 18.87 20.75 14.81 21.5 18.32 16.07 19.28 27.28 21.17 16.94 34.98 20.25 19.97 20.68 34.91 17.61 21.15 20.31 21.57 21.61 20.5 39.36 24.36 21.99 41.55 26.4 26.35 23.05 19.07 22.94 19.14 24.43 19.79 18.15 27.29 22.73 20.36 24.63 22.55 22.73 23.18 18.86 29.35 30.63 22.52 29.63 23.68 20.28 23.57 33.45 22.34 18.94 20.12 17.67 19.89 20.59 48.41 24.16 21.36 23.93 23.92 22.73 26.56 38.48 21.91 17.78 20.38 22.23 19.5 19.45 25.2 19.59 36.02 23.29 17.62 25.15 18.54 15.65 49.42 20.65 22.37 17.86 41.67 23.24 23.4 21.13 18.78 23.12 19.78 21.43 23.52 29.55 22.76 23.71 17.25 14.49 17.03 17.5 17.59 17.92 16.7 17 223852_s_at STK40 151.15 100.96 190.72 137.65 163.18 184.56 127.11 160.38 130.82 200.69 116.14 156.24 127.62 109.49 127.84 148.01 164.03 150.59 170.89 120.3 151.13 153.78 213.39 173.26 363.3 180.95 209.76 160.73 170.32 170.83 178.66 147.33 116.24 131.56 113.02 165.4 187.52 367.93 160.5 169.43 121.33 216.77 230.7 177.87 143.76 157.37 135.84 126.97 142.25 211.98 234.26 162.47 140.31 270.81 124.05 131.41 225.46 124.59 191.79 222.01 236.58 160.34 252.19 102.15 165 188.18 190.23 142.9 192.21 181.82 257.85 236.94 99.41 139.92 159.79 100.9 120.24 156.53 76.38 121.25 119.91 231.91 174.56 190.4 164.71 128.34 202.37 158.96 169.12 344.12 101.11 237.56 234.74 142.58 256.5 211.71 225.29 409.16 236.88 264.31 214.23 227.84 289.01 254.2 170.33 287.23 180.95 235.81 357.6 160.7 221.37 181.87 181.89 164.67 296.38 107.47 160.53 184.38 174.6 462.15 194.81 274.68 234.12 277.18 208.13 150.79 104.1 192.67 118.54 215.9 159.69 162.94 267.99 198.33 151.1 115.38 127.11 132.33 128.01 207.61 93.34 129.88 95.09 144.81 154.13 127.33 130.29 218.85 225.09 211.27 309.77 275.05 168.99 202.88 211.5 159.13 225.44 224.39 180.89 174.21 232.6 180.37 104.79 217.53 146.68 181.03 164.83 216.04 141.39 108.75 267.49 210.84 222.14 239.66 131.61 254.29 249.65 178.84 262.61 153.17 227.52 279.44 182.57 257.87 321.1 145.51 367.52 187.7 305.54 131.44 181.01 169.89 160.17 241.29 196.16 331.81 319.46 204.84 415.22 193.22 276.93 168.82 272.34 128.21 555.27 173.35 246.19 274.58 208.08 170.42 171.98 207.61 219.71 407.25 187.48 189.69 255.41 234.49 175.59 251.67 318.6 230.09 236.51 199.07 245.51 219.78 230.06 223910_at ERN2 31.59 35.41 40.71 45.1 30.32 30.15 51.25 29.99 32.25 38.49 39.08 32.3 31.85 29.02 39.01 44.86 49.59 36.72 34.09 37.28 54.11 40.97 32.43 62.48 38.07 30.35 35.16 34.84 42.74 38.91 39.04 47.38 39.31 39.63 39.88 47.68 42.95 30.63 43.12 44.64 37.54 38.67 41.24 40.28 38.78 24.17 46.42 29.1 49.4 44.53 29.75 38.02 35.97 35.29 40.5 29.74 42.52 52.59 41.26 53.97 49.74 39.45 41.53 53.27 43.29 24.79 38.81 34.16 41.67 30.22 37.51 40.84 34.48 34.23 40.51 39.4 44.52 27.45 54.92 40.32 42.15 42.93 42.8 50.09 39.99 39.02 42.58 43.89 55.54 43.38 88.78 43.5 42.38 34.09 31.66 40.1 42.53 47.81 50.41 48.81 41.54 44.85 52.18 39.87 39.72 36.66 43.53 36.48 38.15 113.14 39.87 26.28 34.15 36.4 38.07 31.78 37.5 48.97 31.38 47.96 32.29 40.25 31.83 46.44 42.95 51.55 39.15 44.87 58.31 50.89 45.24 54.94 65.24 61.23 45.86 48.22 58.24 49.13 49.08 46.96 48.4 52.78 53.85 50.27 56.22 71.76 51.97 48.06 49.42 42.94 53.24 49.67 53.61 18.32 16.07 19.28 27.28 21.17 16.94 34.98 20.25 19.97 20.68 34.91 17.61 21.15 20.31 21.57 21.61 20.5 39.36 24.36 21.99 41.55 26.4 26.35 23.05 19.07 22.94 19.14 24.43 19.79 18.15 27.29 22.73 20.36 24.63 22.55 22.73 23.18 18.86 29.35 30.63 22.52 29.63 23.68 20.28 23.57 33.45 22.34 18.94 20.12 17.67 19.89 20.59 48.41 24.16 21.36 23.93 23.92 22.73 26.56 38.48 21.91 17.78 20.38 22.23 19.5 19.45 25.2 19.59 36.02 23.29 17.62 25.15 18.54 15.65 49.42 20.65 22.37 17.86 41.67 23.24 23.4 21.13 18.78 23.12 19.78 21.43 23.52 29.55 22.76 23.71 17.25 14.49 17.03 17.5 17.59 17.92 28.92 53.47 57.08 62.1 46.23 42.45 59.35 38.04 36.84 50.97 42.44 40.94 49.13 40.71 57.74 51.47 52.93 51.76 49.98 50.14 49.94 102.59 70.77 59.64 43.76 46.53 58.22 44.87 53.46 59.07 49.44 48.67 56.51 57.77 45.84 51.86 52.6 50.51 45.02 40.76 43.56 38.09 38.09 35.38 32.09 35.03 48.26 44.63 36.05 32.76 94.7 34.62 34.31 40.82 35.14 51.79 32.3 33.92 29.17 70.86 67.36 53.4 36.37 46.21 54.43 47.76 38.23 36.78 37.45 36.6 39.29 36.45 33.96 31.27 224412_s_at TRPM6 105.84 5.97 6.15 5.65 6.25 5.05 6.15 5.84 5.99 6.08 6.97 7 7.31 6.13 7.47 29.39 7.11 6.31 7.3 12.81 6 5.51 5.82 7.04 7.45 13.08 6.41 5.31 6.85 8 6.67 5.96 6.2 6.74 6.29 6.12 7.29 7.5 8.25 7.49 10.77 6.99 5.85 11.34 6.85 9.75 6.5 7.07 8.05 6.25 40.65 7.89 5.99 20.59 5.33 6.22 6.57 6.54 7.03 7.23 6.92 6.78 10.18 6 5.24 13.6 5.4 5.75 6.75 6.98 6.41 8.56 7.31 6.07 5.75 5.94 6.98 6.02 7.66 7.05 6.51 6.77 7.5 6.32 8.72 6.53 5.83 6.23 5.73 10.14 8.8 7.44 6.02 5.72 12.28 6.21 6.54 6.63 8.1 10.56 33.14 13.17 6.36 8.91 6.21 7.09 7.42 8.75 7.11 7.78 17.67 9.58 7.61 6.78 9.96 10.67 7.34 15.04 13.61 8.28 9.45 9.27 7.54 8.72 12.8 6.97 7.54 8.94 7.21 6.2 6.19 5.71 9.28 7.04 6.04 6.22 34.02 7.31 6.7 6.42 6.87 6.6 6.81 6.17 7.08 6.25 6.48 12.78 12.46 8.4 8.08 10.19 9.5 7.5 7.63 101.92 7.36 8.14 7.92 7.76 7.45 13.61 7.33 6.87 7.14 8.11 7.56 12.74 7.58 7.97 6.08 6.82 5.74 15.31 6.67 6.18 6.68 6.53 9.9 9.46 8.84 12.35 6.45 8.08 7.19 8 10.11 7.42 17.72 21.43 6.53 8.15 6.3 5.7 8.54 6.33 15.32 7.88 8.61 9.34 6.35 6.8 6.94 5.63 6.24 6.4 7.34 7.76 5.92 6.54 17.38 6.35 6.82 6.05 5.9 6.94 6.34 6.12 6.23 6.75 13.87 5.55 6.88 42.32 8.59 28.59 13.41 224450_s_at RIOK1 390.78 247.67 105.51 294.83 252.72 147.95 228.85 221.4 182.92 242.25 141.05 223.22 160.04 194.57 86.13 208.52 436.08 245.02 544.85 909.17 218.14 388.75 379.13 321.85 311.43 275.64 187.31 109.92 287.45 251.38 158.59 171.11 160.54 246.26 294.24 275.43 404.42 199.09 105.56 114.94 630.22 493.91 395.02 168.97 212.88 103.95 194.5 118.2 289.72 118.65 101.57 132.47 108.1 326.6 239.18 126.41 365.65 242.96 122.07 61.55 80.77 155.51 329.74 101.02 178.76 290.23 197.05 164.98 432.99 212.91 160.74 247.73 215.13 174.13 130.62 94.68 407.37 160.42 136.75 170.75 81.17 123.16 135.95 119.38 122.96 144.54 88.79 125.64 88.89 210.21 40.37 117.8 142.32 199.3 186.23 172.61 120.79 263.28 212.33 490.75 200.12 155.92 183.89 149.51 195.37 135.79 153.86 117.46 70.83 37.41 128.49 141.59 155.59 124.43 369.06 547.13 243.48 129.41 227.09 132.25 111.4 290.73 210.48 391.16 88.84 160.07 73.16 106.03 128.47 225.94 88.31 51.42 69.35 97.32 111.43 108.33 94.33 113.71 107.08 119.82 69.41 91.24 136.34 180.37 104.44 82.94 119.69 121.3 84.6 160.87 233.74 96.97 179.67 132.08 110.21 311.98 109.63 136.32 110.98 161.14 117.77 126.93 92.55 99.41 86.6 105.19 127.86 102.15 99.1 110.56 70.22 60.63 110.3 180.83 56.71 91.13 186.34 92.98 91.47 152.99 104.39 99.7 88.11 100.39 160.05 130.15 81.05 147.16 103.94 243.77 64.35 108.88 80.21 124.53 139.98 154.03 276.87 60.08 169.03 120.9 109 87.41 117.64 64.91 203.17 89.92 350.95 310.12 93.85 147.22 186.88 118.83 87.03 76.43 91.8 124.09 81.49 119.51 82.29 82.03 280.12 500.43 128.12 341.41 214.45 303.11 131.71 224621_at MAPK1 2040.61 1099.06 1200.34 909.72 749.17 832.14 643.4 1643.87 1312.61 809.83 608.96 1207.34 930.5 565.82 690.94 776.47 507.42 634.16 1101.42 1220.84 934.73 692.2 975.95 465.83 1020.51 1002.72 1033.48 821.84 711.95 648.91 2052.78 643.5 824.46 928.63 659.18 717.2 672.12 882.41 592.46 622.93 807.24 714.17 650.72 1189.44 609.36 896.36 608.39 891.98 732.5 711.34 1791.09 920.65 1096.92 1152.71 1142.49 907.92 957.82 503.69 532.23 692.53 1068.28 654.91 1655.13 728.24 705.37 679.31 549.31 682.1 711.34 1007.59 644.5 390.79 976.81 631.66 875.87 842.28 1117.44 805.47 949.25 1301.95 734.91 1020.18 1013.69 563.22 757.12 668.86 973.03 885.88 846.52 1366.96 165.19 925.55 905.9 1269.52 1420.61 853.14 827.01 608.98 2101.35 1446.98 980.19 1006.12 754.98 652.75 895.13 597.44 872.37 830.11 756.71 108.44 462.68 972.71 836.36 796.6 711.79 653.39 639.66 1195.41 649.87 762.15 711.55 952.05 496.42 755.59 997.42 713.31 684.55 995.93 1078.57 545 725.24 650.92 562.65 672.81 923.77 592.15 655.87 769.36 716.84 939.47 769.48 861.36 614.32 754.9 622.41 437.15 1154.74 583.13 655.22 865.95 779.14 682.67 545.94 897.89 961.92 501.21 698.15 659.89 795.68 982.66 401.18 832.08 869.29 462.29 748.45 592.85 600.48 629.11 414.4 520.33 663.66 355.75 491.12 660.8 193.7 521.09 627.93 450.23 862.73 610.81 717.87 579.67 578.69 505.48 591.67 815.49 420.35 1115.39 564.88 560.21 530.27 820.65 557.24 738.84 668.7 552.76 534.73 601.98 513.92 411.12 542.1 589.23 666.85 206.44 394.82 578.58 573.53 425.63 743.67 553.12 421.49 662.82 719.41 381.72 626.76 847.17 739.99 643.59 637.32 678.2 1047.03 589.77 909.95 1217.79 690.8 797.82 801.81 224739_at PIM3 654.08 556.35 538.45 472.63 551.04 517.26 545.25 670.44 1114.77 631.76 313.75 1037.72 560.86 621.89 667.56 378.73 877.51 730.12 656.45 517.01 536.35 430.58 620.44 638.54 1068.79 880.69 387.48 434.95 1026.93 846.33 396.59 487.63 478.04 1004.14 299.19 759.97 893.24 621.56 607.02 495.59 688.43 650.91 1012.09 646.53 882.79 493.74 408.39 498.76 542.36 903.13 536.85 493.72 1085.32 417.94 878.68 543.86 1244.37 964.28 723.1 935.63 1039.81 518.01 472.92 505.44 359.61 797.52 395.6 594.79 877.78 579.91 587.22 531.03 585.15 422.79 518.2 616.73 330.11 323.25 457.94 367.96 1118.22 751.29 406.1 361.28 432.9 374.75 815.8 548.83 389.41 509.23 561.75 577.38 458.2 501.91 739.32 573.75 785.33 324.55 336.79 511.03 297.84 255.47 246.68 622.47 473.35 257.27 363.4 393.59 307.42 520.06 588.82 296.89 250.59 430.3 288.21 306.03 504.87 293.74 450.57 528.13 301.82 336.26 352.97 334.13 350.77 437.12 458.54 302.84 295.05 244.35 324.26 252.03 426.62 371.92 409.32 371.76 460.56 248.48 278.82 351.14 338.44 353.14 181.79 277.38 304.87 598.44 560.84 389.58 729.9 502.83 648.04 1141.2 590.22 277.72 404.38 529.57 416.71 531.78 293.56 650.08 344.11 345.84 551.45 232.48 761.91 335.84 362.58 524.2 684.67 253.95 687.64 434.03 441.31 625.37 771.8 274.28 382.38 197.53 397.37 678.19 352.99 343.31 415.02 466.79 529.6 767.61 457.19 577.64 369.93 421.59 509.72 352.9 291.12 335 369.02 463.69 605.96 795.25 446.16 398.41 570.55 821.08 492.92 486 648.67 511.57 814.52 395.08 480.64 583.39 551.86 610.8 2587.91 473.55 372.65 599.29 535.43 546.09 442.6 547.18 557.18 469.77 509.78 1331.6 594.42 600.87 398.61 224851_at CDK6 332.48 73.56 239.1 91.09 140.56 97.72 17.15 73.37 51.02 151.81 77.49 305.49 118.72 37.29 130.52 1836.5 649.89 285.32 287.6 219.55 81.06 90.21 249.04 309.53 507.68 347.97 74.07 409.52 573 54.79 95.73 48.01 97.69 63.36 110.43 143.22 1100.65 998.2 207.24 106.42 185.79 110.22 394.12 307.92 155.45 210.33 322.82 183.84 136.73 95.12 776.35 245.37 99.77 315.71 87.65 96.19 179.57 78.56 158.06 102.63 42.33 467.48 145.25 180.79 514.83 1869.52 479.22 385.38 211.72 1420.64 1459.13 939.51 597.5 170.43 116.44 180.35 129.38 266.94 39.84 247.05 221.42 183.11 86.48 231.81 377.11 187.52 230.03 352.04 262.71 451.1 75.75 533.05 146.32 219.87 492.43 294.19 166.34 218.84 192.34 660.99 5776.87 264.11 185.24 214.45 177.66 152.68 173.89 226.52 153.58 17.39 721.49 595.79 2766.66 410.82 1000.3 37.27 117.8 366.71 846.47 1158.94 812.97 1243.68 115.77 2116.14 572.08 92.77 341.07 407.14 163.54 812.27 410.72 421.8 363.88 169.09 195.35 144.3 191.5 286.94 363.75 298.73 95.53 121.61 71.78 240.14 323.64 257.11 83.12 122.39 290.52 58.19 593.02 257.66 408.19 214.05 91.79 351.47 30.48 1177.8 200.86 480.92 227.88 318.4 140.27 108.4 527.47 190.85 110.27 162.37 42.15 62.93 131.09 159.4 211.53 146.84 129.36 483.41 457.89 119.08 244.38 64.99 404.86 228.55 80.88 290.8 2263.04 196.91 101.56 150.48 202.35 53.49 264.98 265.25 133.73 145.4 416.72 152.25 349.14 139.27 271.32 950.41 67.59 158.76 215.67 44.7 506.78 165.11 1919.77 290.62 229.44 99.34 221.44 198.86 234.75 224.85 88.84 94.3 201.11 209.97 170.03 139.67 535.78 414.32 77.65 244.43 500.51 481.67 460.04 224960_at SCYL2 389.15 399.28 279.83 374.02 433.65 343.34 455.51 435.43 382.71 669.59 534.19 536.26 309.24 373.51 170.84 213.37 772.58 282.49 470.05 511.41 251.96 664.31 1172.25 203.43 187.73 446.32 601.99 560.38 548.97 758.79 523.48 805.65 640.56 415.53 609.92 266.29 642.62 706.16 257.54 288.1 387.23 353.16 479.24 257.59 354.8 185.97 290.73 235.45 335.46 269.57 665.54 321.9 200.77 391.68 280.51 474.72 265.48 302.94 366.55 266.12 378.03 529.18 809.89 301.49 212.01 372.02 526.15 223.05 447.72 358.34 176.42 243.51 505.6 354.66 422.53 371.59 470.38 367.83 296.87 481.3 239.88 555.3 407.28 360.09 304.42 418.76 328.77 301.14 314.27 395.71 13.32 248.09 305.47 337.29 319.96 624.96 437.17 655.5 510.69 395.71 426.17 552.34 544.59 338.22 443.81 429.63 282.92 267.27 315.6 17.86 179.04 251.33 408.46 426.06 363.91 563.1 189.21 175.59 275.13 288.97 278.9 310.26 173.58 259.55 206.38 365.99 231.98 313.45 373.79 447.62 206.34 495.87 132.62 302.89 495.03 279.41 250.36 381.81 357.43 421.88 279.35 504.21 572.67 409.73 372.79 128.9 219.71 296.67 214.65 501.9 239.96 152.85 314.63 347.77 327.67 268.94 383.85 275.29 354.06 236.05 431.86 310.33 332.31 244.17 414.97 414.7 422.48 267.18 45.05 296.5 264.91 159.94 292.01 305.91 34.81 274.67 213.78 255.31 180.2 98.42 223.13 215.63 330.41 170.11 238.7 206.44 280.99 283.19 278.61 192.33 339.83 282.49 219.16 322.7 196.54 382.18 259.97 193.24 323.17 174.01 212.4 270.48 361.29 34.76 238.56 211.26 352.27 532.99 331.76 305.19 192.26 192.67 178.39 288.55 175.71 204.95 456.46 443.63 238.59 183.99 353.92 335.02 241.81 274.95 372.15 313.15 322.23 225019_at CAMK2D 615.13 226.35 269.65 360.94 189.15 597.76 490.95 195.29 230.28 573.15 264.44 351.93 264.93 77.95 376.34 1543.99 140.1 313.29 467.85 274.72 208.02 358.49 174.32 309.53 376.94 518.84 543.7 177.18 724.11 490.6 647.4 816.89 142.5 73.78 301.6 612.18 588.91 622.03 265.37 248.62 599.36 490.27 317.19 198.82 240.29 358.81 377.28 424.97 191.32 230.38 812.07 403.96 304.24 284.88 38.82 349.6 195.29 409.38 305.03 334.66 100.63 288.93 720.58 134.26 376.48 464.48 97.87 234.87 295.75 199.32 214.77 630.31 267.94 332.44 46.84 281.49 357.14 278.61 88.42 267.07 196.66 135.1 85.24 125.33 296.36 323.91 256.14 311.29 366.84 317.76 45.16 274.24 409.7 292.07 420.37 595.19 273.41 343.11 307.7 844.99 290.61 524.42 172.85 219.62 442.12 363.92 714.11 327.94 178.39 46.17 151.17 363.94 321.79 395.45 495.26 271.19 100.39 132.09 315.21 212.3 351.53 264.67 291.66 300.21 242.15 506.24 150.97 380.35 399.88 158.43 263.6 319.07 192.83 214.76 623.82 185.11 182.25 266.58 262.23 234.43 135.81 385.63 600.05 195.15 175.79 97.98 195.85 137.97 240.06 138.83 180.59 292.21 623 418.68 280.95 172.29 46.86 423.78 204.27 230.76 136.12 245.14 136.34 124.09 214.46 237.11 111.68 308.77 136.89 137.84 232.86 99.5 403.56 156.12 51.86 374.94 272.97 269.05 376.15 163.96 304.6 243.01 321.89 160.85 295.3 152.05 234.18 177.85 239.7 366.18 290.5 396.82 217.74 250.71 235.45 351.78 220.94 205.3 290.71 246.53 87.33 296.58 205.53 56.21 261.33 227.66 249.46 283.55 171.8 521.54 129.25 203.67 223.3 235.5 79.09 126.89 184.59 430.99 189.07 130.5 703.25 262.47 176.29 359.83 442.84 262.73 476.63 225067_at ULK3 123.56 165.66 179.14 133.71 320.16 376.44 187.41 316.89 261.41 207.69 321.78 314.56 280.82 296.83 278.78 177.52 133.52 277.17 280.31 147.11 246.35 168.18 126.4 179.56 150.48 222.73 103.27 201.5 170.39 130.43 133.76 123.35 215.93 558.97 142.89 149.41 134.3 194.2 230.07 232.15 221.81 177.6 121.72 281 128.98 235.64 152.16 163.86 173.59 316.43 168.99 167.63 283.22 153.69 527.31 239.01 120.42 94.1 190.35 201.75 280.3 228.45 184.56 158.18 314.82 155.49 262.65 158.37 130.68 128.95 204.53 233.35 125.07 212.71 154.65 100.35 321.92 270.39 147.04 174.46 146.22 204.96 218.06 188.14 146.56 146.47 131.9 169.36 154.67 177.05 296.11 214.72 356.78 130.11 191.17 136.18 222.31 56.98 204.67 149.5 249.15 246.4 224.09 196.51 212.67 226.89 211.65 209.49 265.12 356.07 191.75 182.65 165.11 159.14 255.37 113.63 717.86 410.21 148.38 250.12 243.08 324.02 226.74 181.28 181.4 227.48 308.35 170.14 165.26 160.39 271.95 231.51 306.02 217.76 257.08 264.96 250.74 199.15 173.84 311.63 224.79 135.6 153.93 227.14 156.01 242.71 222.64 240.97 204.25 238.81 198.63 277.29 125.59 195.39 310.35 139.76 118.34 191.09 196.05 177.67 389.37 247.11 156.1 309.71 125.44 194.06 164.97 236.93 486.73 308.94 287.62 264.24 252.71 188.3 203.81 258.02 213.69 251.73 228.85 301.34 170.46 171.93 389.7 282.93 117.89 367.84 263.54 366.7 199.09 204.67 347.71 153.3 216.96 204.64 190.97 180.09 207.18 270.09 268.75 203.97 340.38 257.58 184.71 323.5 156.58 192.07 231.89 129.02 199.35 216.85 366.93 392.88 222.95 330.53 430.47 285.51 282.71 211.51 220.2 305.19 139.84 188.75 292.96 128.3 186.52 216.9 200.17 225144_at BMPR2 392.27 459.53 383.38 373.2 345.19 354.81 249.93 905.77 515.27 220.34 358.12 366.97 285 382.46 193.38 272.91 281.11 171.45 375.57 516.2 482.34 627.32 481.21 203.06 214.86 502.36 489.02 538.97 237.22 318.67 776.53 388.31 383.71 200.61 486.27 267.57 400.34 297.87 440.64 352.89 330.51 242.3 219.15 361.65 267.61 524.2 369.11 251.76 223.4 202.25 663.23 519.51 468.26 413.78 342.53 482.35 326.47 257.48 268.37 220.23 220.63 147.08 763.22 128.04 82.62 249.14 228.31 182.57 214 424.74 313.63 263.69 274.4 339.79 289.45 302.89 354.32 665.13 115.69 325.18 103.62 527.5 480.87 385.88 570.13 488.99 445.85 520.06 260.78 390.84 19.51 385.1 236.33 406.8 434.83 277.36 297.82 291.3 403.87 344.43 409.93 428.78 283.08 175.04 309 221.31 398.94 330.88 286.53 12.02 233.77 318.44 371.16 278.45 247.88 193.39 239.48 96.9 176.03 177.7 331.32 316.51 69.18 233.1 297 245.71 423.47 698.06 428.78 321.89 363.78 312.7 87.25 382.78 426.64 294.71 226.33 522.11 419.9 434.61 347.37 468.69 364.37 343.97 334.49 55.29 321.52 418.71 423.89 701.76 189.2 225.4 344.18 617.8 495.92 207.04 206.48 277.02 491.56 364.5 278.68 624.3 349.05 217.11 244.39 399.63 558.63 245.94 23.78 162.84 348.12 167.9 367.39 311.23 21.43 320.02 326.44 269.98 394.03 199.19 467.81 207.62 375.29 377.18 331.68 210.58 295.21 440.81 156.01 119.07 260.16 771.45 371.39 334.72 395.52 421.62 281.77 242.28 222.32 267.06 243.53 350.96 327.38 19.86 189.28 275.13 213.74 132.17 183.88 413.93 155.65 312.36 296 192.29 246.3 295.35 432.23 226.05 312.49 331.87 557.84 402.07 427.16 324.78 378.53 104.82 391.08 225164_s_at EIF2AK4 157.97 176.62 148.15 168.59 127.58 207.41 108.87 268.74 222.89 231.96 211.57 314.01 215.15 88.06 231.99 259.32 205.66 148.47 201.23 244.34 124.69 176.87 258.85 74.47 121.6 176.08 195.64 153.18 126.88 119.26 220.31 77.6 119.92 71.28 207.39 113.33 148.85 178.2 131.4 59.17 164.97 177.75 118.38 68.4 103.06 153.42 46.53 128.68 97.47 97.53 128.08 124.45 97.12 158.58 208.12 122.27 141.44 77.06 106.26 115.06 107.32 73.39 205.74 137.31 152.5 168.73 112.67 169.72 137.78 96.06 224.42 126.46 166 280.46 200.31 117.7 138.55 218.68 123.86 160 279.24 226.01 269.13 182.78 185.26 175.49 137.46 127.52 169.36 214.72 31.94 247.03 154.19 212.96 321.72 230.53 300.05 190.43 263.16 212.35 132.05 186.58 113.23 227.18 212.71 124.8 158.63 153.43 286.58 52.53 151.17 176.26 113.94 199.14 188.4 67.77 214.68 58.78 92.85 191.23 76.55 86.38 118.41 181.2 127.54 139.52 141.49 120.97 172.63 173.24 189.24 169.04 86.83 97.12 232.08 154.09 110.12 131.19 155.91 186.32 187.91 190.44 81.63 214.18 143.43 134.21 176.83 74.18 132.86 200.56 124.5 134.55 128.92 126.76 138.62 84.91 93.04 182.11 153.14 117.66 220.97 163.87 136.94 160.44 87.79 146.3 132.12 135.45 186.84 169.34 125.63 73.46 142.3 79.4 65.05 69.93 95.52 114.48 139.62 130.53 73.35 75.44 93.47 142.4 72.73 124.51 84.48 156.52 78.25 88.11 102.1 149.4 104.17 113.17 120.96 155.38 158.6 106.46 86.01 125.51 88.1 139.4 111.12 120.82 218.93 152.79 111.89 102.19 131.9 231.27 92.98 126.74 129.45 80.17 239.66 202.85 123.64 180.2 119.89 180.93 101.2 175.85 133.98 139.87 142.79 126.64 184.13 225207_at PDK4 25.76 289.9 178.83 75.41 377.47 247.97 111.73 411.13 92.6 69.21 278.91 114.72 250.3 116.64 304.98 85.03 89.99 51.03 76.14 99.9 124.62 75.89 146.05 51.54 88.83 106.74 50.86 85.95 92.95 55.25 388.49 160.82 87.12 95.8 152.29 20.08 57.92 165.2 384.29 38.36 56.99 79.55 186.14 117.08 93.71 632.3 143.21 127.04 83.36 98.94 128.39 60.06 82.72 114.86 36.19 444.46 38.76 52.23 352.77 20.26 23.6 67.4 119.16 26.49 35.25 13.89 20.51 75.75 23.6 78.12 20.76 35.19 37.09 140.48 167.88 128.22 55.91 393.22 50.27 145.02 86.7 118.81 398.78 169.85 588.2 557.18 101.11 217.09 35.63 213.4 126.09 158.14 205.56 78.02 293.79 75.27 136.44 30.49 237.15 188.99 282.58 194.53 146.47 425.62 111.46 196.53 329.92 95.68 117.27 34.36 117.48 229.87 86.73 95.97 56.44 34.19 25.32 327.53 205.67 103.34 447.32 71.31 62.41 78.71 395.45 124.74 300.11 409.18 109.4 95.88 233.03 106.15 245.72 149.3 59.5 133.98 126.59 126.88 135.47 258.47 161.7 54.02 111.92 117.33 238.26 76.37 65.72 270.91 303.58 102.59 29.62 166.96 18.17 157.74 94.43 62.47 355.15 28.33 104.98 43.02 246.89 269.21 42.07 129.42 80.8 456.95 204.5 85.54 19.78 80.87 44.41 95.43 152.75 91.14 12.77 86.95 55.01 47.97 183.92 32.8 213.75 26.29 88.08 119.28 69.87 129.93 44.84 136.65 17.15 82.4 138.36 400.58 140.8 31.21 214.66 30.85 14.12 105.81 210.8 35.43 38.98 20.89 118.76 21.75 10.25 206.92 79.39 141.77 41 29.26 281.51 283.25 270.68 26.72 37.83 53.46 26.79 36.03 28.51 123.99 47.35 45.4 177.67 193.41 35.85 31.54 318.25 225364_at STK4 331.45 433.44 585.45 741.46 539.72 449.26 327.16 621.23 410.86 437.58 433.28 310.65 348.25 334.14 303.57 267.46 374.38 856.99 560.33 369.58 427.76 634.78 268.68 584.91 410.1 814.43 394.77 432.28 507.95 348.43 623.69 414.7 1274.54 400.49 350.46 498.44 365.03 259.44 520.46 544.63 530.31 508.05 493.99 345.52 333.98 388.79 376.1 453.08 455.14 325.86 219.36 472.38 394.93 574.38 350.58 384.38 453.99 395.35 667.94 311.32 286.82 438.67 333.34 719.73 1702.54 348.77 373.97 497.73 344.84 361.92 484.68 506.15 355.43 377.86 118.59 146.87 276.84 551.78 343.18 401.22 420.92 467.38 297.25 497.81 399.88 603.46 434.02 585.06 856.03 553.17 90.73 306.42 316.73 257.29 410.05 295.92 188.27 155.89 276.27 284.79 389.19 473.95 374.2 171.59 262.92 651.84 356.04 393.37 478.14 128.06 326.98 295.03 719.02 430.67 686.36 421.56 348.04 327.27 773.93 440.96 550.57 627.14 519.51 639.3 339.19 387.18 341.31 309.83 225.69 206.32 473.89 223.86 281.97 387.88 212.78 333.55 427.41 276.45 228.21 254.74 221.18 244.53 278.18 317.05 346.77 408.81 343.74 442.7 397.59 280.67 332.25 652.73 246.75 367.3 404.52 289.77 246.64 592.38 219.95 426.75 294.5 226.79 275.83 205.47 194.88 458.51 306.53 555.13 191.76 241.37 355.44 270.14 396.24 448.94 168.36 265.67 371.34 216.17 234.16 286.14 507.72 230.49 213.5 233.27 345.76 272.26 242.66 264.9 254.56 211.89 271.26 232.88 161.82 432.4 170.95 223.95 150.24 259.91 319.75 221.07 198.41 229.2 404.73 352.8 199.52 234.24 149.3 244.27 295.93 268.4 241.16 438.99 237.35 253.86 214.13 283.85 279.35 253.93 182.94 219.5 546.99 295.07 241.16 273.07 274.8 314.74 391.51 225368_at HIPK2 1935.53 1205.25 866.45 661.13 392.7 1374.35 362.14 1930.45 1191.36 641.35 1090.23 974.23 995.44 1234.63 956.23 508.09 628.21 498.38 1025.4 397.07 814.59 630.22 876.83 354.87 506.98 530.46 587.77 1160.17 1274.28 551.85 397.23 794.4 553.88 327.25 453.97 397.39 620.08 1627.27 577.59 454.08 855.12 984.46 494.82 220.74 459.87 527.19 665.98 530.88 297.31 208.22 1333.12 513.74 338.94 419.91 1210.74 449.24 608.9 877.26 290.32 273.83 770.25 827.59 843.64 217.94 94.02 409.16 503.24 705.92 1537.03 615.5 748.01 542.07 219.66 279.48 1146.5 141.28 456.05 742.18 335.47 417.93 421.46 316.87 1217.12 656.16 520.45 566.52 304.06 408.51 990.73 817 359.15 637.86 489.4 797.08 521.94 579.94 348.7 535.54 542.85 325.09 1086.17 1116.36 598.7 271.46 497.75 584.21 890.04 587.63 667.25 229.8 341.18 667.04 520.73 245.29 620.82 630.17 480.96 383.03 590.42 997.78 568.27 457.82 990.88 585.48 387.53 248.31 420.61 490.67 151.64 551.88 365.22 331.51 531.9 350.35 387.8 433.96 409.06 351.82 259.57 332.59 549.18 357.37 575.7 566.03 315.42 380.56 348.92 1109.73 563.52 944.26 634.84 484.49 517 425.79 596.12 329.16 775.61 579.67 708.94 270.63 851.82 778.84 620.38 642.24 743.91 330.54 320.36 348.63 221.64 581.64 630.04 435.93 226.83 469.46 562.22 933.69 387.32 808.62 710.98 770.29 689.2 615.6 316.72 512.04 453.15 171.72 864.89 1289.52 774.38 437.23 536.99 1028.3 434.7 436.34 517.15 385 494.62 560.53 352.12 523.27 1105.05 251.1 710.47 682.89 807.4 856.48 631.1 483.43 380.22 474.81 430.53 383.1 639.8 618.64 776.94 850.06 214.15 265.93 241.3 637.18 494.15 345.23 855.57 658.55 385.32 922.82 842.78 225380_at LOC91461 77.23 148.94 144.02 94.26 97.16 152.28 68.6 81.81 58.7 85.57 77.86 102.04 80.74 117.99 125.76 136.3 387.62 232.65 46.63 51.71 77.1 54.86 35.24 76.41 63.51 73.62 70.1 56.91 59.83 54.2 67.51 57.62 46.53 895.75 151.59 184.6 54.68 54.26 163.64 152.31 51.54 210.25 138.8 70.56 607.03 124.3 443.91 282.1 87.31 93.84 71.65 112.55 109.07 96.08 53.02 85.55 70.83 694.87 95.25 83.03 94.73 90.18 79.18 44.17 88.72 98.21 269.59 160.87 184.67 61.06 114.82 116.36 118.49 70.64 234.86 67.29 51.24 94.52 33.77 49.25 68.3 44.56 43.46 47.9 133.22 85.99 56.07 121.66 56.74 98.91 187.42 127.36 75.05 100.51 107.77 71.56 73.79 58.77 111.42 150.77 106.28 68.44 82.04 96.7 115.64 87.25 99.63 128.38 80.55 134.54 463.07 80.46 141.99 115.68 91.73 91.8 103.12 195.62 366.9 89.73 223.1 137.76 90.06 94.22 252.69 75.05 74.93 281.97 64.53 295.5 104.41 90.25 283.09 87.97 65.11 87.38 82.38 72.14 64.61 53.15 103.1 45.22 47.87 26.97 72.2 89.05 246.6 125.16 151.55 85.18 112.93 134.34 71.35 84.8 37.94 85.21 51.09 56.82 116.86 72.42 99.25 96.89 62.53 72.29 46.88 67.64 68.43 102.91 73.91 55.74 170.71 105.89 129.15 76.77 53.09 89.42 74.33 46.74 176 211.14 141.26 58.57 78.82 103.66 136.02 66.08 71.33 75.64 56.12 77.52 90.64 153.25 112.62 56.26 139.58 75.59 41.76 129.41 93.23 130.54 151.27 82.94 80.81 79.35 51.56 103.15 65.29 56.15 79.14 50.77 64.7 224.59 204.19 92.99 73.86 163.72 97.85 69.83 62.32 107.71 97.16 134.03 105.47 128.15 83.77 52.61 112.63 225471_s_at AKT2 207.94 198.22 212.12 178.8 199.93 324.77 224.62 242.22 205.55 143.77 225.62 283.55 204.21 303.67 242.17 177.58 260.26 188.63 166.47 176.56 171.93 151.77 199.14 164.27 232 216.49 191.66 316.86 201.68 216.72 183.14 168.44 126.09 336 178.74 259.32 168.6 204.29 150.17 656.36 214.74 183.16 213.87 179.9 155.51 187.36 217.97 139.49 212.56 199.55 163.22 148.93 235.05 237.42 568.27 215.58 272.54 242.29 214.08 201.15 247.4 259.75 256.99 152.48 171.25 186.87 79.91 76.9 95.11 80.82 139.14 228.9 114.67 99.75 89.11 84.12 127.68 156.42 178.93 166.74 157.83 145.27 183.44 173.7 134.91 128.97 177 150.44 156.51 174.94 219.82 192.78 232.47 141.89 164.77 131.17 267.24 200.15 153.79 190.42 142.06 174.52 238.57 219.62 170.4 210.19 265.27 282.47 318.33 309.47 203.15 261.14 206.65 219.72 202.02 199.02 343.5 720.24 177.56 411.35 289.53 277.83 305.24 221.59 178.85 295.23 208.39 175.54 139.19 195.51 155.11 219.46 258.14 316.83 127.93 240.03 236.28 122.45 181.87 168.26 169.88 159.69 204.03 207.7 170.9 124.02 152.14 258.1 263.53 294.3 279.68 238.09 219.98 187.36 235.8 295.49 230.99 181.62 216.97 179.7 284.39 212.77 179.9 399.64 165.52 222.55 213.09 208.73 183.17 255.76 213 290.23 224.08 305.75 215.49 130.7 152.27 189.72 208.32 276.87 193.96 263.83 305.01 186.5 144.23 218.93 200.58 304.35 293.49 215.92 180.85 193.14 232.27 212.14 199.32 152.42 238.28 175.04 190.26 282.41 272.67 263.59 298.46 215.25 145.21 189.41 144.97 182.9 194.43 167.27 206.05 189.82 270.7 197.89 206.31 281.25 239.79 207.88 145.58 203.44 178.61 166.45 180.15 179.56 201.99 180.38 172.44 225522_at AAK1 398.28 281.52 464.79 343.77 285.19 272.14 275.36 266.57 339.84 421.85 494.13 316.73 263.97 347.2 291.44 303.29 550.1 337.08 411.04 225.26 247.34 370.14 303.92 271.73 512.97 337.85 335.02 497.31 244.24 259.31 255.73 240.04 199.33 240.01 330.3 173.91 194.65 225.7 321.38 191.24 173.42 315.79 180.46 248.04 278.5 317.57 313.62 258.85 335.47 190.6 199.29 310.7 214 318.13 181.44 220.65 166.51 228.45 307.95 260.79 286.2 144.55 424.33 221.87 157.24 237.96 290.39 187.06 183.25 193.34 227.64 242.85 275.71 279.75 132.04 295.19 180.34 353.12 314.31 236.52 283.64 340.66 351.26 325.34 347.24 240.67 290.6 404.81 433.06 293.91 221.77 310.13 236.96 204.41 270.62 255.24 287.08 369.71 449.74 327.52 256.22 584.73 340.69 289.39 297.3 566.86 367.38 395.84 502.44 314.53 311.62 309.16 377.09 265.07 340.17 380.61 242.52 397.8 373.18 380.5 342.88 334.27 348.81 405.55 439.15 266.15 483.42 463.14 292.81 441.73 512.04 430.01 455.82 501.38 341.82 335.98 320.41 380.73 328.44 363.54 409.25 365.26 260.52 311.71 396.35 365.21 453.12 320.37 446.69 326.97 387.53 444.68 309.46 347.96 344.15 254.01 300.71 475.63 326.18 324.52 299.21 343.94 275 236.34 292.41 335.79 316.55 489.41 327.44 267.2 285.06 385.11 309.98 370.15 580.84 479.04 431.9 328.56 370.97 501.62 462.56 334.45 284.16 308.94 394.77 258.3 437.84 305.43 346.62 457.09 333.35 388.95 324.74 325.31 279.24 347.49 237.33 427.08 496.94 296.79 325.11 390.87 401.13 422.53 228.56 365.11 346.33 434.28 337.21 367.56 381.63 359.2 323.87 314.12 334.55 311.44 307.19 370.67 273.38 262.72 426.32 320.07 510.21 379.68 407.75 220.28 408.96 225545_at EEF2K 367.89 427.53 505.49 354.17 502.04 682.07 372.08 568.7 379.25 391.69 387.55 553.27 346.84 825.29 384.97 200.86 1102.29 374.86 372.03 273.33 564.86 451.63 258.04 208.86 441.02 378.49 510.84 367.41 358.42 468.21 574.39 444.48 353.57 724.17 300.39 414.56 429.76 299.74 279.64 338 310.78 314.12 313.54 294.21 403.65 385.17 337.36 268.09 287.79 608.24 206.07 618.01 342.95 553.47 408.32 486.5 391.01 308.54 617.85 428.81 371.41 1077.76 341.8 318.11 255.41 504.27 875.91 338.17 280.28 155.89 365.7 375.48 284.6 286.13 143.84 361.05 366.76 164.44 824.95 680.95 269.86 625.2 539.39 348.24 243.71 450.2 230.89 368.14 296.84 232.75 206.85 259.89 321.63 357.15 617.58 341.04 332.73 290.15 309.24 303.85 252.8 333.15 514.37 333.39 270.93 413.54 513.46 588.25 424.14 229.7 488.64 500.37 263.42 283.97 606.3 647.65 278.22 221.99 232.33 485.04 670.33 430.03 414.78 277.01 493.35 404.42 245.91 324.3 386.17 475.27 282.28 294.38 456.67 381.03 359.18 379.23 226.12 325.45 238.3 303.89 520.21 409.51 283.79 271.73 284.35 281.09 423.51 424.88 377.29 388.11 550.52 249.96 309.61 381.55 300.58 371.21 190.66 196.64 320.2 329.1 226.11 236.41 325.03 165.27 336.25 216.88 508.9 307.45 163.19 268.63 449.35 446.98 382.22 391.28 96.82 544.62 326.85 593.05 436.18 254.09 373.22 204.28 502.52 268.64 271.22 241.02 445.37 315.01 196.62 364.56 536.51 391 359.63 601.7 464.63 379.73 246.63 286.95 297.99 258.6 402.1 232.67 352.81 295.19 179.27 346.78 253.18 176.94 184.75 515.79 204.87 353.4 293.08 237.37 174.24 221.94 139.84 325.3 190.26 217.09 412.71 291.29 359.13 352.59 482.25 290.97 381.17 225648_at STK35 105.84 156.31 197.25 130.02 477.22 131.48 129.52 245.09 147.24 212.12 170.5 209.72 131.41 183.59 172.09 108.73 353.87 149.87 216.5 150.16 175.79 208.51 177.66 162 276.8 182.43 158.08 187.12 136.12 171.45 242.68 193.48 196.64 203.21 212.55 277.32 113.09 177.6 120.6 158.73 71.58 172.06 254.42 144.78 161.55 126.98 174.17 118.59 126.95 144.64 165.12 177.02 214.8 198.69 198.62 203.16 131.92 125.54 221.96 191.65 187.64 269.56 193.65 163.49 102.81 101.14 129.99 101.88 205.75 144.87 179.7 135.72 139.4 208.15 143.5 157.64 66.25 108.03 83.73 136.41 196.38 156.14 113.84 82.21 116.8 117.07 98.9 95.93 144.54 111.11 140.3 92.77 159.86 108.02 119.84 128.1 115.79 163.62 64.33 123.68 157.92 115.31 161.1 96.08 148.5 188.78 146.46 170.37 184.14 123.33 149.88 123.86 117.96 157.39 197.99 125.72 329.14 150.29 102.9 241.04 170.93 155.75 105.37 229.85 108.54 78.79 152.05 143.12 91.1 102.87 133.69 121.95 127.89 146.51 131.63 181.31 133.59 120.51 117.15 200.69 83.36 91.64 164.43 85.34 94.7 160 128.47 250.9 175.37 118.7 156.16 160.83 250.89 112.29 130.71 222.86 142.09 151.75 153.11 108.02 160.6 121.15 113.45 129.4 114.61 239.87 141.32 136.02 110.84 142.32 182.83 170.4 222.32 179.8 202.72 96.5 127.89 145.08 147.47 232.52 131.45 129 95.79 144.94 141.09 134.85 183.64 230.15 148.05 185.21 189.58 115.75 138.5 526.94 121.73 180.93 186.94 139.13 215.45 182.56 214.78 115.99 213.95 66.38 210.25 128.32 131.31 123.97 132.08 125.09 157.75 182.14 177.45 131.97 169.11 140.77 125.08 223.09 124.24 148.86 130.38 199.53 164.32 168.74 137.48 98.6 83.99 225665_at ZAK 217.05 439.35 526.02 253.82 397.97 160.26 173.18 797.36 465.97 200.57 472.88 182.56 286.17 198.42 252.39 242.25 557.61 179.29 795.14 334.79 363.41 615.1 1110.35 246.98 879.6 246.77 284.1 417.54 206.75 329.07 515.06 849.79 256.28 161.44 1192.77 140.43 112.92 342.77 325.92 196.82 328.83 197.6 452.95 168.59 184.85 570.47 255.06 226.39 189.36 749.56 627.81 386.39 596.22 559.52 315.18 352.45 299.9 284.21 160.79 154.2 211.56 748.73 409.94 89.23 84.57 322.83 93.69 190.88 314.88 170.41 557.04 203.7 265.56 274.29 370.91 872.93 120.64 375.39 94.49 239.32 146.75 596.51 397.08 332.25 429.63 313.61 168.14 412.03 159.07 581.31 142.69 462.5 228.26 559.84 633.41 136.18 295.61 575.47 390.6 306.08 405.04 246.69 332.14 402.58 304.88 354.54 288.61 348.1 339.3 76.07 315.92 595.07 261.24 344.47 124.63 298.63 182.05 108.47 169.95 69.45 235.76 134.65 252.61 193.46 932.95 223.11 351.3 645.25 266.1 294.81 168.75 147.41 323.92 260.53 216.21 361.95 222.96 522.58 215.47 387.35 435.26 211.8 289.97 228.18 280.51 68.81 597.36 290.56 278.56 591.18 364.79 205.21 307.56 300.24 244.91 169.49 161.69 420.91 280.5 190.06 181.69 376.44 381.46 165.93 337.71 259.54 234.96 379.38 219.15 159.75 161.79 171.11 196.41 188.91 30.19 131.01 321.99 322.47 570.3 239.51 462.11 327.66 219.19 284.29 259.13 215.58 194.49 295.8 414.72 200.92 143.18 432.62 186.99 140.92 613.89 218.68 460.82 215.97 229.67 264.94 183.84 261.32 395.99 14.53 217.98 408.47 430.84 176.2 150.33 274.11 442.93 330.08 371.16 91.39 388.12 223.14 185.33 179.25 212.96 384.16 230.23 375.72 245.03 174.48 345.29 202.18 423.73 225697_at CRKRS 125.35 142.84 372.25 140.05 105.6 99.6 57.72 260.82 179.86 117.14 148.95 162.91 115.44 121.81 142.42 91.64 111.47 158.37 114.01 136.88 160.4 248.24 154.44 130.78 101.47 134.55 616.35 436.26 77.77 163.89 303.94 128.88 165.67 241.93 135.56 139.12 120.05 84.18 111.69 102.38 96.62 101.92 90.44 1048.2 103.24 143.1 126.79 113.94 141.75 113.87 68.48 220.61 126.4 124.79 134.74 132.01 122.33 102.64 365.51 975.11 84.41 143.62 125.09 308.28 392.44 172.55 101.61 181.05 166.94 150.37 174.28 190.27 188.74 1537.38 201.52 841.96 137.02 162.47 193.24 220.57 222.57 166.66 255.1 258.31 104.33 171.24 283.48 139.13 163.24 163.59 188.98 177.96 258.24 225.85 161.79 289.83 227.16 84.69 181.69 109.67 251.68 120.27 96.56 134.39 155.68 86.4 108.37 93.96 180.36 119.81 98.44 144.7 177.59 173.83 186.35 64.57 284.16 223.82 141.14 198.61 113.36 160.68 154.46 178.68 257.86 223.3 523.27 223.68 1487.22 166.7 183.31 293.08 249.8 175.53 180.09 333.68 176.49 1007.11 905.58 192.97 267.31 297.4 149.05 208.98 180.39 194.07 2400.39 136.58 337.73 129.54 110.89 118.88 125.92 965.21 128.3 148.48 237.24 109.55 142.42 191.72 168.28 105.92 164.76 170.62 158.04 158.91 182.06 193.65 1289.22 169.39 115.47 195.07 172.7 120.72 1242.03 159.7 123.06 181.28 185.96 169.67 163.33 134.76 133.58 142.37 166.02 217 867.11 197.71 162.54 336.28 137.4 129.82 145.92 383.8 97.35 152.02 82.13 139.24 191.79 163.73 164.9 464.74 94.02 128.69 172.84 146.08 175.57 177.89 200.12 166.26 129.55 194.27 224.34 139.55 136.36 168.82 394.41 417.49 202.17 158.67 101.58 131.51 174.38 155.61 93.91 116.01 112.06 225796_at PXK 91.41 216.81 167.67 107.71 130.54 223.2 96.61 164.66 164.09 112.18 140.72 83.07 80.16 109.5 98.89 90.5 52.63 125.68 125.81 131.93 98.19 99.25 110.89 66.28 105.06 160.56 87.52 128.75 102.85 123.25 117.36 37.73 94.94 154.17 99.18 49.85 101.14 136.98 140.71 83.31 74.65 105.59 83.97 78.01 99.02 121.81 37.03 88.72 79.44 115.66 93.02 97.07 88.19 95.3 109.58 116.49 67.71 92.94 112.5 116.13 147.37 30.64 83.83 71.39 698.4 103.37 59.13 80.87 140.41 93.79 73.93 58.49 57.46 98.24 122.69 74.31 120.69 175.11 474.21 80.46 55.8 164.54 108.56 139.53 176.65 176.78 104.62 143.12 85.92 204.4 24.35 215.92 103.48 144.71 265.33 89.1 170.86 51.3 167.48 94.64 111.88 101.02 138.85 176.41 91.47 133.75 147.23 127.65 97.08 50.48 52.78 104.8 116.61 103.54 141.69 99.72 95.35 50.69 74.84 115.34 109.86 58.82 50.82 73.39 168.51 124.38 127.19 138.1 226.79 73.32 131.53 82.76 114.55 78.52 112 120.82 72.41 116.22 121.06 145.35 255.71 64.42 91.74 99.48 108.63 50.85 80.36 106.78 116.77 193.91 118.23 124.66 84.9 151.23 117.09 68.83 210.59 128.12 165.72 129.08 124.61 119.02 217 135.47 152.74 152.15 91.3 137.99 42.81 174.88 101.84 85.62 92.57 113.54 31.34 62.54 88.14 130.19 208.99 95.04 130.72 102.84 58.81 91.72 118.78 126.7 107.88 136.79 120.27 134.71 149.77 202.23 125.22 71.51 99.4 68.24 126.84 136.73 106.56 116.1 138.37 166.35 158.92 29.66 139.54 180.32 100.76 108.73 146.12 103.39 81.52 127.62 143.82 92.69 193.44 130.1 110.66 114.49 116.09 117.73 115.15 76.15 95.34 119.22 101.03 45.03 122.82 225927_at MAP3K1 173.67 637.98 241.05 1269.63 281.75 497.18 275.3 612.5 229.17 361.02 620.64 493.93 872.31 394.15 279.34 296.2 247.91 405.34 580.72 386.58 561.51 567.22 280.43 321.85 374.67 465.29 341.92 179.15 421.63 590.09 437.12 226.6 422.37 154.86 201.12 575.81 284.02 223.37 372.03 351.6 412.3 434.54 366.28 241.1 166.75 574.02 384.16 398.11 454.06 1141.16 321.5 564.72 337.8 637.29 217.16 474.51 453.73 401.87 385.56 191.56 504.28 264.97 291.92 241.98 635.29 330.83 176.96 190.93 316.61 193.51 190.17 278.47 238.8 403.56 368.9 1077.39 604.97 602.62 694.81 360.43 221.69 838.09 873.33 579.81 299.48 709.23 318.75 421.55 488.05 809.75 90.24 699.35 439.53 733.55 617.32 330.6 396.78 287.66 1040.1 291.13 200.74 600.74 579.39 705.65 427.29 734.02 900.58 777.92 606.29 15.56 330.1 828.33 329.28 292.18 535.73 404.77 59.88 179.33 310.21 272.62 395.83 154.94 209.3 367.6 321.43 516.34 231.82 500.59 905.52 616.31 356.22 198.12 311.66 464.09 716.78 393.37 520.53 1128.27 319.66 319.73 765.34 270.63 530.63 681.82 371.45 132.97 189.17 293.23 301.42 1457.06 309.4 332.33 311.29 464.49 939.25 301.59 272.48 289.05 1445.28 425.87 228.35 760.97 289.73 271.29 303.19 375.51 945.05 509.34 64.26 724.62 453.73 329.48 489.08 367.7 70.47 360.16 423.95 541.28 1029.89 825.01 751.9 172.83 382.9 849.41 307.05 378.02 496.71 560.56 238.14 243.55 459.38 1021.76 501.25 544.36 647.68 411.12 288.07 858.96 247.83 332.06 302.75 403.13 1218.87 141.31 198.12 538.49 491.86 317.42 276.71 868.52 173.67 408.72 302.24 314.49 361.11 295.27 1010.3 198.82 263.67 697.66 396.61 209.43 3163.8 343.64 410.53 617.64 522.52 225949_at NRBP2 670.28 334.15 302.01 174.81 372.42 1252.82 351.89 1104.5 143.88 257.79 245.13 551.77 232.24 400.91 452.36 262.91 57.03 445.5 229.28 349.28 237.09 190.81 572.36 421.8 326.68 174.12 173.56 242.3 297.81 414.27 254.05 201.15 247.63 471.73 105.28 168.36 512.97 395.72 200.1 244.57 200.32 285.6 238.68 241.68 320.15 258.24 192.42 268.31 325.88 489.03 262.88 200.54 231.38 364.05 456.75 286.74 750.72 274.89 398.69 310.46 337.41 304.38 258.26 152.01 94.46 417.89 258.71 102.85 325.91 166.75 263.79 118.15 157.22 132.18 143.3 97.94 109.44 137.12 230.91 398.76 209.34 101.56 227.17 166.08 125.13 366.79 178.37 123.59 149.93 198.92 457.43 198.98 309.92 325.47 307.87 176.03 268.56 281.91 409.05 306.12 1542.69 340.55 487.75 513.96 239.14 251.32 259.68 972.1 307.83 1510.86 221.21 390.36 157.87 239.57 375.2 257.62 478.98 332.17 243.03 565.59 261.99 495.72 196.21 191.04 267.48 328.21 238.7 295 367.65 207.61 204.71 230.77 433.84 158.23 267.4 396.44 410.08 203.1 239.34 166.8 245.53 182.01 377.77 163 194.95 162 139.43 308.69 383.36 149.85 335.89 423.42 293.62 380.25 195.29 442.57 480.46 147.77 297.55 234.65 299.03 441.02 247.44 310.85 209.96 289.66 166.27 214.34 311.97 462.46 169.76 277.35 277.9 451.43 151.85 190.82 264.38 352.9 381.11 292.88 219.28 315.49 141.57 314.61 378.53 361.52 175.02 219.54 281.82 119.69 242.12 227.92 304.6 245.64 499.8 194.23 350.09 271.82 272.5 602.58 476.96 384.94 291.31 120.9 275.95 209.55 392.14 558.1 472.68 290.31 418.69 248.66 374.3 498.64 488.99 321.44 138.1 129.51 115.34 408.21 323.65 537.28 547.51 865.54 404.71 429.26 290.56 225984_at PRKAA1 127.49 59.34 93.05 63.13 50.81 56.88 68.94 67.08 85 81.03 91.63 114.48 105.1 32.21 129.2 85.11 212.99 66.06 110.24 145.64 73.17 78.17 50.61 58.06 90.23 119.53 155.12 307.31 107.02 52.78 152.73 64.47 64.87 26.62 58.82 40.36 102.39 58.14 66.24 36.56 97.93 61.75 82.82 47.22 46.66 86.58 43.21 62.02 65.83 82.16 75.26 105.3 40.86 121.82 42.98 41.89 86.75 22.71 36.2 64.94 100.89 73.37 104.84 22.87 63.12 69.83 65.42 51.04 87.33 84.9 66.88 46.01 95.99 95.53 52.06 141.87 79 61.22 15.25 56.77 25.84 86.14 58.1 67.3 102.67 108.57 75.27 73.39 47.96 123.88 13.17 118.56 69.03 136.28 177.5 136.42 71.68 27.82 64.76 64.83 112.43 211.54 43.12 79.06 54.82 48.82 65.37 46.39 29.55 19.37 40.66 61.5 42.6 47.39 72.63 71.39 49.75 47.33 55.82 74.82 61.27 65.09 28.46 43.92 63.47 39.55 57.39 66.95 62.55 49.36 54.5 47.49 45.09 48.36 57.75 33.6 32.68 97.54 41.84 67.84 60.63 168.66 50.48 38.89 53.07 46.76 69.74 40.69 47.65 81.35 44.93 51.44 60.53 64.62 59.93 35.56 24.05 43.47 59.74 57.13 38.94 71.78 49.2 25.96 65.83 45.53 47.87 52.16 50.26 12.83 33.45 26.19 42.77 47.9 16.76 48.61 41.33 17.02 73.86 35.68 48.94 52.68 30.59 41.97 50.96 22.52 36.7 40.14 80.82 44.81 55.92 100.26 30.05 51.59 51.79 33.18 51.19 47.12 42.95 58 15.31 36.51 42.49 23.1 44.45 57.12 84.26 52.65 42.13 60.31 37.55 42.15 30.68 66.09 24.15 37.45 41.49 71.2 79.69 61.13 52.52 60.72 52.07 59.65 58.34 57.08 72.94 226048_at MAPK8 225.88 189.95 100.19 128.87 167.27 190.03 89.81 135.82 183.01 169.13 185.22 190.64 238.9 248.68 126.91 184.07 417.98 180.93 211.34 219.14 147.38 184.16 395.36 128.94 176.84 194.01 181.41 167.03 285.74 104.31 142.2 183.33 160.98 135.19 353.22 80.29 210.86 227.75 91.6 103.27 206.87 103.26 321.34 226.16 181.05 118.68 154.18 136.16 86.52 122.92 397.68 160.25 93.28 258.74 196.68 137.72 232.45 105.1 117.05 82.02 168.14 296.67 137.27 89.99 47.06 125 165.73 154.57 191.16 143.34 142.61 219.12 161.77 105.83 151.07 164.95 192.21 167.8 135.78 161.91 58.56 174.09 130.83 346.31 101.61 130.13 207.16 89.24 75.38 174.43 31.82 176.54 162.15 240.17 141.94 257.89 145.93 294.22 211.74 252.86 171.62 99.5 196.95 163.16 152.81 110.84 152.92 163.91 89.57 35.17 197.94 165.81 179.18 214.18 121.5 149.9 133.21 263.9 132.52 90.58 96.7 120.7 162.32 126.7 97.2 164.96 134.05 132.3 148.69 172.53 89.93 93.47 51.81 84.76 196.38 162.61 84.31 92.68 95.4 109.64 106 108.81 147.6 143.7 97.8 73.38 117.9 200.38 95.38 163.44 342.29 78.08 145.17 113.66 120.59 171.57 138.1 108.18 151.58 82.06 168.59 173.05 149.92 66.28 70.04 96.46 160.01 86.93 26.29 121.19 122.25 105.13 120.18 114.5 59.42 72 127.45 135.33 114.78 79.63 135.52 161.67 113.41 117.01 172.62 99.43 98.47 160.22 169.78 103.33 71.83 127 131.13 101.11 220.68 97.8 211.83 90.62 170.42 176.31 123.25 126.33 178.97 76.99 174.23 161.02 261.58 86.31 114.33 187.47 111.63 121.4 86 49.98 56.8 135.98 53.6 72.7 95.91 81.39 83.51 119.43 118.7 107.71 107.27 121.59 75.8 226053_at MAP2K7 116.96 129.89 146.63 153.7 153.28 186.04 90.41 174.33 113.57 114.25 130.03 123.76 116.51 158.42 115.61 114.61 110.36 139.62 267.95 143.57 139.07 128.32 206.14 141.46 158.01 94 111.13 109.63 92.73 102.25 86.08 90.6 104.76 201.05 94.55 129.43 175.57 165.22 144.01 126.3 129.22 218.1 278.51 137.25 94.05 116.65 101.25 87 117.11 166.41 107.09 89.64 167.18 127.41 178.09 114.22 182.24 67.78 132.9 104.7 132.47 213.25 88.1 81.99 145.87 97.27 198.32 112.51 103.04 118.6 152.06 126.74 109.2 104.71 115.27 112.55 110.57 80.89 100.15 116.61 115.12 103.32 96.56 114.11 99.54 113.45 105.47 100.83 113.16 85.61 93.02 139.17 108.57 64.83 64.26 63.31 92.37 156.1 63.04 83.92 188.8 130.66 147.75 94.04 121.89 148.66 123.26 197.28 109.23 132.48 142.81 145.7 131.03 144.01 147.7 85.78 253.93 108.16 142.18 167.36 141.28 239.9 159.74 199.17 110.44 80.87 101.95 92.89 58.59 90.9 99 94.61 121.22 86.7 95.55 98.18 96.73 94.88 93.88 93.64 75.25 63.85 127.99 101.78 88.27 80.76 125.16 132.22 143.14 101.67 77.22 152.2 134.19 110.29 98.08 135.6 86.83 94.55 105.22 114.43 193.15 121.07 126.25 126.73 131.93 126.02 130.92 102.32 108.07 112.47 163.38 160.62 145.09 158.08 162.62 173.61 117.47 134.37 153.83 166.09 132.04 133.68 141.92 189.88 158.02 149.42 97.8 137.8 148.4 282.95 156.93 117.68 133.64 125.69 136.62 116.38 120.02 139.64 113.35 132.73 171.46 109.32 101.28 141.24 145.18 132.5 159.08 99.43 87.13 96.85 194.64 137.04 173.61 138.95 136.79 144.44 107.44 91.64 89.3 155.55 107.97 161.72 190.08 205.68 179.71 213.56 142.41 226054_at BRD4 192.07 304.77 612.95 432.03 425.9 340.3 169.33 294.96 224.23 674.01 361.46 341.06 308.09 592.09 304.96 290.36 243.6 250.74 448.05 254.73 256.73 151.67 413.94 352.73 240.35 245.14 172.33 216.77 315.59 200.2 135.41 340.09 196.82 638.37 303.43 341.3 423.99 235.63 317.12 183.47 195.42 371.59 250.88 306.34 299.75 257.61 303.89 300.18 218.47 237.1 192.01 206.65 386.22 266.6 263.29 206.95 473.55 268.18 439.42 335.87 369.83 757.1 375.01 503.51 625.68 611.94 793.47 405.56 335.52 349.45 633.87 609.72 384.62 258.46 413.96 317 438.19 217.75 143.48 223.57 360.99 323.48 291.26 285.55 254.33 217.98 302.33 367.27 349.64 483.72 735.51 383.83 430.86 362.03 517.76 301.62 334.89 478.89 426 358.19 519.86 325.93 330.56 377.6 377.6 391.91 209.49 296.55 338.04 446.61 396.39 283.64 309.41 478.53 337.53 196.93 456.92 510.17 478.17 648.87 595.3 785.94 574.18 528.57 246.71 165.64 228.02 267.59 158.3 188.26 266.34 258.12 566.3 249.27 239.36 240.05 209.24 194.58 216.32 241.13 162.28 285.1 224.39 202.86 228.64 404.83 243.6 247.07 353.81 174.48 483.95 396.66 318.88 177.87 250.28 472.83 157.83 249.33 325.84 275.53 332.98 244.46 171.51 292.96 216.32 241.69 285.48 344.63 642.33 297.4 279.52 323.64 291.1 288.48 695.98 396.15 285.52 243.61 338.75 350.43 247.97 336.71 209.5 294 328.11 267.35 241.82 226.83 334.12 295.72 336.53 221.25 228.18 196.9 224.85 258.71 243.3 323.42 284.23 435.28 269.01 230.12 224.4 335.39 450.24 275.45 407.01 239.1 249.91 148.05 295.42 340.02 284.83 437.08 311.49 236.5 444.42 147.98 181.59 395.97 371.95 413.52 566.43 431.84 404.62 428.76 334.77 226068_at SYK 81.93 76.02 148.74 196.5 164.3 141.13 57.88 39.71 145.33 156.38 120.63 129.9 160.64 38.53 73.28 65.17 105.23 249.67 219.43 279.35 170.39 131.35 115.14 174.81 138.65 299.9 326.57 246.37 163.44 104.01 87.28 56.45 61.65 95.33 154.6 166.88 190.48 171.58 260.58 280.53 224.98 280.64 87.67 220.46 86.02 134.43 185.08 249.65 206.33 139.91 44.93 123.07 109.89 216.08 97.64 78.63 143.08 195.95 218.48 89.73 44.55 229.67 258.2 78.15 1186.12 147.34 218.24 347.2 62.53 139.82 140.58 132.58 106.91 115.84 71.66 62.45 72.59 167.52 53.06 97.94 81.17 102.57 93.27 113.63 149.49 126.8 101.56 201.4 186.23 115.62 21.06 108.83 55.01 109.01 99.34 118.58 123.15 75.26 107.01 130.39 47.22 111 114.98 66.36 81.36 174.79 129.9 131.1 140.01 27.82 134.55 70.65 224.91 101.8 261.72 96.76 24.41 118.1 161.28 79.27 138.35 53.27 183.05 161.83 131.47 114.7 63.06 138.71 79.17 86.97 210.01 101.29 74.67 133.69 55.85 79.18 164.86 84.42 39.45 178.2 38.24 76.3 126.67 84.58 101.48 80.84 105.56 77.22 113.9 85.34 94.08 273.85 42.14 128.85 115.55 91.28 48.63 286 99.91 241.8 133.38 92.01 247.14 69.55 105.47 177.74 74.46 242.76 55.32 130.01 176.33 58.33 89.66 149.87 20 59.2 237.4 108.66 90.11 87.33 145.08 70.48 95.52 113.98 114.27 125.69 80.35 64.91 57.85 100.23 74.05 67.93 89.15 202.04 52.23 130.6 127.34 99.2 147.79 182.15 34.34 99.18 178.76 111 94.91 86.62 106.96 91.77 106.98 59.53 149.61 104.88 279.71 55.5 71.28 63.87 140.97 77.9 100.6 50.59 264.53 198.44 74.98 62.95 95.81 115.75 177.79 226101_at PRKCE 68.95 76.5 59.4 78.99 82.5 148.99 81.03 160.81 85.71 56.53 160.88 120.31 157.56 55.8 47.65 96.52 44.5 41.5 110.1 78.22 83.05 58.75 81.56 47.29 191.68 63.52 27.08 320.27 41.72 59.04 53 79.65 85.23 132.68 62.05 64.57 46.94 34.68 37.84 49.51 43.75 61.61 58.76 74.5 67.96 70.16 42.16 69.71 46.02 33.75 41.36 93.21 75.78 79.5 221.71 52.67 71.99 44.7 48.15 49.75 68.69 21.79 75.31 65.78 417.89 88.34 59.96 75.33 61.98 77.99 44.95 74.92 87.22 47.72 124.12 58.79 40.77 66.37 30.83 118.23 54.03 46.96 83.53 69.37 124.99 61.24 43.29 54.62 39.25 76.37 53.79 115.18 114.15 224.7 131.16 58.62 40.62 67.77 63.81 59.47 60.79 59.68 72.69 131.22 65.63 51.49 66.35 48.25 53.91 40.08 46.13 114.86 95.96 91.01 77.83 73.3 30.43 87.01 97.71 83.36 94.52 137.05 59.48 80.83 86.88 53.93 84.54 62.68 31.39 33.53 51.94 60.95 106.57 51 44.29 61.65 98.5 46.72 51.01 57.99 35.92 35.08 48.49 45.74 63.09 47.94 65.43 179.69 57.36 136.35 42.03 62.49 58.39 36.53 33.4 56.02 37.84 35.87 61.85 62.51 109 59.17 87.53 36.35 70.39 51.03 62.32 60.9 77.83 91.1 47.42 39.08 41.68 39.72 44.84 58.08 39.29 86.43 81.02 208.66 54.53 44.79 43.93 62.19 75.19 43.73 70.62 47.93 27.31 37.36 60.96 56.13 52.12 31.26 55.01 31.09 38.53 46.87 63.2 99.46 57.51 49.82 41.7 52.6 43.55 55.58 44.57 41.27 24.75 33.1 44.35 47.98 51.91 44.76 115.85 44.14 59.2 64 55.87 42.65 43.1 62.53 64.25 69.72 63.98 51.94 68.05 226126_at MGC16169 265.93 384.41 382.6 346.49 516.86 529.33 622.14 286.39 714.56 226.64 453.56 571.82 322.11 434.27 317.46 419.33 257.89 231.07 201.02 181.86 235.31 354.56 353.86 244.29 193.08 455.55 322.55 259.2 167.87 242.75 417.58 332.32 243.36 446.66 170.56 508.22 291.13 154.21 323.83 338.82 337.44 269.08 179.65 205.51 329.03 238.53 262 296.84 311.18 257.03 124.15 351.83 281.75 251.12 231.59 382.75 254.59 199.89 248.79 386.46 207.83 152.77 384.11 215.85 219.43 218.89 245.04 270.88 229.75 138.02 207.44 243.81 179.29 387.38 196.5 350.31 85.26 351.32 384.13 399.91 231.6 463.95 313.19 634.96 318.95 433.65 279.31 176.06 216.78 266.97 102.74 347.64 351.35 240.82 418.95 391.39 535.14 210.79 427.42 380.39 289.11 505.46 318.61 445.16 705.83 272.04 621.46 543.59 308.57 467.45 199.16 285.53 275.67 218.53 384.98 399.33 334.68 193.3 273.39 217.85 315.55 230.53 146.63 168.23 310.8 198.83 589.25 296.87 369.75 275.19 323.67 267.3 323.54 174.21 446.03 304.06 228.13 219.41 321.62 164.32 341.12 409.26 330.63 394.33 263.64 209.05 253.96 347.94 197.45 492.79 187.86 310.76 121.6 265.89 356.58 295.55 318.38 201.33 384.85 148.04 303.87 327.01 295.26 371.56 253.68 303.47 279.56 294.43 114.82 582.12 184.69 300.5 275.24 242.35 153.79 218.61 183.96 326.7 405.89 354.19 235.57 142.66 324.07 223.42 227.07 340.52 310.07 183.68 128.36 600.47 355.19 366.98 244.06 236.93 276.82 192.63 238.84 182.99 282.47 183.43 279.95 308.7 356.79 67.02 183.95 374.59 147.7 161.23 188.27 364.79 222.39 318.42 320.85 365.13 337.55 361.11 259.86 157.31 278.01 285.03 334.39 218.57 367.25 376.65 278.79 154.95 291.52 226190_at MAP3K13 785.44 557.05 399.43 543.52 355.8 487.22 285.14 424.34 440.65 481.72 837.37 853.29 450.81 779.36 369.99 410.32 658.19 874.12 939.76 33.99 336.4 373.88 800.02 430.17 330.2 456.87 564.77 1514.01 522.94 904.71 424.1 935.36 420.37 601.16 814.37 1188.7 1938.58 662.42 283.88 342.67 1202.21 475.25 767.68 346.17 355.83 281.66 315.54 1172.71 358.95 641.92 814.47 563.38 206.93 505.27 969.62 421.21 538.87 590.72 494.49 681.92 950.37 1396.31 483.83 537.83 582.17 1816.7 2183.19 809.49 519.91 1246.04 806.12 824.36 1441.03 436.83 694.06 666.3 87.12 973.54 1310.65 628.26 1433.74 1435.51 1313.5 836.21 579.36 563.49 672.62 668.46 741.91 300.79 1555.17 574.24 578.71 744.82 418.07 310.9 747.58 2200.09 343.17 554.65 841.65 481.28 822.85 486.21 753.98 865.91 572.33 549.61 556.36 1163.09 1525.02 703.74 574.3 985.19 893.58 848.51 618.06 893.57 731.43 1049.12 727.75 1391.01 1481.69 855.43 404.55 1049.65 436.41 404.25 491.21 1357.2 559.2 760.15 594.53 762.38 807.09 580.01 1036.82 628.3 629.03 580.11 954.07 581.03 555.93 648.67 450.11 1692.42 640.41 516.31 497.74 202.04 1140.17 286.11 421.44 469.77 562.52 746.95 738.93 642.12 501.96 1118.66 532.16 736.57 683.36 900.17 722.25 375.33 1214.01 400.28 1335.14 520.87 485.59 702.11 573.77 413.97 697.81 1007.74 1023.13 578.36 379.06 1167.95 554.83 442.73 387.95 871.6 1159.62 692.76 503.54 776.27 613.63 786.46 440.73 658.08 430.86 726.95 552.8 590.71 879.64 702.01 1229.46 1080.52 272.17 866.15 565.67 1062.84 644.86 645.59 2009.49 652.79 547.35 665.93 628.84 747.41 441.07 763.92 595.96 641.15 470.79 469.9 342.31 601.23 823.14 660.87 618.04 563.69 994.92 1787.35 358.05 226213_at ERBB3 952.58 2416.32 818.54 1395.8 1961.42 2375.18 1718.55 1456.03 1772.74 1339.42 2855.23 2985.72 2222.14 3533.65 1971.64 728.98 779.58 637.26 768.69 519.64 4463.61 1267.21 671.04 553.01 1920.1 162.55 1296.39 1547.75 184.69 1130.57 1547.57 1564.51 2582.19 3850.22 1387.77 916.88 1935.24 671.98 303.94 816.09 1509.1 670.04 1633.28 561.93 1135.15 1312.55 83.62 2256.99 1027.2 1400.94 1777.14 2757.2 1696.59 1147.59 3369.44 2370.32 818.02 1963.25 2237.07 413.09 2950.13 2829.46 397.44 2320.55 13.3 366.15 199.36 199.51 737.5 1495.44 491.12 757.98 573.06 840.38 508.67 2262.35 562.52 1353.99 2201.51 355.75 1316.2 1309.97 2048.93 1771.88 1380.82 1179.75 1243.12 923.42 1666.58 1237.64 1074.24 1878.53 2991.91 1226.46 3604.53 1315.94 4019.12 2496.06 2830.86 866.88 2379.47 1178.12 2320.61 3923.35 2173.13 2641.91 2383.24 2128.86 2917.04 1390.95 794.32 1451.27 392.27 557.68 1954.79 734.52 1532.19 723.56 1471.93 1573.05 759.48 1905.44 483.09 1124.49 320.21 2291.99 3273.52 1172.53 1152.71 1156.07 929.43 1196.78 595.19 715.13 1643.76 1043.66 406.23 1318.07 1758.59 1625.53 1443.5 1714.85 867.53 1280.81 1330.75 741.28 2560.22 2554.26 1608.55 2456.41 511.88 875.66 711.74 880.09 2005.39 1031.1 1527.42 72.64 1532.04 443.46 3713.88 1158.3 2265.63 3976.96 2151.56 1331.02 2540.43 1334.77 1308.12 2214.83 1303.25 1750.24 2721.4 413.76 1435.25 1452.39 747.42 2549.18 1862.55 2197.83 1036.2 1692.77 2700.36 2029.08 392.03 2206.32 3159.26 2924.6 1751.3 2361.4 2192.35 1491.49 804.68 1316.79 1061.23 677.42 954.55 1060.37 740.53 999.6 2776.02 1050.95 962.29 602.13 625.09 2310.16 447.12 1001.59 776 2214.14 926.95 779.25 2004.01 1073.7 1349.83 1332.31 763.81 614.47 1521.28 1381.16 578.99 729.43 3457.81 900.73 711.12 1095.99 877.85 226299_at PKN3 23.88 41.03 55.11 31.24 107.55 52.83 74.65 75.28 42.66 31.78 92.05 37.52 71.85 156.28 109.96 126.56 43.8 47.92 116.11 113.84 52.14 32.54 52.12 59.1 31 36.52 78.66 36.48 55.58 178.31 34.12 56.05 37.01 77.25 59.67 57.45 38.19 43.79 79.52 48.4 54.46 40.94 40.19 125.98 126.74 58.9 50.44 53.35 109.12 285.54 42.48 56.35 90.95 56.5 38.35 69.72 58.9 50.54 46.63 137.35 156.34 104.25 95.54 47.68 34.96 47.23 78.51 58.87 26.74 27.67 45.54 47.05 33.14 32.65 43.35 27.35 81.04 42.41 39.86 29.97 72.63 36.4 42.49 27.83 45.24 46.5 29.12 58.74 32.83 56.7 112.8 56.84 68.43 41.3 61.94 27.27 53.05 44.98 33.2 56.69 63.89 111.36 35.79 99.97 42.6 131.91 50.76 67.7 55.79 72.81 207.74 67.39 60.88 52.69 55.22 33.64 61.74 53.53 44.88 100.3 100.37 100.41 40.58 48.3 78.02 50.3 79.31 81.03 33.42 55.99 41.41 42.33 128.04 45.16 68.61 56.03 47.98 51.69 46.85 25.15 59.58 33.17 30.97 41.1 43.01 54.41 37.79 76.87 163.55 94.9 75.99 65.35 60.93 51.96 75.9 104.33 43.4 75.28 65.95 153.23 72.92 69.67 87.46 45.9 61.73 50.41 72.74 79.97 55.55 125.27 39.83 56.04 60.2 54.5 46.58 84.47 117.46 41.4 70.36 142.78 49.11 54.04 65.46 84.93 42.32 84.64 57.53 51.09 46.57 46.99 48.6 74.07 60.77 49.1 90.57 149.74 81.53 87.67 67.14 69.98 57.28 75.24 36.4 112.16 94.64 58.24 47.77 48.28 35.18 28.02 79.46 90.01 85.49 120.39 150.63 54.83 39.2 59.75 49.64 116.22 60.4 43.43 75.64 43.85 83.36 73.27 75.07 226335_at RPS6KA3 199.52 112.99 162.97 211.84 239.22 157.64 158.92 331.29 116.7 162.34 98.32 85.33 122.49 53.75 154.22 165.81 240.5 316.16 263.35 335.08 128.95 285.19 361.05 199.79 258.81 435.37 123.87 220.79 266.6 123 144.07 113.39 156.66 54.37 257.05 124.6 395.96 268.06 348.93 160.97 245.44 273.08 146.95 258.81 165.89 273.07 203.59 234.32 180.18 156.3 127.58 187.58 163.45 210.74 65.57 127.49 139.13 156.34 226.89 184.66 67.03 146.67 257.02 79.84 155.52 128.03 109.98 202.86 172.39 126.42 144.93 171.05 143.97 139.92 88.46 58.83 207.88 178.93 28.96 107.68 46.25 87.14 117.94 69.01 197.63 132.87 85.92 179.09 129.82 161.48 27.86 157.79 46.69 71.03 116.41 123.04 126.63 300.28 216.9 173.07 213.73 582.35 264.31 83.29 144.42 170.72 105.67 128.04 109.29 15.52 102.55 135.89 180.98 130.88 194.9 150.65 132 81.59 227.93 93.35 156.98 73 72.3 156.49 222.64 147.8 104.26 232.73 95.51 144.64 237.84 63.1 149.41 258.53 86.33 140.78 210.29 169.4 131.85 326.09 97.87 76.45 104.99 101.42 228.31 51.49 67.7 132.38 288.11 158.51 174.09 239.08 326.95 310.6 330.06 199.45 67.42 368.15 254.92 620.71 143.43 296.38 100.24 95.04 58.22 296.79 88.83 252.67 21.64 75.33 158.71 84.03 140.39 225.35 128.23 126.66 250.85 110.1 255.72 46 430.56 105.22 99.41 159.12 189.41 111.07 170.21 135.26 133.44 38.13 91.32 313.29 108.68 159.35 187.55 124.49 130.01 160.9 193.65 167.77 106.9 101.99 245.66 21.15 177.51 97.28 118.51 163.18 130.36 97.9 120.21 155.4 112.55 85.62 50.31 95.96 98.32 123.84 123.84 81.03 266.98 121.22 125.69 160.15 129.4 173.69 219.72 226452_at PDK1 661.82 152.59 361.56 393.5 189.21 177 138.14 152.04 156.14 970.91 152.49 93.62 136.91 125.33 137.02 314.12 342.15 526.37 742.81 670 91.36 200.81 660.99 341.09 394.84 442.16 305.93 535.17 664.33 503.51 260.56 275.28 142.69 209.09 544.73 367.02 459.6 141.35 397.11 301.41 271.76 370.16 661.92 391.99 141.09 139.07 545.43 312.13 123.46 112.43 91.25 133.72 94.89 372.72 94.18 126.49 338.65 218.58 219.97 257.95 86.95 614.86 561.14 253.33 784.57 746.42 275.76 205.5 514.18 321.5 622.04 230.24 379.66 370.33 248.52 262.6 350.68 152.12 90.38 142.51 136.25 150.82 113.01 153.58 121.22 115.36 112.11 190.67 649 183.51 30 238.4 78.78 76.77 147.25 211.67 58.45 261.08 94.85 282.04 231.08 225.56 247.95 72.94 97.05 110.19 56.91 71.97 62.14 13.75 205.31 141.16 442.62 1450.7 534.95 434.76 114.7 208.63 218.87 94.28 141.04 312.27 644.49 309.99 116.08 122.74 224.06 89.95 303.53 501.33 249.33 94.94 147.91 196.66 93.93 172.12 91.82 317.76 133.79 237.5 340.44 309.15 58.6 85.57 119.78 121.95 238.57 82.25 153.21 216.97 343.45 148.36 300.06 115.75 308.66 243.72 64.96 562.18 89.99 214.49 92.87 137.44 100.25 50.05 160.85 138.26 87.29 206.44 102.08 79.9 219.77 40.43 64.71 187.27 24.64 173.22 197.52 106.02 90.53 73.5 95.66 234.05 77.21 66.89 227.41 51.35 117.4 137.17 319.84 211.87 77.71 128.76 103.83 231.97 202.27 326.2 538.34 101.83 210 221.34 54.83 77 122.47 26.18 558.24 85.19 423.27 393.4 423.21 146.05 95.32 110.26 158.61 123.22 93.19 120.05 304.7 333.88 92.54 130.34 279.62 459.31 57.44 271.98 722.49 257.14 158.69 226498_at FLT1 112.59 97.65 143.48 82.64 137.9 54.64 55.37 117.07 110.39 68.61 75.97 65.7 68.37 84.88 73.29 111.09 201.56 42.67 100.76 135.32 105.45 114.93 96.61 76.94 99.71 50.61 176.48 71.5 123.7 149.28 109.39 74.29 53.25 89.69 44.43 93.03 94.52 67.23 98.44 30.65 36.63 103.26 154.41 124.7 101.24 253.91 83.77 47.65 114.97 98.02 65.08 71.98 76.9 131.13 73.5 82.57 91.07 132.32 56.73 184.67 43.73 82.68 124.69 22.51 11.64 61.87 20.63 55.19 48.68 73.41 39.6 71.82 74.22 61.29 64.07 35.46 54.84 38.82 20.76 31.7 43.93 45.38 16.2 34.06 76.48 36.57 60.02 37.84 18.52 39.6 21.29 35.73 17.31 37.87 21.48 57.74 52.61 55.8 26.5 58.23 50.62 91.92 56.28 32.56 40.86 51.68 59.14 47.28 50.14 10.43 14.38 59.75 32.3 91.03 36.41 87.84 120.31 26.96 16.07 44.31 76.59 36.35 19.98 19.15 81.78 50.01 40.09 99.26 83 48.44 79.41 54.86 92.58 71.47 140.8 35.43 105.37 101.62 62.9 162.09 53.56 111.21 19.83 42.95 100.7 65.23 57.34 71.08 67.15 51.08 77.88 33.44 159.33 101.98 56.1 140.93 42.44 72.92 43.3 38.22 18.79 79.92 40.52 33.33 100.08 44.36 47.03 49.04 12.15 15.92 43.48 57.01 59.14 59.86 9.02 94.17 93.51 67.79 47.84 36.89 45.27 207.89 64.35 76.93 87.15 48.52 50.01 151.91 107.13 106.56 71.63 44.21 91.56 129.23 51.99 136.3 79.03 100.4 71.04 78.03 23.32 55.17 59.17 12.24 154.96 36.72 27.95 74.89 60.87 64.29 48.44 64.1 37.76 35.39 34.21 76.7 77.24 99.74 57.7 42.58 74.82 161.68 111.45 117.34 128.43 110.87 72.06 226507_at PAK1 194.01 372.14 481.4 346.68 468.61 889.73 191.97 1282.32 205.77 296.68 238.97 250.05 212.27 1404.38 145.01 212.63 321.98 299.84 171.35 292.48 285.11 162.48 415.01 272.6 181.74 336.19 144.52 271.02 293.05 131.17 279.7 337.43 311.95 435.75 293.65 164.92 441.12 256.35 180.22 392.29 305.58 241.93 254.96 210.53 259.22 229 415.68 319.63 427.46 235.58 442.85 191.86 201.02 373.28 166.77 424.26 162.29 597.06 225.03 291.05 396.68 766.62 393.06 130.9 125.88 306.68 298.01 217.21 186.79 289.97 331.06 187.12 281.96 496.15 344.88 298.08 233.17 369.56 207.82 228.79 362.73 342.06 2607.79 246.54 248.1 1011.39 230.03 269.79 327.06 377.62 467.11 297.51 376.47 4415.56 474.33 338.58 205.57 361.19 834.96 357.37 437.38 206.25 274.37 405.82 223.39 332.29 254.6 300.45 397.33 140.11 215.42 279.72 300.99 293.4 312.43 479.15 110.09 245.74 237.51 353.38 187.41 316.74 328.49 275.49 211.59 673.81 214.06 249.22 668.51 288.31 639.5 138.17 783.95 203.44 231.89 814.65 242.62 213.71 185.83 214.98 1209.04 155.01 170.72 211.1 154.03 97.98 192.13 236.12 179.51 457.66 328.13 441.74 300.31 208.32 203.86 162.94 151.06 315.25 252.84 393.38 374.34 245.98 327.37 224.19 191.33 183.82 315.68 262.06 224.64 312.15 1164.5 540.61 393.07 314.16 238.18 139.57 344.84 325.84 226.19 2235.79 323.7 257.03 200.44 490.39 210.1 159.99 159.06 216.64 288.04 238.3 184.02 192.68 245.29 188.46 209.56 224.46 217.48 220.52 246.89 343.94 200.82 164.99 389.73 312.82 234.42 272.36 291 219.7 185.09 268.69 273.55 143.66 202.66 152.92 457.16 1068.75 214.39 232.33 163.29 2073.24 355.27 263.2 799.43 141.56 257.13 144.25 273.26 226548_at SBK1 75.78 123.27 158.42 149.21 251.17 289.01 102.4 336.51 133.51 173.18 189.89 180.42 98.39 152.51 91.97 56.96 33.58 191.07 148.01 129.58 242.84 215.4 110.65 106.96 271.01 114.59 91.45 586.69 87.61 136.39 161.27 75.95 96.83 177.8 182.87 207.98 88.44 156.47 46.29 58.95 72.6 59.33 78.09 117.17 174.72 80.62 173.47 71.39 59.57 85.47 100.83 113.07 147.22 137.4 317.32 63.74 37.98 84.8 247.26 49.15 298 387.21 83.06 149.62 40.84 178.28 479.8 71.64 432.14 260.9 236.37 159.67 101.63 102.05 211.19 316.17 180.25 123.88 220.53 187.61 285.88 237.2 70.33 242.62 133.96 194.07 225.6 80.52 270.53 137.31 503.42 132.73 240.4 127.83 423.33 216.66 189.81 482.92 180.16 364.19 503.8 55.42 497.74 173.59 280.6 200.64 162.93 97.96 99.48 148.31 304.14 53.32 64.26 166.32 140.08 52.47 636.75 104.83 150.23 153.26 368.47 841.87 368.99 187.71 42.9 129.02 220.15 80.29 33.48 146.84 202.43 116.78 109.52 164.73 275.98 307.33 249.15 131.32 389.39 225.05 260.7 46.64 254 381.05 108.05 341.86 434.48 254.26 125.54 209.29 231.3 125.18 61.15 105 147.28 250.81 77.16 44.36 94.34 84.92 107.88 94.59 62.74 102.87 77.04 70.57 155.34 287.3 245.24 71.66 167.61 300.38 280.43 119.67 1859.35 265.48 47.23 114.65 180.6 379.79 68.88 81.38 89.11 101.37 74.82 91.23 228.51 166.98 93.35 363.2 322.48 60.14 126.29 266.15 56.36 259.78 168.91 165.04 329.42 198.49 136.72 197.17 159.61 361.19 72.58 89.78 290.98 228.6 52.78 184.81 121.78 177.73 114.71 134.14 235.68 214.42 105.44 320.08 97.22 104.87 71.76 419.24 220.23 342.62 209.23 237.87 47.99 226551_at RIPK1 122.85 211.55 158.91 186.83 207.14 172.15 134.48 226.16 182.54 178.19 188.53 150.48 142.13 206.63 177.9 146.02 176.58 175.61 176 325.15 162.77 206.38 81.24 149.79 119.76 243.18 193.66 104.96 107.86 138.57 136.69 150.86 149.54 204.31 131.54 237.44 122.83 142.28 134.72 122.07 191.37 153.19 81.36 135.1 123.48 120.17 110.53 112.57 241.48 145.68 67.91 181.88 116.33 109.54 122.96 120.73 102.5 122.25 152.92 129.03 119.18 120.99 275.83 126.72 106.43 178.72 123.46 120.49 118.82 135.66 92.26 140.08 152.39 162.22 73.59 90.79 185.18 154.48 89.06 132.15 121.58 200.34 147.61 141.54 147.99 186.3 177.3 147.11 143.86 144 114.77 125.78 141.2 89.96 129.58 145.55 135.76 163.42 106.54 127.01 118.25 139.49 133.81 107.45 127.31 153.9 132.86 133.01 102.91 70.09 66.31 104.13 109.96 132.78 144.8 78.47 167.47 138.85 99.66 130.82 139.29 117.16 132.72 169.76 125.33 135.7 154.08 149.33 97.51 110.83 146.28 118.02 119.3 126.49 143.25 115.9 153.16 140.14 154.68 142.61 119.19 133.25 182.31 191.33 115.05 116.44 118.64 155.46 135.68 163.75 112.52 171.93 118.74 130.04 150.51 193.89 113 146.2 159.76 155.97 180.41 145.52 80.15 133.71 142.02 169.46 168.31 148.7 137.46 128.73 148.11 125.2 139.74 136.1 113.22 121.71 131.87 110.9 113.85 213.35 117.28 62.59 158.63 124.12 133.37 127.38 128.53 110.81 74.22 125.7 102.3 131.02 131.65 137.69 111.4 153.52 124.9 109.46 165.97 92.12 156.28 150.26 145.37 140.56 112.14 123.89 142.1 111.63 87.87 143.99 108.29 176.33 144.32 116.19 132.83 133.54 148.47 175.37 123.67 136 209.8 179.55 131.29 107.08 129.04 130.61 114.82 226653_at MARK1 19.92 10.91 25.64 23.16 50.37 14.91 11.2 39.6 48.49 13.68 19.13 27.48 41.51 12.82 24.09 25.88 36.22 44.53 48.16 55.03 47.83 29.62 151.42 36.57 27.21 32.7 21.53 117.93 26.27 34.73 29.57 8.93 61.94 11.99 34.88 31.97 66.36 17.87 22.56 15.15 71.73 22.79 49.5 53.22 42.38 31.14 90.96 55.84 16.61 63.66 49.59 25.62 28.64 47.1 14.88 17.79 71.11 17.32 17.26 11.08 12.45 27.38 12.23 9.2 12.78 60.85 20.76 63.88 7 43.26 28.99 35.74 14.59 15.19 20.59 25.94 13.24 16.41 13.46 13.99 33.71 18.02 22.7 13.49 23.34 20.71 15.48 17.04 18.22 27.1 16.23 16.51 14.06 10.08 26.01 9.69 13.62 53.64 35.18 22.2 34.93 26.14 72.49 21.66 14.64 31.96 14.2 15.34 19.62 11.09 120.73 30.38 13.56 12.12 19.1 47.16 46.46 16.8 13.61 12.81 24.95 21.17 18.24 10.13 36.6 16.99 30.04 45.54 18.85 72.53 19.42 23.37 29.61 16.11 25.56 24.57 14.67 37.95 28.88 11.4 10.44 12.83 11.41 52.06 17.44 10.6 31.84 18.35 13.49 9.76 48.35 15.65 10.98 10.99 9.56 15.12 8.84 7.79 11.15 12.48 15.2 31.15 6.98 12.49 7.17 13.5 11.41 15.35 18.06 10.17 18.45 12.95 24.36 27.23 24.59 14.35 15.53 45.21 16.38 10.72 39.8 17.7 17.88 28.13 10 11.25 18.37 32.4 21.03 16.79 11.06 19.7 10.27 25.79 27.54 14.81 62.3 13.68 20.48 11.23 9.06 16.8 13.24 24 14.03 22.23 19.33 39.91 16.94 21.11 13.56 23.37 19.54 16.02 12.63 18.14 10.42 35.82 9.17 11.78 35.91 68.56 9.98 53.98 26.98 38.86 22.3 226660_at RPS6KB1 220.99 507.7 619.81 381.66 268.22 268.17 430.04 685.63 552.87 416.12 1420.69 392.33 325.06 428.05 298.38 261.94 158.67 269.64 389.83 494.81 2817.12 462.63 485.79 205.14 196.9 293.6 297.03 596.74 298.02 208.49 2208.34 293.3 3025.51 437.2 204.1 243.91 201.43 177.76 186.67 153.22 313.81 235.96 276.43 238.37 121.93 238.93 259.86 311.02 163.23 148.9 503.7 225.93 622.38 236.22 851.39 395.46 186.64 158.26 219.04 199.58 321.08 215.37 334.84 426.33 229.89 148.83 241.04 155.93 275.32 497.33 120.19 213.97 179.44 190.02 388.84 223.02 251.78 289.51 290.41 218.17 408.23 180.81 504.7 238.2 1108.93 333.73 228.84 427.12 773.28 399.87 51.46 242.07 564.75 602.6 411.66 391.4 631.41 210.23 495.83 217.52 328.8 219.23 353.97 312.53 366.64 319.5 253.91 315.82 515.87 80.02 163.85 305.48 268.18 267.11 302.62 239.73 487.05 146.65 215.38 92.22 129.41 125.08 145.32 170.76 225.35 519.72 1044.81 242.13 320.7 172.71 356.69 275.62 132.24 289.53 512.52 460.4 190.12 272.18 387.11 180.97 323.51 380.66 266.62 534.8 233.2 142.63 346.92 2527.14 203.07 394.68 211.59 224.62 170.41 512.04 297.77 153.19 556.21 174.69 323.98 302.78 311.22 312.32 405.17 236.82 292.34 339.3 313.09 490.18 218.39 324.01 463.62 220.87 250.22 205.93 57.42 122.79 203.97 234.47 194.8 321.26 263.58 218.42 739.41 307.3 186.03 236.88 2133.44 595.32 208.34 340.29 166.95 311.99 231.57 281.78 288.14 399.68 314.8 581.11 197.61 146.81 254.57 534.96 328.63 149.46 191.69 231.84 371.14 236.68 244.76 419.53 158.37 278.76 253.13 282.77 258.63 439.59 291.08 343.91 374.64 237.82 196.58 167.67 227.06 144.03 157.37 206.37 191.91 226705_at FGFR1 176.31 182.94 411.91 752.66 3930.51 1581.16 1899.7 2969.34 2393.34 462.52 234.49 2849.03 176.93 682.4 321.38 785.04 83.69 97.82 133.01 47.35 238.34 147.89 269.14 105.98 66.11 208.66 152.74 297.36 244.74 59.5 209.83 161.69 144.72 150.35 221.46 65.81 177.25 277.27 248.56 168.1 139.48 105.83 77.37 264.38 227.57 400.34 469.87 88.67 215.92 272.64 904.62 339.5 972.46 220.11 298.43 269.15 68.31 127.74 232.54 419.45 304.82 111.59 202.7 173.09 135.26 121.85 855.61 99.76 113.48 261.77 247.08 120.49 141.62 158.69 311.11 96.3 96.94 162.74 109.27 1455.35 343.13 211.7 164.78 154.11 309.53 253.13 295.4 228.44 200.6 266.97 122.69 239.4 123.36 380.31 380.93 107.5 283.92 190.76 529.29 161.37 774.42 306.51 285.82 156.73 439.25 217.41 240.05 260.88 330.56 605.57 231.27 397.17 209.06 182.62 167.3 815.74 5456.5 123.06 173.15 279.09 641.05 283.24 361.31 178.65 455.05 57.55 145.35 253.05 100.36 123.66 196.83 159.27 531.9 144.17 119.24 1995.08 349.99 159.86 185.77 329.08 107.37 118.78 419.36 206.27 176.06 169.31 159.9 250.98 542.26 213.33 320.12 295.26 201.37 193.82 115.48 137.87 67.45 105.98 242.85 58.88 152.04 431.73 251.8 117.38 384.29 225.66 167.4 235.74 213.11 117.14 762.82 256.34 208.35 394.94 125.37 221.79 116.87 133.59 338.63 67.14 249.03 459.71 169.91 202.94 189.61 126.23 91.63 268.28 384.97 105.42 193.9 210.8 256.76 147.91 487.59 173.14 172.86 262.59 188.97 144.8 162.22 174.79 213.73 102.2 115.73 241.58 129.16 304.14 102.03 149.27 180.54 265.8 298.76 223.58 136.6 169.19 218.48 125.53 295.74 111.38 213.41 202.2 410.24 186.26 214.24 85.53 318.83 226853_at BMP2K 141.17 188.55 95.07 298.94 161.5 141.01 185.75 155.17 118.43 149.19 144.42 146.19 341.46 59.66 93.94 196.27 96.3 98.92 218.45 120.09 156.29 203.94 90.4 66.11 118.22 325.38 249.98 134.54 165.26 78.36 129.07 68.78 125.71 42.17 125.42 134.07 160.82 170.31 209.51 182.65 191.36 158.9 63.1 174.74 123.71 200.87 102.24 143.67 180.58 103.47 118.93 93.43 141.22 278.44 99.85 138.62 84.1 201.76 144.48 116.75 75.29 49.55 207.56 59.4 177.09 60.7 82.05 246.8 135.01 88.28 64.19 104.75 101.41 169.03 68.66 84.69 119.62 206.66 65.56 107.63 105.52 324.37 149.14 326.62 136.25 120.56 104.56 152.09 148.75 66.6 35.3 113.13 71.88 137.93 68.53 229.13 151.1 89.21 71.69 120.26 109.91 209.62 157.68 48.77 113.29 182.87 460.19 177.69 210.37 18.88 97.02 255.24 209.75 159.83 326.38 220.23 122.25 73.4 156.08 150.69 170.45 109.46 165.79 141.28 121.39 112.98 104.96 130.28 130.04 134.58 178.22 83.31 87.05 186.54 129.51 95.79 138.45 238.03 111.83 348.28 244.92 119.75 82.04 133.65 133.52 57.31 117.38 118.73 99.99 152.8 114.55 137.11 121.12 181.97 196.6 98.05 84.24 226.33 156.96 103.74 112.91 213.3 293.25 59.83 147.3 121.94 93.91 180.3 38.39 99.54 151.43 227.57 155.3 175.68 82.21 171.72 201.25 175.74 194.33 65.24 157.69 139.03 300.07 101.23 118.77 128.59 74.27 87.35 113.71 124.79 198.26 318.14 156.31 214.2 127.6 187.7 157.63 106.52 282.9 9 80.31 192.52 595.65 105.57 85.72 212 111.85 84.37 186.22 245.16 120.55 118.81 113.2 147.35 92.77 165.41 145.68 152.14 251.45 144.59 276.84 204.86 176.52 169.13 139.36 116.26 182.45 226888_at CSNK1G1 127.24 219.05 162.2 121.45 253.11 164.96 257.95 326.79 211.26 94.86 268.1 247.74 162.72 195.52 110.63 137.09 163.44 131.28 294.35 207.23 185.95 225.2 207.39 107.76 75.24 178.84 107.03 183.79 125.12 129.59 142.82 121.56 266.5 187.58 215.02 104.19 119.56 214.8 98.73 122.98 144.12 133.08 95.52 108.43 75.79 107.97 181.35 207.62 106.97 106.07 169.57 201.83 659.2 119.97 227.43 100.82 121.79 112.19 112.12 109.33 220.23 99.97 156.54 159.45 113.84 100.08 165.49 127.26 106.73 111.86 114.94 150.48 74.44 129.92 99.37 90.19 151.06 178.41 129.42 110.94 76.11 249.32 216.84 206.55 175.38 178.11 156.4 189.94 126.04 287.62 32.32 243.4 262.3 137.59 283.76 238.29 254.38 110.75 484.79 148.72 186.37 159.7 143.69 234.56 140.32 235.59 144.84 151.76 337.56 34.85 90.39 122.34 101.51 126.12 155.92 160.99 124.68 119.33 107.04 133.41 146.77 113.08 94.92 126.32 99.38 214.23 167.01 142.77 179.01 110.54 169.79 128.21 116.77 120.91 239.9 161.78 84.91 96.21 73.48 133.83 106.53 86.94 218.52 100.05 137.15 105.69 111.9 250.26 128.04 222 167.19 150.17 116.29 134.8 171.29 113.03 173.46 129.85 157.72 118.96 167.5 98.05 143.65 105.34 117.23 126.21 173.57 136.8 81.38 124.35 151.92 91.64 130.81 99.25 40.45 89.92 79.02 129.89 140.84 114.62 116.97 101.68 103.17 125.2 81.27 135.1 146.23 225.8 101.9 93.61 101.67 120.81 101.83 144.98 88.86 117.61 106.64 94.01 111.05 66.42 129.76 106.71 124.18 68.29 105.62 133.5 101.62 95.72 68.81 152.5 60.68 99.28 88.14 89.27 115.58 123.69 76.59 82.96 132.4 131.08 150.41 135.79 240.63 145.49 145.35 136.06 113.77 226979_at MAP3K2 919.42 1113.71 937.44 1236.03 999.56 1030.73 517.71 1007.36 1156.85 1591.47 894.07 879.88 729.95 1580.82 679.5 767.65 632.77 1057.4 1020.59 853.24 980.45 848.48 886.28 793.92 897.99 1131.32 967.06 999.18 944.86 965.48 903.16 949.95 797.84 952.04 1135.59 779.67 601.53 578.79 788.95 609.17 921.75 679.49 961.29 844.28 511.9 702.8 854.63 619.49 694.78 610.07 818.81 847.71 642.8 1113.43 939.09 700.68 803.39 624.56 1017.98 825.45 611 713.99 1204.68 1091.13 1864.69 2113.89 1227.53 1283.8 1631.7 1352.7 1454.43 1440.27 1360.05 1239.96 1116.11 1221.73 1356.19 1405.36 1059.01 1314.76 1779.38 1433.16 1664.72 1467.89 1206.69 1450.5 1120.3 1269.18 1105.25 1438.56 1463.37 1982.48 1956.55 1764 1843.98 1531.61 945.37 1236.54 1436.57 1413.18 1155.56 1063.27 1154.34 1366.37 1016.29 1320.22 1056.67 1088.16 1332.38 757 1003.33 891.01 1318.12 1379.19 1571.12 1413.2 1200 1124.51 1121.77 1040.43 1190.34 1328.45 815.23 1226.19 1057.52 1158.78 1410.44 1236.21 1042.48 2166.89 1689.41 1274.69 1141.5 1125.98 1610.94 1670.46 826.72 1270.96 1320.34 1381.1 1595.45 1263.99 1331.98 1160.27 1167.76 825.87 921.83 1007.45 679.36 675.18 998.31 889.98 563.08 842.37 1005.69 885.27 584.87 831.22 815.5 899.92 837.02 813.38 519.12 581.55 584.06 1085.7 880.73 792.28 1262.33 851.8 1034.78 1070.04 1447.69 1227.64 900.12 809.17 1209.9 1095.84 1227.88 1091.25 1319.6 883.3 1037.67 1151.99 1419.58 1122.84 969.75 1106.22 878.14 1014.98 1083.42 1126.58 1056.34 767.81 774.07 1090.15 992.6 995 1019.2 804.42 1183.96 1158.85 972.14 831.23 1416.43 1082.67 1322.56 763.87 1169.04 768.72 1055.6 1266.79 846.56 906 943.81 1130 997.35 914.66 797.24 1080.72 906.01 1077.83 883.23 997.48 946.22 879.54 869.91 227004_at CDKL5 123.09 85.18 113.94 62.82 60.34 51.82 77.44 161.92 95.44 77.42 67.78 120.14 49.38 27.11 68.12 49.53 49.11 48.62 78.27 131.14 46.63 47.23 48.26 31.8 82.33 148.39 127.29 110.16 74.6 47.81 53.52 81.9 29.65 24.38 60.1 49.09 88.31 127.97 41.71 19.51 21.03 48.65 69.22 42.11 77.81 49.67 37.38 52.12 38.31 58.8 75.36 92.11 38.64 47.98 80.42 48.62 34.93 26.76 44.51 55.16 46.96 33.85 166.52 18.44 13.54 111.07 37.99 39 39.32 47.43 58.16 63.89 56.05 49.83 18.61 40.98 54.07 40.47 24.33 79.67 37.9 69.35 18.76 92.02 46.48 34.34 56.28 26.59 39.41 54.57 8.08 63.63 28.51 110.3 140.27 65.13 176.97 63.52 68.05 183.34 85.21 128.33 68.02 139.12 122.77 25.41 45.35 44.65 54.13 5.93 36.06 45.76 51.62 72.11 30.02 62.67 51.74 11.04 38.12 48.23 35.37 31.41 23.34 63.62 35.09 9.69 42.64 39.07 70.83 55.45 35.45 38.67 20.36 45.06 47.08 40.71 29.96 53.42 82.42 86.43 31.45 51.37 16.68 98.96 30.68 15.61 60.7 42.92 33.5 24.26 305.21 26.41 67.26 60.24 20.48 45.47 35 25.57 72.23 23.27 26.39 65.56 50.19 22.34 51.76 29.9 30.67 34.47 9.73 24.08 25.33 40.28 28.67 27.77 9.49 64.74 47.81 24.16 59.02 14.49 32.36 80.61 27.66 36.93 139.49 18.89 77.73 54.19 83.13 95.06 58.49 91.15 35.08 67.79 33.46 57.11 45.89 29.34 56.54 168.16 13.12 54.32 37.83 5.38 61.05 47.98 43.41 36.55 37.05 49.17 24.55 33.47 60.03 74.04 34.46 41.24 36.7 49.03 70.67 19.46 48.18 78.29 67.1 107 105.49 75.91 52.13 227013_at LATS2 420.98 523.31 718.37 440.51 282.2 368.57 431.73 245.26 249.33 551.82 457.24 285.19 258.21 273.26 362.27 434.73 291.47 263.37 437.6 273.95 360.46 289.73 363.41 353.04 278.7 542.83 508.61 289.58 290.53 277.03 532.62 149.81 289.22 204.18 617.28 289.5 450.47 316.56 468.51 419.21 347.21 165.89 360.27 569.26 530.94 512.43 356.23 383.53 552.5 333.24 631.14 349.1 480.87 631.18 105.59 421.94 393.89 301.86 323.12 316.61 204.68 68.64 617.06 324.39 108.05 361.93 220.28 256.8 163 483.53 430.6 230.92 277.45 327.67 84.31 180.96 422.26 215.78 133.51 244.77 280.78 208.13 253.67 241.43 337.83 322.56 449.94 367.05 127.04 325.31 93.55 299.56 181.03 469.29 500.72 298.67 357.58 162.65 236.72 403.88 331.45 274.86 222.55 336.78 253.67 401.15 448.7 328.46 517.07 40.97 296.64 274.78 329.34 351.1 281.61 585.14 106.17 149.9 273.04 324.48 588.99 205.26 241.48 279.29 668.26 223.39 254.96 518.9 259.24 228.2 281.11 342.09 526.84 345.94 299.52 157.49 179.18 318.66 330.09 280.06 161.14 204.81 115.47 233.3 290.69 132.45 199.74 237.7 271 172.03 259.97 235.9 313.58 437.43 181.07 336.29 111.39 346.38 357.81 287.61 171.18 270.46 201.53 206.41 239.98 328.79 202.05 237.37 114.29 135.5 171.74 264.65 302.87 404.91 47.6 329.58 402.54 237.04 393.87 97.37 350.09 331.75 245.12 295.4 411.93 282.21 212.93 222.7 221.38 247.24 455.99 478.95 415.98 340.78 290.73 347.51 416.39 305.25 299.6 279.79 173.59 241 230.15 49.01 185.2 395.7 281.55 279.98 411.89 246.51 429.45 355.49 227.35 259.45 150.51 169.18 234.18 218.7 267.12 220.38 402.54 462.41 255.49 426.46 444.35 270.11 398.47 227131_at MAP3K3 114.63 118.59 195.13 156.52 130.83 197.82 190.99 178.55 139.69 166.64 100.81 105.67 147.61 147.19 250.73 160.76 85.57 129.41 164.1 173.57 162.02 156.34 137.92 156.87 94.81 140.12 137.23 145.96 128.63 133.11 193.98 113.89 223.09 156.76 92.35 95.14 101.94 176.16 209.19 142.81 129.46 117.08 101.38 118.14 111.46 215.48 104.45 235.22 149.14 216.87 130.71 157.14 227.63 129.06 142.88 154.51 143.23 98.59 162.86 139.06 171.32 91.56 166.47 171.22 181.08 106.71 96.2 131.28 107.65 160.7 98.14 157.05 107.36 116.35 101.44 94.8 126.29 122.48 84.03 108.2 172.13 93.46 99.83 105.34 207.68 139.81 243.68 159.04 145.85 155.53 140.08 195.79 215.01 107.08 276.57 167.44 176.09 94.95 203.42 137.72 147.84 124.61 169.61 208.81 120.73 171.13 120.64 132.44 197.6 214.05 118.85 158.1 149.8 135.36 147.7 192.6 144.39 159.77 160.06 205.28 168.87 102.8 131.04 91.57 209.16 239.4 146.61 156.31 71.93 62.47 184.2 139 218.81 148.05 118.64 121.21 150.71 128.49 170.89 111.09 77.77 86.61 124.14 104.52 123.62 98.83 148.03 202.49 177 110.33 96.48 166.18 71.29 158.79 114.55 154.82 7 104.79 112.28 98.56 169.87 128.32 111.2 106.13 94.36 145.05 102.94 153.05 137.31 83.47 164.73 199.55 145.03 127.93 88.26 186.8 131.29 125.3 196.52 165.14 162.58 102.45 142.95 153.68 141.33 173.18 119.42 186.9 68.4 125.95 147.52 145.02 169.51 170.2 146.32 175.79 170.28 231.45 181.47 180.76 127.06 174.92 149.27 192.42 92.89 160.77 125.67 111.58 176.51 103.47 165.81 171 188.61 185.69 104.36 128.95 126.9 151.96 107.29 170.01 143.53 114.99 155.12 118.44 122.74 192.06 170.19 227217_at WNK2 71.28 56.17 64.67 59.77 47.36 50.43 96.14 102.29 50.72 65.51 87.7 57.24 63.8 247.79 65.62 299.88 116.89 123.74 122.16 43.75 80.06 36.42 268.06 108.05 175.33 53.77 96.12 153.65 70.09 109.73 71.1 73.12 65.52 167.26 85.23 86.57 442.8 75.02 58.74 66.84 80.96 72.86 99.19 48.45 92.77 68.32 158.8 96.53 44.18 51.74 90.77 53.99 48.33 125.47 67.03 46.36 91.08 85.28 69.71 110.86 118.66 111.87 231.32 64.66 32.85 99.76 48.7 76.4 60.67 67.25 155.11 111.54 63.53 45.81 74.58 92.94 63.54 66.79 68.91 69.78 50.86 66.66 49.29 52.5 50.57 55.58 35.99 39.34 47.28 55.6 76.64 99.01 61.69 115.98 82.78 70.94 46.26 97.59 54.52 96.62 708.87 63.66 61.74 72.96 49.12 116.01 54 52.73 88.2 79.3 139.72 168.89 66.79 68.88 79.13 74.38 71.38 476.46 43.96 57.5 83.86 230.86 136.9 355.53 105.31 66.89 72.05 59.19 70.28 105.94 51.86 95.69 96.25 72.38 71.22 55.43 76.9 64.32 61.91 68.03 124.44 65.42 80.09 52.17 82.47 101.81 90.83 109.57 55.29 119.68 102.01 62.65 76.95 94.38 71.42 114.13 76.14 46.25 42.54 65.76 85.48 85.26 69.28 83.85 64.78 53.3 67.25 93.36 108.03 93.93 62.81 86.82 107.22 68.5 260.57 74.06 96.31 93.67 69.49 105.78 59.56 164.48 64.45 100.42 154.66 54.04 125.56 76.82 179.74 91.22 126.89 82.9 78.83 48.02 171.58 54.37 124.2 64.7 118.15 184.31 55.37 66.47 59.32 67.84 67.46 70.59 180.59 62.46 60.92 82.47 88.6 48.92 48.19 71.52 112.48 47.18 37.58 60.49 48.29 69.39 49.32 85.4 33.44 66.58 65.26 106.31 64.36 227255_at PDIK1L 88.77 164.29 49.64 110.85 140.77 145.04 145.45 279.39 154.38 63.18 84.07 187.42 116.12 125.65 60.75 130.08 55.89 78.91 183.27 141.73 113.1 259.17 93.84 59.08 91.66 93.53 96.81 99.23 108.18 59.49 168.38 83.15 211.68 85.5 96.68 74.83 92.71 172.05 70.85 64.32 135.05 132.92 73.96 67.79 53.21 112.84 78.15 184.49 50.29 73.21 104.88 120.27 104.85 126.05 190.65 115 60.11 41.06 69.96 35.75 60.46 84.48 49.58 62.58 101.01 158.36 127.2 56.22 157.13 100.26 86.63 107.89 63.47 137.97 161.77 83.3 153.69 149.88 93.5 84.9 113.89 342.58 130.07 183.17 164.06 158.36 74.55 117.17 145.36 172.79 19.65 83.75 195.38 110.53 169.52 237.81 152.87 115.59 120.76 108.78 171.35 153.68 165.47 88.73 86.71 112.11 109.76 96.98 122.85 35.75 47.04 137.56 99.51 80.56 113.77 82.88 147.95 36.69 107.34 50.65 57.76 57.33 105.42 69.86 57.78 143.66 114.71 46.28 185.11 47.59 102.03 74.82 49.09 89.88 161.72 98.93 82.04 131.27 76.56 142.7 98.52 80.86 180.92 150.8 85.8 60.07 109.08 150.23 38.02 191.97 119.99 49.56 56.5 106.21 121.12 68.54 143.15 59.72 101.27 74.99 86.28 72.14 74.26 38.51 73.93 93.9 82.77 82.54 27.9 85.55 104.79 51.93 147.98 101.55 53.32 60.97 76.65 89.74 79.22 134.51 84.24 45.25 96.77 124.82 54.49 82.59 54.76 94.7 63.4 77.41 84.14 138.06 64.44 101.52 105.49 128.92 120.39 60.52 76.25 71.18 66.83 175.4 150.3 49.88 81.32 102.38 119.37 86.66 62.04 140.76 43.69 68.78 58.11 88.66 143.55 111.29 125.89 87.04 147.05 100.63 78.82 68.81 140.38 86.19 80.01 98.56 101.99 227324_at ADCK4 114.44 97.69 119.04 145.46 121.37 143.29 146.52 108.08 120.19 121.15 146.28 128.81 116.57 121.09 109.91 128.85 124.46 118.86 98.22 89.06 136.45 95.22 90.62 150.71 105.63 111.33 117.44 155.26 90.94 113.72 102.22 112.22 112.5 141.66 110.87 108.37 115.74 106.96 130.12 126.6 105.05 111.83 93.66 142.45 122.89 108.32 129.68 135.86 129.31 123.41 107.27 128.89 169.26 123.11 169.67 114.13 160.84 154.6 105.99 117.24 114.84 122.78 95.54 104.63 104.57 92.64 100.52 92.57 138.72 96.3 132.75 122.57 114.22 107.27 105.55 109.68 102.79 97.63 139.38 130.52 127.1 120.21 161.42 177.41 107.62 94.73 135.35 116.11 141.48 93.12 265.1 86.33 138.61 81.38 96.4 98.77 152.05 129.53 87.09 95.92 81.89 112.93 141.84 121.04 112.9 168.68 168.86 145.38 186.34 299.42 137.53 110.59 106.44 113.65 120.1 141.41 157.97 165.54 115.65 139.16 127.03 168.22 180.63 132.72 7 130.07 95.91 94.66 86.29 101.78 81.62 94.98 116.51 101.91 79.29 96.05 94.65 91.36 102.12 76.68 98.83 102.6 108.23 95.88 92.33 93.1 112.71 126.87 100.08 141.65 115.3 127.47 118.72 109.59 138.55 120.03 157.97 119.04 108.3 101.07 107.15 108.81 118.65 184.65 125.71 114.99 119.71 116.07 171.65 121.17 128.38 139.54 127.47 121.76 151.85 118.94 91.46 115.7 105.38 114.65 96.63 112.59 103 113.36 109.78 106.67 116.98 119.7 111.68 141.52 113.06 91.32 117.81 85.81 91.01 92.65 100.55 120.97 96.97 113.47 113.55 104.82 101.61 189.46 92.25 94.64 107.83 103.96 122.12 113.71 110.63 109.27 119.99 128.79 117.79 112.36 92 95.78 94.13 109.03 109.09 96.17 110.89 102.68 146.97 116.18 96.6 227438_at ALPK1 189.72 95.77 95.71 94.51 84.23 103.52 106.37 58.75 91.36 96.61 156.97 89.17 129.86 34.94 137.73 111.54 73.14 144.92 107.57 104.43 90.78 144.82 201.77 111.93 135.85 303.39 130.31 146.91 155.36 111.4 105.75 178.78 108.56 58.09 105.21 238.17 193.28 146.12 165.92 149.35 200.35 221.73 111.59 92.45 107.24 103.64 150.52 213.67 109.25 103.11 225.56 105.36 61.72 100.3 35.13 79.11 158.21 127.09 160.12 162.61 243 75.06 113.86 58.7 125.46 111.48 70.36 191.84 128.11 69.12 94.3 214.27 94.74 148.47 92.82 228.53 86.02 104.72 44.51 73.12 80.06 128.48 99.65 113.87 70.9 121.76 85.75 94.26 78.6 98.02 90.24 113.29 104.35 74.72 103.59 184.57 62.33 56.69 94.51 234.62 227.11 105.67 67.2 111.47 130.45 144.76 115.73 152.29 116.58 96.03 66.15 143.73 147.79 102.65 135.36 113.67 37.86 156.63 118.44 86.35 116.82 104.34 102.27 107.59 104.5 67.02 82.39 110.49 96.74 97.01 154.48 84.62 101.49 94.48 127.26 54.28 110 100.4 65.16 79.73 136.25 76.36 91.82 81.72 69.42 97.93 67.45 100.5 115.25 128.18 117.21 111.91 82.64 128.03 170.5 71.39 74.55 179.35 122.65 214.4 92.38 107.59 66.38 91.49 62.21 127.12 95.56 117.1 68.06 97.25 94.93 114.91 126.6 107.25 33.18 68.51 130.3 113.5 113.08 73.92 103.62 97.66 88.62 60.76 90.14 57.2 64.3 67.26 99.31 101.68 92.48 111.4 58.31 92.37 79.78 73.53 117.32 91.91 95.69 88.55 58.7 86.75 106.51 34 81.04 86.64 214.48 97.05 86.46 79.5 101.85 118.94 88.05 95.19 70.6 80.79 71.87 63.34 59.82 146.11 152.04 124.67 66.98 119.8 91.52 122.73 105.4 227454_at TAOK1 112.4 374.65 306.43 166.2 102.15 162.26 24.8 408.09 257.49 262.82 426.86 339.73 221.88 379.06 256.27 192.14 83.32 252.29 134.59 246.83 267.23 341.63 288.58 180.25 114.6 224.21 165.94 609.4 136.56 251.06 144.58 193.22 189.37 228.68 246.9 108.96 89.18 116.21 123.72 130.06 155.51 127.52 117.49 236.11 137.05 267.85 233.64 146.85 228 185.11 172.13 200.54 255.22 115.22 197.26 227.27 148.28 90.15 307.62 183.82 114.44 201.46 304.71 604.64 439.03 387.84 320 468.59 427.04 515.96 245.38 567.79 530.56 380.57 224.5 986.64 357.92 413.01 406.99 423.42 352.91 445.78 988.19 687.38 463.35 314.31 416.11 251.15 615.25 256.32 236.55 318.4 372.36 293.18 328.8 468.51 246.58 115 201.57 124.68 229.15 157.99 161.39 118.88 230.68 154.99 259.85 136.49 249.95 102.05 207.43 341.02 483.18 350.71 332.43 80.05 645.57 480.64 264.36 388.17 288.41 435.17 300.51 238.51 544.38 314.32 554.3 619.91 303.97 295.09 449.54 388.03 367.99 709.75 579.55 665.43 460.02 660.86 1039.16 542.32 446.51 248.82 352.88 506.93 453.39 794.59 1163.34 274.05 251.12 283.64 242.21 245.25 216.86 292.84 238.64 230.03 341.11 210.85 246.94 242.42 363.63 266.87 259.27 263.07 264.21 313.39 270.07 284.26 807.19 323.11 351.89 508.41 467.96 370.54 554.56 376.26 305.13 495.53 474.66 443.53 417.28 442.69 1283.73 675.03 299.28 511.73 480.29 598.34 462.75 873.97 379.11 358 156.46 186.3 180.23 190.43 205.13 233.74 206.24 228.21 157.46 142.25 179.47 187.63 162.56 393.35 376.09 297.94 376.37 234.33 240.8 341.74 329.1 276.22 230.46 276.61 171.53 373.92 194.87 252.79 173.63 216.62 322.34 267.32 282.43 196.53 186.51 227482_at ADCK1 87.23 106.7 113.94 136.72 152.18 119 190.79 75.87 74.95 128.19 112.35 105.36 117.38 87.44 115.37 119.43 115.94 105.58 115.62 80.05 111 86.31 66.77 99.78 86.79 89.93 74.72 66.15 83.17 84.99 54.86 84.91 81.75 133.67 85.34 95.48 109.58 65.88 102.63 97.84 91.13 82.66 82.66 96.93 102.02 85.33 87.83 76.79 88.53 60.03 66.56 85.6 117.1 106.81 81.4 89.86 90.45 101.66 120.29 109.42 155.7 177.93 75.27 88.54 102.5 88.56 146.15 111.26 90.13 70.4 112.65 117.7 92.52 96.73 71.62 86.1 78.58 128.11 89.56 107.35 96.47 90.4 76.19 136.64 79.73 98.73 63.97 77.79 93.7 72.36 163.63 114.02 95.8 90.43 108.45 119.18 117.03 103.78 100.66 70.09 150.18 125.55 117.16 92.26 117.85 109.33 96.61 125.45 126.66 114.03 112.77 103.23 96.21 138.61 163.72 127.38 158.51 148.46 128.15 222.78 154.29 158.63 235.25 141.23 82.79 77.85 115.22 84.51 93.78 97.73 110.79 120.72 119.32 81.15 79.72 70.79 82.74 74.66 97.37 81.82 79.84 91.78 71.96 94.04 67.19 167.13 73.31 111.64 111.28 134.52 101.71 103.64 134.37 112.17 127 146.55 97.57 122.2 109.18 118.78 146.39 111.89 96.66 137.55 161.69 207.44 108.48 126.49 185.72 135.8 103.04 130.26 130.32 96.45 145.81 149.72 74.49 83.34 89.62 97.48 95.55 145.47 171.14 95.78 94.66 96.48 161.57 96.49 136.16 128.21 203.41 85.94 106.62 99.05 97.62 110.12 134.86 99.75 166.23 89.38 98.32 114.82 116.82 123.4 108.04 91.1 107.07 95.7 146.71 118.92 143.54 132.51 105.42 261.21 9 99.64 90.47 141.94 104.12 104.07 118.18 62.79 78.75 96.44 70.78 81.74 96.27 227627_at SGK3 955.16 731.46 194.34 453.54 285.84 635.68 347.08 263.57 226.11 246.06 436.83 459.22 736.1 525.15 477.99 453.99 267.45 475.51 1668.91 1617.21 436.36 730.82 575.11 442.31 366.52 355.03 305.03 193.47 352.62 442.64 972.55 785.12 1334.54 3732.98 260.99 389.49 282.94 413.06 288.03 249.44 477.21 525.87 291.4 169.4 453.15 399.72 289.16 414.62 342.44 327.37 330.6 212.47 368.26 520.13 1188.55 540.97 405.07 371.79 288.49 280.79 317.49 555.52 250.24 1183.82 173.5 624.43 220.16 372.58 701.12 450.61 141.21 336.91 267.35 242.57 653.38 604.6 563.41 666.94 1489.1 883.23 144.74 241.48 1153.58 1020.1 229.96 847.6 307.07 274.77 1879.33 611.67 228.8 970.06 2226.86 528.48 688.57 473.23 1030.54 348.4 3884.09 618.26 628.81 374.29 239.75 980.18 523.31 635.07 1272.9 2979.73 696.74 1251.51 376.43 470.01 513.29 569.64 701.63 458.13 131.03 231.29 406.67 510.98 288.14 552.71 363.48 269.94 329.03 733.11 189.94 352.17 691.56 316.64 310.54 223.71 184.23 161.28 297.67 1523.24 542.38 237.15 314.3 375.48 1614.03 167.04 3991.35 515.79 893.31 166.52 233.43 307.62 290.77 413.21 355.71 973.57 131.21 359.24 725.52 141.49 3301 470.25 344.68 277.71 507.2 480.63 405.68 1445 241.46 623.96 547.63 213.73 239.02 752.62 235.77 419.15 478.42 1201.95 11.97 198.35 330.6 919.06 558.44 897.13 777.33 523.22 1155.29 433.02 431.06 290.69 192.01 644.18 535.3 246.65 353.11 391.79 329.44 333.51 593.06 218.61 362.16 220.56 327.65 260.24 420.25 339.64 515.64 241.36 318.92 414.81 554.06 352.84 1255.64 579.12 320.54 305.31 404.58 325.22 842.05 688.55 186.4 231.69 185.81 792.06 326.67 221.63 321.96 433.13 196.64 339.48 301.5 227666_at DCAMKL2 60.85 41.93 84.55 33.13 44.49 38.49 44.15 39.54 36.67 66.55 29.25 39.7 31.6 31.95 75.78 28.44 27.98 38.92 47.35 36.43 43.34 90.55 83.61 36.91 44.72 32.11 54.24 67.06 22.91 42.66 36.88 35.41 30.42 27.92 27.78 45.02 44.29 38.68 61.12 39.13 46.17 40.83 48.35 138.53 53.16 50.15 42.45 41.9 100.94 51.34 29.17 45.62 55.6 113.83 36.03 27.72 32.43 124.8 43.33 63.41 53.65 157.64 60.24 26.25 29.5 51.54 25 27.2 28.43 27.87 69.14 34.57 25.6 42.32 28.39 43.8 26.72 20.34 19.86 26.77 22.14 16.06 13.47 18.29 25.65 26.55 59.91 37.53 18.6 22.28 45.63 24.41 16.59 37.61 34.54 17.17 25.66 28.51 15.88 33.49 44.72 26.25 25.11 28.86 18.54 33.21 39.84 30.88 38.41 33.87 15.6 21.39 21.72 57.52 27.59 29.17 53.55 39.8 25.82 55.94 30.66 68.89 25.51 23.53 26.98 19.83 17.2 25.83 21.74 24.36 25.99 44.14 49.37 20.59 30.28 19.71 37.85 19.1 34.18 25.12 13.5 26.46 13.16 17.3 14.86 22.88 15.6 25.14 40.3 16.52 37.52 30.78 28.68 31.2 20.21 42.18 18.22 19.5 30.97 24.82 26.11 47.49 20.46 29.16 24.49 24.39 24.96 27.07 29.62 20.82 23.46 29.67 25.65 37.16 20.32 45.69 32.92 26.4 57.25 14.64 23.84 31.81 20.28 26.02 21.63 29.37 34.1 19.71 26.53 24.86 35.8 26.83 50.29 52.41 36.06 38.99 76.47 35.22 48.86 33.95 28.67 36.13 34.36 45.21 34.86 30.73 30.95 32.45 53.41 31.62 81.38 58.11 22.67 31.54 23.96 28.16 28.7 41.82 32.01 22.08 62.08 105.48 67.5 62.1 64.36 33.49 42.89 227677_at JAK3 109.74 46.74 284.11 163.11 176.04 150.33 51.57 93.12 62.91 179.14 50.52 41.35 157.59 67.04 157.78 254.8 228.63 465.47 202.22 89.37 133.4 301.84 49.32 331.86 109.73 586.35 95.73 136.86 308.58 61.78 89.34 100.56 134.4 157.69 156.07 335.36 339.31 112.93 912.06 421.43 224.66 329.19 151.22 123.84 175.83 270.53 185.15 189.13 339.32 117.47 48.89 233.67 127.42 345.74 44.53 73.97 567.86 196.23 549.32 133.17 50.36 145.8 118.71 134.5 847.92 106.53 149.59 218.94 119.93 93.19 85.09 243.31 115.99 106.67 70.43 42.53 56.82 107.68 27.23 29.99 51.22 116.31 71.71 97.8 100.56 101.57 135.61 333.13 371.07 272.98 79.54 131.74 78.99 39.46 127.29 143.72 30.21 47.86 107.92 102.95 48.92 175.91 101.54 51.61 35.95 305.26 58.84 155.33 177.4 54.07 128.85 75.05 232.78 107.49 287.15 37.58 50.84 415.12 425.81 218.87 216.19 177.59 232.91 312.79 129.75 131.84 57.36 173.93 46.01 67.76 360.94 58.61 136.44 211.04 36.88 56.16 95.72 81.99 66.43 65.12 26.24 80.94 35.95 57.28 139.76 159.41 47.84 94.02 494.47 83.27 82.45 466.98 78.19 171.93 212.2 97.86 26.19 508.46 56.38 368.95 109.66 131.23 31.9 65.47 48.2 244.13 36 470.35 74.43 27.06 180.34 99.25 63.89 179.96 47.21 62.27 352.96 42.04 102.12 48.56 270.71 131.2 42.17 65.58 151.45 61.73 87.19 39.46 83.04 23.58 53.32 82.89 49.48 158.58 77.32 140.34 69.34 267.36 254.94 108.96 90.46 146.85 275.65 117.27 332.93 38.86 74.93 100.66 120.43 107.66 143.96 280.97 194.54 109.1 24.56 114 136.01 97.14 98.63 69.66 246.97 100.33 33.33 77.87 83.29 101.82 189.02 227740_at UHMK1 212.25 304.83 316.9 434.05 309.38 182.94 59.62 319.72 374.16 352.02 694.82 504.2 578.34 489.14 252.42 238.82 317.14 383.02 338.54 393.15 299.42 299.35 395.57 378.07 170.16 383.04 135.68 324.78 376.62 517.95 344.75 280.43 497.04 341.51 251.89 199.64 147.76 139.23 137.29 214.04 320.63 483.25 220.59 251 164.69 268.3 321.53 444.1 214.66 305.56 437.68 311 587.42 333.13 351.69 310.16 170.52 191.04 151.33 175.25 322.48 299.75 340.87 900.45 1133.5 1009.35 814.81 748.2 1218.25 836.38 328.39 1547.82 882.62 715.97 999.08 603.49 987.75 635.48 1217.15 551.27 839.95 1892.88 1233.23 1099.58 688.47 969.98 592.33 424.48 844.33 988.75 102.86 578.8 935.53 579.15 693.81 629.03 377.45 153.79 582.86 403.02 225.77 335.32 414.39 357.02 638.27 548.78 512.6 222.63 375.48 35.78 476.27 399 535.11 438.11 501.2 43.76 409.44 379.69 383.96 561.96 564.07 380.44 365.7 440.53 493.24 592.92 920.6 681.33 569.87 919.76 617.76 826.82 410.27 290.39 771.53 856.21 1176.64 690.6 360.69 602.99 1001.92 515.55 775.97 598.68 791.16 343.41 646.57 495.11 415.83 554.42 567.76 419.85 385.37 212.59 521.23 431.3 556.52 255.82 280.94 339.55 565.74 295.62 526.76 224.74 458.18 327.97 444.07 238.83 408.61 743.44 708.25 299.55 516.69 605.24 358.91 755.77 459.61 835.4 726.52 1203.54 608.82 483.87 794.95 478.94 391.44 841.91 916.99 1076.19 726.94 802.32 1011.21 479.45 200.33 531.38 244.22 317.21 249.85 691.2 342.09 335.83 384.49 393.54 416.69 331.03 669.36 656.88 646.5 680.66 280.92 640.22 590.11 762.85 366.78 327.73 688.29 451.07 528.29 298.21 344.31 410 321.84 434.65 521.98 458.27 584.85 305.03 354.23 227750_at KALRN 261.12 78.31 147.09 478.97 112.97 71.79 149.83 65.32 129.13 91.96 105.68 145.87 62.59 66.38 111.68 103.86 70.52 73.32 151.11 133.82 54.78 114.76 104.74 93.97 202.33 107.37 164.67 151.23 95 80.04 64.85 43.81 165.41 313.45 67.62 119.41 99.05 79.4 92.22 44.67 94.49 123.21 154.66 99.11 116.12 111.37 124.21 202.61 101.52 82.32 120.4 93.33 133 189.71 78.54 90.48 120.8 79.85 177.4 90.74 71.16 95.68 141.77 99.83 41.29 100.19 67.21 54.73 145.66 102.63 78.84 100.45 103.32 83.69 99.94 103.58 64.4 55.85 62.86 127.33 128.06 114.69 69.38 78.77 82.46 81.69 152.45 106.39 90.9 149.49 101.09 88.83 125.33 221.15 78.15 90.35 113.33 127.56 174.58 115.95 284.35 62.18 107.94 77.4 160.6 88.52 94.91 78.67 147.38 69.2 103.98 131.5 60.98 96.02 78.98 215.91 54.81 95.34 78.82 57.89 226.13 126.98 98.54 68.47 86.36 54.35 71.83 88.46 59.19 73.78 140.34 79.41 99.55 88.26 74.9 64.16 73.42 89.27 80.11 96.75 100.69 57.27 73.46 96.65 86.99 98.14 54.23 104.39 115.06 110.02 92.92 140.31 100.42 101.19 101.29 161.18 98.57 83.27 75.03 66.41 91.91 110.13 75.66 102.15 79.33 168.42 77.5 123.24 73.12 307.48 43.01 58.28 64.62 89.25 114.15 67.92 83.76 78.57 71.3 66.08 70.95 157.63 127.28 89.3 125.9 106.2 53.96 92.21 163.5 135.55 66.81 71.86 53.89 56.65 93.89 76.56 74.68 60.74 91.21 136.7 96.39 57.95 69.73 67.89 58.56 54.98 72.2 68.91 43.49 51.82 91.33 45.42 144.83 64.83 65.08 92.52 224.66 70.62 141.7 68.86 120.17 97.89 110.91 118.99 125.38 154.78 79.19 227767_at CSNK1G3 90.49 314.76 87.4 199.26 125.1 182.26 101.23 445.7 317.04 112.74 207.59 227.58 177.51 282.18 82.5 169.94 104.57 112.61 325.88 237.84 164.97 125.75 83.17 60.35 130.49 122.8 84.44 161.53 74.54 160.92 165.28 49.72 149.54 271.91 66.91 38.47 72.42 68.04 115.14 59.77 99.23 93.49 71.57 83.04 67.54 88.22 82.02 75.95 51.4 39.8 105.14 123.25 103.95 118.97 215.24 135.51 70.87 59.65 74.07 43 97.08 52.71 56.75 65.53 144.88 89.95 54.96 86.44 77.82 124.77 78.82 82.83 73.34 120.85 75.87 123.44 113.11 192.72 270.04 79.47 76.8 238.23 260.23 223.92 192.8 268.89 176.03 163.2 184.72 267.11 30.7 245.08 167.43 466.76 387.36 134.78 257.13 137.89 240.77 136.48 116.2 151.35 120.64 193.44 185.7 107.01 126.51 94.82 69.08 12.39 52.08 147.85 98.88 90.86 106.29 74.18 196.18 38.41 83.96 54.37 88.79 59.07 76.69 69.4 132.91 153.99 120.56 169.3 153.26 98.08 130.76 76.12 87.94 92.04 144.21 132.24 131.66 119.34 145.72 152.06 167.24 110.93 117.92 150.59 108.16 78.61 116.44 123.16 77.57 194.64 79.17 74.99 164.99 122.47 118.11 67.77 106.51 72.17 162.48 111.74 109.92 167.04 288.94 46.09 188.12 111.37 185.78 79.92 56.44 99.43 242.92 79.06 152.89 120.26 39.67 90.25 125.81 122.5 146.2 189.93 152.57 44.36 139.82 142.15 137.81 123.5 71.33 180 44.21 103.56 151.31 250.85 142.07 92.82 148.95 79 125.53 111.83 83.16 69.8 137.11 138.27 122.74 56.21 46.13 109.06 97.25 84.51 94.19 184.33 85.27 114.61 94.55 60.38 140.78 237.73 80.34 113.86 151.96 170.43 82.58 104.14 104.51 156.79 93.13 101.95 104.24 227817_at PRKCB1 21.45 17.84 30.79 36.58 32.19 28.49 15.1 17.92 18.99 24.89 15.57 16.38 47.51 17.29 37.92 26.52 38.25 58.27 29.28 17.42 18.71 32.18 14.67 31.9 21.25 84.87 19.64 24.77 28.5 15.65 18.45 19.85 16.95 30.84 20.73 27.31 39.32 39.95 83.14 65.01 37.46 64.16 18.43 28.47 26.74 43.69 25.36 36.79 41.72 24.05 20.35 54.91 29.4 38.58 19.01 21.82 25.59 27.88 69.79 21.28 16.98 17.67 19.17 39.38 767.96 32.12 51.87 88.28 40.26 36.37 32.07 87.52 71.58 39.98 19.5 20.91 26.83 57.63 27.37 19.88 20.76 28.47 27.56 26.59 52.19 45.04 42.66 137.96 94.72 121.59 36.81 73.94 29.66 22.71 45.2 32.63 24.65 26.32 44.74 41.47 24.49 53.38 43.66 30.18 22.99 58.77 34.22 67.42 38.18 33.28 54.05 36.06 160.22 48.35 97.84 37.75 31.8 185.69 182.83 58.15 79.29 68.25 66.01 143.97 168.6 96.45 30.14 58.92 22.93 31.35 104.38 28.24 40.37 59.82 19.61 33.5 48.16 39.88 34.42 17.52 29.76 28.12 27.14 34.67 52.5 61.38 25.01 31.82 84.84 29.15 26.39 207.44 29.25 42.48 57.19 40.68 23.31 85.07 22.64 87.5 42.61 37.57 23.82 25.6 29.22 88.91 27.28 113.46 38.12 36.7 49.97 29.67 29.11 65.17 28.02 28.72 77.43 23.88 40.88 22.26 95.13 75.88 31 27.35 121.55 30.84 32.9 20.67 100.09 19.42 30.89 43.08 29.24 29.21 26.32 27.37 21.8 119.34 78.46 42.67 33.27 43.46 102.63 46.56 32.78 29.59 34.16 35.33 36.02 29.73 40.23 86.4 55.03 28.46 23.33 42.12 42.87 31.89 31.18 34.04 92.8 21.65 19.46 20.18 22.44 35.8 102.76 227971_at NRK 6.62 23.27 13.85 18.18 12.17 14.18 11.71 7.4 8.68 245.68 13.85 335.56 10.98 8.05 9.79 140.48 6.97 6.27 8.5 14.14 7.89 106.47 6.25 9.35 7.24 8.1 8.04 6.3 5.56 7.55 8.25 10.44 29.02 8.7 8.64 7.25 6.75 6.87 9.34 6.66 7.51 7.44 7.81 6.5 8.81 7.49 6.55 7.27 7.92 10.98 7.49 13.5 7.63 6.96 7.73 6.57 6.68 4.99 38.15 7.35 108.55 13.46 9.2 6.86 5.92 7.4 6.34 7.37 6.47 8.03 7.29 6.37 8.01 31.96 8.23 12.47 7.7 19.05 9.3 9.53 11.78 20.68 8.01 10.58 22.46 10.1 44.85 10.53 12.55 24.6 13.17 31.85 8.2 89.48 16.43 10.44 16.7 37.52 37.02 7.39 7.46 18.4 99.14 130.09 18.34 10.26 22.88 64.31 10.48 10.88 9.16 8.12 8.22 7.91 7.61 13.01 62.84 9.54 8.88 9.16 8.81 6.91 7.91 7.89 9.46 8.03 8.81 10.32 13.33 7.55 15.82 15.09 11.28 53.4 266.44 26.23 7.42 8.13 9.31 42.76 10.54 1085.82 14.67 10.12 8.33 5.93 7.7 9.83 15.83 19.92 9.88 11.3 29.36 115.17 8.8 8.12 9.47 7.45 50.25 7.4 9.07 12.9 8.76 7.95 7.73 17.73 9.22 13.71 11.01 19.12 9.06 12.41 31 7.92 11.31 27.16 10.55 10.79 47.01 25.01 14.76 9.1 14.35 9.75 11.53 12.12 8.44 12.96 9.16 11.98 13.15 32.31 15.76 14.14 14.64 25.85 10 32.25 16.64 10.46 7.42 175.8 29.55 12.09 100.94 21.89 9.47 15.16 65.75 50.01 8.97 43.04 78.81 179.97 11.7 11.01 284.81 35.98 12.49 14.07 7.38 7.15 127.7 6.8 6.4 5.17 9.78 228139_at RIPK3 35.17 45.37 62.03 51.33 168.91 44.57 38.72 26.94 81.29 44.97 100.56 36.11 68.08 97.14 61.52 31.32 31.68 57 37.62 30.03 76.25 51.85 33.35 54.82 30.77 82.49 47.08 41.41 55.65 32.3 59.5 76.47 46.97 73.18 38.59 66.67 45.78 40.24 69.36 80.38 109.09 47.48 34.65 34.5 40.55 51.66 31.4 68.99 58.03 34.25 37.67 41.42 44.47 57.07 36.15 93.31 47.3 47.16 72.19 46.72 65.39 38.58 36.11 50.4 85.68 41.44 30.83 60.34 40.06 32.54 35.45 55.3 25.58 45.78 50.79 33.03 37.34 89.44 41.92 77.67 35.77 175.76 80.24 152.14 49.61 67.14 49.25 63.67 63.94 56.96 58.44 78.95 53.46 44.93 49.54 38.24 74.4 59.45 61.71 56.09 44.56 54.98 47.81 79.58 34.45 52.64 82.22 78.93 129.14 94.77 38.54 27.84 49.88 27.71 69.38 33.71 21.79 53.48 54.14 86.26 72.38 30.61 46.14 62.9 41.29 102.09 42.29 28.49 40.33 30.67 59.84 68.08 48.51 46.69 36.01 44.59 32.69 32.15 31.52 28.45 78.15 30.87 31.58 33.11 44.49 33.28 20.85 90.36 58.13 34.16 33.69 78.8 32.03 52.77 91.03 35.02 47.67 78.67 60.14 57.72 55.47 51.48 115.2 32.66 68.35 50.84 99.39 64.09 40.18 85.72 52.9 86.35 60.56 40.04 39.36 43.89 72.1 103.75 60.3 40.76 59.94 25.65 94.59 75.04 38.96 61.45 64.88 53.52 35.22 65.16 44.85 52.1 83.11 51.04 51.5 47.76 37.32 59.84 94.06 58.05 49.67 75.85 79.12 50.59 37.33 64.23 37.46 46.8 57.77 51.6 66.04 104.94 55.86 37.08 65.48 38.76 55.43 43.2 38.33 63.39 88.85 71.99 96.99 35.82 37.82 34.95 56.19 228367_at ALPK2 38 44.67 43.96 51.18 13.79 16.74 46.96 19.03 23.24 59.7 11.31 36.04 16.9 17.85 17.57 22.67 30.85 16.91 34.2 15.21 50.56 27.85 18.82 19.4 17.09 55.78 41.13 18.26 28.95 41.69 21.18 20.98 12.14 23.15 28.4 24 32.02 33.69 151.28 37.24 22.73 33.6 34.91 58.93 75.79 15.84 60.3 28.65 55.94 24.84 18.74 46.48 38.53 23.11 16.29 13.29 24.62 75.37 34.49 42.14 27.46 16.97 47.12 17.17 117.02 86.83 20.44 293.39 15.96 16.49 79.08 37.99 30.83 55.27 21.95 19.7 12.95 14.47 12.31 21.47 39.33 13.96 13.18 14.47 11.44 13.79 24.28 74.53 11.46 34.73 13.55 12.36 10.64 68.77 12.18 48.56 53.46 16.17 18.37 82.79 17.33 15.56 85.48 15.43 53.57 19.29 39.61 18.57 21.52 27.15 44.73 16.55 42.57 32.44 42.3 63.1 18.14 96.53 23.37 101.69 133.24 34.74 26.1 29.12 55.78 20.37 10.96 91.07 17.12 28.96 26.69 31.09 12.57 16.78 33.96 11.36 13.87 22.64 14.21 66.86 10.79 38.97 13.26 19.47 13.2 48.67 17.52 11.79 20.15 19.46 17.25 13.91 34.83 33.85 16.38 14.98 10.06 31.2 38.36 43.82 14.53 25.89 21.44 11.39 31.07 14.77 18.5 17.48 14.77 20.63 21.95 23.52 42.93 18.81 20.49 29.17 45.09 50.23 17.52 15.3 16.13 45.87 21.54 15.7 80.38 16.2 12.95 14.97 29.02 16.07 61.32 26.56 45.55 65.68 33.88 64.93 12.26 31.51 15.04 16.82 14.06 25.96 12.26 18.44 26.48 15.97 14.95 18.24 17.2 15.55 13.17 26.77 16.06 30.2 15.44 23.61 112.13 32.63 72.21 11.65 34.44 106.54 9.62 24.86 40.48 26.34 20 228468_at MASTL 213.79 86.54 25.06 79.22 59.17 31.21 104.26 130.78 57.87 94.95 83.68 30.33 26.84 105.44 29.71 40.11 183.39 132.66 156.75 147.53 70.5 142.75 199.17 65.03 95.23 89.19 121.77 140.29 152.09 213.79 70.71 99.14 69.11 99.92 232.83 139.85 96.06 87.34 23.45 187.46 145.01 205.7 118.38 50.42 90.29 29.8 24.22 97.93 75.21 41.33 38.41 60.48 27.87 66.11 50.22 34.05 294.69 74.79 81.8 28.29 48.56 55.77 59.51 59.92 43.01 202.12 107.11 178.44 110.1 61.72 84.01 118.44 59.36 281.75 99.78 55.17 241.1 63.69 32.41 48.07 71 41.32 42.49 24.97 17.48 31.96 17.34 31.95 38.65 73.19 10.13 46.9 51.41 63.4 40.83 131.55 35.85 108.84 96.43 138.15 82.49 46.68 60.45 65.28 36.45 58.04 28.82 43.44 75.79 20.63 75.38 27.93 35.98 189.62 64.34 107.76 58.32 87.53 56.11 47.68 33.68 179.26 43.2 115.89 31.85 104.09 32.79 37.02 54.8 155.51 29.79 36.6 24.25 35.43 74.26 56.05 31.6 101.18 56.21 88.37 54.81 74.71 139.34 50.05 5 21.45 164.29 45.46 73.54 36.04 171.79 21.41 101.99 51.08 31.01 79.82 45.75 53.27 28.9 73.95 57.96 41.89 44.81 17.61 30.59 47.8 89.84 46.61 24.28 69.91 46.9 36.4 62.18 38.47 27.73 31.94 73.82 42.81 35.68 46.68 25.34 156.92 47.33 42.47 50.54 24.96 56.64 36.64 207.94 32.35 80.43 28.7 40.18 60.12 37.46 24.72 88.15 41.7 72.29 47.47 38.35 61.18 39 26.75 121.58 22.63 88.28 139.11 33.71 62.11 125.98 50.5 27.75 24.09 61.13 50.12 21.68 144.33 50.24 37.64 41.18 155.41 15.96 126.7 106.52 73.37 22.18 228524_at ADCK5 220.54 69.01 67.78 59.1 100.18 177.06 79.9 119.8 44.44 64.91 69.04 71.51 59.01 69.1 56.63 48.87 43.76 60.78 60.5 56.73 54.68 53.74 63.05 95.56 49.03 44.24 50.81 51.66 53.02 71.72 87.84 89.04 100.98 85.34 42.18 75.62 46.66 59.42 51.09 49.72 68.55 81.4 92.35 56.29 58.49 56.6 35.88 53.3 54.75 114.17 48.78 45.82 105.25 96.93 175.57 70.89 105.23 46.72 65.24 60.8 89.08 69.41 54.44 64.66 30.45 53.15 41.35 48.47 55.25 45.25 78.21 41.32 59.98 48.02 85.53 35.09 28.06 32.32 55.9 118.93 72.64 40.86 28.4 43.8 28.62 73.39 36.45 29.52 45.44 21.45 88.31 28.9 46.82 22.42 23.29 36.82 30.12 78.67 41.63 35.48 32.59 28.63 51.26 32.46 40.93 39.85 34.56 69.02 52.48 157.4 50.38 40.05 38.77 57.68 39.54 26.61 83.82 58.98 43.91 45.5 49.3 70.55 46.19 55.97 21.41 35.83 39.22 32.82 33.07 25.76 27.08 52.24 31.77 26.4 24.23 43.26 35.42 26.78 25.92 41.72 19.45 30.15 69.13 22.14 27.97 36.98 19.7 52.71 51.15 50.68 79.99 64.15 61.06 59.06 47.61 86.19 85.52 53.35 43.98 41.4 72.19 57.56 44.42 56 36.29 54.8 41.47 46.82 116.05 77.15 55.62 58.62 48.04 64.5 62.39 45.12 45.84 51.07 32.56 81.28 34.65 53.86 45.13 48.16 44.31 52.92 42.19 36.76 61.03 37.92 42.46 28.1 63.66 39.07 32.67 55.87 125.96 42.75 52.96 161.84 63.7 83.97 46.93 59.83 47.29 30.16 45.26 57.65 52.32 49.98 74.11 34.85 79.69 73.47 56.52 31.98 23.94 22.54 33.96 34.42 40.07 51.91 53.33 67.03 54.83 53.05 39.47 228565_at KIAA1804 179.44 51.13 27.6 66.97 19.29 19.87 86.12 43.91 68.42 56.65 127.41 66.56 41.27 18.19 30.61 20.3 124.66 95.35 354.97 399.8 50.53 14.93 345.92 70.82 569.68 53.68 22.23 90.04 125.15 122.31 90.78 94.5 82.42 24.98 103.77 193.17 125.92 60.34 17.59 100.92 150.97 135.5 376.01 71.32 115.78 31.07 47.67 279 70.34 50.8 140.58 29.29 24.23 367.58 12.45 18.41 163.98 73.85 17.94 31.35 17.92 211.26 10.24 37.96 11.69 149.25 95.37 117.4 371.22 124.07 137.5 102.42 162.29 40.41 129.96 79.85 117.82 24.33 70.54 34.16 28.97 27.91 41.4 15.88 29.28 31.63 87.56 19.67 10.44 17.86 13.17 23.65 52.69 22.88 28.35 53.98 36.46 109.66 54.33 121.18 127.24 39.81 12.4 27.93 88.06 62.45 57.96 61.91 46.83 17.79 140.13 68.97 66.52 101.46 165.37 114.02 16.56 56.2 85.58 17.91 21.83 115.64 74.31 97.81 21.84 38.4 18.36 38.62 58.17 120.44 22.61 76.13 28.95 25.84 37.8 26.81 73.09 32.26 35.26 35.64 24.04 49.58 46.38 29.26 36.02 19.39 72.6 28.04 24.73 17.24 192.38 23.74 98.69 29.57 38.42 86.63 16.84 44.45 30.83 72.06 41.18 160.9 83.5 32.39 13.6 34.09 23.87 25.37 36.26 45.22 24.68 20.05 34.98 137.63 16.26 44.06 49.88 38.56 28.2 17.26 39.92 92.64 29.62 36.24 40.01 38.16 44.07 32.15 42.36 132.72 25.55 30.55 15.08 23.54 55.56 29.14 142.29 32.18 27.46 56.09 25.21 28.65 34.67 27.97 90.82 52.88 182.66 125.02 27.94 63.54 115.44 16.12 14.75 20.92 22.57 49.18 45.42 110.6 24.06 32.21 100.18 75.41 9.78 87.23 58.34 222.72 26.62 228627_at TLK2 51.38 89.18 62.46 69.17 69.81 57.79 55.28 146.38 89.28 83.68 125.69 61.7 48.07 108.88 42.27 52.73 63.38 66.81 111.44 116 69.43 67.02 69.67 62.11 46.96 51.09 46.42 73.91 50.77 39.14 80.03 83.71 134.97 82.41 55.24 66.3 48.65 43.67 38.73 44.69 44.34 63.88 64.91 59.55 40.79 51.31 46.18 59.71 59.2 90.88 59.03 61.79 125.67 40.83 100.08 58.57 60.45 33.44 49.53 46.67 68.56 47.82 44.5 81.31 60.57 50.52 54.81 38.3 64.63 100.69 32.65 53.12 51.7 44.6 97.96 53.36 41.71 41.25 41.9 51.42 95.66 29.93 78.85 49.73 112.5 39.53 529.25 41.05 63.5 93.04 75.95 63.13 118.56 146.99 94.28 55.66 127.35 76.22 138.33 74.96 102.23 54.06 86.49 123.54 83.34 79.2 61.33 57.92 119.69 98.66 51.21 45.54 44.67 90.38 47.28 56.12 94.95 40.67 42.47 35.16 36.28 54.56 51.23 37.89 58.07 153.5 181.48 52.92 47.64 41.97 62.05 75.92 61.99 60.09 67.5 76.86 47.98 53.6 71.81 51.07 45.44 58.6 111.59 58.81 62.56 47.85 107.69 80.13 46.5 51.47 39.55 52.76 37.82 47.38 54.23 37.82 60.7 37.48 50.98 57.99 94.8 48.44 56.28 36.53 41.81 53.68 57.26 70.37 66.98 46.12 112.61 74.79 70.63 41.49 67.65 61.22 54.78 48.17 44.61 97 45.08 56.12 67.91 72.95 55.71 42.86 92.44 130.59 45.76 79.79 57.06 46.48 52.6 54.38 57.48 49.98 63.53 63.52 64.63 36.07 54.52 88.92 44.21 107.31 45.63 57.03 50.56 52.65 54.14 51.76 49.24 56.92 58.51 51.58 65.1 65.11 70 76.88 55.85 65.47 67.45 65.33 80.14 81.79 66.84 81.56 47.54 228751_at CLK4 36.99 134.26 56.46 59.36 120.74 139.77 95.42 109.96 71.63 55.29 84.57 110.53 90.97 235.35 94.89 119.98 28.2 82.79 64.12 71.83 60.64 67.97 26.31 33.48 61.51 116.55 53.8 27.77 36.16 29.76 52.28 23.74 72.95 131.89 45.63 37.36 96.54 32.67 78.9 39.92 64.49 36.86 27.28 37.05 33.77 63.89 128.59 35.39 38.96 41.78 30.31 46.31 28.49 50.37 65.55 44.77 27.86 24.16 67.11 39.96 23.99 19.82 23.83 19.89 86.7 32.76 29.27 51.27 21.1 36.65 19.24 50.7 24.28 30.17 13.41 34.78 68.93 125.34 65.18 30.17 21.64 62.01 64.8 49.03 70.89 65.06 45.89 58.29 46.15 62 20.02 130.67 87.06 83.7 131.71 72.09 80.07 72.25 57.42 40.72 50.62 90.79 66.27 113.02 75.81 46.18 65.85 67.13 28.65 12.35 40.11 68.3 40 33.67 29.1 31.19 106.32 32 56.06 22.48 47.6 22.83 26.09 34.06 52.16 61.79 43.83 51.94 34.22 31.99 72.11 46.05 41.72 45 48.17 27.2 61.6 44.53 48.94 48.04 90.93 60.88 41.7 42.3 47.75 26.06 30.17 40.63 37.62 49.62 48.84 42.6 46.77 52.51 52.18 22.83 35.94 53.67 61.83 36.75 75.36 86.33 36.05 36.93 33.09 64.43 63 61.96 16.42 48.88 29.07 36.22 76.22 44.94 20.78 31.71 32.23 42.01 44.74 62.28 72.69 22.16 49.33 91.62 43.51 35.77 36.75 49.09 20.23 23.46 45.34 50.17 36.16 51.67 66.43 41.17 34.64 63.63 81.06 35.89 42.81 35.45 74.8 23.99 50.12 52.69 41.76 29.03 31.28 70.94 37.6 62.43 29.52 108.19 60.14 75.45 63.32 32.78 34.17 110.34 43.82 50.11 56.31 49.03 29.97 62.27 100.39 228771_at ADRBK2 85.83 192.4 115.32 143.35 130.09 184.14 124.47 136.51 277.6 93.61 114.96 180.17 245.16 83.68 169.24 192.35 296.75 168.95 145.04 72.38 120.58 103.61 73.47 88.48 121.58 222.4 84.57 65.41 110.72 55.47 237.36 110.36 103.13 42.83 85.79 55.31 183.41 123.64 181.92 107.1 105.24 144.46 41.37 78.31 88.64 196.84 78.43 88 74.45 60.21 87.75 103.33 105.26 109.33 193.42 69.49 42.7 115.66 164.89 43.95 172.08 34.77 84.32 36.57 555.51 75.84 61.99 270.98 87.71 94.04 72.81 53.35 62.29 68.57 70.37 124.02 193.78 159.63 54.79 108.83 90.55 118.8 157.28 90.9 180.03 124.64 106.2 141.7 179.74 307.63 112.51 317.68 189.47 122.37 334.64 101.5 137.17 64.81 253.53 123.56 218.03 163.6 103.26 168.81 153.16 141.63 180.32 358.74 95.67 19.7 82.7 243.68 136.48 53.02 97.86 244.69 110.25 143.1 144.62 163.8 159.36 91.77 73.62 132.16 254.22 195.55 71.48 219.75 173.04 80.75 228.42 81.09 290.87 150.23 114.97 103.6 140.28 78.64 104.54 139.82 311.88 66.86 138.21 88.94 118.44 79.57 76.72 108.15 106.86 157.5 61.47 217.61 27.36 113.45 132.15 38.67 81.21 147.81 116.68 141.98 98.46 137.88 22.94 68.35 51.66 113.68 111.09 130.73 86.47 40.08 205.7 91.81 118.74 138.25 16.33 66.94 88.94 118.71 252.56 255.57 188.67 136.96 94.24 94.33 133.65 61.91 51.14 124.79 82.65 141.16 105.5 167.54 79.12 118.27 261.91 92.53 72.92 116.7 136.11 160.23 98.18 108.33 266.8 54.59 77 174.65 111.44 56.79 113.08 88.52 137.09 151.5 179.18 107.89 183.23 222.19 70.78 50.34 72.46 218.82 177.76 92.99 157.34 119.97 57.06 65.05 178.1 228849_at NTRK3 46.1 52.58 58.24 75.26 54.29 84.57 53.42 152.95 61.19 55.03 60.34 63.37 176.14 73.89 85 68.58 72.14 102.87 59.92 82.43 64.61 54.37 37.58 64.62 56.97 48.29 59.03 59.45 417.95 59.72 49.7 116 64.6 69.26 79.07 92.28 80.54 55.7 75.73 65.75 97.66 59.33 70.53 87.37 75.85 61.16 107.81 71.32 65.59 54.88 162.34 61.06 75.91 87.12 66.2 59.18 110.73 52.58 50.84 60.99 114.6 66.71 50.73 65.7 84.07 71.24 129.05 76.56 64.04 56.85 95.45 57.59 55.93 48.37 55.77 60.65 65.13 153.18 73.17 82.14 61.19 61.59 56.25 61.25 54.27 108.54 59.56 46.7 33.98 44.4 88.39 46.81 45.42 48.33 54.73 50.76 47.09 94.63 48.81 61.37 48.96 50.81 45.57 60.88 51.74 39.25 42.87 45.69 49.25 59.61 82.7 69.73 71.17 44.72 40.97 50.43 80.16 78.36 64.11 44.03 56.88 62.26 56.97 46.78 59.36 49.22 55.14 59.8 61.47 47.18 59.08 46.39 91.52 63.65 50.12 60.35 58.69 56.61 68.32 64.08 59.61 65.17 54.75 61.62 59.25 58.34 60.03 87.89 67.88 47.25 99.26 65.83 65.56 59.01 53.15 77.25 61.1 59.88 74.59 62.72 62.76 71.91 69.55 82.48 64.65 75.13 90.32 68.09 69.84 54.54 48.6 59.83 53.92 49.79 62.9 62.29 44.31 52.07 142.97 46.21 87.76 50.71 77.59 62.62 57.06 67.86 59.88 61.78 53.21 60.24 61.8 62.71 66.37 38.64 58.29 46.96 77.55 53.95 45.33 58.06 49.68 38.53 43.33 76.5 60.85 100.91 58.34 68.3 56.53 43.78 80.05 50.16 65.82 57.31 73.17 53.33 69.71 46.4 55.55 57.4 43.46 35.14 45.5 76.8 35.06 46.89 59.92 229158_at WNK4 14.05 41.51 11.97 110.35 394.74 114.95 174.23 198.7 27.13 13.19 71.5 305.66 26.78 30.67 45.7 82.01 10.7 7.52 8.76 9.63 13.13 10.68 9.33 9.86 9.5 7.81 14.28 9.36 11.03 9.49 20.99 26.72 9.23 40.78 7.59 12.23 7.98 8.58 8.61 11.32 9.63 8.14 8.43 10.86 9.24 24.75 9.79 8.7 10.93 57.28 7.17 29.24 51.96 12.42 756.24 9.4 11.26 10.59 14.52 8.52 20.05 9.24 8.42 35.88 10.61 8.65 7.81 7.69 8.8 13.52 6.58 10.05 9.28 11.54 25.32 12.74 10.42 16.78 40.85 10.98 284.69 48.75 558.96 52 59.91 32.87 59.84 53.95 48.06 43.67 815.23 53.93 57.21 190.38 175.72 17.26 119.02 35.41 51.76 14.04 13.37 11.26 17.27 401.37 60.64 415.41 57.69 584.58 81.28 2035.35 17.21 34.06 17.79 21.29 26.95 23.74 113.88 19.87 51.26 17.52 24.57 16.97 25.51 19.5 12.18 17.88 22.63 60.02 16.36 8.92 10 11.93 40.26 12.07 51.49 113.61 8.84 7.25 484.59 13.67 41.37 15.83 34.27 56.47 14.14 18.31 9.49 28.22 57.89 15.95 11.28 43.63 15.3 13.71 72.84 15.35 16.96 13.85 81.07 21.35 92.54 40.08 16.46 364.84 21.18 64.3 400.3 47.93 53.43 14.09 19.18 49.69 34.66 34.84 36.14 40.82 16.83 9.65 160.73 36.9 30.3 19.69 36.44 36.63 24.49 287.58 15.1 37.2 24.37 20.32 33.26 63.79 43.02 18.1 90.26 26.27 20.46 36.65 52.97 51.1 263.02 584.99 27.61 740.28 21.83 146.2 16.45 23.89 54.51 21.12 130.16 890.92 322.57 19.09 93.43 176.41 20.12 11.97 48.48 197.29 17.12 32.87 1186.45 57.47 12.04 18.61 22.75 229285_at RNASEL 38.8 61.28 90.02 107.5 154.86 66.99 128.04 79.09 160.27 61.63 119.23 75.26 134.15 61.8 61.71 48.7 31.11 80.78 70.89 46.75 49.96 114.9 26.23 38.77 25.9 131.06 114.21 177.55 39.85 61.2 98 46.76 79.93 43 42.92 44.55 43.05 46.22 87.07 79.02 61.16 50.59 33 34.96 59.5 55.74 69.29 70.55 46.57 79.22 26.83 89.76 80.44 49.59 87.54 95.18 45.44 27.41 50.54 29.79 25.72 24.18 35.52 29.33 115.38 80.78 34.79 72.6 39.11 59.42 44.59 66.48 46.4 123.63 19.08 72.44 37.18 110.21 46.81 50.93 30.44 114.22 62.54 69.95 89.21 123.94 60.04 42.68 75.29 128.43 14.96 122.86 169.26 182.8 187.2 97.09 123.5 71.23 133.76 101.83 20.94 85.58 82.66 164.77 104.32 91.64 72.77 52.02 104.63 8.42 48.89 64.07 65.49 41.31 56.22 51.24 13.91 27.99 51.6 38.07 98.76 25.75 27.9 38.49 98.74 48.86 97.48 84.58 129.38 83.6 138.41 86.2 78.69 42.42 74.8 81.56 33.63 91.85 61.18 112.05 74.96 83.55 62.49 106.29 77.07 83.51 70.69 46.39 52.9 71.82 26.04 57.44 25.19 61.9 76.95 44.37 57.26 46.8 72.4 49.3 81.67 45.18 73.42 42.87 61.57 78.98 93.55 59.75 14.11 54.35 61.4 57.1 58.65 37.63 13.96 40.72 63 52.78 84.37 58.35 75.57 12.55 93.78 75.26 65.1 62.8 63.29 98.31 15.77 13.59 58.19 95.32 64.78 57.08 66.35 103.98 16.65 82.49 66.21 32.3 27.02 89.91 99.66 8.01 37.63 67.99 28.56 44.59 44.26 117.99 40.54 78.46 61.53 43.8 63.19 67.21 83.12 59.27 85.18 49.58 69.43 66.48 41.96 62.04 61.47 45.74 72.1 229411_at PNCK 50.04 37.78 41.95 54.09 34.19 77.42 48.16 45.05 40.35 50.38 57.18 59.85 33.2 44.9 36.62 87.45 56.16 42.02 37.92 32.44 56.35 38.06 41.97 48.75 37.58 34.6 43.49 54.69 43.79 55.72 41.69 82.47 54.19 41.66 52.39 39.19 44.2 41.98 44.91 48.62 34.32 40.24 38.47 49.73 51.83 36.95 77.64 54.72 44.38 55.16 35.45 49.19 50.98 70.41 35.72 71.02 49.48 51.26 45.09 45.37 69.64 229.2 98.94 57.02 35.74 45.54 38.95 35.17 42.63 38.85 56.48 45.45 38.37 35.98 89.37 46.33 39.29 43.09 57.38 49.34 49.42 29.67 33.77 30.49 29.17 34.33 61.23 34.6 42.01 32.98 53.14 36.62 39.66 35.78 44.63 32.17 48.26 47.01 37.02 44 39.74 42.39 45.32 47.03 104.62 43.06 37.62 35.51 33.32 103.4 42.61 38.55 42.07 49.6 54.47 37.44 47.9 79.1 45.89 44.75 50.74 55.78 50.39 42.01 52.14 28.94 32.6 27.66 36.06 35.92 31.25 51.54 34.66 38.83 30.35 30.41 41.31 34.92 37.02 31.54 44.45 39.3 36.35 33.36 37.87 41.73 34.6 55.81 40.41 45.11 46.88 51.04 71.72 49.85 84.2 36.92 40.77 42.85 41.38 44.52 53.33 31.62 50.51 45.68 47.66 38.6 39.12 36.47 64.46 42.28 45.59 50.32 57.42 48.48 76.68 50.25 51.91 51 57.92 59.02 44.12 56.12 49.67 45.28 50.07 48.14 91.25 42.43 49.25 43.84 51.1 43.74 53.48 46.68 36.26 47.64 132.54 66.68 36.67 70.81 73.41 44.95 46.78 83.18 39.18 48.05 46.72 57.82 46.9 46.28 43.03 32.02 82.46 44.63 54.44 34.93 64.4 40.02 42.83 36.17 39.23 56.4 57.03 34.52 60.07 35.96 26.55 229468_at CDK3 79.28 89.52 168.38 66.52 153.8 157.14 142.54 125.05 95.97 192.22 143.11 150.37 137.05 116.87 222.98 123.88 82.42 151.25 129.69 213.83 249.78 99.59 147.11 133 103.48 109.13 82.22 218.95 120.04 194 67.93 83.26 130.36 97.56 72.81 162.94 113.41 119.68 151.76 113.38 89.91 95.26 110.02 133.73 82.9 151.87 210.47 133.54 139.12 120.07 123.1 95.14 160.99 77.84 77.15 117.99 118.68 69.97 121.77 132.49 201.47 219.15 136.35 54.93 148.67 150.27 72.54 78.92 53.51 186.77 259.65 77.13 115.19 142.46 69.75 91.39 66.16 82.05 49.11 105.2 167.42 62.98 112.17 82.5 86.51 87.25 82.15 129.2 82.46 149.58 222 123.05 107.03 126.52 107.53 124.56 133.34 166.38 145.4 171.14 474.45 83.82 218.05 264.07 161.85 171.88 93.29 153.37 137.08 265.33 126.87 119.84 130.79 276.68 155.47 150.24 331 408.73 140.64 144.15 156.01 252.52 246.64 85.97 118.88 50.92 122.17 119.93 54.82 232.6 244.5 100.23 225.76 190.59 116.9 144.55 152.76 125.65 128.37 197.41 87.02 125.09 109.68 139.73 89.08 175.79 128.72 127.3 132.72 90.5 97.57 168.33 95.94 135.77 127.6 214.03 38.99 82.03 133.28 165.26 290.4 142.02 86.8 171.26 149.76 97.61 151.99 291.95 315.54 82.43 140.61 166.84 244.61 114.97 436.85 243.19 206.82 140.22 189.49 122.7 170.88 95.81 409 173.5 207.09 176.58 96.92 204.81 86.07 120.77 257.83 103.39 141.6 104.18 131.36 157.46 204 139.67 140.48 328.24 145.99 104.38 114.83 149.31 237.2 94.53 391.14 123.79 135.92 89.67 229.7 219.47 132.18 262.13 151.36 159.2 195.28 76.1 91.77 172.36 123.53 153.81 224.68 120.17 139.13 122.33 74.57 229584_at LRRK2 12.89 16.89 16.1 35.53 19.57 20.6 13.99 12.58 23.69 57.66 12 12.44 14.4 8.46 14.66 7.24 11.59 38.68 22.44 37.42 9.71 56.03 7.59 16.95 13.57 59.5 21.3 31 27.61 11.09 20.11 12.33 12.15 10.81 11.6 89.91 99.66 8.01 37.63 67.99 28.56 44.59 44.26 117.99 40.54 78.46 61.53 43.8 63.19 67.21 83.12 59.27 85.18 49.58 69.43 66.48 41.96 62.04 61.47 45.74 72.1 229411_at PNCK 50.04 37.78 41.95 54.09 34.19 77.42 48.16 45.05 40.35 50.38 57.18 59.85 33.2 44.9 36.62 87.45 56.16 42.02 37.92 32.44 56.35 38.06 41.97 48.75 37.58 34.6 43.49 54.69 43.79 55.72 41.69 82.47 54.19 41.66 52.39 39.19 44.2 41.98 44.91 48.62 34.32 40.24 38.47 49.73 51.83 36.95 77.64 54.72 44.38 55.16 35.45 49.19 50.98 70.41 35.72 71.02 49.48 51.26 45.09 45.37 69.64 229.2 98.94 57.02 35.74 45.54 38.95 35.17 42.63 38.85 56.48 45.45 38.37 35.98 89.37 46.33 39.29 43.09 57.38 49.34 49.42 29.67 33.77 30.49 29.17 34.33 61.23 34.6 42.01 32.98 53.14 36.62 39.66 35.78 44.63 32.17 48.26 47.01 37.02 44 39.74 42.39 45.32 47.03 104.62 43.06 37.62 35.51 33.32 103.4 42.61 38.55 42.07 49.6 54.47 37.44 47.9 79.1 45.89 44.75 50.74 55.78 50.39 42.01 52.14 28.94 32.6 27.66 36.06 35.92 31.25 51.54 34.66 38.83 30.35 30.41 41.31 34.92 37.02 31.54 44.45 39.3 36.35 33.36 37.87 41.73 34.6 55.81 40.41 45.11 46.88 51.04 71.72 49.85 84.2 36.92 40.77 42.85 41.38 44.52 53.33 31.62 50.51 45.68 47.66 38.6 39.12 36.47 64.46 42.28 45.59 50.32 57.42 48.48 76.68 50.25 51.91 51 57.92 59.02 44.12 56.12 49.67 45.28 50.07 48.14 91.25 42.43 49.25 43.84 51.1 43.74 53.48 46.68 36.26 47.64 132.54 66.68 36.67 70.81 73.41 44.95 46.78 83.18 39.18 48.05 46.72 57.82 46.9 46.28 43.03 32.02 82.46 44.63 54.44 34.93 64.4 40.02 42.83 36.17 39.23 56.4 57.03 34.52 60.07 35 96 26 55 44.63 54.44 34.93 64.4 40.02 42.83 36.17 39.23 56.4 57.03 34.52 60.07 35.96 26.55 229468_at CDK3 79.28 89.52 168.38 66.52 153.8 157.14 142.54 125.05 95.97 192.22 143.11 150.37 137.05 116.87 222.98 123.88 82.42 151.25 129.69 213.83 249.78 99.59 147.11 133 103.48 109.13 82.22 218.95 120.04 194 67.93 83.26 130.36 97.56 72.81 162.94 113.41 119.68 151.76 113.38 89.91 95.26 110.02 133.73 82.9 151.87 210.47 133.54 139.12 120.07 123.1 95.14 160.99 77.84 77.15 117.99 118.68 69.97 121.77 132.49 201.47 219.15 136.35 54.93 148.67 150.27 72.54 78.92 53.51 186.77 259.65 77.13 115.19 142.46 69.75 91.39 66.16 82.05 49.11 105.2 167.42 62.98 112.17 82.5 86.51 87.25 82.15 129.2 82.46 149.58 222 123.05 107.03 126.52 107.53 124.56 133.34 166.38 145.4 171.14 474.45 83.82 218.05 264.07 161.85 171.88 93.29 153.37 137.08 265.33 126.87 119.84 130.79 276.68 155.47 150.24 331 408.73 140.64 144.15 156.01 252.52 246.64 85.97 118.88 50.92 122.17 119.93 54.82 232.6 244.5 100.23 225.76 190.59 116.9 144.55 152.76 125.65 128.37 197.41 87.02 125.09 109.68 139.73 89.08 175.79 128.72 127.3 132.72 90.5 97.57 168.33 95.94 135.77 127.6 214.03 38.99 82.03 133.28 165.26 290.4 142.02 86.8 171.26 149.76 97.61 151.99 291.95 315.54 82.43 140.61 166.84 244.61 114.97 436.85 243.19 206.82 140.22 189.49 122.7 170.88 95.81 409 173.5 207.09 176.58 96.92 204.81 86.07 120.77 257.83 103.39 141.6 104.18 131.36 157.46 204 139.67 140.48 328.24 145.99 104.38 114.83 149.31 237.2 94.53 391.14 123.79 135.92 89.67 229.7 219.47 132.18 262.13 151.36 159.2 195.28 76.1 91.77 172.36 123.53 153.81 224.68 120.17 139.13 122.33 74.57 229584_at LRRK2 12.89 16.89 16.1 35.53 19.57 20.6 13.99 12.58 23.69 57.66 12 12.44 14.4 8.46 14.66 7.24 11.59 38.68 22.44 37.42 9.71 56.03 7.59 16.95 13.57 59.5 21.3 31 27.61 11.09 20.11 12.33 12.15 10.81 11.6 12.55 18.5 21.87 42.32 27.97 33.2 28.52 9.04 17.69 11.14 39.06 11.43 20.41 10.27 12.55 9.5 17.96 10.28 28.66 9.92 20.85 14.3 18.45 75.21 11.65 13.25 8.97 11.87 17.4 76.46 19.7 13.61 55.38 12.23 14.85 12.09 9.8 19.32 17.31 7.63 8.52 14.74 37.02 13.41 9.53 15.98 238.48 17.34 21.23 30.79 57.68 15.18 118.16 31.81 34.36 8.22 36.73 17.6 179.95 54.36 33.21 45.06 17.67 41.16 25.3 10.99 47.17 194.63 36.14 19.63 43.59 52.44 33.43 37.76 16.32 15.95 18.83 38.51 19.69 76.02 19.83 10.53 15.94 63.82 18.04 25.08 11.08 18.67 22.27 42.74 17.34 18.13 55.04 21.6 21.95 43.18 34.77 20.28 38.12 16.98 20.72 16.67 38.97 20.36 63.45 22.69 23.58 13.9 24.79 19.19 12.64 86.87 13.75 23.77 18.84 14.71 21.87 17.18 44.69 73.98 11.03 14.73 39.15 21.93 39.12 26.24 22.36 32.76 15.31 11.61 36.39 22.72 26.6 9.67 14.21 45.05 19.58 42.04 21.6 8.05 138.33 25.02 19.67 44.82 14.85 31.76 11.06 19.87 18.41 28.51 13.7 13.5 19.29 11.91 14.08 29.25 35.51 21.62 48.25 25.94 29.12 10.04 38.24 21.6 27.28 11.65 101.68 57.32 27.1 9.63 24.56 10.24 16.47 37.99 22.21 14.2 19.54 31.64 22.42 17.52 30.73 36.8 26.63 18.17 19.55 51.04 24.14 24.3 11.57 14.51 22.43 38.89 229854_at OBSCN 79.38 29.87 51.48 28.61 40.33 36.09 43.26 40.94 26.1 74.76 38.14 47.05 60.68 72.26 60.03 84.82 60.28 50.48 66.73 72.5 58.1 33.73 236.26 47.81 71.54 35.36 25.29 42.02 51.79 55.3 39.53 77.34 48.9 60.95 48.78 64.64 150.3 35.96 43.21 69.59 62.75 56.25 104.76 35.48 41.42 38.46 122.26 31.52 145.46 42.24 45.28 43.93 34.38 44.42 27.5 55.32 51.5 48.86 39.17 36.14 42.56 122.21 32.06 28.85 48.44 46.56 27.84 41.7 44.25 54.68 59.58 37.23 48.71 23.18 46.46 46.54 35.46 31.04 46.64 42.09 56.49 30.75 41.61 23.44 33.94 28.53 37.2 35.26 31.84 57.58 102.71 43.45 47.24 32.62 34.72 23.95 30.88 79.24 60.35 40.65 48.37 34.14 33.36 76.75 58.76 48.76 43.78 60.09 82.05 101.92 47.69 58.33 60.27 62.86 46.14 48.59 44.68 262.7 67.38 34.2 35.1 77.11 38.74 70.77 35.89 30.65 31.38 27.31 27.68 70.15 35.1 54.21 58.46 24.78 36.47 35.74 36.62 26.47 45.47 32.98 90.49 38.88 25.44 23.31 36.69 31.39 30.38 38.66 66.65 28.52 38.79 51.95 49.5 27.46 29.05 73.55 39.93 34.99 33.3 48.88 188.79 58.32 54.03 38.36 26.99 31.65 28.94 31.96 125.22 60.29 38.24 37.28 45.74 73.38 91.46 61.4 42.27 41.38 44.29 81.68 44.23 68.6 36.43 61.4 96.38 35.04 48.01 47.09 81.97 29.15 45.95 34.62 58.75 23.54 56.55 27.16 70.51 30.48 33.1 69.61 50.54 27.93 36.06 30.41 34.42 30.55 60.12 75.69 33.11 27.65 144.05 29.14 33.85 23.59 90.28 40.03 35.47 77.5 32.41 114.39 42.91 126.44 45 113.24 41.74 48.77 27.81 230191_at TTBK1 51 47.83 50.65 78.62 62.8 43.95 65.22 53.73 48.24 57.74 130.11 44.79 39.93 47.81 36.81 66.05 64.16 49.38 41.44 38.65 46.48 52.03 39.84 57.77 46.27 41.02 53.8 47.42 56.47 72.19 41.05 82.44 51.9 46.02 57.41 60.94 57.76 43.44 46.03 48.88 42.61 38.07 49.76 52.31 46.49 45.51 49.79 36.88 63.86 46.21 48.43 40.27 48.73 52.7 72.12 38.01 68.58 63.41 65.16 47.86 57.29 76.61 50.01 73.68 49.26 46.02 42.5 46.08 50.72 45.85 57 46.83 45.28 43.54 92.82 51.37 44.61 37.64 60.24 51.72 44.6 45.96 47.79 48.82 46.25 44.88 59.47 52.17 61.05 42.65 141.11 41.49 53.87 52.13 49.3 38.38 47.16 43.99 48.58 43.37 41.87 46.66 46.69 38.82 40.38 53.15 38.54 37.7 46.05 115.21 33.21 33.37 41.7 39.12 38.43 45.6 94.42 48.65 34.99 36.3 34.88 51.69 46.19 44.59 37.58 48.17 41.29 32.52 43.21 41.83 34.17 35.2 59.88 41.4 41.35 47.47 48.78 50.22 45.4 45.24 46.09 45.49 42.07 41.97 45.31 47.36 66.88 41.01 30.67 34.53 45.33 48.71 51.76 41.64 48.14 55.28 48.45 43.58 38.04 60.93 39.72 40.19 48.56 63.33 60.54 50.28 47.27 52.43 161.64 50.67 39.85 47.54 38.87 42.83 75.75 55.42 45.92 39.16 27.61 35.79 42.04 41.3 52.26 51.46 55.97 48.57 41.76 38.89 50.09 48.51 44.96 36.3 41.85 40.5 32.63 41.37 39.71 42.48 43.95 42.93 55.65 38.76 39.09 79.72 48.96 40.99 37.79 38.31 59.43 37.96 42.44 48.78 40.92 42.09 32.47 42.44 43.64 45.24 40.9 34.21 48.48 37.65 45.89 38.88 38.52 42.52 27.5 230239_at ROCK1 129.77 127.03 141.93 161.3 117.39 122.24 137.85 109.61 141.61 151.17 128.68 119.77 122.39 126.14 87.56 134.35 130.1 126.19 121.17 134.79 138.75 121.21 118.52 176.66 131.19 98.03 122.27 116.87 121.3 127.23 123.36 142.59 130.52 128.95 151.52 137.07 140.7 129.97 134.46 115.93 140.27 131.2 139.44 128.65 134.55 119.89 112.51 116.69 121.67 126.94 126.78 129.66 145.26 112.88 139 107.09 185.65 156.04 123.8 156.9 137.94 112.64 149.35 193.23 136.82 129.75 132.02 138.28 147.35 125.77 152.45 128.57 133.56 127.12 153.46 127.01 118.62 102.51 133.83 114.44 125.72 140.17 134.43 140.91 113.34 97.82 131.21 113.48 129.88 133.51 228.11 116.8 128.28 130.59 127.65 130.18 143.99 165.72 146.08 158.87 139.52 139.42 145 103.26 143.34 127.01 95.97 101.18 144.93 238.49 159.58 114.8 127.37 148.49 162.98 136.67 125.65 99.57 151.09 131.16 119.91 119.71 170.21 136.14 155.96 154.69 143.75 128.78 151.72 163.44 136.02 152.38 173.11 179.88 139.68 179.23 158.07 147.08 141.27 143.36 145.43 184.3 150.15 159.89 166.49 172.49 158.85 149.83 141.87 133.28 165.49 159.87 171.84 141.9 156.84 137.96 163.04 160.02 128.89 159.29 145.69 127.3 158.38 177.75 143.72 154.73 147.04 182.29 160 162.59 164.27 150.03 149.8 135.86 272.58 163.7 142.92 156.55 153.54 155.34 135.37 155.32 158.57 149.86 136.3 163.28 178.86 133.64 158.52 173.22 142.56 141.96 164.71 131.76 125.12 143.05 138.31 89.69 137.84 144.8 173.48 135.8 127.38 167.58 153.94 148.69 163 158.65 161.43 131.32 127.07 141.95 122.14 124.83 123.88 117.33 118.61 145.21 139.89 115.9 116.72 92.99 113.37 96.02 103.78 79.82 92.81 230240_at DYRK3 75.65 53.65 60.7 42.17 28.8 41 36.05 80.6 54.15 42.66 43.92 64.52 41.87 36.01 37.49 43.31 74.87 26.55 56.86 55.48 33.19 33.45 39.7 27.03 174.09 38.14 27.83 31.45 27.4 43.83 32.15 86.8 73.19 27.43 45.74 43.58 68.37 33.35 44.23 30.65 55.14 48.35 54.19 25.81 84.81 49.96 110.11 48.59 37.62 39.55 34.22 33.86 67.88 82.09 53.93 64.67 57.68 40.63 39.18 56.24 43.09 22.77 51.83 40.19 33.77 33.51 31.65 41.6 56.81 42.34 38.05 26.79 60.35 42.73 136.4 47.37 58.5 38.26 40.86 45.89 45.54 55.07 40.81 45.01 38.04 41.44 42.86 43.14 36.77 47.65 41.88 48.65 49.82 46.11 88.52 41.65 49.29 74.31 95.88 90.59 43.32 35.72 30.87 37.31 41.04 61.06 51.43 49.8 89.77 49.08 102.79 51.16 41.15 28.08 33.93 78.72 30.32 36.09 30.95 45.21 53.33 38.69 43.09 50.32 47.2 30.08 34.61 51.3 64.37 73.56 37.48 44.86 66.58 41.62 40.47 36.31 51.57 44.92 47.39 43.99 47.14 37.79 60.35 41.47 78.09 47.79 45.53 50.76 39.18 48.25 73.89 44.1 52.72 40.27 28.86 45.03 52.95 35.83 41.1 40.66 38.11 43.81 48.98 42.27 55.4 35.64 52.55 35.66 50.33 28.84 53.32 49.52 32.5 49.8 31.46 32.6 30.25 74.84 47.63 32.26 59.44 60.14 39.91 46.98 61.62 42.05 33.78 82.52 57.62 48.13 38.47 47.67 39.87 26.79 31.76 35.59 41.41 45.98 38.05 56.64 30.75 35.28 35.45 56.73 38.43 34.15 30.7 46.56 36.25 28.87 32.5 38.17 33.61 33.61 67.47 36.02 40.38 32.52 44.25 36.91 42.12 49.91 38.34 48.59 48.28 74.78 42.7 230425_at EPHB1 80.42 7.69 8.75 12.76 10.41 8.16 14.62 7.38 9.85 7.36 8.52 7.41 11.18 8.86 11.15 15.59 9.78 23.61 30.4 35.24 9.27 9.46 36.29 19.39 162.66 23.04 8.84 8.36 98.85 9.28 12.21 8.9 7.47 9.5 28.4 58.37 50.44 8.58 21.75 18.5 10.39 49.23 43.77 13.04 11.16 21.57 8.65 30.74 13.22 11.09 10.83 8.91 10.73 68.49 6.64 12.9 42.19 7.38 24.26 9.12 8.34 67.88 8.59 9.74 10.5 13.33 25.28 11.09 33.44 32.78 10.15 14.69 44.66 61.11 10.87 10.11 23.37 12.52 10.05 8.78 8.68 9.85 7.89 7.52 23.13 11.56 8.44 8.79 8.78 19.09 9.14 11.01 8.12 9.61 27.6 7 10.2 9.02 14.69 168.04 14.76 12.56 8.57 13.52 8.85 9.68 9.38 9.7 11.44 10.49 30.9 101.78 32.97 14.49 77.41 64.92 12.44 11.3 39.98 14.03 17.23 8.9 12.44 48.19 15.34 17.66 9.93 12.23 8.02 7.93 8.83 7.13 41.29 9.85 9.47 9.91 18.65 16.05 8.79 7.79 6.78 8.59 8.16 6.95 10.2 6.95 8.78 15.86 17.14 9.9 47.59 19.62 20.06 9.72 7.26 12.89 7.66 7.71 8.88 10.42 8.96 21.28 7.9 8.42 11.01 8.91 8.52 25.75 8.87 8.13 7.56 9.69 7.62 114.96 7.46 11.15 10.01 6.91 8.69 9.32 11.14 38.77 7.04 13.83 18.29 8.77 9.67 10.97 40.27 12.75 10.47 14.68 8.01 7.92 60.7 8 8.17 9.88 13.51 89.45 7.96 8.91 17.2 8.49 13.89 10.95 9.97 14.04 7.5 9.21 23.89 10.27 8.85 7.36 7.19 7.14 6.97 6.65 7.92 7.94 60.14 9.33 10.12 108.86 9.63 7.18 56.03 230934_at STK32C 53.14 49.26 54.01 72.46 49.07 44.17 72.1 47.28 45.32 68.35 60.3 52.07 68.63 56.64 44.45 51.63 78.5 65.54 46.02 55.03 69.01 66.51 37.65 75.95 58.62 46.65 42.72 46.62 51.86 85.36 61.57 87.78 56.71 69.17 62.74 71.23 52.6 84.65 52.58 49.72 61.96 69.33 89.48 53.55 65.51 38.83 42.29 49.79 77.96 50.43 72.92 48.83 68.47 76.76 80.26 52.48 62.63 79.14 62.48 37.55 62.11 62.68 34.71 62.35 56.74 63.72 42.45 37.05 58.79 52.6 59.75 54.23 41.36 49.26 55.54 50.3 54.55 43.97 59.04 39.38 58.45 32.12 58.18 33.53 35.05 31.76 61.89 43.14 53.33 40.24 102.32 39.68 58.95 42.28 48.81 38.22 44.54 54.59 44.75 55.26 43.73 32.51 57.86 48.24 49.37 47.72 48.44 51.36 54.84 147.79 69.14 46.19 51.69 88.57 63.56 70.5 58.37 63.77 43.45 54.88 47.89 56.78 80.68 42.41 29.7 63.97 35.57 30.14 33.72 36.85 38.69 39.56 35.63 44.93 42.22 44.38 64.72 37.05 28.73 34.21 33.04 34.31 39.02 41.85 39.33 43.01 33.49 36.25 36.9 50.5 71.8 42.35 46.5 59.29 48.19 67.89 52.03 42.55 41.01 42.39 38.26 37.43 71.88 56.46 49.3 36.96 44.84 37.62 102.81 47.44 52.18 60.11 49.91 57.19 121.91 66.68 52.16 69.72 55.63 72.27 44.83 61.37 42.97 52.89 50.38 58.99 60.06 55.58 69.2 81.45 61.35 48.75 66.25 42.72 54.97 60.94 109.92 51.35 64.74 59.51 73.01 43.56 42 90.17 51.45 37.74 58.71 58.55 43.51 45.39 76.24 53.13 41.45 35.55 36.84 45 31.89 46.75 39.33 32.99 75.18 40.82 55.3 33.37 58.26 40.76 29.52 231779_at IRAK2 51.85 78.59 67.36 52.65 43 60.95 56.12 33.36 50.35 117.11 33.19 32 39.61 63.19 50.13 33.31 62.97 45.22 112.81 93.64 37.28 53.3 37.05 48.7 44.87 93.62 35.33 43.59 92.31 32.8 57.4 76.32 43.48 41.2 62.39 56.55 82.66 185.38 65.26 53.16 63.8 66.17 41.64 41.84 63.08 51.71 42.62 94.26 49.98 62.78 49.08 289.34 35.33 64.29 27.77 49.17 140.87 92.56 71.63 40.02 43.64 31.72 67.83 52.55 401.16 67.01 70.18 71.06 237.36 61.03 33.32 66.21 56.65 119.73 54.23 94.14 25.23 43.41 26.14 37 28.8 63.97 33.89 43.36 42.04 44.74 50.43 49.36 49.03 84.22 42.48 64.49 59.74 72.88 76.18 60.9 34.09 46.03 76.91 131.95 28.82 94.31 49.59 70.28 42.98 46.77 40.99 38.35 52.59 40.03 27.12 27.55 38.36 38.56 50.97 53.66 29.44 57.87 61.74 43.89 46.58 29.3 37.68 41.91 50.12 126.89 29.86 42.33 45.34 37.78 57.78 79.21 76.19 42.23 46.96 30.16 44.86 50.42 37.48 38.55 36.61 81.11 23.98 34.55 49.1 38.57 24.81 36.9 127.91 60.48 49.65 53.81 237.01 50.5 41.58 36.67 78.05 94.7 36.78 51.49 47.84 39.49 55.22 26.24 28.91 58.83 36.63 61.28 29.74 33.75 35.82 30.77 97.58 36.64 29.61 39.92 39.16 36.17 42.67 40.79 57.35 30.07 29.14 28.78 38.51 31.4 37.41 39.56 37.45 66.29 39.33 29.88 27.06 29.13 45.24 51.11 26.51 46.46 52.62 22.7 46.47 34.44 150.54 44.92 231792_at MYLK2 69.17 72.28 79.19 98.4 65.31 72.53 91.49 69.18 76.61 82.42 87.24 66.63 66.18 73.81 68.63 77.75 75.41 67.52 66.36 66.33 74.58 62.07 77.29 109.27 80.43 68.67 75 75.99 90.2 76.84 69.05 82.96 66.05 78.83 90.09 98.91 88.24 77.19 85.53 101.21 85.2 71.54 77.6 82.07 74.3 73.1 87.68 74.1 87.78 75.68 69.76 74.9 74.56 84.58 88.16 73.57 91.96 95.65 64.29 86.82 92.97 86.32 93.87 87.47 94.94 70.05 76.19 67.08 72.82 63.67 86.46 75.38 70.94 64.08 71.19 75.39 71.69 67.86 93.97 82.52 71.72 64.38 78.61 80.02 72.2 80.93 102.18 78.01 85.97 82.8 145.35 77 77.09 84.22 69.81 83.24 92.8 87.1 83.42 74.14 72.67 79.93 71.37 70.33 77.06 83.12 75 70.33 86.42 85.51 63.02 50.66 81.78 60.74 63.93 77.68 53.69 85.96 67.43 69.84 65.79 69.66 86.89 76.67 70.63 85.26 62.31 76.09 70.84 64.59 71.21 72.41 77.7 69.53 67.66 71.25 86.7 72.26 76.79 72.8 79.95 61.8 73.02 70.56 76.64 82.61 76.93 81 66.47 79.15 77.16 78.38 92.51 91.97 85.39 80.14 85.49 94.11 69.87 81.81 80.6 73.02 79.55 101.17 78.35 86.32 73.84 89.59 92 85.37 72.99 66.27 67.39 73.24 101.29 80.45 65.87 59.59 72.74 78.22 65.99 76.38 81.28 60.31 83.19 71.89 73.27 63.27 70.35 68.36 57.62 58.96 84.11 77.74 61.76 80.47 69.93 76.18 75.2 71.78 70.9 68.63 64.84 108.29 80.81 62.82 73.9 86.26 70.23 63.52 74.51 69 72.23 81.08 73.6 62.71 73.52 78.58 80.86 66.28 68.63 64.61 66.38 66.71 93.98 62.11 64.48 231806_s_at STK36 121.98 266.69 283.71 112.6 320.58 482.52 208.88 259.1 201.55 190.25 339.26 368.59 349.59 501.09 370.95 191.96 87.8 256.8 122.92 154.24 291.98 272.58 234.51 192.18 109.69 162.67 195.13 248.29 142.22 203.27 199.54 236.96 273.76 396.53 168.04 211.97 287.32 204 233.32 169.25 138.93 218.18 201.24 185.3 200.96 157.68 242.19 121.46 165.21 235.23 136.47 251.96 410.9 111.1 276.98 244.68 159.39 114.15 275.54 329.37 317.4 309.37 160.05 123.96 118.28 111.48 309.41 109.65 111.61 147.79 124.05 171.12 152.22 101.03 163.66 206.63 43.77 147.58 133.49 185.73 379.11 159.99 172.88 320.87 123.21 259.35 202.78 112.73 142.35 186.51 555.24 205.65 233.82 128.31 179.2 177.78 169.6 315.52 210.2 121.7 574.27 149.94 253.36 245.67 286.91 444.02 341.85 282.98 284.66 825 177.39 163.46 306.85 830.57 150.04 228.89 494.46 437.43 154.3 169.92 288.65 465.55 198.61 211.01 156.98 140.8 314.09 182.51 148.91 178.69 344.73 169.95 244.03 195.31 207.35 242.78 155.16 162.76 222.54 233.28 191.72 157.66 280.21 346.19 131.77 282.32 210.23 238.59 241.02 185.55 142.02 206.39 113.87 260.61 307.65 217.26 155.13 117.52 275.62 130.74 491.86 229.22 129.77 323.06 208.05 273.12 352.49 212.24 455.58 330.27 214.1 331.57 438.65 103.61 403.23 266.44 148.36 298.32 436.87 286.85 189.84 144.6 358.01 246.44 193.46 300.62 249.79 298.61 130.53 513.35 395.7 167.15 335.41 247.16 202.64 293.17 135.88 260.79 349.3 334.03 193.77 365.36 247.77 427.84 188.49 248.39 131 129.08 198.09 222.33 136.88 483.7 202.76 446.77 338.89 256.5 210.13 135.88 118.72 342.69 99.86 189.32 267.47 155.91 113.89 74.52 112.05 232153_at SPEG 77.68 46.05 70.22 67.71 47.52 55 64.48 40.74 62.39 66.45 52.95 54.3 48.21 41.7 51.17 52.64 43.79 66.52 67.07 52.3 47.47 54.19 58.36 70.05 66 46.38 53.36 56.34 60.44 122.18 49.69 69.01 56.66 46.69 68.84 51.26 52.44 52.55 60.18 76.13 57.22 56.99 74.85 67.07 60.44 49.68 72.33 55.3 66.42 54.14 85.19 56.89 51.32 61.17 67.18 47.42 67.9 57.73 56.22 75.38 62.41 58.78 57.7 60.99 52.02 46.43 48.71 56.07 49.67 49.19 53.89 59.63 58.49 43.91 57.5 52.88 41.58 47.76 62.8 54.17 57.44 37.97 26.81 39.77 32.51 28.49 35.54 33.43 36.13 48.52 109.56 41.59 50.03 41.25 44.74 44.16 39.76 50.48 46.83 75.86 47.13 43.49 48.88 34.58 51.32 47.81 43.48 49.51 46.16 86.52 73.92 48.38 48.85 80.91 62.14 89.61 58.25 89.49 44.48 51.44 61.26 88.8 57.46 50.04 37.21 35.47 35.61 36.8 29.12 38.9 31.31 38.46 42.47 36.07 26.46 33.44 38.15 33.24 33.35 37.51 36.31 30.52 34.79 32.98 32.12 37.89 33.52 46.16 39.57 34.96 54.73 49.87 49.33 40.63 47.06 82.44 43.3 44.12 36.98 50.77 39.35 47.17 43.28 55.44 36.8 40.38 43.07 43.24 82.4 41.05 53.78 52.71 54.4 54.83 135.54 66.81 66.83 58.36 60.6 44.26 48 65.4 61.22 61.03 46.32 60.2 58.64 48.13 63.32 51.09 50.63 50.6 53.43 52.9 51.95 56.84 59.19 41.27 60.02 66.15 55.96 55.64 48.14 65.67 41.81 45.05 57.04 56.57 54.54 42.33 84.33 51.52 49.89 51.25 37.55 37.83 40.88 41.21 39.39 40.02 33.49 42.84 21.39 39.27 39.75 35.69 29.86 232205_at RYK 49.85 84.25 64.99 22.22 54.19 68.42 41.66 37.36 41.31 72.4 55.4 64.09 54.11 118.15 62.73 103.99 39.27 39.36 50.34 64.69 53.31 44.4 33.9 23.83 66.54 37.42 41.05 41.27 49.42 22.35 26.03 20.85 47.1 68.83 27.65 22.09 54.35 58.38 37.19 30.03 53.74 33.51 24 47.49 36.1 39.11 34.06 35.97 17.76 34.04 56.52 21.12 43.77 30.84 50.18 31.6 30.78 15.74 43.01 46.95 54 62.33 27.78 39.34 67.85 45.66 34.31 37.58 55.19 45.29 54.53 41.54 108.57 51.12 42.44 44.42 61.5 31.75 40.31 37.6 68.99 35.76 63.08 49.42 46.77 46.01 56.17 29.02 40.2 58.38 79.54 104.56 66.31 45.95 147.93 63.56 46.7 40.38 50.54 70.43 110.41 66.21 44.8 162.23 62.42 45.49 44.97 45.52 41 32.43 28.3 87.01 37.68 53.03 47.64 89.35 54.57 74.93 43.02 64.73 84.35 79.27 59.28 57.21 70.52 36.45 64.19 106.92 27.35 41.79 71.64 66.27 122.43 43.06 71.06 83.56 70.57 45.32 82.91 47.12 65.11 44.18 43.71 37.07 72.18 138.01 208.32 48.84 36.44 28.63 28.83 34.87 14.24 29.13 38.1 37.86 39.81 28.24 41.62 18.84 41.86 39.54 32.61 35.34 41.78 32.59 43.28 35.58 56.53 28.81 27.8 38.23 37.64 101.62 81.03 55.84 40.13 45.63 74.62 87.79 60.47 33.79 32.25 71.64 49.35 41.59 57.23 49.45 33.48 70.67 41.17 42.38 49.67 42.31 106.44 39.33 118.56 77.6 68.24 76.21 153.91 97.72 71.09 86.39 41.67 60.73 103.75 54.71 55.39 39.83 125.49 51.57 61.4 76.96 79.46 50.29 75.72 52.88 45.38 76.23 42.2 50.16 52.93 128.97 68.5 41.4 46.63 232206_at ULK4 15.37 18.73 21.03 35.14 23.16 21.06 28.2 18.9 30.21 16.1 20.37 19.16 16.92 56.86 19.33 23.05 23.64 11.27 19.46 11.38 22.12 29.32 17.88 19.61 15.19 12.98 19.44 13.99 18.97 18.48 13.46 14.52 12.31 14.63 14.11 16.78 20.39 16.85 21.11 23.05 13.08 15.49 14.57 11.83 17.09 19.45 13.87 10.43 19.09 24.13 19.78 23.61 17.15 17.4 43.6 28.07 18.05 18.44 20.75 20.26 30.03 20.26 17.87 20.99 21.15 15.1 18.01 13.77 15.72 16.61 22.29 16.13 13.84 15.25 22.82 45.44 24.8 18.52 21.71 19.52 17.94 38.16 23.34 36.45 19.28 22.34 19.3 14.97 17.52 22.11 25.52 21.99 15.7 20.79 30.26 16.52 23.95 25.8 27.78 19.3 18.73 18.44 26.52 20.98 21.94 28.37 28.06 22.82 16.12 33.2 11.9 13.71 17.04 17.49 18.89 19.53 16.95 14.19 17.99 22.08 22.54 15.77 20.23 25.44 16.57 16.01 12.57 24.93 19.23 13.01 18.36 12.32 141.11 12.71 18.78 18.53 14.45 13.82 15.72 24.48 29.45 17.23 23.93 17.09 15.38 19.46 23.15 23.87 18.87 18.67 15.55 19.09 20.83 19.75 25.85 12.71 18.97 23.32 17.37 12.9 16.93 16.57 20.31 20.5 20.67 17.02 26.94 17.11 18.06 19.01 21.95 25.65 20.01 17.4 27.52 22.68 15.17 16.53 16.75 26.47 21.63 22.14 22.79 15.42 26.22 21.63 32.36 18.55 22.32 24.91 28.85 22.08 19.01 14.61 14.79 19.23 18.51 19.34 24.59 12.9 23.41 19.13 17.46 46.45 11.97 20.39 18.7 14.82 18.45 28.3 12.9 18.44 17.6 19.95 20.54 20.62 36.06 16.74 23.77 30.78 13.97 16.67 48.49 15.13 13.98 14.48 14.62 232282_at WNK3 18.82 13.7 13.29 9.06 11.24 14.36 8.15 72.12 74.02 13.28 8.06 30.94 16.3 84.66 16.83 307.64 47.32 81.9 94.71 119.67 5.88 6.25 45.77 15.18 7.78 21.15 7.85 29.69 130.04 36.94 8.17 6.2 8.18 7.19 28.61 42.12 41.69 64.77 8.78 50.28 42.88 85.86 13.22 7.68 62.51 16.53 50.37 7.56 17.9 9.72 49.09 11.99 9.43 48.28 63.95 14.33 8.81 99.24 9.25 11.77 10.2 112.58 76.86 7.38 9.33 134.85 31.95 14.51 103.86 43.94 55.44 97.17 9.41 8.12 31.87 10.93 8.08 12.76 9.29 16.65 13.25 12.37 58.18 15.25 31.64 10.8 15.89 10.59 65.95 9.92 10.13 9.87 7.09 15.27 9.55 6.87 18.85 18.29 12.06 12.1 82.87 14.57 7.62 11.32 14.13 11.59 28.56 12.26 25.46 16.7 24.42 29.76 17.72 9.71 10.39 25.3 151.16 10.03 18.29 10.91 14.43 43.49 48.82 15.16 11 9.81 9.83 13.47 9.98 12.53 7.02 11.05 14.31 9.44 11.22 18.24 102.02 10.9 12.05 11.54 10.37 9.9 11.43 13.3 10.5 9.2 15.08 18.18 20.74 9.99 124.21 15.88 12.75 12.14 11.01 51.11 12.48 10.41 18.78 20.02 17.21 18.57 11.27 18.24 11.78 14.32 11.56 15.26 11.46 11.49 7.41 7.71 8.63 30.95 6.1 8.85 7.92 8.5 7.04 9.56 9.49 64.23 9.45 9.69 10.18 7.09 7.94 22.36 73.68 9.5 7.06 9.15 9.65 15.48 11.44 7.62 26.48 9.97 6.78 7.26 9.57 6.19 9.03 8.72 50.87 9.13 8.99 10.85 7.67 8.04 8.61 7.12 8.74 10.91 6.96 102.15 7.38 10.19 9.81 19.37 6.72 26.38 6.47 66.06 62.24 48.25 12.27 232470_at SNF1LK 70.1 66.16 86.39 113.06 67.76 72.7 87.5 64.87 70.61 84.6 69.02 72.89 90.68 74.03 83.57 93.92 88.41 88.87 64.66 74.18 99.43 73.75 66.82 114.64 80.48 64.63 92.22 72.93 90.78 115.77 84.91 100.94 96.99 90.31 88.14 107.46 88.75 73.02 82.5 101.66 80.32 94.84 142.13 98.86 102.09 88.2 90.23 98.57 87.92 86.68 84.14 92.02 109.05 74.71 109.81 90.81 98.79 123.11 78.38 99.49 66.02 113.59 84.37 84 84.08 75.35 88.32 72.51 86.62 73.77 80.37 86.06 77.66 78.62 75.93 76.77 80.2 83.04 129.06 100.02 103.43 63.74 71.57 82.97 51.08 43.29 54.27 56.5 56.92 58.21 158.09 65.73 80.37 60.86 66.17 62.06 53.54 76.98 68.91 65.43 64.53 63.92 77.62 55.88 63.39 67.77 82.41 55.55 72.13 159.5 112.92 65.31 73.02 90.61 91.56 96.77 91.77 83.03 80.05 104.85 77.78 89.77 101.85 80.83 49.8 82.69 63.8 49.01 56.87 56.86 54.56 60.19 67.4 74.75 60.13 65.12 67.54 70.15 63.11 59.62 60.35 50.84 63.41 64.05 64.39 67.34 56.5 66.09 66.86 67.53 96.21 71.64 84.93 75.17 89.28 95.18 114.9 87.18 67.6 72.44 72.64 70.04 78.96 101.73 67.2 76.24 66.29 67.27 99.47 70.76 81.76 91.79 77.05 83.78 115.36 98.24 85.24 87.15 85.04 95.02 76.28 98.19 84.17 72.25 89.36 82.34 84.67 73.55 81.22 95.58 71.92 76.02 90.57 86.64 71.58 93.97 73.09 71.6 72.19 74.42 89.38 65.46 58.53 86.5 74.72 65.66 66.58 83.1 95.87 74.21 76.07 62.48 86.25 88.24 63.19 67.99 66.9 75.5 67.63 62.82 67.66 54.85 84.28 58.34 68.98 61.74 49.35 232541_at EGFR 426.46 45.42 305.85 7.7 122.33 36.29 27.88 19.25 13.79 383.39 27.98 35.46 130.96 15.74 331.66 382.02 302.57 175.05 128.27 13.85 15.03 15.49 7.38 111 92.85 73.72 59.95 25.91 65.22 282.53 22.05 14.15 19.67 16.25 139.2 61.49 105.81 76.74 133.38 29.1 81.81 69.49 94.53 366.38 53.54 196.84 1354.87 105.09 110.74 187.96 64.86 35.5 90.35 185.47 6.77 142 124.03 47.87 49.03 519.59 36.89 190.29 215.4 11.36 104 334.34 108.52 229.54 89.21 278.67 228.74 283.19 246.01 43.81 115.33 178.96 330.56 101.27 10.75 101.56 101.53 18.13 11.7 25.48 184.98 137.92 80.94 142.95 17.86 124.04 395.14 308.39 47.9 36.93 145.7 267.79 22.28 98.97 50.54 200.75 381.48 193.5 30.59 169.96 34.9 30.47 56.95 63.88 37.44 26.31 158.29 516.06 263.83 303.49 85.09 102.72 22.96 502.46 489.76 290.51 162.53 772.44 20.66 134.47 656.01 13.61 90.04 394.07 25.34 334.53 130.93 79.56 643.19 157.29 73.31 82.42 285.86 278.05 254.31 20.09 70.8 602.81 30.93 62.72 177.65 59.3 314.18 65.88 153.56 8.89 146.55 119.47 392.11 83.39 43.35 214.96 10.92 1096.88 99.77 221.03 91.28 208.01 11.14 76.6 22.11 59.36 33.24 159.08 40.19 50.14 9.61 67.33 65.26 163.68 8.65 241.47 356.65 23.17 262.35 13.93 156.93 224.53 45.4 238.44 156.7 65.25 90.62 35.6 140.38 10.89 55.82 129.82 85.91 101.33 401.6 215.63 648.23 128.44 285.18 1639.35 45.2 33.26 64.32 9.51 137.99 105.74 473.37 156.28 99.75 23.92 426.75 88.73 96.73 437.93 24.44 23.26 37.21 33.78 12.49 145.77 78.82 5950.6 37.03 74.82 318.32 162.97 173.12 233055_at PRKD3 36.09 16.88 23.19 19.43 15.43 26.32 29.01 13.35 16.41 30.34 22.6 23.7 33.04 24.41 42.24 17.04 29.64 58.75 45.31 30.12 13.32 20.04 31.2 25.73 45.67 27.39 24.77 34.2 31.29 26.81 16.38 26.68 21.85 17.82 45.24 44.61 61.37 48.56 58 43.28 51.96 41.76 51.8 37.15 31.33 29.77 30.35 51.6 26.14 27.99 30.41 24.54 24.49 26.29 21.17 19.52 51.73 34.38 30.16 18.15 13.62 32.48 22.07 43.11 170.33 176.28 128.65 57.59 91.59 62.08 64 51.37 54.5 35 26.05 40.69 39.64 21.03 19.32 22.84 30.26 12.88 38.46 24.11 25.4 18.46 22.89 37.18 34.49 27.21 40.49 47.46 27.01 28.11 23 30.34 46.52 68.51 44.17 78.74 32.66 29.96 46.07 45.1 58.39 26.27 36.89 45.89 37.96 62.39 43.67 41.64 37.69 98.72 88.45 31.05 31.03 50.04 92.1 26.59 27.25 42.06 39.21 39.11 57.86 35.43 51.6 35.4 30.95 82.62 65.4 36.6 18.8 32.54 37.04 41.05 75.24 49.32 56.58 43.95 42.49 40.59 51.77 38.43 40.02 44.14 86.89 16.61 38.54 17.37 44.87 25.62 36.28 36.53 26.91 61.99 27.99 41.6 37.98 31.53 36.97 48.09 25.27 35.26 17.74 37.9 30.95 37.38 42.15 29.83 26.85 24.31 24.4 45.88 70.73 20.42 31.92 27.8 51.01 21.37 36.08 50.64 29.48 30.05 34.83 27.43 28.03 31.73 43.68 33.06 41.09 24.33 36.92 45.77 41.37 38.18 65.51 54.85 25.78 51.96 32.69 30.92 34.07 30.28 51.14 18.95 181.8 58.1 51.08 30.01 76.61 39.48 46.32 33.91 35.1 32.24 40.02 44.72 27.83 34.1 32.39 66.72 32.64 59.86 66.2 141.96 29.43 233057_at HSPB8 14.1 15.99 12.73 26.06 238.16 15.82 21.73 19.55 17.69 15.28 13.71 12.99 20.49 221.36 32.69 13.93 17.23 14.42 12.2 11.91 16.97 13.12 12.32 18.68 12.76 16.06 21.84 13.5 11.78 11.9 20.02 22.4 39.74 22.9 16.17 16.1 14.12 14.1 14.61 15.07 12.41 13.64 13.15 10.33 12.57 49.06 13.92 16.84 17.49 14.66 13.28 14.13 27.57 11.23 13.54 75.34 14.23 11.29 16.99 20.24 15.74 13.05 15.09 20.34 17.35 10.52 11.63 13.63 15.71 65.82 20.6 17.02 15.26 14.97 18.83 10.9 15.4 22.05 52.76 40.35 35.67 15.86 17.51 38.14 15.68 13.74 23.86 16.14 15.94 35.87 25.12 15.41 16.04 25.75 13.23 20.42 96.24 41.24 32.52 14.58 16.45 15.42 18.22 19.15 156.84 29.23 30.66 62.6 155.27 217.54 25.33 23.21 29.21 20.24 19.19 19.39 22.55 28.32 22.84 33.99 31.21 23.31 18.13 18.89 64.53 21.35 13.18 24.43 23.94 18.09 17.39 38.72 34.74 18.44 11.52 23.16 25.07 17.64 16.52 18.33 20.09 14.56 14.81 36.54 35.46 16.35 19.8 15.34 20.44 14.72 15.6 25.09 16.06 15.85 13.82 13.74 18.01 15.32 15.69 22.58 21.83 16.91 24.35 15.52 19.35 19.81 14.72 17.66 29.43 68.6 19.31 75.83 16.62 16.57 17.88 16.63 13.89 42.19 17.29 15.96 34.39 14.21 15.73 17.97 31.6 24.54 21.13 20.19 15.58 14.53 15.73 16.51 20.57 10.14 12.73 14.86 9.54 109.91 27.49 12.33 44.73 13.82 18.94 14.02 13.3 35.05 13.54 10.02 11.33 12.88 13.26 19.75 144.74 21.86 17.22 14.8 20.84 12.78 23.06 27.72 25.86 26.96 26.92 49.03 27.58 25.6 34.23 233498_at ERBB4 10.18 303.96 61.64 35.21 259.41 813.93 32.38 286.94 97.55 17.13 179.13 801.03 221.05 355.44 125.32 48.51 8.92 14.98 8.62 15.08 260.38 84.88 16.63 22.79 27.57 9.24 12.32 10.04 13.54 14.07 289.31 397 162.89 389.58 11.62 8.6 11.18 12.83 15.09 14.33 13.43 12.87 8.6 13.8 8.37 130.98 13.18 12.79 10.99 694.3 28.62 55.53 489.49 9.52 243.2 263.37 12.47 11.4 658.41 13.52 409.56 79.14 23.79 81.57 14.76 12.54 11.46 11.94 7.88 17.38 20.76 11.78 51.28 15.13 197.19 176.78 21.21 269.64 43.01 35.79 199.46 533.11 359.8 242.66 418.09 141.84 61.85 208.28 260.39 216.31 250.59 94.96 223.5 494.7 123.47 22.77 401.01 442.79 187.35 14.34 58.37 64.84 602.64 310.47 1189.28 485.27 296.76 1047.3 489.07 2963.64 16.38 211.48 13.83 17.01 13.11 30.65 523.99 18.78 31.9 34.92 19.32 17.81 45.08 17.83 28.29 23.24 209.4 26.6 93.42 16.23 101.42 133 83.35 51.09 321.99 451.33 18.62 21.55 173.26 45.62 298.68 12.6 427.02 203.74 120.31 237.5 114.25 689.45 132.96 205.87 20.48 215.35 27.24 49.84 64.3 24.42 58.56 19.5 278.29 63.2 308.35 229.33 136.26 611.14 155.45 91.7 956.13 69.88 115.87 353.97 682.46 212.72 633.08 38.31 38.35 57.54 50.12 54.17 439.58 18.27 223.59 19.49 57.05 1067.34 28.09 528.3 300.15 72.57 21.85 71.98 221.16 279.55 259.05 34.57 195.31 25.9 20.84 31.79 59.74 101.87 74.89 479.99 97.19 366.13 32.67 854.47 20.41 11.53 152.26 189.64 21.23 837.21 228.96 35.26 391.01 227.43 17.73 103.04 98.53 46.72 45.72 7.22 94.79 10.45 11.09 9.63 31.91 233611_at PRKG1 21.63 73.56 212.6 22.36 142.41 92.76 81.7 81.7 47.36 43.06 42.47 49.04 50.51 103.11 106.3 76.17 16.11 14.09 29.51 22.24 144.05 28.41 20.02 27.12 27.67 27.3 70.43 28.09 26.25 29.43 24.14 23.38 63.31 48.66 23.22 16.52 17.99 45.45 58.51 13.9 12.55 18.33 11.74 42.44 31.47 105.01 29.73 22.97 22.27 45.99 22.01 44.69 34.01 31.2 29.49 87.57 18.09 17.35 46.8 50.16 138.51 23.81 58.52 31.29 16.79 40.4 22.71 33.48 21.51 58.36 43.99 42.41 36.42 74.62 23.79 37.83 20.63 41.71 40.53 93.28 346.88 88.03 15.46 25.88 93.68 62.14 161.53 77.8 42.58 95.22 121.17 108.6 49.05 298.72 72.13 47.35 104.62 145.08 59.11 67.28 51.14 114.56 57.78 103.07 186.41 47.7 59.07 101.53 195.57 140.59 39.31 127.92 88.14 66.33 34.17 94.08 181.69 130.83 67.59 106.4 305.88 51.33 32.4 38.6 318.02 55.51 87.6 237.27 68.91 31.35 268.69 118.5 211.82 72.03 236.98 214.75 72.18 124.76 128.02 223.7 130.13 107.8 67.92 141.56 124.53 132.63 83.07 71.5 60.64 23.58 23.33 58.96 56.03 72.61 28.44 27.93 32.66 35.33 76.42 20.97 49.12 157.39 41.92 47.14 56.68 47.6 43.82 54.82 83.61 43.82 35.07 205.38 93.08 38.92 90.56 83.51 86.75 97.49 230.8 34.97 77.38 61.6 117.82 85.01 157.15 77.88 64.92 74.99 31.92 68.33 254.44 95.36 162.14 49.86 153.68 95.46 45.7 111.76 69.56 65.38 31.15 141.24 67.57 81.98 71.24 144.12 78.16 41.48 185.6 44.9 63.71 94.13 105.29 108.01 84.38 78.09 267.23 41.93 81.12 151.99 26.12 101.26 65.88 49.67 90.11 26.01 70.42 235085_at DKFZp761P0423 168.03 242.14 436.27 391.79 338.16 322.74 487.8 251.94 220.91 210.24 787.79 446.32 1409.01 617.47 714.91 317.02 205.99 387.02 315.93 152.65 489.33 481.77 1256.93 624.44 425.16 403.38 209.47 316.47 466.41 160.87 222.14 196.58 199.1 1600.76 431.62 427.59 255.33 457.39 386.57 520.75 328.1 471 323.43 295.49 444.6 493.34 324.29 353.92 414.97 247.94 301.98 380.12 386.14 937.12 299.6 463.26 400.16 365.13 414.7 407.65 536.51 442.13 819.85 860.44 663.76 579.43 310.31 399.98 175.25 377.99 246.73 610.8 500.61 216.66 211.44 285.46 212.97 536.28 131.82 363 473.95 413.41 464.56 256.63 242.78 338.18 245.92 336.67 261.89 775.51 554.54 1233.65 266.07 826.65 486.73 313.6 142.46 471.54 440.17 545.08 830.8 367.56 477.91 1480.63 501.21 650.38 848.17 479.86 477.35 216.85 421.12 420.22 331.1 275.36 423.29 435.79 987.28 439.45 480.2 439.83 936.67 1172.68 207.03 514.12 444.66 1046.59 359.22 614.05 155.24 175.17 345.13 242.73 581.45 304.26 248.27 188.86 448.03 265.83 536.15 269.62 439.14 383.76 141.06 348.51 444.84 361.53 341.62 300.33 578.44 523.82 664.5 588.19 506.81 475.01 473.19 429.33 294.27 317.49 800.15 624.84 287.62 465.13 507.65 386.7 94.85 327.22 851.67 423.75 431.43 885.82 169.79 454.5 340.43 261.59 970.64 287.83 434.73 337.78 1274.63 3042.33 1008.15 987.46 232.2 550.62 502.83 547.51 420.15 592.69 956.89 757.51 598.36 565.17 328.3 482.34 546.37 279.77 509.34 334.31 410.09 435.32 286.55 250.38 477.09 217.21 160.54 267.28 214.69 221.66 228.32 315.6 551.93 384.98 351.94 300.28 1072.87 127.19 540.17 121.75 450.15 739.22 535.51 484.32 2525.83 309.94 676.31 313.26 409.13 235192_at TP53RK 221.57 124.3 70.7 157.91 127.98 143.83 140.16 192.31 164.6 67.09 156.04 182.19 143.18 170.23 70.92 115.13 83.34 79.28 107.52 85.22 129.69 158.27 39.02 83.81 102.61 122.29 105.37 78.7 84.16 128.24 247.92 76.86 140.32 156.35 66.52 111.71 73.79 68.73 104.9 88.68 115.99 95.59 101.22 74.26 89.83 110.94 104.36 69.13 81.53 120.07 94.33 163.07 175.18 118.82 182.3 128.47 94.38 78.53 138.55 64.03 48.36 88.57 52.99 153.54 124.59 76.02 81.33 87.01 72.76 118.51 182.87 69.85 79.91 81.53 61.22 111.38 58.76 108.77 128.1 125.48 58.06 148.05 98.18 194.8 101.17 125.95 105.71 88.21 158.94 148.88 84.94 95.01 155.42 138.46 203.86 101.21 118.78 72.35 143.18 81.54 88.87 99.04 105.09 125.43 127.1 77.57 105.29 113.41 127.78 67.94 60.08 84.39 78.63 59.36 76.61 36.2 95.02 44.85 66.25 126.86 81.53 73.82 76.02 106.46 67.99 124.81 123.4 62.78 153.26 73.7 82.35 87.8 59.88 65.71 83.2 142.9 69.71 75.31 78.25 112.18 72.46 138.91 73.58 91.9 72.19 45.98 131.9 149.01 73.06 272.22 50.38 112.17 91.63 118.34 114.74 105.12 109.53 89.29 118.39 65.63 132.78 97.74 89.87 79.64 110.64 108.28 91.71 99.21 37.86 85.73 162.85 68 126.09 107.49 85.13 94.17 99.98 77.7 87.18 158.85 115.14 82.44 120 103.42 92.82 112.46 81.12 246.34 93.22 77.07 97.38 95.27 141.12 79.18 96.45 90.85 70.96 125.34 97.71 64.53 172.91 110.81 108.16 74.35 61 107.14 3 70.84 51.07 147.84 71.41 71.28 114.55 62.73 107.16 130.54 113.47 80.48 123.06 73.39 88.17 87.76 117.61 128.16 78 22 93 19 99 75 235252_at KSR1 164.7 83.66 141.68 69.96 116.32 152.37 81.46 300.52 55.43 92.06 82.56 42.47 92.21 201.15 120.53 97.91 302.07 141.41 96.53 75.3 57.23 150.98 90.26 101.26 50.7 146.54 89.56 49.94 72.32 66.2 49.22 57.24 83.3 197.13 106.09 148.49 64.76 66.05 253.63 229.98 185.88 155.93 101.58 68.76 436.1 135.44 117.71 76.2 125.27 77.1 37.7 80.5 80.58 95.57 34.12 99.22 197.05 143.22 137.18 68.41 69.6 176.83 77.14 40.23 89.97 67.51 387.25 165.89 104.59 52.97 113.37 99.22 75.38 161.28 82.9 627.33 121.36 81.07 49.29 42.41 46.18 69.53 34.21 57.44 76.47 107.59 71.65 103.02 95.54 144.86 153.67 117.39 108.14 152.5 192.13 107.39 68.07 47.11 164.19 131.5 396.25 105.76 57.43 201.87 105.29 166.78 125.82 129.96 121.91 166.2 290.48 108.15 115.93 58.4 88.72 150.82 58.68 199.24 135.48 97.26 197.4 76.66 104.05 297.97 166.81 72.94 81.72 220.15 42.17 63.91 172.11 61.43 285 102.28 66.18 97.91 99.26 102.1 154.38 63.49 71.79 246.17 61.87 70.41 156.52 117.48 253.47 91.66 224.71 64.95 76.21 163.71 121.52 100.22 59.76 106.37 28.27 125.07 114.49 206.67 143.58 77.76 84.1 69.77 50.45 106.85 52.35 172.97 93.46 91.62 75.18 88.82 102.29 55.79 36.73 58.93 74.33 106 189.14 97.26 127.34 138.18 51.65 118.37 79.21 142.86 80.74 112.93 147.55 29.96 73.26 114.17 142.05 86.16 119.92 77.34 184.59 129.97 160.09 120.18 70.08 70.68 132.47 49.83 152.1 101.78 76.21 132.86 125.68 50.77 155.18 135.53 301.37 114.1 109.54 75.93 85.86 60.39 55.9 135.8 118.96 169.63 76.2 276.62 123.26 83.97 118.18 235421_at MAP3K8 170.42 92.28 125.34 24.71 127.58 173.43 35.33 69.95 44.35 128.05 66.39 263.04 91.58 301.41 194.23 30.09 55.71 149.87 107.23 119.81 91.51 102.34 83.36 157.12 42.58 128.03 89.73 60.7 57.44 90.76 52.41 47.78 66.54 148.45 80.64 112.78 102.24 85.04 187.71 122.25 97.57 65.98 219.55 122.91 120.06 206.88 34.97 224.47 112.41 177.15 24.17 48.69 44.65 88.24 21.83 124.63 187.53 72.72 207.66 65.92 82.85 74.22 64.71 178.19 516.85 256.21 124.53 132.05 81.69 91.4 169.58 132.64 101.01 103.29 91.47 48.34 121.09 106.39 130.89 71.52 322.63 302.76 123.19 125.75 99.04 120.99 159.28 163.59 104.28 101.97 326.92 73.07 76.79 75.73 58.41 76.96 39.12 81.57 39.64 81.87 62.42 103.69 132.97 168.11 59.81 94.48 136.53 149.58 71.54 96.79 123.67 104.56 109.32 172.78 95 48.41 164.72 953.57 155.2 134.11 171.85 151.21 115.7 85.44 378.63 90.89 73.64 184.52 78.95 84.68 231.49 106.04 170.31 124.23 185.48 61.47 64.39 130.47 132.45 61.76 229.56 124.9 93.53 100.19 145.07 144.07 140.3 35.97 249.84 59.11 66.43 196.66 231.4 89.85 99.31 111.19 98.62 169.64 143.83 117.33 132.31 105.02 120.83 161.1 20.41 88.64 45.53 131.54 88.33 37.94 74.36 167.57 176.71 84.59 35.35 76.67 153.26 96.6 121.1 515.87 164.07 247.86 245.15 339.56 167.67 219.32 128.8 37.45 188.53 94.46 114.26 59.42 81.31 88.18 80.61 86.74 59.78 148.19 160.79 176.98 103.73 223.3 197.1 231.38 94.35 284.68 287.5 131.02 323.37 78.5 391.2 452.59 138.62 161.32 78.01 53.28 174.27 80.31 73.26 279.2 75.19 165.22 33.16 197.08 109.61 142.21 82.9 235601_at MAP2K5 60.47 62.37 92.65 37.72 139.94 223.35 136.26 155.56 70.11 94.81 114.09 279.09 155.27 155.65 241.35 115.52 27.67 95.55 78.05 47.14 101.46 59.22 55.61 61.97 41.52 70.04 49.17 55.61 72.58 54.91 46.75 48.25 110.23 204.74 64.27 39.43 56.97 61.94 73.59 49.57 79.7 52.94 34.91 45.3 57.8 98.37 80.78 75.06 45.89 110.06 56.48 69.06 67.75 44.71 47.44 50.75 42.99 37.92 147.19 83.97 238.05 82.94 35.05 155.15 229.61 93.34 140.13 122.85 68.34 57.88 77.96 118.86 53.19 120.45 46.04 80.5 224.23 149.01 171.36 149.81 178.14 117.31 185.47 129.69 92.73 120.89 142.11 203.02 87.23 204.18 350 197.96 529.75 136.28 326.11 109.9 130.73 111.13 188.88 114.78 314.15 119.52 90.18 338.05 182.33 137.23 147.7 200.98 146.51 182.83 74.66 174.48 144.03 175.79 106.14 90.33 701.22 534.1 254.37 163.06 272.83 216.69 94.92 118.04 244.89 127.23 237.36 201.48 130.86 91.71 217.44 115.92 349.93 124.64 211.69 298.32 86.54 120.41 143.39 77.12 210.07 122.27 88.2 120.7 96.91 398.04 129.77 69.81 117.4 41.66 35.82 118.09 41.47 64.06 164.88 74.74 55.03 53.01 129.56 71.47 103.29 81.6 76.68 151.07 60.47 62.69 92.39 93 183.36 176.37 83.83 117.65 251.44 95.12 133.94 107.27 125.89 186.44 224.25 173.98 170.44 105.39 127.06 149.86 70.25 246.92 145.02 163.49 71.91 124.12 179.02 141.31 163.52 123.14 126.55 68.72 73.17 142.1 197.31 135.09 165.65 124.56 77.48 228.46 77.35 148.72 204.79 103.99 119.32 119.94 216.88 242.67 116.04 184.53 153.32 108.44 118.85 48.22 57.28 215.11 49.75 90.14 188.91 85.78 55.06 88.92 95.85 235626_at CAMK1D 274.97 107.96 303.01 253.69 146.02 144.63 78.24 108.4 314.28 393.97 106.48 339.95 123.97 329.71 146.2 697.76 85.53 164 364.49 117.34 253.83 177.28 131.09 268.78 224.13 186.03 136.78 772.78 139.14 520.87 130.1 61.46 75.29 85.3 285.58 181.56 117.47 134.1 179.31 228.88 122.73 111.02 116.64 215.23 88.77 150.48 133.42 201.7 191.08 180.61 97.18 129.41 259.55 196.04 141.81 180.36 199.47 105.12 191.88 101.76 57.48 146.5 435.52 160.57 815.32 396.34 975.39 567.5 102.56 423.76 162.36 394.38 148.38 248.03 403.88 192.84 1658.97 208.84 124.71 115.03 283.23 185.81 259.54 164.27 242.96 263.6 440.01 303.49 343.19 299.01 169.35 286.03 140.21 181.15 181.54 282.93 123.88 216.24 159.68 216.91 149.24 146.72 323.29 144.8 316.07 150.43 191.34 179.8 109.29 352.32 310.78 144.26 323.67 356.47 235.16 92.52 807.38 662.76 267.91 299.35 476.73 150.12 277.5 330.68 260.59 112.41 110.39 262.14 162.11 174.14 286.18 138.38 236.58 185.71 120.88 305.42 256.41 246.61 134.71 252.65 111.93 130.43 78.03 121.21 135.96 132.32 709.55 266.04 238.37 82.64 382.26 262.52 201.34 211.06 212.33 624.68 78.87 152.8 164.59 305.99 207.99 194.79 70.14 87 74.3 195.71 143.75 168.86 87.77 100.14 235.8 166.96 220.33 186.52 122.55 218.95 233.45 140.22 215.79 289.95 291.73 835.02 99.32 173.86 256.08 209.88 348.18 143.18 1007.46 340.67 357.81 225.81 180.5 115.23 92.45 139.31 213.79 196.8 341.71 318.43 75.79 137.97 216.39 146.42 163.5 200.28 171.52 578.87 180.33 111.14 458.62 329.42 279.2 164.66 132.51 281.21 137.42 322.87 215.45 237.12 235.32 401.16 96.5 145.71 367.05 301.83 139.08 235705_at TRIO 493.69 281.14 677.06 54.83 530.49 645.68 357.23 289.1 161.19 354.83 548.3 614.85 814.57 240.22 654.09 193.75 252.71 544.5 210.03 164.61 498.35 405.4 417.97 360.38 222.48 188.84 607.47 444.37 410.92 296.38 362.15 344.44 584.63 417.65 227.45 368.31 264.21 242.41 344.5 130.23 172.69 199.92 469.35 405.12 132.86 355.6 396.38 252.51 232.58 291.63 284.03 251.24 266.11 251.22 65.05 315.61 248.72 179.1 261.45 330.97 1054.36 783.57 365.67 197.04 578.85 453.45 456.2 273.13 498.28 287.16 665.76 399.38 628.32 323.61 224.14 531.82 371.92 238.49 540.98 413.56 659.43 233.59 211.09 219.38 249.13 280.74 371.28 256.69 220.02 255.9 687.45 340.94 238.91 443.94 190.88 232.53 276.81 266.1 177.93 381.09 562.66 331.5 423.64 457.42 444.31 398.54 430.81 482.7 315.4 913.56 190.13 267.73 352.71 412.46 144.63 474.54 485.71 2031.66 254.99 469.51 878.5 779.77 202.54 169.36 285.56 142.51 336.63 306.89 179.85 453.93 515.74 285.76 288.31 274.67 334.22 314.7 119.88 271.97 325.39 344.51 448.18 325.58 354.21 246.8 204.48 318.4 325.44 184.27 278.46 159.63 406.03 325.41 232.97 297.08 327.95 224.8 184.52 302.98 356.98 270.03 248.39 335.69 265.56 378.74 319.15 193.28 457.7 263.94 436.27 158.74 149.26 421.87 273.07 111.12 359.75 417.38 195.57 390.78 364.16 164.7 345.08 334.99 280.86 220.68 279.53 205.3 262.88 197.56 218.52 773.91 419.33 198.98 240.67 203.74 197.29 275.87 205.09 385.97 439.22 295.68 193.5 195.1 192.76 405.96 175.06 203.9 613.71 204.25 569.37 143.33 216.41 274.23 209.27 478.6 486.8 219.05 331.57 162.91 187.03 589.72 125.1 428.4 328.95 297.69 413.84 275.29 210.78 235745_at ERN1 83.51 31.61 35.85 39.46 26.89 47.08 43.34 22.27 63.41 95.25 50.4 35.69 31.3 18.92 32.71 32.48 75.14 96.2 38 28 16.74 37.32 32.77 52.54 25.94 94.71 47.73 43.26 32.96 92.02 42.27 55.21 53.91 24.7 45.97 54.61 41.16 29.5 48.02 41.91 60.97 93.06 74.69 40 33.23 37.33 28.79 105.01 27.63 18.92 48.06 25.61 28.24 44.71 19.87 34.29 76.24 44.67 84.38 65.92 40.31 27.86 37.8 27.94 168.09 51.35 25.92 42.63 65.58 28.95 33.35 46.01 28.72 66.87 61.52 46.54 26.23 55.28 36.67 24.12 19.7 23.21 28.94 51.11 20.76 25.77 53.53 46.34 45.51 67.35 21.31 28.5 77.7 13.03 26.01 34.27 19.1 21.72 47.49 45.35 17.79 28.33 23.68 17.44 41.01 30.16 27.78 40.8 26.44 22.8 43.15 33.84 59.19 62.5 50.69 109.66 18.8 27.64 64.83 28.53 30.77 23.26 61.45 21.38 21.55 43.39 22.82 14.02 21.96 17.93 20.07 21.43 44.61 29.51 26.34 13.23 33.31 21.94 45.11 25.01 22.86 37.04 59.43 22.16 19.88 31.45 58.25 20.27 25.23 24.13 35.98 36.53 34.81 55.9 24.48 40.68 73.99 61.48 28.63 32.49 21.85 21 34.31 30.8 62.24 38.01 13.48 95.54 13.91 10.72 46.69 26.2 17.5 44.44 20.1 39.32 30.32 24.52 13.58 12.38 23.55 25.72 64.84 22.41 23.07 21.3 17.49 27.88 26.95 38.79 20.15 24.74 11.59 46.54 27.58 29.65 27.75 28.21 71.56 23.76 18.03 31.64 39.86 36.51 42.63 14.95 19.24 33.83 46.69 27.81 17.35 29.56 18.19 29.53 15 28.21 64.01 147.46 19.62 17.97 31.5 26.38 14.72 23.54 26.98 40.64 25.29 236073_at EPHA10 39.11 41.28 36.17 43.03 71.13 80.56 70.36 57.82 65.93 62.65 64.38 62.66 47.2 65.97 45.09 34.25 38.21 57.89 48.06 41.87 94.74 29.54 69.47 47.63 57.52 32.96 42.36 27.29 34.05 33.51 33.8 62.03 57.12 56.51 46.28 58.1 36.88 52.9 38.74 47.02 57.95 77.81 38.84 36.71 47.86 30.11 42.86 29.02 37.76 69.4 34.88 25.61 53.8 41.36 89.1 56.37 31.26 50.34 34.61 67.73 85.14 32.55 37.19 51.9 30 61.86 63.52 31.86 43.97 34.85 34.65 58.98 33.92 32.12 77.03 47.36 37.69 34.59 46.87 47.57 51.92 45.49 54.6 60.93 26.51 38.54 52.77 41.16 51.46 49.12 79.1 35.92 59.92 45.39 62.22 29.31 52.11 60.21 45.69 46.99 33.45 36.56 87.54 47.65 54.14 112.14 62.83 59.86 119.57 92.36 61.04 33.97 34.3 42.76 29.34 38.48 144.17 32.28 36.68 30.53 38.22 27.08 38.74 36.47 36.58 66.52 43.66 34.09 37.42 45.17 40.56 44.47 36.09 40.27 41.73 56.16 42.9 42.43 38.88 36.39 45.14 33.25 54.92 44.25 46.93 42.07 60.56 61.2 37.44 49.95 51.25 58.93 39.68 49.88 61.76 33.05 66.24 54.3 40.71 59.23 101.73 40.18 62.49 79.01 51.16 34.34 36.94 54.68 63.01 50.65 75.76 46.65 68.59 51.62 53.63 50.51 38.3 65.28 35.94 62.01 50.35 42.45 62.95 67.64 38.06 62.96 93.89 56.03 40.28 58.36 42.32 39.39 56.81 37.89 39.29 39.63 27.34 45.59 46.86 31.97 60.5 41.55 45 48.91 43.88 69.62 44.57 35.36 43.85 49.7 35.49 30.89 31.44 38.04 71.38 55.65 43.42 48.09 46.33 52.27 52.35 38.79 56.31 29.08 33.95 34.67 25.03 236561_at TGFBR1 120.46 93.43 329.08 49.3 273.43 185.76 310.07 70.24 92.72 239.08 196.22 481.92 243.09 147.49 610.84 172.73 59.63 153.54 167.1 102.82 152.14 152.51 112.85 119.18 94.36 184.82 260.6 107.38 96.24 64.42 95.67 157.3 149.58 114.72 53.83 85.4 86.8 139.64 251.51 79.15 66.05 71.16 59.96 113.99 172.46 184.76 101.69 137.69 136.38 1424.03 73.54 118.03 113.44 155.89 43.88 113.37 66.95 65.1 240.8 182.26 220.3 219.54 131.68 176.54 457.69 195.49 95.75 206.84 71.27 201.98 271.54 130.72 195.91 177.92 61.87 186.68 111.58 257.13 138.43 209.33 433.62 258.06 163.25 193.91 129.86 145.89 406.74 235.66 167.42 303.17 421.36 347.1 66.96 221.51 135.21 161.22 162.33 196.28 139.72 209.77 297.26 253.71 163.76 406.12 324.91 215.34 183.21 391.48 215.59 71.67 131 170.37 259.79 277.9 157.33 253.63 153.85 558.2 372.63 425.57 621.54 203.26 201.15 150.8 269 105.77 227.36 405.37 137.88 132.47 294.25 153.32 324.45 388.29 179.76 143.92 136.04 199.82 201.92 323.6 285.2 202.86 78.75 131.85 149.44 231.08 155.43 77.61 196.23 50.38 139.49 179.77 129.28 213.03 140.4 87.85 31.57 192.69 235.81 123.2 170.8 156.27 60.01 110.81 89.43 70.03 123.68 194 176.55 77.03 63.85 200.9 219.77 95.56 88.47 218.53 191.68 215.26 147.76 51.09 241.05 150.07 154.91 136.86 216.74 198.64 103.17 98.75 86.89 219.69 220.37 127.11 238.25 253.2 149.1 196.55 155.16 180.28 329.11 255.91 85.42 231.15 170.59 116.85 92.24 436.39 289.82 76.44 270.36 83.08 278.69 254.46 185.29 342.38 191.05 117.1 174.61 114.06 194.33 315.74 128.18 242.07 164.44 178.9 233.01 132.14 117.99 237227_at NEK10 12.48 15.7 17.51 18.71 14.77 21.16 16.27 13.35 16.15 15.65 31.92 23.82 79.1 15.66 43.41 25.98 14.65 14.31 12.82 12.16 13.8 15.4 14.55 19.48 13.58 11.28 11.41 12.5 16.27 18.41 21.01 29.32 28.16 20.88 14.27 14.17 12.26 12.44 13.81 13.49 11.88 13.87 19.51 10.56 16.16 19.87 14.28 10.78 14.89 52.07 16.86 17.2 18.51 11.23 19.8 15.25 19.96 19.65 13.24 17.05 30.64 14.17 17.46 13.34 12.6 11.13 12.52 10.47 13.1 11.7 16.87 11.78 13.19 11.58 13.37 39.07 12.03 42.31 32.2 60.3 17.83 199.11 12.16 82.1 12.37 25.66 10.61 16.23 17.53 37.35 18.64 38.77 25.63 14.97 12.75 17.39 12.47 12.8 18.65 9.59 13.95 44.26 11.99 16.21 12.37 35.81 60.66 37.45 21.63 145.52 14.2 18.64 15.34 11.93 16.69 15.77 12.88 14.77 12.88 20.79 14.76 15.41 17.48 13.5 10.99 12.14 11.65 10.9 13.3 10.15 9.11 11.08 11.6 11.28 11.44 13 10.09 10.95 10.24 9.56 15.83 11.06 52.13 11.34 12.55 13.13 9.44 15.71 13.09 165.94 14.49 16.43 13.5 12.58 19.01 16.85 331 14.5 16.26 12.17 15.71 13.77 20.22 27.23 37.78 14.42 72.77 16.59 23.82 15.65 15.72 27.16 37.12 11.83 35.41 18.2 20.84 25.9 29.95 26.45 25.52 17.54 17.44 26.39 14.96 17.84 13.08 20.78 16.76 18.19 21 17.45 14.79 11.49 17.33 13.63 11.09 13.19 14.31 10.47 14.79 13.12 18.36 15.01 11.62 63.8 12.5 12.8 20.77 14.08 11.62 21.17 14.63 28.96 60.54 14.25 11.47 12.84 13.41 24.1 13.15 9.4 11.84 10.89 13.5 12.85 22.31 238025_at MLKL 172.7 141.13 252.25 198.56 133.18 173.97 77.47 166.15 125.26 239.41 85.69 55.19 110.41 196.06 105.39 43.95 169.35 652.64 207.12 174.55 106.55 316.25 149.5 322.62 133.7 477.87 149.79 166.13 222.34 138.16 138.4 133.19 128.65 128.94 310.61 477.66 317.73 170.32 403.92 451.17 270.14 396.83 240.19 182.4 458.84 163.98 107.01 196.92 305.73 129.98 90.51 164.91 118.37 245.12 31.52 188.93 288.18 200.49 325.4 198.9 92.05 59.82 185.96 128.78 504.66 245.67 106.9 426.13 180.6 149.99 131.34 343.66 153.57 247.03 104.22 78.74 124.44 188.48 56.52 187.15 136.25 85.5 71.42 89.16 117.21 165.24 144.57 220.39 273.67 137.87 135.82 194.61 150.31 129.41 118.43 217.04 111.74 140.16 108.77 256.8 80.09 168.59 125.35 128.69 86.71 231.77 118.88 164.08 142.78 35.23 186.14 121.93 293.28 291.36 359.39 274.27 38.01 335.82 193.84 162.64 264.49 88.83 333.3 260.05 238.32 102.6 87.76 201.99 99.73 190.52 241.96 174.28 233.44 264.49 149.94 97.52 128.74 162.51 120.67 156.78 72.55 110.76 136.12 122.24 184.67 186.35 149.43 89.09 173.21 144.14 210.49 267.58 469.67 189.34 167.1 161.26 90.25 585.08 81.46 238.18 124.21 150.52 140.52 95.34 162.85 294.31 87.9 192.6 83.16 113.99 178.06 167.03 136.59 249.72 28.93 87.99 358.19 129.1 117.08 108.22 223.08 265.5 90.29 80.79 231.5 204.61 108.41 81.37 257.01 142.39 130.23 122.43 166.97 296.95 151.16 242.77 107.47 288.18 269.1 148.39 104.86 185.11 195.27 100.25 410.67 107.09 124.3 328.35 358.34 133.16 200.74 323.89 181.29 185.01 96.35 189.28 171.42 257.83 160.24 153.34 376.34 278.28 82.9 90.62 151.59 146.37 149.59 238769_at MAP4K4 31.55 22.79 90.94 12.87 42.2 34.25 13.05 18.87 19.65 110.28 58.84 25.34 71.69 42 145.24 52.49 19.09 59.66 39.31 29.22 26.98 30.34 74.5 39.1 32.94 48.29 26.94 35.11 25.5 23.6 20.04 27.33 31.03 22.74 30.65 51.83 27.74 41.37 114.92 32.74 27.89 43.47 17.17 36.18 20.72 73.29 29.9 32.62 34.33 29.37 43.33 33.07 31.8 21.72 10.23 42.51 30.6 14.59 46.77 45.93 18.25 36.18 42.12 52.89 494.57 101.28 41.74 134.1 73.82 146.65 78.47 116.09 92.22 119.46 22.11 56.7 55.05 131.73 29.94 96.38 135.83 40.02 51.61 52.07 78.72 35.66 52.75 77.84 78.67 149.83 88.32 80.01 34.72 51.04 23.4 43.03 33.94 23.58 28.51 20.59 162.5 27.46 19.45 21.64 40.66 24.22 29.82 47.86 29.5 21.52 66.66 99.03 102.67 123.66 43.22 15.24 230.85 193.05 120.58 83.33 129.92 122.26 47.78 55.85 204.45 80.55 118.63 227.05 40.75 137.35 155.03 120.2 207.94 81.4 79.02 126.63 142.67 118.09 68.8 130.64 75.28 54.83 53.7 60.42 118.19 112.66 101.59 33.7 76.34 35.35 29.37 119.47 71.71 65.9 34.53 61.55 16.49 122.71 63.31 44.37 48.85 77.13 18.81 39.43 31.99 39.63 36.2 67.34 79.99 39.09 23.49 68.88 49.7 57.33 74.52 93.15 95.33 55.97 138.45 18.57 111.87 58.02 61.21 75.72 95.34 68.14 78.13 61.31 56.6 34.44 85.86 54.21 67.53 26.52 91.15 83.07 56.85 100.42 56.36 199.93 32.16 47.28 68.76 21.7 81.29 52.77 151.22 66.86 102.97 43.19 139.79 108.01 75 84.76 42.98 40.25 42.65 31.26 40.72 97.26 50.13 114.53 61.73 49.67 65.23 35.04 64.92 239166_at MARK3 112.1 133.17 218.09 35.28 206.33 140.6 77.88 122.64 57.05 155.59 182.87 143.49 103.18 234.04 229.63 90.08 36.65 139.35 133.68 100.12 179.71 118.9 116.26 118.4 68.2 118.99 77.46 102.19 91.71 72.03 60 118.57 179.11 173.33 93.66 83.81 114.57 85.74 122.08 82.86 72.92 113.12 54.97 99.34 63.42 136.61 87.72 74.98 71.93 81.24 44.18 107.13 151.32 83.21 27.06 102.17 68.08 40.98 167.53 113.8 249.05 432.32 86.38 152.6 424.34 115.83 160.31 254.22 117.96 129.53 115.51 149.26 83.99 150.52 100.29 156.41 130.21 250.93 163.42 153.94 207.14 142.99 145.67 219.15 98.96 157.66 154.7 146.61 171.37 239.22 313.95 275.02 183.37 201.74 144.2 150.64 112.28 212.93 198.92 103.25 265.88 131.04 187.57 233.5 244.82 209.08 114.05 155.64 141.46 256.39 114.43 160.89 184.17 285.47 131.42 88.39 597.52 620.46 284.24 224.75 280.41 141.21 197.03 153.2 227.71 111.77 244.15 248.39 117.42 190.12 304.73 264.25 283.27 228.48 187.09 170.12 192.02 132.04 200.68 136.46 219.42 234.44 150.53 128.17 151.41 590.37 241.54 99.24 206.82 94.98 135.38 201.26 113.87 137.93 150.87 142.83 62.97 118.2 142.67 137.16 179.06 132.7 53.96 123.6 95.56 139.76 120.4 200.79 201.39 183.29 109.39 271.22 193.72 122.78 311.83 211.81 142.35 182.53 193.39 154.08 183.79 212.26 158.19 148.47 126.01 138.59 173.15 110.6 156.39 95.83 215.83 101.05 153.5 162.66 160.45 175.38 118.06 170.59 241.83 119.42 71.36 174.39 133.63 154.51 194.85 144.76 357.3 157.79 262.32 124.77 146.21 282.12 134.27 309.41 225.73 93.52 106.12 101.4 112.18 267.38 84.73 147.58 207.34 118.49 111.09 116.18 102.18 239811_at SRPK2 133.19 95.54 82.56 45.09 121.44 93.41 73.47 88.49 29.87 113.45 104.06 85.97 90.35 183.09 105.78 59.2 59.28 119.55 106.59 47.38 107.16 83.82 126.76 67.82 112.68 68.28 46.04 85.12 67.51 67.13 37.52 47.87 122.89 155.42 87.45 69.09 57.01 90.81 102.66 57.23 66.06 92.49 90.16 65.44 73.68 85.93 66.47 80.45 73.48 85.83 205.06 66.1 45.12 52.68 75.22 85.29 32.27 26.53 66.97 60.7 162.25 141.79 80.81 86.2 131.66 75.47 58.71 85.24 104.95 67.88 133.62 109.84 87.34 85.32 115.35 91.54 81.15 91.05 214.02 184.67 330.26 51.45 95.07 95.41 53.44 64.8 102.8 88.48 124.73 228.99 309.55 104.12 148.75 270.78 85.77 90.6 133.38 197.34 226.52 97.3 241.9 105.01 135.52 239.52 180.23 153.06 88.91 190.73 199.9 471.09 70.13 85.55 102.99 100.56 82.7 138.32 238.4 249.7 115.92 121.43 137.92 157.09 116.77 69.64 132.49 76.35 146.28 117.15 72.85 132.27 114.38 172.56 113.1 78.54 137.05 220.94 107.38 59.58 160.02 105.4 181 61.33 102.85 91.32 115.89 215.58 82.89 61.68 110.12 51.75 96.25 122.27 84.07 80.02 81.08 85.19 99.26 157.36 84.5 74.3 132.94 115.88 93.61 89.61 81.45 76.15 94.32 108.73 316.35 125.38 84.52 104.85 83.15 135.18 258.98 74.38 62.29 143.17 143.11 134.52 77.51 186.65 112.65 107.47 131.5 97.36 137.94 97.59 132.43 86.74 191.51 70.44 109.65 83.73 80.2 63.13 93.22 132.06 83.75 129.78 150.52 110.4 97.2 117.98 146.51 67.71 126.22 98.94 149.17 57.74 72.77 109.84 129.22 95.47 138.14 142.3 114.78 54.11 58.69 187.45 58.11 113.68 151.08 70.52 61.47 85.9 61.68 240331_at BMPR1B 23.32 95.56 11.55 8.51 8.61 217.8 14.03 51.64 84.63 18 52.98 20.13 102.45 407.99 26.79 10.91 8.38 8.05 14.98 10.33 43.15 6.22 6.64 8.66 37.4 8.2 8.87 6.6 22.31 9.72 16.75 58.64 143.99 536.2 5.99 8.6 7.71 26.98 6.24 6.12 6.6 6.9 14.8 9.38 10.36 5.94 6.9 8.24 10.35 31.46 21.6 9.62 15.83 17.1 33.71 7.15 12.32 7.03 7.04 7.66 98.74 6.94 7.51 10.61 6 7 7.22 7.15 28.61 15.26 8.79 7.11 7.21 9.66 6.87 15.01 10.01 7.66 399.31 15.59 28.99 144.82 138.25 153.71 47.74 38.15 12.13 19.33 9.54 42.77 445.38 14.61 113.68 11.16 116.89 7.77 10.62 61.29 11.14 16.97 782.25 11.8 8.47 10.38 18.52 30.09 31.16 24.92 76.13 21.37 10.62 8.74 9.42 14.75 11.94 15.62 10.17 10.44 9.73 15.15 14.48 18.14 15.42 7.96 10.99 58.77 223.3 9.41 114.57 14.47 12.91 24.97 17.75 9.78 26.59 38.94 13.44 8.69 9.83 9.12 340.79 10.83 14.03 10.69 17.77 69.79 16.73 7.7 20.21 48.29 13.12 12.35 13.6 10.47 8.43 8.76 87.94 9.11 11.87 10.79 33.45 7.85 9.86 92.02 53.41 9.78 14.27 10.01 425.75 60.26 113.6 16.21 140.62 17.9 12.47 16.38 10.87 219.87 12.14 166.28 18.12 10.16 9.95 69.34 13.27 47.94 13.71 198.3 9.89 10.36 226.77 15.23 114.33 9.84 8.84 10.86 108.32 12.86 12.39 7.54 13.45 34.21 78.09 328.24 10 52.87 38.97 8.39 64.57 45.82 28.38 12.63 14.21 27.05 142.45 183.46 5.92 9.4 7.21 290.38 5.77 8.22 83.92 31.86 11.21 34.71 7.08 240651_at MAPK4 27.3 21.85 31.7 33.87 21.51 28.73 38.14 26.08 27.32 32.67 29.04 27.79 30.65 36.56 29.95 25.9 38.03 36.38 38.34 30.67 27.93 28.24 35.57 41.11 33.89 26.69 34.75 31.12 37.7 42.99 29.92 34.49 38.33 35.17 32.59 41.31 40.37 32.34 33.7 38.08 29.58 29.46 32.67 29.33 114.69 32.49 35.89 37.86 33.47 28.64 41.63 37.61 29.83 34.6 41.34 26.98 39.46 46.88 25.97 28.55 31.32 61.12 29.66 36.93 38.16 32.93 53.75 30.15 30.48 34.08 51.19 35.64 61.03 30.56 28.54 34.21 25.72 25.42 29.25 32.56 27.53 22.59 22.73 25.83 31.09 26.07 32.66 33.65 34.14 23.4 33.61 23.36 29.93 29.79 27.07 26.77 26.78 29.22 29.29 32.55 99.44 29.88 27.85 29.25 26.83 26.48 27.66 26.43 31.43 65.96 65.94 34.06 36.75 43.89 47.22 57.15 37.56 43.73 40.44 35.32 34.94 99.9 41.81 30.53 49.45 35.43 29.97 31.12 32.45 30.9 30.83 35.98 35.78 28.31 32.13 30.37 48.05 38.34 32.83 30.82 33.96 32.45 34.57 31.69 35.23 32.86 48.19 22.95 20.69 20.99 29.38 27.63 28.63 25.38 27.47 41.37 31.38 26.04 23.29 22.15 26.45 32.6 25.81 38.2 25.07 27.58 25.97 27.4 39.36 31.52 29.68 35.05 35.4 37.58 58.97 33.42 33.51 34.21 30.4 34.29 29.21 37.71 30.9 31.56 32 34.32 32.54 30.42 32.19 36.72 31.26 33.82 27.94 31.44 26.87 27.17 59.63 31.04 28.8 38.25 37.74 26.71 26.35 36.22 30.14 26 85 31.99 29.07 29.79 48.78 30.5 36.38 36.2 36.21 32.16 32.97 31.76 36.14 34.39 42.09 47.9 43.28 45.83 41.31 43.05 31 240913_at FGFR2 14.03 8.28 10.01 8.59 35.87 8.72 11.6 10.47 7.45 11.37 21.97 54.12 17.98 17.34 22.73 10.74 12.27 12.43 11.35 20.09 9.69 7.88 21.26 11.96 13.14 12.75 9.13 15.07 12.67 7.7 10.62 8.55 32.63 12.04 9.45 8.22 15.59 9.04 11.59 11.33 14.3 13.2 10.53 9.29 15.23 10.18 12.62 13.56 8.84 9.82 23.58 65.21 8.33 11.08 10.62 7.14 8.31 9.27 6.94 8.8 12.51 13.28 10.45 11.22 9.78 16.09 10.19 7.97 9.83 14.45 14.92 11.29 30.68 10.41 9.43 12.85 11.84 14.1 18.27 15.88 11.24 12.02 14.9 10.14 8.89 14.43 12.8 10.53 17.78 10.94 24.99 21.63 16.03 20.87 10.78 8.4 10.14 11.01 10.12 14.21 319.79 10.55 10.6 18.24 11.64 19.07 14.31 14.06 13.95 28.55 49.37 20.64 11.57 15.96 10.68 10.6 10.34 20.29 22.71 11.2 11.37 15.36 11.81 11.63 10.92 8.3 31.45 9.38 8.16 36.64 9.31 14.31 20.02 10.97 10.31 9.68 10.61 10.95 10.93 9.72 43.68 12.2 45.5 10.37 13.2 65.23 9.88 10.61 11.04 8.43 19.45 10.48 12.74 11.14 9.42 10.25 11.1 9.32 13.17 10.87 12.62 12.48 17.01 12.5 12.34 8.73 14.51 11.44 14.52 11.23 10.17 9.69 50.53 15.8 11.84 10.13 12.34 14.65 18.6 13.91 10.59 12.12 11.13 19.63 9.02 10.78 9.24 13.05 11.59 8.65 21.76 13.58 13.73 7.38 15.68 9.44 14.59 8.91 9.47 34.82 12.15 9.27 9.55 11.96 15.66 19.13 43.31 8.22 9.44 11.92 18.55 21.44 7.4 9.85 12.69 8.51 8.29 8.06 9.83 13.59 8.89 8.33 6.39 10.83 9.6 9.59 9.36 241357_at MAPK15 151.78 89.5 82.73 81.29 160.51 872.11 145.23 682.69 61.24 94.96 73.15 157.93 91.81 313.75 70.99 62.7 64.21 63.77 88.89 70.72 121.69 81.53 48.88 96.5 151.21 51.4 56.04 56.98 82.7 90.23 69.65 94.91 101.77 61.94 73.3 86.49 222.99 83.69 68.36 132.44 145.51 107.89 120.51 62.44 118.35 67.52 67.32 63.55 74.07 61.44 74.89 95.18 77.76 98.82 466.29 97.34 167.01 113.53 252.82 72.94 206.57 63.7 61.26 92.8 85.71 76.58 110.85 83.59 88.3 47.44 81.41 59.24 48.68 59.13 84.65 68.4 53.19 55.98 116.18 209.35 77.37 76.61 162.18 62.95 41.3 147.01 45.41 33.69 55.77 51.58 283.68 51.81 94.04 65.61 73.96 73.71 93 56 83.1 72.35 73.6 40.02 55.6 73.32 38.13 51.02 56.46 55.7 76.24 409.74 52.05 51.34 54.39 52.73 84.74 101.14 262.08 85.56 59.84 99.94 98.9 119.19 88.36 112.57 47.22 95.61 60.63 42.59 64.99 65.86 48.59 68.6 70.56 61.23 39.48 69.94 69.14 54.97 66.49 69.47 61.08 55.51 76.42 57.86 69.07 133.94 62.21 114.45 64.94 44.17 80.92 151.03 111.41 49.28 117.99 101.65 106.84 50.5 46.6 96.79 137.61 63.24 92.85 75.4 56.98 116.72 61.85 60.25 154 126.45 78.09 80.83 111.8 81.55 139.72 84.7 63.14 97.21 78.41 116.45 74.78 141.05 84.94 64.72 108.86 91.62 85.53 74.22 128.32 104.86 169.96 61.14 76.06 42.22 52.38 53.42 90.42 59.64 45.27 94.07 110.87 74.97 84.32 126.85 55.98 47.48 77.95 116.53 119.43 68.48 66.49 53.31 138.33 61.67 315.99 191.39 55.49 57.54 66.21 202.15 37.65 70.66 115.07 168.32 36.81 74.54 24.37 241928_at CDKL1 42.1 51.41 75.83 21.46 51.7 63.76 27.36 28.69 24.54 29.54 16.57 36.93 30.33 28.89 59.35 35.44 18.9 42.11 45.86 33.97 36.68 35.41 43.77 30.9 37.23 45.37 28.57 30.4 56.94 21.98 19.74 31.99 28.09 29.63 24.24 37.49 28.5 45.21 35.28 20.37 39.77 31.54 30.15 20.96 29.54 30.27 33.3 38.99 30.48 22.74 30.58 38.72 29.17 35.13 18.21 35.56 38.11 27.35 40.37 33.44 42.72 23.67 51.33 30.5 147.43 45.82 22.15 46.87 33.26 3 25.93 48.56 28.81 41.91 26.25 29.93 41.66 49.03 31.88 31.09 38.38 26.41 32.63 34.19 36.02 46.36 30.28 56.97 38.41 51.66 55.36 51.4 36.13 52.36 47.66 34.16 35.49 34.31 43.71 40.1 41.99 44.18 31.16 52.94 35.79 54.13 26.71 26.35 36.22 30.14 26 85 31.99 29.07 29.79 48.78 30.5 36.38 36.2 36.21 32.16 32.97 31.76 36.14 34.39 42.09 47.9 43.28 45.83 41.31 43.05 31 240913_at FGFR2 14.03 8.28 10.01 8.59 35.87 8.72 11.6 10.47 7.45 11.37 21.97 54.12 17.98 17.34 22.73 10.74 12.27 12.43 11.35 20.09 9.69 7.88 21.26 11.96 13.14 12.75 9.13 15.07 12.67 7.7 10.62 8.55 32.63 12.04 9.45 8.22 15.59 9.04 11.59 11.33 14.3 13.2 10.53 9.29 15.23 10.18 12.62 13.56 8.84 9.82 23.58 65.21 8.33 11.08 10.62 7.14 8.31 9.27 6.94 8.8 12.51 13.28 10.45 11.22 9.78 16.09 10.19 7.97 9.83 14.45 14.92 11.29 30.68 10.41 9.43 12.85 11.84 14.1 18.27 15.88 11.24 12.02 14.9 10.14 8.89 14.43 12.8 10.53 17.78 10.94 24.99 21.63 16.03 20.87 10.78 8.4 10.14 11.01 10.12 14.21 319.79 10.55 10.6 18.24 11.64 19.07 14.31 14.06 13.95 28.55 49.37 20.64 11.57 15.96 10.68 10.6 10.34 20.29 22.71 11.2 11.37 15.36 11.81 11.63 10.92 8.3 31.45 9.38 8.16 36.64 9.31 14.31 20.02 10.97 10.31 9.68 10.61 10.95 10.93 9.72 43.68 12.2 45.5 10.37 13.2 65.23 9.88 10.61 11.04 8.43 19.45 10.48 12.74 11.14 9.42 10.25 11.1 9.32 13.17 10.87 12.62 12.48 17.01 12.5 12.34 8.73 14.51 11.44 14.52 11.23 10.17 9.69 50.53 15.8 11.84 10.13 12.34 14.65 18.6 13.91 10.59 12.12 11.13 19.63 9.02 10.78 9.24 13.05 11.59 8.65 21.76 13.58 13.73 7.38 15.68 9.44 14.59 8.91 9.47 34.82 12.15 9.27 9.55 11.96 15.66 19.13 43.31 8.22 9.44 11.92 18.55 21.44 7.4 9.85 12.69 8.51 8.29 8.06 9.83 13.59 8.89 8.33 6.39 10.83 9.6 9.59 9.36 241357_at MAPK15 151.78 89.5 82.73 81.29 160.51 872.11 145.23 682.69 61.24 94.96 73.15 157.93 91.81 313.75 70.99 62.7 64.21 63.77 88.89 70.72 121.69 81.53 48.88 96.5 151.21 51.4 56.04 56.98 82.7 90.23 69.65 94.91 101.77 61.94 73.3 86.49 222.99 83.69 68.36 132.44 145.51 107.89 120.51 62.44 118.35 67.52 67.32 63.55 74.07 61.44 74.89 95.18 77.76 98.82 466.29 97.34 167.01 113.53 252.82 72.94 206.57 63.7 61.26 92.8 85.71 76.58 110.85 83.59 88.3 47.44 81.41 59.24 48.68 59.13 84.65 68.4 53.19 55.98 116.18 209.35 77.37 76.61 162.18 62.95 41.3 147.01 45.41 33.69 55.77 51.58 283.68 51.81 94.04 65.61 73.96 73.71 93 56 83.1 72.35 73.6 40.02 55.6 73.32 38.13 51.02 56.46 55.7 76.24 409.74 52.05 51.34 54.39 52.73 84.74 101.14 262.08 85.56 59.84 99.94 98.9 119.19 88.36 112.57 47.22 95.61 60.63 42.59 64.99 65.86 48.59 68.6 70.56 61.23 39.48 69.94 69.14 54.97 66.49 69.47 61.08 55.51 76.42 57.86 69.07 133.94 62.21 114.45 64.94 44.17 80.92 151.03 111.41 49.28 117.99 101.65 106.84 50.5 46.6 96.79 137.61 63.24 92.85 75.4 56.98 116.72 61.85 60.25 154 126.45 78.09 80.83 111.8 81.55 139.72 84.7 63.14 97.21 78.41 116.45 74.78 141.05 84.94 64.72 108.86 91.62 85.53 74.22 128.32 104.86 169.96 61.14 76.06 42.22 52.38 53.42 90.42 59.64 45.27 94.07 110.87 74.97 84.32 126.85 55.98 47.48 77.95 116.53 119.43 68.48 66.49 53.31 138.33 61.67 315.99 191.39 55.49 57.54 66.21 202.15 37.65 70.66 115.07 168.32 36.81 74.54 24.37 241928_at CDKL1 42.1 51.41 75.83 21.46 51.7 63.76 27.36 28.69 24.54 29.54 16.57 36.93 30.33 28.89 59.35 35.44 18.9 42.11 45.86 33.97 36.68 35.41 43.77 30.9 37.23 45.37 28.57 30.4 56.94 21.98 19.74 31.99 28.09 29.63 24.24 37.49 28.5 45.21 35.28 20.37 39.77 31.54 30.15 20.96 29.54 30.27 33.3 38.99 30.48 22.74 30.58 38.72 29.17 35.13 18.21 35.56 38.11 27.35 40.37 33.44 42.72 23.67 51.33 30.5 147.43 45.82 22.15 46.87 33.26 33 25.93 48.56 28.81 41.91 26.25 29.93 41.66 49.03 31.88 31.09 38.38 26.41 32.63 34.19 36.02 46.36 30.28 56.97 38.41 51.66 55.36 51.4 36.13 52.36 47.66 34.16 35.49 34.31 43.71 40.1 41.99 44.18 31.16 52.94 35.79 54.13 68.49 68.02 50.78 41.18 19.52 36.81 51.67 47.29 40.62 55.46 37.74 86.69 61.2 34.88 62.9 33.48 49.76 32.36 81.58 31.09 43.53 66.17 25.16 42.1 59.9 29.76 64.83 41.06 34.16 36.35 38.71 40.93 39.35 29.35 40.58 23.71 27.21 33.85 31.39 38.18 26.46 26.94 33.68 22.85 24.22 47.5 20.8 38.24 21.34 30.21 15.93 30.6 46.09 32.86 27.68 33.48 31.94 25.6 20.19 26.1 35.33 34.75 30.01 33.04 23.24 36.96 47.71 23.8 20.86 20.73 39.93 29.59 78.82 23.09 55.57 47.05 36.08 32.86 52.93 33.3 24.31 32.68 43.41 23.57 57.1 43.02 61.22 35.95 56.28 26.42 118.34 67.39 36.88 51.63 31.72 39.97 52.59 43.33 25.17 52.22 55.48 31.44 38.84 19.78 70.55 56.91 37.54 29.56 47.22 40.54 40.9 39.27 30.03 56.82 31.66 29.53 33.72 28.37 27.1 41.02 31.89 242693_at CDC2L5 21.7 55.78 63.36 13.13 49.63 77.91 30.53 51.71 24.39 104.52 67.08 96.96 118.65 87.76 126.81 28.02 15.81 49.62 22.64 18.98 46.02 80.29 48.77 32.01 36.02 52.89 39.96 62.98 22.54 50.3 56.15 29.13 55.46 22.13 71.32 21.26 35.45 72.87 52.56 21.92 27.46 38.87 54.95 39.11 33.6 58.05 45.39 36.56 26.12 67.08 35.14 71.14 31.31 31.65 24.68 57.28 22.93 23.94 32.42 39.45 56.23 36.28 28.09 43.51 178.16 82.77 106.19 191.07 117.03 304.33 73.05 80.35 120.41 183.36 154.24 174.27 363.32 222.14 201.92 126.39 175.36 133.07 117.95 139.72 208.07 98.35 97.18 121.19 106.33 264.56 82.84 219.19 156.38 237.02 167.63 217.03 90.41 103.65 72.86 56.29 62.7 44.38 51.71 150.06 59.76 50.73 65.63 80.56 61.7 58.65 77.48 142.33 180.02 61.38 89 30.66 246.94 245.09 99.95 66.35 65.01 191.18 51.66 57.47 174.15 92.1 172.31 169.22 162.49 126.45 138.73 218.66 128.67 177.5 131.71 214.41 183.87 198.17 209.54 151.99 318.97 154.37 108.01 174.91 152.94 203.46 119.35 35.91 49.62 39.62 38.43 52.89 50.35 77.47 72.49 32.81 87.87 59.1 69.25 80.65 82.98 85.03 32.98 130.04 50.1 74.25 72.48 43.01 42.17 70.06 49.99 123.19 82.8 72.47 57.46 35.41 100.29 119.79 221.96 61.33 184.53 54.43 138.69 157.27 93.93 210.28 82.53 141.7 54.62 101.51 141.41 141.87 79.04 81.31 114.92 65.02 46.83 53.96 82.14 101.1 50.47 82.66 107.67 36.65 64.98 173.69 479.9 77.59 181.59 156.44 123.85 184.56 76.66 113.85 70.7 52.62 83.8 53.38 48.31 118.96 23.96 75.71 38.16 26.53 39.03 38.99 57.52 243002_at INSR 18.17 34.78 25.49 25.93 24.33 43.93 31.6 19.97 17.23 26.1 86.91 26.32 42.45 121.45 55.99 23.09 19.53 30.92 28.19 22.54 34.25 32.97 53.7 36.75 27.61 15 27.51 16.84 22.64 31.4 25.34 34.1 31.74 70.16 18.74 28.3 52.39 29.29 31.74 17.22 23.15 22.62 40.81 19.49 37.62 36.56 30.34 42.96 26.52 34.87 29.17 28.43 25.2 20.65 19.67 28.72 24.12 20.72 36.31 24.41 38.85 43.35 25 29.91 16.46 22.7 16.62 21.68 15.68 30.03 20.96 27.14 33.84 24.72 16.29 31.58 23.9 25.72 46.89 24.81 30.92 27.09 25.67 30.44 12.29 16.95 28.17 13.95 25.84 25.1 74.12 27.03 40.43 46.99 21.62 31.4 20.11 23.11 23.44 25.22 50.69 19.9 26.66 75.56 25.37 42.68 19.29 38.35 21.76 88.73 31.06 52.16 28.68 30.12 25.33 40.07 83.26 49.22 34.33 36.44 46.13 32.39 32.76 50.04 17.54 33.75 27.51 14.46 16.95 17.75 21.18 22.58 22.32 21.09 20.47 14.84 18.71 17.34 19.2 19.45 28.86 21.53 20.93 26.23 20.86 27.08 27.48 19.58 24.17 16.88 17.63 23.56 20.69 16.38 18.57 26 28.75 20.8 19.38 14.01 34.65 30.09 33.64 68.47 16.4 31.15 28.52 23.9 43.5 63.23 35.41 45.42 38.34 24 95.39 38.88 32.23 56.23 38.44 33.06 30.21 55.64 30.77 38.2 37.39 43.05 26.83 29.17 30.15 35.09 41.67 22.93 33.29 29.37 48.12 28.26 29.04 37.7 22.57 42.67 32.01 29.64 17.32 23.2 27.73 31.32 50.03 21.45 45.3 19.29 48.1 26.63 35.12 30.01 33.5 16.01 15.62 15.73 16.31 52.72 12.5 22.74 27.92 21.07 16.72 23 25 13 5 243006_at FYN 43.28 19.49 52.61 14.32 60.01 52.74 39.82 19.51 18.9 36.63 20.66 14.38 31.06 22.31 36.51 16.75 34.59 70.9 26.19 30.94 24.02 38.01 17.72 33.67 28.15 59.98 31.25 28.92 40.34 24.96 20.71 19.03 28.38 56.14 23.12 25.22 49.4 24.58 93.95 27.03 19.94 47.48 22.83 33.66 45.9 72.52 20.86 30.29 30.55 28.39 13.82 26.74 20.91 29.72 15.31 26.81 21.26 29.18 60.24 18.2 22.04 151.03 22.56 22.11 254.09 30.07 146.27 70.96 25.22 49.52 43.53 62.36 29.5 37.87 35.41 28.28 16.97 57.93 18.2 27.51 44.32 20.36 18.87 61.66 52.48 54.35 49.95 116.37 96.54 198.81 116.3 98.09 30.04 46.07 46.04 75.94 25.05 43.97 63.78 63.07 93.61 69.28 44.24 63.93 39.62 74.07 29.36 54.59 37.41 49.14 50.81 32.77 100.95 43.39 67.56 106.75 46.29 240.91 185.78 59.53 123.91 68.27 69.07 68.9 128.01 58.88 33.63 91.48 30.47 33.33 143.96 43.76 171.08 86.18 28.74 44.95 75.37 45.32 68.45 41.44 44.36 37.9 26.13 43.45 82.98 132.32 48.08 46.75 124.3 20.41 40.8 133.04 51.49 56.75 55.76 38.87 25.17 106.17 38.41 59.74 45.24 52.11 25.35 40.85 26.2 65.01 37.52 105.74 41.13 31.01 37.2 47.36 49.7 38.41 32.52 332.14 60.98 24.59 66.44 20.37 116.4 66.57 33.84 36.21 64.4 35.23 35.75 32.07 44.25 29.72 44.05 34.73 45.14 40.3 53.97 37.7 36.82 84.62 98.39 44.35 32.18 46.25 89.64 48.3 47.09 30.33 65.62 54.08 54.99 24.02 81.13 94.77 62.91 56.64 29.95 54.04 71.81 58.63 37.58 54.16 39.71 49.16 22.16 43.02 30.25 30.22 50.8 243256_at MKNK1 76.8 69.76 85.13 53.01 179.92 159.16 131.73 100.31 60.9 174.88 97.64 219.13 168.95 168.18 296.47 60.75 59.69 129.62 151.68 90.64 119.41 82.4 74.46 128.5 171.78 154.16 80.72 90.57 77.93 66.19 64.23 108.06 63.66 293.13 80.18 73.31 119.77 90.8 339.51 81.73 65.55 82.22 40.93 126.91 71.73 137.93 72.67 144.88 109.97 64.66 77.26 65.26 74.08 72.99 40.13 90.85 65.81 74.4 115.87 151.26 189.56 66.9 108.06 92.34 263.95 151.74 152.96 113.62 86.42 136.1 108.6 211.86 169.09 100.38 63.78 78.26 69.43 109.28 48.08 114.9 198.33 120.04 102.89 83.26 89.91 97.51 124.76 127.52 120.54 251.03 469.44 306.06 135.04 187.91 155.62 204.51 144.55 168.36 213.84 244.64 141.46 153.85 265.99 241.87 143.77 204.63 159.75 137.59 246.73 703.77 174.29 122.21 196.52 178.62 228.63 146.06 170.68 452.13 185.27 244.78 274.1 163.56 148.13 159.47 307.31 57.44 206.56 262.96 76.11 191.83 300.2 165.21 407.46 166.75 160.24 184.39 232.89 231.81 156.48 148.24 206.83 116.61 181.82 158.89 209.65 334.64 153.92 64.54 169.67 80.9 104.62 115.44 45.76 80.47 122.08 84.81 50.91 141.51 109.44 123.25 164.06 86 46.15 109.7 51.11 59.04 79.26 138.74 234.5 97.19 101.64 169.24 212.3 111.66 260.27 127.04 150.07 159.08 140.06 119.33 147.91 109.17 102.57 120.96 100.98 95.18 201.49 84.87 69.77 174.09 266.83 89.67 139.98 117.2 158.54 115.88 64.75 164.15 233.28 182.99 85.74 137.73 139.57 319.71 153.1 133.08 165.91 129.41 147.35 98.99 137.06 240.17 129.62 298.46 239.14 112.51 117.91 89.44 122.87 249.86 153.92 174.09 169.85 84.92 96.77 140.48 137.18 243829_at BRAF 70.71 69.26 28.53 28.22 16.1 52.91 30.27 99.78 34.39 22.03 18.99 32.01 24.24 40.35 30.94 14.88 46.82 46.09 19.07 33.6 26.37 48.84 95.98 17.46 34.44 38.53 27.27 57.6 42.78 27.67 27.84 51.48 62.51 60.75 33.56 34.87 22.57 51.76 37.26 25.09 55.32 36.43 33.69 61.18 26.55 36.21 45.35 29.13 23.88 41.65 57.64 25.47 20.78 21.52 54.94 48.27 18.36 47.23 48.55 17.03 34.44 16.04 99.15 15.71 32.88 59.64 33.68 50.5 68.57 42.41 95.28 18.01 49.25 35.88 65.63 43.86 96.99 77.66 192.15 118.77 47.37 21.38 74.46 78.07 51.02 58.26 49.47 65.08 65.08 47.74 8.67 45.64 48.24 82.11 18.3 20.62 44.59 50.38 102.69 18.86 17.98 24.5 29.3 51.9 50.63 21.99 65.1 48.71 41.53 18.01 37.77 81.52 35.59 40.99 28.8 40.83 103.61 34.9 37.28 30.63 19.3 28.55 16.06 17.08 32.87 57.97 86.41 16.56 33.74 40.83 24.69 35.15 11.8 16.4 18.34 40.75 19.99 31.8 47.18 31.05 72.14 27.23 27.97 46.57 30.68 32.55 27.78 38.44 33.02 28.12 60.8 34.32 36.13 43.33 54.2 33 138.31 40.79 39.36 41.59 51.01 43.74 38.17 44.83 23.82 35.35 48.85 28.41 13.46 49.37 63.87 29.91 32.91 34.24 10.31 53.21 20.15 49.88 55.13 32.89 34.67 123.98 31.06 33.17 22.07 37.05 12.84 37.82 111.15 32.6 25.51 28.87 21.85 33.72 59.32 20.88 33.05 36.39 25.58 38.23 62.58 47.36 48.93 9.05 29.55 53.81 39.02 42.35 32.94 30.97 26.19 34.18 27.27 14.18 33.11 48.59 72.52 23.86 35.46 24.6 29.07 41.69 32.04 25.3 30.88 33.02 25.14 244332_at ACVR2A 55.73 51.07 105.11 50.19 218.37 75.99 58.54 56.79 36.25 69.3 55.64 54.94 71.6 45.44 124.54 93.29 37.57 56.08 67.08 39.73 69.98 55.22 82.71 76.07 106.56 61.84 91.22 137.02 50.32 42.98 32.3 40.16 60.36 76.9 34.76 57.75 54.28 46.92 120.91 43.54 39.77 46.97 35.64 102.89 62.2 139.73 62.36 56.07 43.12 64.43 49.95 85.42 80.9 58.42 72.17 60.02 34.56 40.24 105.72 76.28 97.23 183.62 41.33 58.23 89.3 77.67 80.74 57.42 50.85 103.13 104.83 71.85 81.37 77 41.19 37.77 44.06 86.68 60.18 80.26 97.02 37.22 62.85 61.32 126.73 83.21 86.41 95.6 86.88 130.29 188.09 136.85 60.07 103.3 95.56 77.49 43.5 52.75 64.02 65.92 259.23 241.64 74.14 83.81 65.64 81.58 59.02 61.39 117.91 76.29 80.4 180.93 127.56 110.31 71.63 75.25 129.54 202.8 157.57 253.96 277.34 237.35 118.11 80.73 280.71 56.55 164.28 237.14 69.32 102.54 196.98 75.35 192.11 132.91 86.44 105.82 76.11 156.09 71.41 90.87 88.06 98.63 66.24 80.89 99.21 102.97 89.52 67.44 144.94 43.19 49.08 90.18 35.72 73.97 60.15 55.36 42.84 57.42 70.37 56.08 72.04 139.38 44.9 71.45 42.92 54.4 54.14 112.44 84.98 44.24 52.35 126.31 134.8 70.36 192.27 86.1 97.94 114.49 200.99 82.44 136.45 51.78 69.39 113.34 72.59 79.32 63.65 82.86 55.69 73.92 192.47 102.23 127.09 57.44 185.12 96.5 55.22 85.25 122.71 143.41 66.65 92.38 76.38 63 50.12 118.38 137.81 93.55 133.41 73.66 136.13 90.12 101.37 210.82 88.57 80.5 132.35 69.05 55.14 179.82 70.02 71.24 87.02 51.06 88.25 52.09 71.1 244394_at BLK 40.89 37.35 45.84 56.1 43.3 45.32 55.06 37.46 44.22 45.89 33.79 33.69 42.9 42.22 36.59 43.59 49 50.75 35.52 34.6 45.07 39.35 29.04 64.22 43.32 43.76 47.17 43.48 43.88 39.63 34.09 44.57 38.61 41.88 47.49 41.71 47.27 40.8 51.22 53.93 41.26 48.49 43.86 36.82 41.72 42.2 39.27 43.43 47.54 31.94 42.1 44.8 45.76 46.78 48.19 35.04 47.73 42.99 51.75 45.31 42.56 46.48 50.8 56.92 683.08 47.9 45.8 45.4 47 38.7 47.11 54.23 40.54 47.66 44.2 37.63 38.35 60.52 48.18 54.49 50.95 64.4 60.79 71.12 47.55 44.97 46.91 54.92 63.11 64.22 89.5 50.33 56.23 46.25 46.91 52.91 39.54 37.97 36.97 36.93 43.25 40.93 44.62 31.74 28.43 43.52 41.8 41.2 43.61 68.79 62.35 47.53 75.02 56.16 73.4 89.6 65.54 104.67 67.66 62.46 91.44 57.54 78.81 80.83 66.52 89.11 61.8 62.21 59.02 60.43 69.31 56.13 68.04 80.24 58.09 62.15 84.72 58.79 61.17 57.17 58.3 59.64 60.93 51.09 71.83 86.28 57.26 66.77 71.03 49.34 50.1 96.91 61.54 65.57 58.63 56.65 62.54 57.91 46.9 94.63 50.12 63.22 56.39 62.67 59.87 81.78 59.76 80.01 73.75 74.95 61.22 60.24 54.71 56.9 86.73 59.91 77.83 62.16 50.47 51.67 62.69 66.14 63.48 49.89 141.46 58.99 55.5 60.44 53.34 52.42 45.62 44.6 48.95 49.4 49.18 52.67 43.76 79.58 68.78 74.85 56.94 46.6 53.9 79.28 58.51 52.45 53.68 53.43 62.22 46.22 56.98 55.07 57.34 62.48 50.7 54.78 61.87 64.55 56.75 60.95 47.88 36.54 33.44 37.94 30.6 40.42 36.67 244450_at MAK 114.4 228.99 205.87 81.92 294.61 301.11 187.05 142.75 90.93 188.97 130.07 146.35 175.1 363.43 301.09 143.99 56.16 176.53 140.52 182.03 184.68 169.59 96.44 163.26 297.58 128.78 163.42 220.22 241.85 135.36 111.26 95.8 177.46 242.18 106.79 117.61 107.91 137.44 111.77 48.95 153.86 74.65 88.09 145.79 117.28 165.06 216.82 114.41 126.07 124.26 126.93 113.67 82.85 155.3 60.23 100.38 105.24 70.74 203 178.05 165.18 236.69 108.72 149.85 205.68 143.28 142.47 143.17 164.7 187.08 129.52 185.8 235.94 131.82 82.14 249.92 156.13 149.88 196.91 165.24 204.28 112.77 171.38 179.92 105.56 182.73 184.68 136.27 137.66 227.18 497.64 215.44 203.77 711.45 132.41 138.04 144.76 311.55 140.3 178.94 576.05 156.28 199.35 271.46 225.06 321.41 215.01 320.91 195.81 395.2 143.6 254.79 186.85 110.27 142.76 530.24 686.71 513.85 277.29 170.35 325.11 327.18 242.44 234.46 224.51 166.42 124.83 184.61 135.19 175.38 137.01 115.79 311.31 132.77 145.65 194.07 148.87 132.59 327.1 131.41 302.14 116.76 114.47 174.73 139.8 245.91 134.82 158.78 187.59 58.44 173.56 182.84 84 151.06 216.58 231.67 121.38 123.9 253.32 146.37 168.05 235.67 93.83 258.11 100.82 190.33 218.58 162.92 261.7 211.85 57.82 229.02 186.17 84.78 240.76 145.89 220.31 260.95 353.28 181.87 234.18 105.74 142.75 239.91 166.17 284.61 177.16 170.86 65.45 351.71 236.6 135.11 300.18 230.4 252.11 111.92 288.09 308.84 127.43 193.12 273.77 151.29 186.94 211.76 113.11 259.43 395.31 237.55 270.93 132.46 235.53 268.33 138.33 159.65 210.06 190.15 140.64 179.13 104.93 248.08 134.96 195.27 258.65 160.3 142.95 178.33 130.48 244547_at FLJ25006 74.27 122.38 57.99 24.16 58.06 112.31 62.87 76.81 47.34 68.37 148.76 102.89 97.24 87.84 211.37 27.21 40.39 220.73 139.44 173.3 48.46 77.93 136.57 58.82 99.39 59.38 136 150.42 43.61 63.94 27.8 52.77 458.14 49.37 104 61.17 52.43 56.3 73 53.22 85.21 64.6 55.86 93.53 29.58 84.2 73.35 72.19 74.33 92.84 47.97 81.42 95.3 57.42 41.27 64.8 59.83 61.8 372.14 126.66 61.97 81.03 61.38 88.41 233.43 66.65 51.68 132.52 82.09 136.34 68.34 85.6 110.76 83.98 66.24 351.36 46.88 77.56 56.61 74.68 173.8 41.61 154.91 82.68 83.11 55.91 69.72 60.52 437.29 81.94 174.32 79.01 178.14 166.37 75.54 77.88 147.31 69.62 114.91 31.53 42.49 19.93 66.5 106.36 96.41 69.44 57.86 58.98 66.85 168.11 28.02 30.01 54.64 54.08 31.17 44.15 108.24 127.64 126.23 30.18 40.45 61.78 72.22 41.17 74.29 67.63 220.63 76.47 45.49 200 120.88 60.55 22.32 117.92 111.39 140.28 85.58 76.45 403.58 121.43 101.23 198.69 93.64 77.92 108.37 242.29 393.26 24.1 69.84 37.85 30.57 84.13 38.17 62.35 73.16 66.23 64.87 53.78 67.25 55.3 197.48 73.25 68.28 102.34 87.65 52.42 75.24 144.84 1201.96 94.87 56.18 74.06 81.95 65.93 297.9 41.15 52.18 92.51 70 79.04 72.88 77.58 323.15 191.87 51.97 181.3 108.55 73.98 81.73 122.59 99.72 56.87 77.58 127.17 52.81 45.48 186.07 109.68 76.17 63.84 104.34 117.46 106.6 47.83 94.63 74.57 211.86 107.96 84.09 57.17 80.87 107.5 164.82 27.45 100.02 81.86 62.13 101.43 73.72 115.05 41.96 69.19 79.15 45.88 54.19 58.45 34.26 32029_at PDPK1 186.13 177.12 217.93 207.24 189.52 247.13 224.82 191.72 202.52 177.2 214.89 203.51 244.96 263.15 250.71 236.41 196.65 194.42 158.91 199.36 268.41 190.07 174.31 236.13 192.66 177.59 217.76 195.28 196.87 199.71 168.66 223.67 214.23 273.53 204.54 176.72 180.69 172.39 178.24 187.94 169.21 155.8 182.89 161.64 180.7 177.41 174.34 169.48 216.39 178.86 217.88 218.71 236.24 172.98 220.64 211.58 237.74 199.57 195.32 213.63 188.76 194.82 188.88 184.42 199.96 174.23 168.46 156.74 259.88 159.87 223.25 198.31 206.34 212.13 164.55 187.91 195.64 255.4 290.49 306.28 281.63 268.11 254.13 294.99 173.79 217.8 220.15 184.12 209.62 214.17 445.38 210.64 236.43 238.88 272.06 230.26 286.29 273.42 251.1 271.56 262.93 280.95 280.65 208.03 287.69 317.83 321.99 307.08 355.2 691.1 227.16 204.27 218.08 206.02 211.66 247.39 253.87 274.27 228.46 266.39 289.17 273.14 262.31 267.23 226.53 225.14 241.24 256.33 236.34 229.78 246.37 250.84 288.68 241.11 236.88 265.88 248.34 262.16 282.3 254.41 249.7 300.22 256.89 281.08 246.86 327.57 228.77 248.79 243.68 178.83 205.81 289.91 234.88 243.15 208.72 239.98 241.99 201.08 217.94 253.9 249.77 227.43 237.33 260.26 245.39 256.23 251.14 266.72 335.71 259.64 277.68 309.19 266.08 201.84 498.99 312.33 250.9 245.38 263.38 296.91 251.74 243.79 275.41 273.14 258.79 277.06 285.58 243.07 242.13 299.17 274.15 234.23 245.62 198.79 174.05 219.86 188.23 228.7 192.6 211.34 224.91 183.15 205.42 405.23 211.43 226.58 211.08 198.84 244.49 207.62 197.76 209.88 250.08 356.55 240.88 251.19 280.19 252.45 232.33 230.75 244.21 205.5 274.17 288.07 249.9 255.47 256.48 32032_at DGCR14 426.07 210.69 305.7 297.03 215.34 138.69 262.48 271.91 297.12 346.95 239.5 200.36 212.17 159.3 185.89 258.06 219.57 213.36 255.31 226.47 113.66 124.45 228.87 163.89 315.61 245.86 178.6 151.56 142.12 147.93 244.99 147.17 159.27 381.29 195.16 245.78 166.77 138.52 185.84 203.25 283.09 299.99 237.46 196.86 222.54 205.12 164.18 179.82 260.27 182.48 150.7 151.47 279.23 293.7 284.57 205.64 286.89 149.64 240.35 174.74 274.12 203.12 317.26 159.92 173.1 133.57 152.77 266.05 192.68 183.84 152.43 141.32 221.96 156.37 234.3 119.84 173.8 136.09 147 234.87 256.87 153.5 167.25 132.74 143.93 144.45 237.94 183.38 229.3 337.5 212.65 237.54 311.18 195.73 345.94 219.7 214.05 319.31 501.92 305.52 239.18 214.4 141.86 311.71 185.37 215.95 204.69 255.51 224.77 421.84 187.11 202.45 166.81 197.96 371.9 286.47 155.42 215.56 214.68 422.34 193.99 286.09 356.17 263.29 151.06 121.61 98.33 132.33 134.52 146.27 138.85 203.5 254.25 136.74 170.01 104.38 118.15 122.27 127.86 158.96 99.2 170.2 112.12 139.16 112.54 266.59 234.83 154.35 166.94 202.37 183.31 196.77 193.61 178.58 162.22 195.81 135.75 172.7 173.34 178.6 156.83 186.66 116.32 123.07 168.95 226.41 120.02 191.12 298.31 136.25 217.39 159.92 132.54 157.4 282.85 256.18 143.74 92.39 155.1 322.66 128.97 176.46 85.93 129.34 220.97 134.24 116.59 284.24 170.64 196.83 122.22 140.92 194.53 206.2 186.48 260.37 139.75 269.03 341.64 164.71 201.55 228.37 184.58 245.57 180.11 141.24 159.77 182.68 172.64 160.04 174.23 252.52 188.32 236.59 175.53 197.93 211.38 204.48 131.57 190.41 295.37 214.29 220.7 388.65 203.02 233.36 230.69 33814_at PAK4 273.27 255.94 386.4 473.79 391.07 293.61 308.15 357.6 304.59 300.42 355.19 304.33 192.55 266.64 324.85 209.94 378.29 258.47 179.89 153.72 337.8 232.4 495.53 238.06 242.68 240.75 329.97 552.27 262.6 326.84 280.86 427.4 273.49 531.06 303.33 263.75 205.54 333.77 157.39 1019.79 284.31 269.38 258.24 388.21 244.23 248.78 217.08 151.02 317.97 312.31 207.64 249.93 560.03 345.54 847.42 322.45 448.38 366.44 313.07 335.96 385.14 254.63 503.82 282.57 114.14 194.72 279.76 182.05 216.3 101.04 184.82 309.06 210.7 235.56 179.26 159.94 183.75 203.07 239.94 208.77 222.82 355.55 308.04 283.68 267.53 195.95 613.49 227.7 244.39 201.86 345.37 188.79 236.26 132.14 131.66 203.98 303.06 332.92 177.38 170.1 193.07 246.77 406.29 167.39 222.93 382.99 273.57 320.56 669.28 342.38 246.13 176.74 176.64 203.96 222.55 349.37 275.16 265.85 201.28 439.53 317.1 370.91 502.26 200.54 100.17 258.96 210.25 140.66 211.18 227.5 131.17 177.64 200.95 250.56 117.13 262.11 395.78 145.16 195.85 281.02 141.74 270.05 188.86 232.98 158.95 178.68 287.31 556.14 302.22 288.48 383.97 279.06 289.89 322.4 345.38 513.05 328.67 197.32 232.9 181.57 253.53 286.32 250.84 468.03 388.98 310.6 348.1 300.59 328.82 324.73 395.3 365.05 302.01 506.7 596.63 226.17 210.14 235.11 278.74 476.61 185.7 376.59 332.2 320.17 205.39 271.2 475.43 374.42 426.21 428.64 321.19 198.18 175.75 371.5 220.68 344.83 544.49 306.89 459.47 447.34 453.04 311.82 414.9 451.98 214.78 246.56 190.67 267.51 203.16 311.03 248.41 214.98 342.03 405.22 279.65 380.62 490.55 349.87 276.73 326.37 252.14 179.01 533.79 216.01 359.25 257.94 250.47 34846_at CAMK2B 85.36 78.47 94.7 101.07 73.12 103.97 80.51 99.16 95.84 80.04 75.51 74.44 89.53 85.76 81.75 100.02 95.38 81.07 74.44 77.76 90.58 83.19 81.88 111.38 85.65 84.72 94.52 85.13 100.99 99.67 88.49 105.7 94.6 93.31 101.87 103.43 95.59 101.17 112.21 106 78.48 75.81 101.79 92.54 99.21 88.39 121.24 100.65 116.97 91.07 108.26 104.34 106.71 85.99 85.48 83.13 109.09 102.28 91.82 117.01 97.81 92.83 94.86 89.47 93.5 81.34 78.8 79.11 86.54 78.41 100.87 86.29 73.72 80.77 104.2 89.29 74.5 82.39 96.02 93.27 74.63 75.46 67.76 76.38 55.01 61.53 69.27 63.62 63.44 69.7 122.77 71.72 76.26 70.77 84.66 70.45 84.23 67.14 66.29 60.59 71.71 71.73 80.55 65.98 66.06 75.93 76.47 74.12 93.24 113.92 87.47 68.3 89.37 67.81 78.36 76.74 74.86 83.37 77.55 91.38 80.48 92.5 98.42 90.92 57.49 70.24 59.3 67.94 63.45 51.76 55.58 63.58 64.04 69.23 58.63 61.87 67.81 65.47 65.12 65.58 67.13 67.43 59.9 64.16 64.52 70.62 72.69 77.92 65.15 65.24 78.5 82.84 87.11 74.17 77.13 79.08 82.69 75.06 79.56 87.17 82.8 82.07 88.62 85.36 86.22 87.46 80.13 83.9 85.86 74.6 86.51 86.38 80.07 81.43 128.45 85.26 83.6 80.31 80.19 89.23 84.51 91.84 94.61 78.06 83.46 104.8 82.2 82.69 85.94 92.85 81.82 88.87 88.17 77.48 71.01 81.37 73.03 75.4 74.7 73.48 92.75 74.74 79.28 105.63 80.29 84.07 77.53 83.94 90.95 80.32 82.76 92.62 88.76 73.48 68.8 70.82 70.87 72.66 87.33 63.26 62.23 58.56 60.31 63.4 60.51 59.14 54.74 35617_at MAPK7 62.59 48.59 86.87 86.4 83.43 58.46 86.97 116.87 55 160.12 77.82 47.87 60.24 87.6 46.42 104.45 61.43 64.99 76.13 105.76 47.49 39.87 130.11 68.4 59.93 62.71 74.98 60.73 77.59 75.27 50.11 55.12 58.59 78.35 78.2 77.26 58.98 57.88 68.97 73.99 50.32 76.35 66.78 139.6 65.19 70.34 103.94 55.27 137.92 134.62 55.18 74.03 101.43 90.24 77.68 92.71 80.19 77.33 73.94 68.8 121.56 139.6 118 53.7 56.59 63.87 66.3 43.84 49.05 61.69 87.63 69.49 70 72.02 40.89 41.36 69.4 42.75 37.97 51.9 58.48 48.99 49.35 59.96 60.89 67.79 53.17 64.91 62.17 65.27 68.96 71.76 53.18 89 100.48 45.64 61.86 65.25 73.04 77.16 115.08 63.99 92.96 70.91 83.76 137.62 95.92 80.93 104.38 100.46 82.33 77.07 93.07 97.9 84 78 62.68 84.71 75.69 115.94 111.69 129.88 158.32 89.31 90.81 58.74 94.45 77.69 44.37 73.15 63.84 65.45 117.16 79.95 56.61 77.12 61.18 107.11 77.22 63.64 51.24 48.88 70.98 55.33 58.78 63.26 64.51 111.51 90.89 89.25 75.85 111.77 94.84 100.39 63.99 117.12 65.49 81.96 72.1 73.1 85.59 78.7 70.28 54.98 97.95 72.03 115.2 90.24 91.91 59.23 73.95 101.74 95.34 78.84 79.39 79.85 82.23 69.85 72.98 130.99 82.81 85.24 66 71.06 86.75 58.23 73.6 111.9 94.03 100.76 76.9 98.25 102.18 96.22 101.79 114.08 124.38 92.14 106.13 104.34 82.07 71.77 106.73 79.59 106.11 94.3 110.99 89.79 72.57 69.12 96.11 90.79 97.15 89.52 75.75 57.5 82.91 96.91 66.11 99.68 120.88 92.46 72.26 95.25 97.69 86.86 74.76 37996_s_at DMPK 66.42 95.36 146.82 204.63 70.67 98.84 73.85 64.61 99.73 130.58 184.44 103.51 89.45 136.09 92.13 146.19 67.8 103.62 99.3 91.74 66.31 50.66 71.69 73.42 54.83 59.31 130.54 68.65 55.18 102.22 62.39 83.95 75.62 116.28 112.46 117.93 70.58 96.35 129.44 192.04 80.06 70.14 69.15 200.05 63.43 132.55 77.16 53.93 118.28 97.16 55.66 60.18 132.23 66.8 123.93 147.06 55.39 102.98 108.52 265.14 181.52 108.4 158.65 31.83 44.85 29.09 100.61 36.28 38.37 50.8 40.92 97.27 56.56 51.69 34.16 37.44 38.45 35.32 42.66 35.27 96.8 68.81 76.36 66.97 71.23 58.43 87.69 49.81 39.91 59.01 202.24 46.43 41.5 24.83 21.79 70.78 102.73 130.98 23.26 32.38 133.28 63.06 63.64 49.78 88.49 252.49 160.79 113.58 324.59 519.63 58.97 70.76 73.03 110.79 42.18 33.82 103.78 113.55 89.72 72.52 131.38 77.97 52.22 74.45 89.26 33.57 59.11 74.59 31.79 117.34 53.67 70 81.25 76.11 37.17 39.93 53.81 47.35 63.8 61.26 41.35 46.04 46.52 51.3 28.21 54.71 35.54 71.18 174.41 80.81 81.37 147.24 98.27 77.31 72.02 130.62 46.5 60.09 149.27 49.6 115.72 86.95 57.35 117.99 121.68 94.68 75.83 117.61 272.13 86.83 79.74 111.64 63.87 47.13 130.77 45.77 79.87 57.84 102.17 97.73 51.78 59.19 58.96 66.66 49.79 106.89 119.65 92.04 88.6 108.13 83.37 62.81 102.15 67.03 63.9 69.4 60.39 139.84 88.38 116.04 104.39 123.1 68.26 63.96 60.77 94.73 96.97 73.04 65.48 60.6 88.2 145.88 211.07 109.37 148.83 132.01 224 155.82 99.34 141.33 114.71 97.08 178.95 112.24 121.68 95.62 91 38269_at PRKD2 414.78 428.78 653.17 421.14 426.34 550.97 364.65 582.12 329.51 624.07 520.33 358.25 504.07 465.12 571.12 727.21 466.26 1014.25 480.75 472.39 359.72 507.04 445.21 703.06 300.93 609.77 483.22 521.71 383.53 384.62 302.97 402.4 358.23 468.21 960.97 720.68 484.88 493.09 641.16 739.15 493.09 644.61 507.21 525.52 365.87 614.75 445.63 354.8 553.86 472.01 277.85 384.13 500.59 523.94 334.28 468.78 494.69 799.61 782.85 535.06 533.77 416.38 605.44 549.82 1066.69 506.46 537.42 416.75 364.42 305.84 388.23 698.19 380.75 348.36 257.95 280.34 163.91 467.53 223.77 302.72 323.64 353.25 329.4 411.4 429.75 459.98 358.41 543.78 669.98 377.68 610.33 561.73 427.18 344.86 336.15 389.38 593.67 590.95 421.84 372.05 496.14 447.11 485.84 403.64 337.81 878.61 532.82 725.31 1008.9 865.83 575.25 586.65 700.56 658.86 542.05 336.19 441.8 696.13 934.47 601.94 712.4 628.56 747.63 846.09 357.24 404.64 352.51 362.85 222.22 390.45 459.97 494.43 621.63 528.9 278.8 362.68 423.95 231.77 277.86 326.22 226.25 315.39 470.47 242.48 323.14 368.8 369.25 426.59 857.7 424.21 522.92 1118.88 468.66 548.83 553.78 593.2 342.18 661.5 412.64 640.19 608.25 441.38 300.04 665.62 428.66 635.17 572.87 845.1 669.84 385.66 454.99 505.91 374.4 399.51 552.1 364.46 541.78 395.77 506.52 473.55 492.29 327.36 412.87 444.37 356.72 474.68 562.48 346.62 369.19 476.47 517.87 364.19 425.01 554.79 453.55 441.14 244.19 646.57 544.96 552.14 635.66 582.36 581.7 489.38 447.62 375.11 419.12 601.31 431.08 338.08 337.41 614.16 812.47 437.02 391.8 498.21 378.79 378.28 287.29 494.27 662.23 522.47 587.98 375.11 625.92 378.22 478.05 38447_at ADRBK1 130.1 121.65 161.82 153.74 133.08 136.07 146.27 152.86 159.46 130.55 121.06 122.22 116.96 150.64 158.25 147.63 161.87 139.96 110.67 133.04 144.63 129.02 127.95 187.85 138.74 144.82 147.64 155.75 175.1 128.87 120.92 144.04 126.5 138.66 158 178.35 146.14 158.87 179.11 164.43 133.19 120.04 132.95 114.71 122.52 133.22 185.51 140.16 172.79 151.03 157.3 130.69 141.04 133.7 140.09 120.76 165.61 145.73 135.71 211.18 181.82 196.38 175.39 124.32 156.97 109.06 119.71 116.13 137.76 116.94 126.4 115.37 138.63 122.51 127.28 116.93 134.76 138.68 151.27 164.64 128.62 152.99 154.2 150.48 123.27 134.36 167.29 123.73 154.08 128.47 191.29 127 131.47 121.95 103.93 139.04 111.44 116.85 117.47 117.5 115.41 106.21 113.53 110.89 93.25 145.25 132.58 170.57 171.11 219.55 139.95 111.03 145.84 142.91 135.25 150.21 147.07 191.62 142.76 136.27 147.53 146.84 173.62 191.19 131.03 160.17 140.94 154.45 150.68 140.57 129.23 145.06 163.94 179.18 138.92 148.78 164.67 150.46 154.49 172.62 166.44 180.07 164.8 146.25 161.85 206.31 168.81 134.36 129.24 113.63 131.01 165.39 156.79 151.39 143.1 149.64 148 146.32 133.18 192.48 152.1 154.2 149.63 181 181.98 162.48 156.94 176.82 197.22 181.97 151.86 152.6 140.7 138.97 174.47 163.82 130.74 141.25 108.28 139.58 135.34 163.38 163.09 148.59 166.85 161.4 153.71 141.51 185.67 149.48 147.5 143.35 155.29 136.23 122.14 139.31 129.11 140.33 130.75 153.45 155.08 143.28 156.86 233.13 187.93 169.73 182.77 153.33 166.89 158.58 167.95 186.19 153.54 158.27 148.55 156.92 155.61 144.9 172.78 165.35 136 104.76 110.59 102.9 113.66 117.32 102.81 40225_at GAK 722.48 654.91 605.93 896.85 1173.66 1269.44 1310.64 850.95 960.41 830.35 905.9 837.27 903.39 1458.48 1167.58 897.84 554.95 801.15 637.39 490.4 1249.57 544.45 715.47 559.55 505.52 655.22 585.28 576.78 610.87 619.56 646.99 411.82 945.49 1330.23 731.69 824.88 506.71 405.6 732.92 569.03 400.45 444.45 459.16 390.15 842.95 561.85 539.66 385.53 687.22 421.88 297.76 703.25 1002.97 542.36 542.86 622.19 363.13 301.39 733.77 670.84 525.9 629.71 506.87 476.88 697.87 400.36 728.85 430.64 370.05 429.55 333.75 569.57 337.06 331.41 353.43 364.82 226.01 456.17 442.67 531.67 705.25 628.48 511.71 653.57 538.97 515.4 557.01 500.89 640.05 1189.96 1433.12 781.52 1701.8 735.08 1306.07 563.71 1231.84 897.04 1085.77 645.13 994.04 489.56 594.24 1200.98 938.59 638.37 627.83 756.64 1000.63 1231.72 661.05 532.92 468.19 545.82 594 494.8 985.27 885.76 897.41 974.01 877.41 661.75 516.94 519.8 446.48 603.16 660.92 410.91 272.56 372.58 658.63 355.7 713.52 531.22 456.25 526.77 298.26 321.6 398.02 340.31 457.31 493.15 321.2 512.09 436.28 614.8 417.97 949.55 743.11 562.97 339.04 854.62 331.12 550.92 716.23 758.54 609.8 534.89 559.28 571.72 862.16 550.38 346.3 831.2 427.35 700.3 1058.02 701.54 758.59 841.41 1054.98 766.77 894.81 483.27 1200.8 621.93 558.44 635.39 668.91 615.4 526.46 428.39 1030.65 613.7 574.46 621.08 509.01 442.12 420.37 659.09 761.12 375.31 489.6 480.48 387.28 465.49 257.97 533.58 473.89 359.99 824.86 590.61 587.09 396.95 456.21 420.27 431.83 352.52 512.54 340.78 319.54 492.66 764.39 635.03 542.08 759.45 646.8 368.95 619.37 703.73 564.25 337.52 860.14 663.04 483.52 465 542.29 40420_at STK10 129.22 127.98 193.64 261.39 167.51 180.98 185.42 144.64 201.7 244.11 93.99 77.28 128.02 133.83 118.72 165.51 122.19 274.1 188.64 100.5 132.12 222.39 55.74 208.06 111.45 341.17 139.07 129.69 182.55 113.16 125.22 135.14 136.61 174.43 148.52 212.2 138.96 139.78 304.61 249.25 149.27 256.94 143.84 127 113.56 172.66 114.92 154.19 283.82 103.39 53.57 154.95 150.82 187.17 108.69 133.5 213.38 189.81 301.21 106.71 65.24 80.99 183.57 143.35 356.64 132.87 118.51 199.1 163.68 113.02 105.57 178.1 102.65 162.71 97.73 112.63 95.47 224.28 65.89 112.57 55.26 152.64 102.79 166.78 140.8 135.57 130.42 210.21 225.12 292.21 107.79 232.92 146.94 178.81 191.86 248.96 146.7 96.7 222.62 171.63 57.9 151.15 118.72 129.73 99.86 231.88 119.33 141.62 247.59 63.13 113.42 85.13 188.35 129.79 233.53 54.95 54.42 185.73 262.28 162.62 146.89 120.39 152.19 213.09 142.26 165 106.51 154.21 87.1 102.07 239.44 146.93 176.94 174.88 114.74 86.7 100.61 111.47 84.32 112.6 65.51 174.03 136.14 96.49 154.21 130.98 83.32 138.97 235.89 125.03 105.13 351.8 246.03 210.78 186.72 129.09 88.84 345.77 126.25 213.97 177.44 144.36 106.82 90.88 98.69 286.49 134.81 294.49 57.83 109.76 191.99 136.4 130.98 125.19 67.82 152.44 196.83 95.79 90.28 100.13 207.61 163.98 93.26 147.83 173.92 136.15 101.82 91.89 164.79 64.5 124.41 116.76 133.68 149.3 79.51 183.43 97.8 206.7 189.55 93.32 125.46 158.53 223.68 77.81 246.25 77.67 62.41 119.11 152.51 91.83 115.42 190.01 126.08 105.08 96.64 160.9 166.06 153.04 128.53 136.66 279.59 196.03 89.37 210.38 145.81 240.12 215.26 41329_at SCYL3 139.73 186.44 130.15 144.32 265.72 163.27 287.02 142.01 126.15 154.17 302.46 318.24 272.61 175.44 200.3 109.02 145.67 191.11 99.22 160.35 144.04 264.61 191.53 133.22 102.09 180.09 155.65 97.55 86.58 163.18 141.56 133.82 142.1 214.98 132 151.07 122.55 89.43 81.83 96.63 233.91 107.34 174.5 112.29 129.91 149.15 111.56 87.88 58.56 262.76 128.52 137.86 107.18 198.61 169.41 174.46 105.01 112.26 142.69 76.99 266.32 96.87 41.48 80.56 140.59 131.26 57.25 114.69 120.03 101.62 59.34 197.48 127.83 159.08 148.58 177.93 286.51 185.4 205.56 92.25 139.68 247 112.22 113.74 100.05 207.22 99.16 110.42 139.51 177.85 33.56 179.3 306.85 247.11 220.78 329.19 231.45 106.75 274.07 263.01 161.58 204.25 639.33 424.47 315.82 227.15 250.27 208.92 132.15 62.33 141.96 114.03 112.63 88.33 142.1 204.98 106.84 110.43 124.74 127.03 171.79 100.76 91.97 81.01 80.49 101.53 211.4 98.07 209.19 187.21 170.25 165.24 106.56 94.3 178.74 94.7 112.31 109.18 89.19 158.41 203.93 176.87 77.41 130.54 66.3 83.73 140.97 103.33 106.36 259.82 101.66 107.89 98.37 118.18 187.02 196.73 122.04 93.05 115.07 76.78 171.56 109.19 217.34 84.72 140.39 160 210.58 200.34 81.7 246.73 73.63 103.31 129.39 129.75 123.68 87.46 95.08 160.67 161.58 225.27 129.39 76.14 170.16 95.04 83.37 107.88 181.56 184.2 80.87 131.06 220.07 130.04 95.42 118.3 98.01 168.39 108.16 126.82 359.39 107 117.89 154.03 121.68 14.59 115.1 96.66 122.91 144.92 39.99 166.46 93.78 97.47 109.81 200.57 170.74 89.33 174.23 79.25 84.52 120.68 110.28 162.76 171.34 162.64 138.59 113.38 110.42 41657_at STK11 153.69 191.99 147.09 163.48 164.49 252.29 199.36 187.3 192.6 141.07 213.11 197.27 196.93 221.18 188.54 225.66 130.93 229.59 131.48 193.93 209.13 150.56 128.26 202.65 179.17 145.06 139.98 142.74 127.21 176.08 121.78 151.65 168.86 193.19 199.49 209.14 133.96 136.16 176.47 144.58 210.14 275.19 126.26 145.91 87.52 189.5 234.41 159.11 256.42 175.2 125.91 167.32 279.29 165.1 319.58 195.95 264.33 188.73 201.54 189.2 245 213.87 188.52 126.15 108.69 102.41 89.85 167.72 149.17 115.19 109.61 165.64 160.7 116.88 136.54 134.67 143.74 133.59 140.69 141.18 99.12 192.55 139.11 164.09 146.61 185.06 153.11 146.39 161.98 115.35 256.23 137.03 133.63 103.71 101.88 103.13 120.67 122.52 122.24 127.72 122.49 108.05 99.14 115.29 132.63 200.02 184.74 155.87 169.99 228.29 108.99 154.09 119.35 179.72 91.54 81.42 188.39 129.23 126.07 112.55 128.43 106.7 132.46 154.91 90.14 92.86 93.97 85 91.88 90.78 103.43 94.06 91.65 91.04 80.97 86.33 100.19 95.45 89.01 88.79 76.23 90.44 94.59 90.73 93.03 115.09 110.35 163.93 183.77 166.61 180.79 198.26 189.97 151.25 181.92 191.55 146.21 138.74 150.66 164.6 212.56 166.23 180.89 194.32 180.38 131.97 228.17 158.2 212.24 159.68 158.99 155.46 125.15 152.78 147.5 110.12 86.53 116.87 112.21 125.02 118.57 122.59 136.42 118.55 111.38 106.85 115.97 108.46 121.1 114.51 123.91 103.05 116.03 124.14 109.51 105.43 112.72 123.03 105.43 101.96 138.35 120.69 119.43 170.61 107.71 120.58 94.22 92.77 86.74 107.87 99.43 134.57 138.8 130.29 140.78 148.15 113.07 109.97 136.83 151.72 196.54 161.36 185.23 172.82 153.32 205.6 157.09 44120_at ADCK2 169.29 162.33 150.9 277.64 226.85 202.32 294.86 402.21 229.48 234.33 256.47 148.28 231.65 235.54 131.16 185.35 127.4 110.5 164.43 134.05 232.21 188.39 296.52 140.46 130.64 152.47 149.64 112.64 143.56 152.74 139.92 112.65 189.22 234.61 79.56 192.5 95.56 170.58 107.3 223.93 296.75 132.63 157.83 135.71 140.26 140.99 118.15 172.82 261.84 174.38 238.14 154.77 184.22 144.69 393.91 202 116.12 160.07 186.42 76.38 208.52 69.92 308.76 155.25 164.87 197.61 147.4 134.12 279.79 149.87 195.73 149.4 143.74 177.99 289.8 130.47 360.89 196.22 243.57 286.83 139.11 188.51 269.09 245.11 166.05 210.69 243.37 192.36 199.24 240.92 110.67 233.85 300.02 213.6 262.98 303.12 249.15 201.68 315.37 185.08 121.41 163.97 157.62 222.35 191.52 232.97 153.8 232.44 238.47 185.85 152.47 125.5 98.33 105.88 190.18 119.07 219.27 78.39 127.78 221.28 166.96 137.96 196.66 158.89 120.53 254.54 175.66 132.77 106.45 177.68 144.56 348.92 143.27 127.27 181.12 196 186.58 119.78 193.81 161.83 127.39 158.96 164.39 188.33 152.83 117.17 153.21 250.19 149.49 182.96 216.23 143.29 177.2 133.08 183.87 169.6 170.9 155.74 169.16 118.37 264.2 153.71 160.46 117.38 174.79 200.44 125.92 144.5 219.98 235.96 184.45 129.88 215.93 180.53 88.49 264.06 139.8 173.93 147.78 189.63 123.15 206.36 205.35 165.89 132.14 205.36 166.35 172.65 199.06 154.42 186.99 125.13 185.22 130.78 141.86 155.58 215.18 170.21 160.17 132.45 602.45 193.43 144.63 178.79 155.07 135.52 86.29 116.83 123.21 141.31 151.87 101.11 203.07 73.23 161.93 147.74 248.77 101.67 169.27 143.93 189.4 165.25 130.17 129.39 112.62 260.82 184.92 52169_at LYK5 220.37 276.15 319.81 293.53 351.76 359.05 391.42 381.6 315.1 347.05 205.49 336.93 252.23 284.25 425.2 335.22 232.33 279.31 232.07 251.47 364.34 325.22 287.14 335.28 195.2 386.56 361.05 322.65 304.06 299.27 273.57 368.92 581.28 527.07 191.27 296.51 246.67 297.74 323.36 295 291.97 264.11 215.9 323.19 210.56 330.98 431.45 372.7 330.46 280.45 209.11 235.6 434.51 204.2 314.4 305.56 262.4 235.46 339.76 276.37 293.69 392.8 260.01 313.4 458.45 265.37 255.91 302.56 221.74 336.98 212.58 303.43 229.73 212.69 345.39 261.5 197.67 281.61 274.02 368.03 414.23 229.11 299.82 308.94 313.45 271.45 994.83 260.62 318.43 481.33 333.97 380.75 614.08 428.98 460.84 434.26 531.01 348.56 554.24 340.95 526.36 314.16 431.95 475.77 538.94 491.43 380.97 399.06 697.08 1365.44 313.32 281.28 297.04 408.47 389.15 569.84 404.68 510.93 436.72 322.91 408.4 379.13 369.9 308.43 381.29 579.55 670.28 283.79 266.17 189.26 413.72 346.58 406.39 374.98 393.9 360.84 392.94 316.66 583.46 271.07 293.57 280.82 394.99 519.34 306.77 401.23 494.58 399.81 310.05 321.63 223.42 382.56 204.65 489.86 295.41 298.49 317.52 300.7 293.77 303.14 457.28 323.65 303.95 407.27 304.98 382.71 344.11 377.98 584.91 331.15 363.17 414.73 329.66 208.74 316.42 431.76 249.16 347.39 273.12 349.22 262.98 144.68 367.22 259.62 239.44 289.8 225.56 385.35 126.02 299.83 274.63 225.65 337.06 334.07 264.04 457.43 261.96 424.71 348.57 201.64 293.82 420.33 340.25 401.99 180.6 265.36 278.31 264.77 309.48 237.98 243.04 395.83 332.24 439.34 363.41 302.86 515.19 412.34 263.08 347.91 285.37 312.59 406 276.94 249.99 412.55 311.88 55065_at MARK4 345.04 289.87 414.98 771 296.84 518.23 354.39 609.68 471.71 437.27 493.51 409.92 281.01 449.82 272 387.69 523.41 327.15 231.87 337.46 285.33 268.01 314.18 371.16 318 306.17 341.36 476.98 462.2 414.75 327.25 215.25 234.44 409.07 447.34 391.16 248.37 458.18 297.67 375.66 294.18 260.28 467.56 394.29 419.47 352.76 325.92 175.7 417.5 290.86 427.02 236.59 637.66 381.91 644.26 354.88 584.75 396.18 537.66 291.87 336.27 358.74 530.06 168.5 136.03 313.62 350.04 130.25 273.08 164.93 345.05 416.6 225.31 174.49 170.18 168.73 163.13 215.75 245.93 292.75 304.16 477.25 267.03 450.58 275.84 234.61 244.45 332.83 296.77 1200.78 628.33 416.86 457.72 404.58 835.88 379.05 668.36 611.26 427.12 396.49 387.51 326.57 368.4 440.76 373.73 436.69 322.48 375.82 795.09 518.23 291 265.57 284.2 276.51 407.31 738.87 419.51 182.57 249.8 563.11 370.48 388.26 505.83 452.11 181.28 252.32 201.33 166.22 227.62 320.55 150.62 282.19 386.39 248.34 144.31 361.87 344.69 133.86 214.27 305.74 129.03 184.71 265.79 333.41 162.97 218.91 233.06 318.62 421.89 266.14 405.66 352.44 357.18 263.94 294.69 943.63 278.9 271.76 315.03 238.86 302.69 338.38 253.33 449.9 329.05 370.88 379 358.31 467.12 308.64 547.49 477.37 386.99 393.35 738.87 420.39 265.2 234.17 322.55 431.87 266.81 462.72 306.35 347.32 264.05 298.92 439.98 447.78 559.9 403.9 308.76 300.11 283.69 291.41 283.11 300.36 220.67 392.04 401.76 489.92 628.25 300.45 451.52 570.24 372.33 310.66 355.8 347.02 286.03 268.75 212.97 307.42 495.96 359.31 309.58 555.79 377.3 323.92 248.05 404.69 450.66 312.93 533.2 474.1 501.62 291.13 324.06 632_at GSK3A 87.1 86.76 103.01 119 94.85 85.03 104.08 104.02 107.37 100.67 82.09 90.75 80.64 91.54 68.65 112.61 116.7 91.33 84.01 76.37 95.36 84.54 87.5 130.71 109.07 83.74 83.82 104.87 99.49 114.77 88.98 98.44 83.03 115.25 121.66 147.41 141.31 129.17 106.2 113.95 104.9 113.2 125.5 141.19 127.5 112.48 144.84 87.43 142.69 123.18 99.83 99.62 133.44 120.33 134.93 111.6 163.17 166.99 115.66 152.69 147.05 157.2 157.35 113.22 83.35 77.01 137.91 64.79 90.28 72.87 108.62 113.52 93.08 88.67 84.61 103.75 74.5 81.84 96 109.91 102.95 99.63 81.43 101.84 91.97 79.25 109.26 78.51 98.39 89.69 131.86 82.4 98.54 74.36 71.49 84.77 127.77 115.49 71.31 86 99.93 105.21 117.66 63.5 97.62 161.1 92.92 116.95 173.12 209.79 130.53 78.23 96.67 86.37 110.64 129.75 102.26 97.28 82.48 119.8 114.43 124.39 183.03 140.03 73.62 102.91 78.75 73.44 85.77 81.38 64.95 89.06 93 103.39 71.33 97.34 112.04 89.97 88.26 105.12 75.41 117.29 92.93 100.94 86.34 102.99 103.66 127.19 100.39 108.24 148.55 130.67 168.88 100.53 90.15 177.88 136.43 119.78 108.68 102.14 116.21 113.94 113.23 150.84 159.86 136.49 126.99 125.93 156.58 111.41 116.68 118.32 91.17 114.3 154.75 121.07 92.2 84.28 82.4 141.42 76.7 109.22 119 92.87 95.17 105.86 118.54 117.23 118.57 125.45 97.15 98.71 138.35 133.38 99.1 131.76 110.78 117.68 140.07 142.22 161.75 107.53 137.89 136.6 129.49 127.81 95.95 141.53 129.13 105.51 120 99.89 178.83 185.53 109.61 116.11 134.52 126.68 133.66 117.86 98.4 104.59 100.11 109.11 105.36 105.32 93.49
https://openalex.org/W2161770015	https://europepmc.org/articles/pmc1489944?pdf=render	English	null	Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms	BMC genomics	2,006	cc-by	13,388	BioMed Central BioMed Central BioMed Central This article is available from: http://www.biomedcentral.com/1471-2164/7/127 This article is available from: http://www.biomedcentral.com/1471-2164/7/127 © 2006 Sørlie et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Gene expression profiling has been used to define molecular phenotypes of complex diseases such as breast cancer. The luminal A and basal-like subtypes have been repeatedly identified and validated as the two main subtypes out of a total of five molecular subtypes of breast cancer. These two are associated with distinctly different gene expression patterns and more importantly, a significant difference in clinical outcome. To further validate and more thoroughly characterize these two subtypes at the molecular level in tumors at an early stage, we report a gene expression profiling study using three different DNA microarray platforms. Results: Expression data from 20 tumor biopsies of early stage breast carcinomas were generated on three different DNA microarray platforms; Applied Biosystems Human Genome Survey Microarrays, Stanford cDNA Microarrays and Agilent's Whole Human Genome Oligo Microarrays, and the resulting gene expression patterns were analyzed. Both unsupervised and supervised analyses identified the different clinically relevant subtypes of breast tumours, and the results were consistent across all three platforms. Gene classification and biological pathway analyses of the genes differentially expressed between the two main subtypes revealed different molecular mechanisms descriptive of the two expression-based subtypes: Signature genes of the luminal A subtype were over-represented by genes involved in fatty acid metabolism and steroid hormone-mediated signaling pathways, in particular estrogen receptor signaling, while signature genes of the basal-like subtype were over- represented by genes involved in cell proliferation and differentiation, p21-mediated pathway, and G1-S checkpoint of cell cycle- signaling pathways. A minimal set of 54 genes that best discriminated the two subtypes was identified using the combined data sets generated from the three different array platforms. These predictor genes were further verified by TaqMan® Gene Expression assays. Conclusion: We have identified and validated the two main previously defined clinically relevant subtypes, luminal A and basal- like, in a small set of early stage breast carcinomas. Signature genes characterizing these two subtypes revealed that distinct molecular mechanisms might have been pre-programmed at an early stage in different subtypes of the disease. Our results provide further evidence that these breast tumor subtypes represent biologically distinct disease entities and may require different therapeutic strategies. Finally, validated by multiple gene expression platforms, including quantitative PCR, the set of 54 predictor genes identified in this study may define potential prognostic molecular markers for breast cancer. Received: 03 March 2006 Accepted: 26 May 2006 Received: 03 March 2006 Accepted: 26 May 2006 Published: 26 May 2006 BMC Genomics 2006, 7:127 doi:10.1186/1471-2164-7-127 BMC Genomics Open Access Open A Research article Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms Therese Sørlie†1, Yulei Wang†2, Chunlin Xiao3, Hilde Johnsen1, Bjørn Naume4, Raymond R Samaha2 and Anne-Lise Børresen-Dale1,5 Address: 1Department of Genetics, Institute for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Center, N-0310 Oslo, Norway, 2Applied Biosystems, Foster City, CA 94404, USA, 3Celera Genomics, Rockville, MD 20850, USA, 4Department of Oncology, Rikshospitalet- Radiumhospitalet Medical Center, N-0310 Oslo, Norway and 5Medical Faculty, University of Oslo, N-0310 Oslo, Norway Email: Therese Sørlie - tsorlie@labmed.uio.no; Yulei Wang - wangyy@appliedbiosystems.com; Chunlin Xiao - Chunlin.Xiao@celera.com; Hilde Johnsen - hjohnsen@ulrik.uio.no; Bjørn Naume - bjorn.naume@medisin.uio.no; aha - Raymond.Samaha@appliedbiosystems.com; Anne-Lise Børresen-Dale - a.l.borresen-dale@medisin.uio.no th †Eq l t ib t Raymond R Samaha - Raymond.Samaha@appliedbiosystems.com; Anne-Lise Børresen-Dale - a.l.borresen-dale@ * C di h †E l ib Raymond R Samaha - Raymond.Samaha@appliedbiosystems.com; Anne-Lise Børresen-Dale - a.l.borresen-dale@medisin.uio.no * Corresponding author †Equal contributors * Corresponding author †Equal contributors * Corresponding author †Equal contributors Received: 03 March 2006 Accepted: 26 May 2006 Results Identification and validation of tumor subtypes in early breast cancer g Breast cancer is a complex disease and although recent research has emphasized the heterogeneity of the disease, much of its biology remains poorly understood. In partic- ular, genomic tools such as DNA microarrays hold great potential for the deciphering of the molecular patterns of tumors and the identification of new and improved clini- cal markers. Gene expression profiling has been used extensively over the last few years to analyze breast tumors and has resulted in several gene signatures associated with different clinical parameters [1-7]. Using an unsupervised approach, we have identified five clinically relevant sub- types of breast tumors [4,8], which have been further val- idated in independent data sets [3,9-14]. Of these, the two main subtypes are associated with the most significant dif- ference in clinical outcome: Patients with luminal A type tumors are facing a relatively good prognosis, whereas patients with basal-like tumors experience a much shorter overall-and disease-free survival period [10]. They are also associated with differences in pathologic response to chemotherapy [9,10,15]. Our first approach for identifying the previously described tumor subtypes was to correlate the expression patterns of the 20 early stage breast carcinomas analyzed by Applied Biosystems Expression Array System with the previously published expression centroids of the five tumors sub- types [10]. The correlation matrix summarized as a heat map is shown in Figure 1A. The previously identified sub- types were evident also in this small tumor set. Using a correlation coefficient cutoff of 0.2, six tumors were defined as basal-like, seven were luminal A, three tumors were ERBB2+, one luminal B and finally, one was identi- fied as normal breast-like. Two tumors remained unclassi- fied using the 0.2 threshold. Unsupervised hierarchical clustering of the 20 tumors using the 526 mapped intrinsic genes identified three sub- clusters of samples based on their expression patterns (Figure 1B). Individual dendrogram branches are colored according to the strongest correlation of the correspond- ing tumor with a subtype centroid. Among these sub- groups, the clearest distinction was observed between the luminal A (ER+) and the basal-like (ER-) subtypes, as has been repeatedly reported. Our earlier findings suggested that the distinct expression patterns of the tumor subtypes and the significant differ- ences in disease outcome are likely to be caused, at least in part, by alterations in specific cellular pathways and/or different cell type origin. Results Identification and validation of tumor subtypes in early breast cancer The luminal A type tumors are characterized by high expression of the estrogen receptor (ESR1) and a handful of other genes generally co- expressed with ESR1, many of which are genes typically expressed in the luminal epithelium that lines the ducts. The basal-like tumors on the other hand, are characterized by high expression of some basal epithelial markers such as KRT5, KRT17 and LAMC2 (laminin), and many cell cycle-regulated genes [16,17]. A more thorough character- ization of the molecular basis underlying these subtypes, in particular in breast carcinomas at an earlier stage, will help us to better understand breast cancer diseases at cel- lular levels and hopefully provide new molecular prog- nostic markers and targets for therapy. As a second approach to validate the tumor subtypes in this data set, we applied a supervised analysis using "Near- est Shrunken Centroid Classifier" and the PAM software. We took the previously published 122 Norway/Stanford data set [10] and the mapped 526 genes as the training set to identify predictor genes for the five subtypes. With a threshold (∆) of 1 and 10-fold cross validation, we built a classifier containing 428 genes which gave < 5% misclas- sification error (data not shown). We then used this clas- sifier to predict the subtypes of the 20 tumors analyzed in this study. The prediction results from the supervised analysis were overall consistent with the unsupervised analysis using hierarchical clustering and centroid correla- tion analysis: 6 tumors were predicted to be basal-like, 8 tumors were predicted to be luminal A, 4 were determined as ERBB2+, 1 as luminal B, and 1 was unclassifiable (Fig- ure 2, top panel). In other words, all but one sample were assigned to a subtype with high prediction probability; however, it is worth to note that the prediction accuracy may be over-estimated as the predictor genes are a subset of those used to define the subtypes in the first place. In this study, we profiled 20 samples from early breast car- cinomas (T1/T2) using three different microarray plat- forms to address the question of uniformity of the breast cancer phenotypes with different technologies. We identi- fied differentially expressed genes between the two main tumor subtypes, luminal A and basal-like, and subjected these to protein classification and pathway analyses. Results Identification and validation of tumor subtypes in early breast cancer Finally, we identified a minimum set of genes with the best possible predictive power for an expression-based prognostic assay for the two clinically relevant subtypes of breast cancer. Abstract Page 1 of 15 (page number not for citation purposes) Page 1 of 15 (page number not for citation purposes) BMC Genomics 2006, 7:127 http://www.biomedcentral.com/1471-2164/7/127 http://www.biomedcentral.com/1471-2164/7/127 Confirmation of the luminal A and basal-like subtypes using different array platforms Branches are color-coded according to the subtype h which the corresponding tumor sample showed the highest correlation Tumors with low correlation (< 0 2) with a spe Luminal A Basal-like ERBB2+ Luminal B Normal-like Luminal A Basal-like ERBB2+ Normal-like Luminal B A B 5.7 4 2.8 2 1.4 1 1.4 2 2.8 4 5.7 M icM a088_AB_REP2 M icM a088_AB_REP1 M icM a065_AB_REP2 M icM a065_AB_REP1 M icM a101_AB_REP2 M icM a101_AB_REP1 M icM a263_AB_REP2 M icM a263_AB_REP1 M icM a632_AB_REP2 M icM a632_AB_REP1 M icM a122_AB_REP2 M icM a122_AB_REP1 M icM a148_AB_REP2 M icM a148_AB_REP1 M icM a085_AB_REP2 M icM a085_AB_REP1 M icM a132_AB_REP2 M icM a132_AB_REP1 M icM a053_AB_REP2 M icM a053_AB_REP1 M icM a146_AB_REP2 M icM a146_AB_REP1 M icM a079_AB_REP2 M icM a079_AB_REP1 M icM a020_AB_REP2 M icM a020_AB_REP1 M icM a067_AB_REP2 M icM a067_AB_REP1 M icM a031_AB_REP2 M icM a031_AB_REP1 M icM a042_AB_REP2 M icM a042_AB_REP1 M icM a709_AB_REP2 M icM a709_AB_REP1 M icM a267_AB_REP2 M icM a267_AB_REP1 M icM a185_AB_REP2 M icM a185_AB_REP1 M icM a091_AB_REP2 M icM a091_AB_REP1 Luminal A Basal-like ERBB2+ Luminal B Normal-like Luminal A Basal-like ERBB2+ Luminal B Normal-like Luminal A Basal-like ERBB2+ Normal-like Luminal B A ERBB2+ Unsupervised approach to identify breast cancer subtypes Figure 1 Unsupervised approach to identify breast cancer subtypes. (A) Correlation of breast tumor samples with the previ- ously identified five subtypes of breast tumors. 526 out of 552 previously identified "intrinsic" genes were cross-mapped to Applied Biosystems Human Genome Survey Microarray and used for centroid correlation analysis and hierarchical clustering. Correlations with the centroids of the five subtypes were calculated for each sample from this study (the two microarray rep- licates for each sample were averaged). Samples were assigned to a subtype with which it showed the highest correlation using a cutoff value of 0.2. (B) Unsupervised hierarchical clustering of 20 breast tumor tissues analyzed by AB arrays using the 526 mapped intrinsic genes (the two microarray replicates for each sample are shown). The level of expression of each gene in each sample, relative to the median level of expression of that gene across all the samples, is represented using a red-black- green color scale as shown in the key (green: below median; black: equal to median; red: above median). (Left panel): Scaled down representation of the entire cluster of the 526 intrinsic genes and 20 tissue samples. Confirmation of the luminal A and basal-like subtypes using different array platforms Correlations with the centroids of the five subtypes were calculated for each sample from this study (the two microarray rep- licates for each sample were averaged). Samples were assigned to a subtype with which it showed the highest correlation using a cutoff value of 0.2. (B) Unsupervised hierarchical clustering of 20 breast tumor tissues analyzed by AB arrays using the 526 mapped intrinsic genes (the two microarray replicates for each sample are shown). The level of expression of each gene in each sample, relative to the median level of expression of that gene across all the samples, is represented using a red-black- green color scale as shown in the key (green: below median; black: equal to median; red: above median). (Left panel): Scaled down representation of the entire cluster of the 526 intrinsic genes and 20 tissue samples. (Right panel): Experimental dendro- gram displaying the clustering of the tumors into three distinct subgroups. Branches are color-coded according to the subtype with which the corresponding tumor sample showed the highest correlation. Tumors with low correlation (< 0.2) with a spe- cific subtype are indicated by gray branches. Unsupervised approach to identify breast cancer subtypes Figure 1 Unsupervised approach to identify breast cancer subtypes. (A) Correlation of breast tumor samples with the previ- ously identified five subtypes of breast tumors. 526 out of 552 previously identified "intrinsic" genes were cross-mapped to Applied Biosystems Human Genome Survey Microarray and used for centroid correlation analysis and hierarchical clustering. Correlations with the centroids of the five subtypes were calculated for each sample from this study (the two microarray rep- licates for each sample were averaged). Samples were assigned to a subtype with which it showed the highest correlation using a cutoff value of 0.2. (B) Unsupervised hierarchical clustering of 20 breast tumor tissues analyzed by AB arrays using the 526 mapped intrinsic genes (the two microarray replicates for each sample are shown). The level of expression of each gene in each sample, relative to the median level of expression of that gene across all the samples, is represented using a red-black- green color scale as shown in the key (green: below median; black: equal to median; red: above median). (Left panel): Scaled down representation of the entire cluster of the 526 intrinsic genes and 20 tissue samples. (Right panel): Experimental dendro- gram displaying the clustering of the tumors into three distinct subgroups. Confirmation of the luminal A and basal-like subtypes using different array platforms We also analyzed the same 20 tumor samples on Stanford Human cDNA microarrays and Agilent Whole Human Genome Oligo Microarrays. Centroid correlation analysis was performed as described above using the 510 common Page 2 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/127 BMC Genomics 2006, 7:127 upervised approach to identify breast cancer subtypes ure 1 supervised approach to identify breast cancer subtypes. (A) Correlation of breast tumor samples with the previ- y identified five subtypes of breast tumors. 526 out of 552 previously identified "intrinsic" genes were cross-mapped to lied Biosystems Human Genome Survey Microarray and used for centroid correlation analysis and hierarchical clustering. relations with the centroids of the five subtypes were calculated for each sample from this study (the two microarray rep- es for each sample were averaged). Samples were assigned to a subtype with which it showed the highest correlation using toff value of 0.2. (B) Unsupervised hierarchical clustering of 20 breast tumor tissues analyzed by AB arrays using the 526 pped intrinsic genes (the two microarray replicates for each sample are shown). The level of expression of each gene in h sample, relative to the median level of expression of that gene across all the samples, is represented using a red-black- en color scale as shown in the key (green: below median; black: equal to median; red: above median). (Left panel): Scaled wn representation of the entire cluster of the 526 intrinsic genes and 20 tissue samples. (Right panel): Experimental dendro- m displaying the clustering of the tumors into three distinct subgroups. Confirmation of the luminal A and basal-like subtypes using different array platforms (Right panel): Experimental dendro- gram displaying the clustering of the tumors into three distinct subgroups. Branches are color-coded according to the subtype with which the corresponding tumor sample showed the highest correlation. Tumors with low correlation (< 0.2) with a spe- cific subtype are indicated by gray branches. B 5.7 4 2.8 2 1.4 1 1.4 2 2.8 4 5.7 M icM a088_AB_REP2 M icM a088_AB_REP1 M icM a065_AB_REP2 M icM a065_AB_REP1 M icM a101_AB_REP2 M icM a101_AB_REP1 M icM a263_AB_REP2 M icM a263_AB_REP1 M icM a632_AB_REP2 M icM a632_AB_REP1 M icM a122_AB_REP2 M icM a122_AB_REP1 M icM a148_AB_REP2 M icM a148_AB_REP1 M icM a085_AB_REP2 M icM a085_AB_REP1 M icM a132_AB_REP2 M icM a132_AB_REP1 M icM a053_AB_REP2 M icM a053_AB_REP1 M icM a146_AB_REP2 M icM a146_AB_REP1 M icM a079_AB_REP2 M icM a079_AB_REP1 M icM a020_AB_REP2 M icM a020_AB_REP1 M icM a067_AB_REP2 M icM a067_AB_REP1 M icM a031_AB_REP2 M icM a031_AB_REP1 M icM a042_AB_REP2 M icM a042_AB_REP1 M icM a709_AB_REP2 M icM a709_AB_REP1 M icM a267_AB_REP2 M icM a267_AB_REP1 M icM a185_AB_REP2 M icM a185_AB_REP1 M icM a091_AB_REP2 M icM a091_AB_REP1 Luminal A Basal-like ERBB2+ Luminal B Normal-like B 5.7 4 2.8 2 1.4 1 1.4 2 2.8 4 5.7 M icM a088_AB_REP2 M icM a088_AB_REP1 M icM a065_AB_REP2 M icM a065_AB_REP1 M icM a101_AB_REP2 M icM a101_AB_REP1 M icM a263_AB_REP2 M icM a263_AB_REP1 M icM a632_AB_REP2 M icM a632_AB_REP1 M icM a122_AB_REP2 M icM a122_AB_REP1 M icM a148_AB_REP2 M icM a148_AB_REP1 M icM a085_AB_REP2 M icM a085_AB_REP1 M icM a132_AB_REP2 M icM a132_AB_REP1 M icM a053_AB_REP2 M icM a053_AB_REP1 M icM a146_AB_REP2 M icM a146_AB_REP1 M icM a079_AB_REP2 M icM a079_AB_REP1 M icM a020_AB_REP2 M icM a020_AB_REP1 M icM a067_AB_REP2 M icM a067_AB_REP1 M icM a031_AB_REP2 M icM a031_AB_REP1 M icM a042_AB_REP2 M icM a042_AB_REP1 M icM a709_AB_REP2 M icM a709_AB_REP1 M icM a267_AB_REP2 M icM a267_AB_REP1 M icM a185_AB_REP2 M icM a185_AB_REP1 M icM a091_AB_REP2 M icM a091_AB_REP1 Luminal A Basal-like ERBB2+ Luminal B Normal-like B B Unsupervised approach to identify breast cancer subtypes Figure 1 Unsupervised approach to identify breast cancer subtypes. (A) Correlation of breast tumor samples with the previ- ously identified five subtypes of breast tumors. 526 out of 552 previously identified "intrinsic" genes were cross-mapped to Applied Biosystems Human Genome Survey Microarray and used for centroid correlation analysis and hierarchical clustering. Confirmation of the luminal A and basal-like subtypes using different array platforms Branches are color-coded according to the subtype with which the corresponding tumor sample showed the highest correlation. Tumors with low correlation (< 0.2) with a spe- cific subtype are indicated by gray branches. Page 3 of 15 (page number not for citation purposes) Page 3 of 15 (page number not for citation purposes) BMC Genomics 2006, 7:127 http://www.biomedcentral.com/1471-2164/7/127 BMC Genomics 2006, 7:127 http://www.biomedcentral.com/1471-2164/7/127 Prediction of tumor subtype by Prediction Analysis of Microarrays (PAM) Figure 2 Prediction of tumor subtype by Prediction Analysis of Microarrays (PAM). 428 genes were selected at a threshold of 1.0 that separated the two subtypes with the lowest overall misclassification rate of 5% (data not shown). Predicted probabili- ties of subtype for each tumor sample analyzed on Applied Biosystems Human Genome Survey Microarrays (top panel), Stan- ford Human cDNA arrays (middle panel) and Agilent Whole Human Genome Microarrays (bottom panel) were computed using the predictive model built using these 428 predictor genes. Prediction Figure 2 yp y y y ( ) g Prediction of tumor subtype by Prediction Analysis of Microarrays (PAM). 428 genes were selected at a threshold of 1.0 that separated the two subtypes with the lowest overall misclassification rate of 5% (data not shown). Predicted probabili- ties of subtype for each tumor sample analyzed on Applied Biosystems Human Genome Survey Microarrays (top panel), Stan- ford Human cDNA arrays (middle panel) and Agilent Whole Human Genome Microarrays (bottom panel) were computed using the predictive model built using these 428 predictor genes. multiple testing corrections were performed on six lumi- nal A samples and six basal-like tumor samples (to be most stringent in this analysis, samples with centroid cor- relation coefficient > 0.3 were used, therefore, MicMa088 and MicMa 132 were excluded, see Figure 1A). 1210 genes represented by 1244 probes were identified as the "signa- ture" genes meeting the following criteria: (1) Detectable (signal to noise > 3) in > 50% samples; (2) > 2-fold change between the two subtypes, and (3) False discovery rate < 5% (see Additional file 1). Figure 4 displays a hier- archical clustering diagram of the 12 tumor samples using these 1210 signature genes. Among the signature genes, 613 probes (603 genes) were specifically over-expressed in luminal A type tumors (luminal A "signature" genes), which included some previously identified markers, such as ESR1, GATA3 and LIV1 [10], as well as many other potential marker genes for this subtype. Confirmation of the luminal A and basal-like subtypes using different array platforms One example is the EMP2 gene, which encodes a tetra-span membrane protein that has been reported to suppress B-cell lym- phoma tumorigenicity [18]. In the basal-like tumors, 631 intrinsic genes mapped among all three platforms. Unsu- pervised hierarchical clustering of the three data sets revealed the exact same subgroups of tumors, with the luminal A and the basal-like subtypes as the most pre- dominant and distinct (Figure 3A). PAM analysis showed consistent prediction of subtype for each tumor sample from the three data sets except for two samples (MicMa148 and MicMa 020; see Figure 2). Overall, there was consistency in identifying these biological subtypes between all three platforms. Page 4 of 15 (page number not for citation purposes) Molecular characterization of luminal A and basal-like subtypes of breast tumors The data from each platform were first transformed independently and then combined for clustering: the level of expression of each gene in each sample (Applied Biosystems microarrays: normalized sig- nal intensity; Stanford cDNA and Agilent microarrays: normalized log2 ratio of the sample vs. the reference (UHR)) was trans- formed into a log2 ratio relative to the median level of expression of that gene across all the samples within the data set of the given platform. The experimental dendrogram displays the clustering of the tumors into distinct subgroups. Branches are color-coded according to the subtype with which the corresponding tumor sample showed the highest correlation. Tumors with low correlation (< 0.2) with a specific subtype are indicated by gray branches. Luminal A subtype (dark blue) and basal-like subtype (red). (B) Venn Diagram of the most differentially expressed genes identified by all three different array platforms; 319 genes were identified as the common signature genes using ANOVA analysis and the following criteria: (1) > 2-fold change between the two subtypes, and (2) False discovery rate < 5%. (C) PCA analysis of luminal A and basal-like samples using a min- imum set of 54 genes identified by PAM analysis. Data sets generated from three array platforms (48 arrays total, two repli- cates per sample in the Applied Biosystems data set) on 6 luminal A and 6 basal-like tumor samples using the 319 common signature genes were used as training set for the PAM analysis. B 0 1 0 1 Y: [PCA component 2 (6.579% variance)] Z: [PCA component 3 (4.439% variance)] 0 1 0 1 Z: [PCA component 3 (4.439% variance)] X: [PCA component 1 (67.24% variance)] 0 1 0 1 X: [PCA component 1 (67.24% variance)] Y: [PCA component 2 (6.579% variance)] Luminal A Basal-like C 0 1 0 1 Y: [PCA component 2 (6.579% variance)] Z: [PCA component 3 (4.439% variance)] 0 1 0 1 Z: [PCA component 3 (4.439% variance)] X: [PCA component 1 (67.24% variance)] 0 1 0 1 X: [PCA component 1 (67.24% variance)] Y: [PCA component 2 (6.579% variance)] Luminal A Basal-like C C B B yp g y p g Validation of the luminal A and basal-like subtypes using three microarray platforms. (A) Unsupervised hierarchi- cal clustering of 20 breast tumor tissues analyzed by Applied Biosystems (two replicates per sample), Stanford cDNA and Agi- lent arrays using the 510 mapped intrinsic genes. Molecular characterization of luminal A and basal-like subtypes of breast tumors To molecularly characterize these two subtypes, we first identified the most differentially expressed genes as the "signature" genes using data from the Applied Biosystems Expression Arrays, as this system provides a comprehen- sive coverage of the genome including genes not covered by other commercial microarrays. ANOVA analysis cou- pled with Benjamini and Hochberg False Discovery Rate Page 4 of 15 (page number not for citation purposes) Page 4 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/127 BMC Genomics 2006, 7:127 MicMa 065_Stanford cDNA MicMa065_Agilent MicMa065_AB_REP2 MicMa065_AB_REP1 MicMa 122_Stanford cDNA MicMa122_Agilent MicMa122_AB_REP2 MicMa122_AB_REP1 LuminalA Centroid Micma 101_Stanford cDNA MicMa101_Agilent MicMa101_AB_REP2 MicMa101_AB_REP1 MicMa 263_Stanford cDNA MicMa263_Agilent MicMa263_AB_REP2 MicMa263_AB_REP1 MicMa 632_Stanford cDNA MicMa632_Agilent MicMa632_AB_REP2 MicMa632_AB_REP1 MicMa 088_Stanford cDNA MicMa088_Agilent MicMa088_AB_REP2 MicMa088_AB_REP1 MicMa 085_Stanford cDNA MicMa085_Agilent MicMa085_AB_REP2 MicMa085_AB_REP1 MicMa 148_Stanford cDNA MicMa148_Agilent MicMa148_AB_REP2 MicMa148_AB_REP1 MicMa132_AB_REP2 MicMa132_AB_REP1 MicMa132_Agilent MicMa 132_Stanford cDNA MicMa267_AB_REP2 MicMa267_AB_REP1 MicMa267_Agilent MicMa 267_Stanford cDNA MicMa 709_Stanford cDNA MicMa709_Agilent MicMa709_AB_REP2 MicMa709_AB_REP1 MicMa 031_Stanford cDNA MicMa031_Agilent MicMa031_AB_REP2 MicMa031_AB_REP1 MicMa 067_Stanford cDNA MicMa067_Agilent MicMa067_AB_REP2 MicMa067_AB_REP1 Basal Centroid MicMa 042_Stanford cDNA MicMa042_Agilent MicMa042_AB_REP2 MicMa042_AB_REP1 MicMa185_Agilent MicMa 185_Stanford cDNA MicMa185_AB_REP2 MicMa185_AB_REP1 Normal-like Centroid MicMa 020_Stanford cDNA MicMa020_Agilent MicMa020_AB_REP2 MicMa020_AB_REP1 MicMa 079_Stanford cDNA MicMa079_Agilent MicMa079_AB_REP2 MicMa079_AB_REP1 MicMa 053_Stanford cDNA MicMa053_Agilent MicMa053_AB_REP2 MicMa053_AB_REP1 ERBB2+ Centroid MicMa 146_Stanford cDNA MicMa146_AB_REP2 MicMa146_AB_REP1 MicMa146_Agilent MicMa091_Agilent MicMa 091_Stanford cDNA MicMa091_AB_REP2 MicMa091_AB_REP1 LuminalB Centroid Luminal A Basal-like A A A Validation of the luminal A and basal-like subtypes using three microarray platforms Figure 3 Validation of the luminal A and basal-like subtypes using three microarray platforms. (A) Unsupervised hierarc cal clustering of 20 breast tumor tissues analyzed by Applied Biosystems (two replicates per sample), Stanford cDNA and A lent arrays using the 510 mapped intrinsic genes. The data from each platform were first transformed independently and the combined for clustering: the level of expression of each gene in each sample (Applied Biosystems microarrays: normalized s nal intensity; Stanford cDNA and Agilent microarrays: normalized log2 ratio of the sample vs. the reference (UHR)) was tra formed into a log2 ratio relative to the median level of expression of that gene across all the samples within the data set of t given platform. The experimental dendrogram displays the clustering of the tumors into distinct subgroups. Branches are color-coded according to the subtype with which the corresponding tumor sample showed the highest correlation. Tumor with low correlation (< 0.2) with a specific subtype are indicated by gray branches. Molecular characterization of luminal A and basal-like subtypes of breast tumors Luminal A subtype (dark blue) and basal-li subtype (red). (B) Venn Diagram of the most differentially expressed genes identified by all three different array platforms; 3 genes were identified as the common signature genes using ANOVA analysis and the following criteria: (1) > 2-fold change between the two subtypes, and (2) False discovery rate < 5%. (C) PCA analysis of luminal A and basal-like samples using a m imum set of 54 genes identified by PAM analysis. Data sets generated from three array platforms (48 arrays total, two repli- cates per sample in the Applied Biosystems data set) on 6 luminal A and 6 basal-like tumor samples using the 319 common signature genes were used as training set for the PAM analysis. MicMa 065_Stanford cDNA MicMa065_Agilent MicMa065_AB_REP2 MicMa065_AB_REP1 MicMa 122_Stanford cDNA MicMa122_Agilent MicMa122_AB_REP2 MicMa122_AB_REP1 LuminalA Centroid Micma 101_Stanford cDNA MicMa101_Agilent MicMa101_AB_REP2 MicMa101_AB_REP1 MicMa 263_Stanford cDNA MicMa263_Agilent MicMa263_AB_REP2 MicMa263_AB_REP1 MicMa 632_Stanford cDNA MicMa632_Agilent MicMa632_AB_REP2 MicMa632_AB_REP1 MicMa 088_Stanford cDNA MicMa088_Agilent MicMa088_AB_REP2 MicMa088_AB_REP1 MicMa 085_Stanford cDNA MicMa085_Agilent MicMa085_AB_REP2 MicMa085_AB_REP1 MicMa 148_Stanford cDNA MicMa148_Agilent MicMa148_AB_REP2 MicMa148_AB_REP1 MicMa132_AB_REP2 MicMa132_AB_REP1 MicMa132_Agilent MicMa 132_Stanford cDNA MicMa267_AB_REP2 MicMa267_AB_REP1 MicMa267_Agilent MicMa 267_Stanford cDNA MicMa 709_Stanford cDNA MicMa709_Agilent MicMa709_AB_REP2 MicMa709_AB_REP1 MicMa 031_Stanford cDNA MicMa031_Agilent MicMa031_AB_REP2 MicMa031_AB_REP1 MicMa 067_Stanford cDNA MicMa067_Agilent MicMa067_AB_REP2 MicMa067_AB_REP1 Basal Centroid MicMa 042_Stanford cDNA MicMa042_Agilent MicMa042_AB_REP2 MicMa042_AB_REP1 MicMa185_Agilent MicMa 185_Stanford cDNA MicMa185_AB_REP2 MicMa185_AB_REP1 Normal-like Centroid MicMa 020_Stanford cDNA MicMa020_Agilent MicMa020_AB_REP2 MicMa020_AB_REP1 MicMa 079_Stanford cDNA MicMa079_Agilent MicMa079_AB_REP2 MicMa079_AB_REP1 MicMa 053_Stanford cDNA MicMa053_Agilent MicMa053_AB_REP2 MicMa053_AB_REP1 ERBB2+ Centroid MicMa 146_Stanford cDNA MicMa146_AB_REP2 MicMa146_AB_REP1 MicMa146_Agilent MicMa091_Agilent MicMa 091_Stanford cDNA MicMa091_AB_REP2 MicMa091_AB_REP1 LuminalB Centroid Luminal A Basal-like A B 0 1 0 1 Y: [PCA component 2 (6.579% variance)] Z: [PCA component 3 (4.439% variance)] 0 1 0 1 Z: [PCA component 3 (4.439% variance)] X: [PCA component 1 (67.24% variance)] 0 1 0 1 X: [PCA component 1 (67.24% variance)] Y: [PCA component 2 (6.579% variance)] Luminal A Basal-like C Validation of the luminal A and basal-like subtypes using three microarray platforms Figure 3 Validation of the luminal A and basal-like subtypes using three microarray platforms. (A) Unsupervised hierarchi- cal clustering of 20 breast tumor tissues analyzed by Applied Biosystems (two replicates per sample), Stanford cDNA and Agi- lent arrays using the 510 mapped intrinsic genes. Molecular characterization of luminal A and basal-like subtypes of breast tumors Page 5 of 15 (page number not for citation purposes) Page 5 of 15 (page number not for citation purposes) Page 5 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/127 BMC Genomics 2006, 7:127 Two-dimensional cluster diagram of the 1210 signature genes characterizing the luminal A and basal-like subtypes Figure 4 Two-dimensional cluster diagram of the 1210 signature genes characterizing the luminal A and basal-like sub- M icM a185_REP2 M icM a185_REP1 M icM a267_REP2 M icM a267_REP1 M icM a031_REP2 M icM a031_REP1 M icM a709_REP2 M icM a709_REP1 M icM a067_REP2 M icM a067_REP1 M icM a042_REP2 M icM a042_REP1 M icM a065_REP2 M icM a065_REP1 M icM a101_REP2 M icM a101_REP1 M icM a122_REP2 M icM a122_REP1 M icM a263_REP2 M icM a263_REP1 M icM a632_REP2 M icM a632_REP1 M icM a085_REP2 M icM a085_REP1 Luminal A (603 genes) Basal-like (607 genes) LIV-1 GATA3 ESR1 LBR DSC2 KRT17 7.3 4 2.8 2 1.4 1 1.4 2 2.8 4 7.3 ESR1 GATA3 LIV1 EMP2 PGDS ACOX2 PTE2B HMGCL CROT IVD DECR2 FLJ20920 SLC27A2 ELOVL5 MCCC2 AR MGC32124 TFF1 KRT17 LBR DSC2 MRAS CDCA7 MCM3 MCM4 MCM7 MAD2L1 SKP2 EN1 NCK1 MSN IFNAR2 DAPK1 CYP39A1 EMP2 MRAS CDCA7 Two-dimensional cluster diagram of the 1210 signature genes characterizing the luminal A and basal-like subtypes Figure 4 Two-dimensional cluster diagram of the 1210 signature genes characterizing the luminal A and basal-like sub- types. ANOVA analysis was performed on 6 luminal A samples and 6 basal-like samples, coupled with Benjamini and Hochberg False Discovery Rate multiple testing corrections. The two subtypes of breast tumors (two replicates per sample) were clus- tered into distinct clusters with reversed gene expression patterns: highly expressed in luminal A (bottom, 613 probes repre- senting 603 genes) and highly expressed in basal (top, 631 probes representing 607 genes). The color scheme of the heat map was described in legend to Figure 1B and is shown in the key. Branches in the dendrogram are color-coded according to the subtypes: blue, luminal A; red, basal-like. Molecular characterization of luminal A and basal-like subtypes of breast tumors M icM a185_REP2 M icM a185_REP1 M icM a267_REP2 M icM a267_REP1 M icM a031_REP2 M icM a031_REP1 M icM a709_REP2 M icM a709_REP1 M icM a067_REP2 M icM a067_REP1 M icM a042_REP2 M icM a042_REP1 M icM a065_REP2 M icM a065_REP1 M icM a101_REP2 M icM a101_REP1 M icM a122_REP2 M icM a122_REP1 M icM a263_REP2 M icM a263_REP1 M icM a632_REP2 M icM a632_REP1 M icM a085_REP2 M icM a085_REP1 Luminal A (603 genes) Basal-like (607 genes) LIV-1 GATA3 ESR1 LBR DSC2 KRT17 7.3 4 2.8 2 1.4 1 1.4 2 2.8 4 7.3 ESR1 GATA3 LIV1 EMP2 PGDS ACOX2 PTE2B HMGCL CROT IVD DECR2 FLJ20920 SLC27A2 ELOVL5 MCCC2 AR MGC32124 TFF1 KRT17 LBR DSC2 MRAS CDCA7 MCM3 MCM4 MCM7 MAD2L1 SKP2 EN1 NCK1 MSN IFNAR2 DAPK1 CYP39A1 EMP2 MRAS CDCA7 M icM a185_REP M icM a185_REP M icM a267_REP M icM a267_REP M icM a031_REP M icM a031_REP M icM a709_REP M icM a709_REP M icM a067_REP M icM a067_REP M icM a042_REP M icM a042_REP M icM a065_REP M icM a065_REP M icM a101_REP M icM a101_REP M icM a122_REP M icM a122_REP M icM a263_REP M icM a263_REP M icM a632_REP M icM a632_REP M icM a085_REP M icM a085_REP 7.3 4 2.8 2 1.4 1 1.4 2 2.8 4 7.3 M icM a185_RE M icM a185_RE M icM a267_RE M icM a267_RE M icM a031_RE M icM a031_RE M icM a709_RE M icM a709_RE M icM a067_RE M icM a067_RE M icM a042_RE M icM a042_RE M icM a065_RE M icM a065_RE M icM a101_RE M icM a101_RE M icM a122_RE M icM a122_RE M icM a263_RE M icM a263_RE M icM a632_RE M icM a632_RE M icM a085_RE M icM a085_RE Luminal A (603 genes) Basal-like (607 genes) LIV-1 GATA3 ESR1 LBR DSC2 KRT17 7.3 4 2.8 2 1.4 1 1.4 2 2.8 4 7.3 ESR1 GATA3 LIV1 EMP2 PGDS ACOX2 PTE2B HMGCL CROT IVD DECR2 FLJ20920 SLC27A2 ELOVL5 MCCC2 AR MGC32124 TFF1 KRT17 LBR DSC2 MRAS CDCA7 MCM3 MCM4 MCM7 MAD2L1 SKP2 EN1 NCK1 MSN IFNAR2 DAPK1 CYP39A1 EMP2 MRAS CDCA7 7.3 4 2.8 2 1.4 1 1.4 2 2.8 4 7.3 g g g g yp g Two-dimensional cluster diagram of the 1210 signature genes characterizing the luminal A and basal-like sub- types. Molecular characterization of luminal A and basal-like subtypes of breast tumors The data from each platform were first transformed independently and then combined for clustering: the level of expression of each gene in each sample (Applied Biosystems microarrays: normalized sig- nal intensity; Stanford cDNA and Agilent microarrays: normalized log2 ratio of the sample vs. the reference (UHR)) was trans- formed into a log2 ratio relative to the median level of expression of that gene across all the samples within the data set of the given platform. The experimental dendrogram displays the clustering of the tumors into distinct subgroups. Branches are color-coded according to the subtype with which the corresponding tumor sample showed the highest correlation. Tumors with low correlation (< 0.2) with a specific subtype are indicated by gray branches. Luminal A subtype (dark blue) and basal-like subtype (red). (B) Venn Diagram of the most differentially expressed genes identified by all three different array platforms; 319 genes were identified as the common signature genes using ANOVA analysis and the following criteria: (1) > 2-fold change between the two subtypes, and (2) False discovery rate < 5%. (C) PCA analysis of luminal A and basal-like samples using a min- imum set of 54 genes identified by PAM analysis. Data sets generated from three array platforms (48 arrays total, two repli- cates per sample in the Applied Biosystems data set) on 6 luminal A and 6 basal-like tumor samples using the 319 common signature genes were used as training set for the PAM analysis. Molecular characterization of luminal A and basal-like subtypes of breast tumors ANOVA analysis was performed on 6 luminal A samples and 6 basal-like samples, coupled with Benjamini and Hochberg False Discovery Rate multiple testing corrections. The two subtypes of breast tumors (two replicates per sample) were clus- tered into distinct clusters with reversed gene expression patterns: highly expressed in luminal A (bottom, 613 probes repre- senting 603 genes) and highly expressed in basal (top, 631 probes representing 607 genes). The color scheme of the heat map was described in legend to Figure 1B and is shown in the key. Branches in the dendrogram are color-coded according to the subtypes: blue, luminal A; red, basal-like. Page 6 of 15 (page number not for citation purposes) Page 6 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/127 BMC Genomics 2006, 7:127 Table 1: Annotation and biological pathway analysis. Protein Classifications and Pathways Number of Overlapping Genes Random Overlapping p value PANTHER™ Protein Classification System (p < 0.001) Luminal A subtype Fatty acid metabolism 11 1.22E-04 Steroid hormone-mediated signaling 5 8.09E-04 Basal-like subtype Cell cycle 55 2.50E-13 Cell proliferation and differentiation 40 9.62E-08 Protein phosphorylation 34 2.31E-07 B-cell-and antibody-mediated immunity 17 2.63E-07 Cell cycle control 25 2.38E-06 Jubilant PathArt™ Pathways (p < 0.01) Luminal A subtype ER Signaling Pathway 6 2.80E-04 Retinoic Acid Signaling Pathway 4 4.46E-04 Nucleotide Excision Repair Pathway 4 1.92E-03 IL6 Signaling Pathway 6 4.73E-03 EGF Signaling Pathway 7 4.92E-03 Basal-like subtype p21 Mediated Pathway 15 9.14E-13 G1-S Checkpoint Pathway 10 1.03E-08 FAS Mediated Pathway 3 7.49E-04 p53 Signaling Pathway 7 8.45E-04 Upper part: The most significant biological processes (PANTHER™ Protein Classification System) over-represented by the luminal A subtype and the basal-like subtype (p < 0.001, top 5 are shown); Lower part: The most significant cellular pathways (PathArt™) over-represented by the luminal A subtype and the basal-like subtype (p < 0.01). Upper part: The most significant biological processes (PANTHER™ Protein Classification System) over-represented by the luminal A subtype and the basal-like subtype (p < 0.001, top 5 are shown); Lower part: The most significant cellular pathways (PathArt™) over-represented by the luminal A subtype and the basal-like subtype (p < 0.01). Upper part: The most significant biological processes (PANTHER™ Protein Classification System) over-represented by the luminal A subtype and the basal-like subtype (p < 0.001, top 5 are shown); Lower part: The most significant cellular pathways (PathArt™) over-represented by the luminal A subtype and the basal-like subtype (p < 0.01). Molecular characterization of luminal A and basal-like subtypes of breast tumors and differentiation, protein phosphorylation, B-cell-and antibody-mediated immunity. probes (607 genes) were specifically over-expressed (basal-like "signature" genes), and these included markers such as KRT17, the Lamin B receptor (LBR) and DSC2. Two interesting genes specifically overexpressed in these tumors were MRAS, a well known oncoprotein of the RAS superfamily whose mutant forms may transform mam- mary epithelial cells [19], and CDCA7, a direct target of the MYC oncogene [20]. In a similar fashion, we also analyzed which cellular path- ways played critical roles for defining the two distinct sub- types using Jubilant's PathArt™ database. Table 1 (lower panel) shows the top five PathArt™ pathways over-repre- sented (p < 0.01) by the genes characteristic for luminal A and the basal-like subtypes, respectively. Again, quite dis- tinct pathways were found to underlie the two subtypes of breast tumors. As expected, the most over-represented pathway activated in the luminal A subtype is the ER sig- naling pathway (see Additional file 2A): 6 genes within the ER signaling pathway were significantly up-regulated in these tumors, including the estrogen receptor 1 (ESR1) and the estrogen-induced gene trefoil factor 1 (TFF1). This is consistent with the previous findings that the luminal A type tumors over-express ESR1 and other estrogen- responsive genes and therefore are responsive to adjuvant hormonal treatment [10,21]. On the other hand, the bio- logical pathways underlying the basal-like subtype are well known cancer-associated pathways. For example, fif- teen genes in the p21 (CDKN1A) pathway were coordi- nately over-expressed in the basal-like tumors, many of these, such as MCM3, MCM4, MCM7 and MAD2L1 play To depict more detailed molecular portraits of these two subtypes, we analyzed which biological processes were over-represented by these signature genes using the PAN- THER™ Protein Classification System analysis. The most significantly over-represented biological processes are listed in Table 1 (upper panel). Not surprisingly, very dif- ferent processes are underlined by the signature genes of the two subtypes: For luminal A, the most over-repre- sented biological processes (p < 0.001) include fatty acid metabolism (e.g. PGDS, ACOX2, PTE2B, HMGCL, CROT, IVD, DECR2, FLJ20920, SLC27A2, ELOVL5, and MCCC2) and steroid hormone mediated signaling (e.g. Discussion l f In an effort to identify a minimal set of genes that best characterize the two subtypes and can form the basis for a prognostic gene profile, we performed ANOVA analysis on the data sets generated on each of the three array plat- forms using the 16,611 common genes (see Additional file 3) and the same six luminal A and six basal-like tumor samples. Differentially expressed genes between the two subtypes were determined for each platform using the same criteria: (1) > 2-fold change between the two sub- types, and (2) False discovery rate < 5%. From these, 319 genes were identified as the common signature genes (Fig- ure 3B). The combined data sets generated from the three platforms using expression data from these 319 common genes was then used as the training set to perform PAM analysis for the identification of the minimal set of genes to best discriminate luminal A and basal-like tumors. Ten- fold cross validation and a threshold of ∆ = 1.9 identified 54 genes with a misclassification error of ~ 16.7%. The genes are listed in Additional file 4. Principle Component Analysis on the 12 tumor samples profiled on all three array platforms using these 54 genes clearly separated the two subtypes of breast tumors (Figure 3C). Analyses of gene expression patterns from thousands of genes using DNA microarrays have demonstrated great diversity among tumors arising in the same organ and with apparently similar histopathology. This has raised hopes that classification schemes based on molecular pro- filing may better capture the complex behavior of tumors and lead to improved prognostication and tailor-made therapeutic strategies. We were the first to identify that specific subclasses of breast cancer, based on gene expres- sion profiling, were distinct biological entities and associ- ated with significant differences in outcome for patients with locally advanced breast cancer [4]. Subsequently, this has been validated both by us and other groups in differ- ent types of breast cancer patient cohorts [3,9-13]. Here, we could confirm the existence of the molecular subtypes of breast tumors also in early breast cancer (T1/T2) using three different microarray platforms. Due to the small sample size reported here, only the luminal A and basal- like groups could be robustly identified, although the other less represented subtypes could also be recognized. Molecular characterization of luminal A and basal-like subtypes of breast tumors CRABP2, AR, MGC32124, ESR1), whereas for the basal-like sub- type, the most over-enriched processes (p < 0.001, only top five listed) include ones that involve many cancer "hallmark" genes, such as the cell cycle, cell proliferation Page 7 of 15 (page number not for citation purposes) Page 7 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/127 BMC Genomics 2006, 7:127 BMC Genomics 2006, 7:127 Table 2: Pearson correlation between gene expression profiles determined by TaqMan real-time PCR and three DNA microarray platforms Pearson Correction with TaqMan Assay Applied Biosystems Stanford cDNA Array Agilent Good (cor.coeff. > 0.8) 75 (88%) 69 (81%) 73 (86%) Consistent (0 < cor.coeff. < 0.8) 8 (9%) 15 (18%) 9 (11%) Anti-Correlation (cor.coeff. < 0) 2 (2%) 1(1%) 3 (4%) Median 0.91 0.90 0.91 2: Pearson correlation between gene expression profiles determined by TaqMan real-time PCR and three rms lation between gene expression profiles determined by TaqMan real-time PCR and three DNA microarray Good (cor.coeff. > 0.8) Consistent (0 < cor.coeff. < 0.8) Anti-Correlation (cor.coeff. < 0) Median the PAM analysis, 10 previously identified markers (from the intrinsic gene list) and 7 selected G-protein coupled receptors (GPCRs) and secreted proteins) and performed real-time PCR using TaqMan® Gene Expression Assays (see Additional file 5). Profile correlation analysis showed that the expression profiles across the 20 tumor samples deter- mined by each array platform and by TaqMan® assays are highly correlated (Figure 5) (median correlation coeffi- cient R > 0.9, the rate of good correlation (Pearson corre- lation coefficient, R > 0.8) varied from 81–88% for the three array platforms (Table 2). Hierarchical clustering analysis not only demonstrated excellent separation of the luminal A and basal-like subtypes, but that the same tumor sample analyzed by three microarray platforms and the TaqMan® assays were clustered together by tumor sam- ple rather than by method (Figure 6). critical roles in cell proliferation and DNA replication (see Additional file 2B). Among many genes in this pathway, SKP2 was found over-expressed in basal-like tumors; it encodes a protein involved in the degradation of another cyclin-dependent kinase inhibitor p27 (CDKN1B) and recently reported to be over-expressed in many tumor types and to correlate with poor prognosis [22,23]. Molecular characterization of luminal A and basal-like subtypes of breast tumors Our findings confirm the existence of the intrinsic tumor subtypes in these early breast cancer specimens, as has been reported by others [3,13] and indicate that the cellu- lar processes revealed by gene expression profiling have been programmed at earlier stages of tumorigenesis. Discussion l f These two subtypes are easily distinguishable in several tumor data sets and their expression profiles seem to be anti-correlated, as also has been shown for breast cancer cell lines [24], but the cellular pathways affected are not known in detail. We show here that the differences in gene expression patterns between the two main subtypes reflect levels of activation of distinct signaling pathways. These Page 8 of 15 (page number not for citation purposes) Page 9 of 15 (page number not for citation purposes) Real-time PCR validation using TaqMan® Gene Expression Assays To further validate these prognostic markers, we selected altogether 85 genes (the 54 minimal set of predictor genes plus an additional 14 top-ranking predictor genes from Page 8 of 15 (page number not for citation purposes) Page 8 of 15 (page number not for citation purposes) BMC Genomics 2006, 7:127 http://www.biomedcentral.com/1471-2164/7/127 Validation of potential prognostic markers by Taqman® assay-based real-time PCR Figure 5 Validation of potential prognostic markers by Taqman® assay-based real-time PCR. 85 marker genes including the minimal set of 54 genes identified to best distinguish the luminal A and the basal-like subtypes were validated by TaqMan® Gene Expression assays. Profile correlation analysis showed the expression profile across the 20 tumor samples determined by the three microarray platforms and by TaqMan® Gene Expression assays are highly correlated with median correlation coefficient R > 0.9 and a rate of good correlation (R > 0.8) of 81–88%. Applied Biosystems TaqMan® assays Agilent Stanford cDNA Expression level (z-score) Validation Figure 5 Validation Figure 5 p p g y q y g Validation of potential prognostic markers by Taqman® assay-based real-time PCR. 85 marker genes including the minimal set of 54 genes identified to best distinguish the luminal A and the basal-like subtypes were validated by TaqMan® Gene Expression assays. Profile correlation analysis showed the expression profile across the 20 tumor samples determined by the three microarray platforms and by TaqMan® Gene Expression assays are highly correlated with median correlation coefficient R > 0.9 and a rate of good correlation (R > 0.8) of 81–88%. and substantiate the value of gene expression-based clas- sification in prognosis of breast cancer at an early stage. changes might have been pre-programmed already at a relatively early stage in the progression of the cancer and hence, imply that the fate of the tumor is already set. This is in accordance with previous reports on breast cancer [3,9,10,25-27]. Other groups have analyzed gene expres- sion in DCIS (ductal carcinoma in situ) for comparison with invasive carcinomas and highlighted transcripts that may be important for transformation and invasion [13,28,29]. Extensive studies of DCIS and other pre-inva- sive stages of tumors will further enlighten this hypothesis Specifically in this study, a more in-depth molecular char- acterization of these phenotypes of breast cancer was car- ried out and provided new insights into the biology of the disease at the molecular level. The distinct and character- istic molecular mechanisms revealed by the protein classi- fication and biological pathway analysis, provided further evidence that these molecular subtypes represent biologi- cally distinct disease entities and may require different Page 9 of 15 (page number not for citation purposes) Page 9 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/127 BMC Genomics 2006, 7:127 Hierarchical clustering analysis of expression data from the TaqMan® assays and the three microarray platforms across the 85 marker genes Figure 6 Hierarchical clustering analysis of expression data from the TaqMan® assays and the three microarray plat- forms across the 85 marker genes. The same tumor sample analyzed by the three microarray platforms and the TaqMan® Gene Expression assays were clustered together except for one sample (MicMa185). For the Applied Biosystems microarrays, the mean expression values of the two microarray replicates were presented. The transformed z-scores were represented using a red-black-green color scale as shown in the key (green: below mean; black: equal to mean; red: above mean). Validation Figure 5 M icM a085_Stanford cDNA M icM a085_Agilent M icM a085_TaqM an M icM a085_AB M icM a101_AB M icM a101_TaqM an M icM a101_Stanford cDNA M icM a101_Agilent M icM a122_TaqM an M icM a122_Agilent M icM a122_Stanford cDNA M icM a122_AB M icM a632_TaqM an M icM a632_Stanford cDNA M icM a632_Agilent M icM a632_AB M icM a263_Stanford cDNA M icM a263_TaqM an M icM a263_Agilent M icM a263_AB M icM a065_TaqM an M icM a065_AB M icM a065_Stanford cDNA M icM a065_Agilent M icM a267_TaqM an M icM a267_AB M icM a267_Stanford cDNA M icM a267_Agilent M icM a185_TaqM an M icM a709_TaqM an M icM a709_Agilent M icM a709_Stanford cDNA M icM a709_AB M icM a067_Agilent M icM a067_Stanford cDNA M icM a067_TaqM an M icM a067_AB M icM a042_TaqM an M icM a042_Stanford cDNA M icM a042_Agilent M icM a042_AB M icM a031_TaqM an M icM a031_Stanford cDNA M icM a031_Agilent M icM a031_AB M icM a185_AB M icM a185_Stanford cDNA M icM a185_Agilent Luminal A Basal-like -4.7 -4 -3 -2 -1 0 1 2 3 4 4.7 -4.7 -4 -3 -2 -1 0 1 2 3 4 4.7 g y p q y y p g g Hierarchical clustering analysis of expression data from the TaqMan® assays and the three microarray plat- forms across the 85 marker genes. The same tumor sample analyzed by the three microarray platforms and the TaqMan® Gene Expression assays were clustered together except for one sample (MicMa185). For the Applied Biosystems microarrays, the mean expression values of the two microarray replicates were presented. The transformed z-scores were represented using a red-black-green color scale as shown in the key (green: below mean; black: equal to mean; red: above mean). and cell proliferation and possibly hormonal treatment in this subtype of breast cancer. Indeed, it has been specu- lated that some lipids may modulate steroid metabolism [32]. therapeutic strategies. For example, our results indicated that the luminal A subtype showed coordinated activation of genes involved in steroid/estrogen signaling and fatty acid metabolism. Applied Biosystems expression array analysis pp y p y y The Applied Biosystems Human Genome Survey Microar- ray (P/N 4337467) contains 31,700 60-mer oligonucle- otide probes representing 27,868 individual human genes. Digoxigenin-UTP labeled cRNA was generated and amplified from 2 µg of total RNA from each sample using Applied Biosystems Chemiluminescent RT-IVT Labeling Kit v 1.0 (P/N 4340472) according to the manufacturer's protocol (P/N 4339629). Array hybridization was per- formed for 16 hrs at 55 °C. Chemiluminescence detec- tion, image acquisition and analysis were performed using Applied Biosystems Chemiluminescence Detection Kit (P/N 4342142) and Applied Biosystems 1700 Chemi- luminescent Microarray Analyzer (P/N 4338036) follow- ing the manufacturer's protocol (P/N 4339629). Images were auto-gridded and the chemiluminescent signals were quantified, corrected for background, and finally, spot- and spatially-normalized using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer software v 1.1 (P/N 4336391). A total of 40 microarrays were used for the analysis: Two process replicates (independent labe- ling and independent hybridization process) were gener- ated for each of 10 samples, and two technical replicates (the same pool of labeled cRNA and then split into two independent array hybridizations) were generated for the remaining 10 samples. For inter-array normalization, we applied global median normalization across all microar- rays to achieve the same median signal intensities for each array. The minimal set of 54 genes that best characterized lumi- nal A and basal-like subtypes was identified based on dif- ferential expressed genes on all three platforms and validated by using TaqMan® assays. Convincingly, cluster- ing of expression data from all four methods grouped the experiments together by tumor sample of origin and not by platform. Hence, these genes provide a robust set of potential prognostic molecular markers, but which covers only the two main subtypes. More thorough characteriza- tion on significantly larger sample sizes is needed to pro- vide prognostic predictor sets for all subtypes. Validation Figure 5 Fatty acid synthase (FAS)-dependent endogenous fatty acid synthetic activity has been found to be abnormally elevated in a subset of aggressive breast car- cinomas [30], in particular ERBB2-overexpressing tumors [31], whereas here, high expression of many genes involved in fatty acid/lipid metabolism and degradation were coupled to the luminal A phenotype, know to be associated with a relatively good prognosis [10]. Although no correlation between fatty acid metabolism and estro- gen and progesterone receptor expression status of tumors has been documented in cancer, our results may indicate some level of cross-talk between fatty acid metabolism and steroid signaling that may have effects on apoptosis Such molecular profiling of clinically relevant subtypes of breast tumors provide opportunities for identification of novel targets that can be exploited for targeted therapeu- tics of the disease. Among the 1210 genes most differen- tially expressed between luminal A and basal-like tumors, 145 are secreted proteins based on the prediction method- ology published in a recent paper [33]. A variety of bio- molecules are secreted proteins such as cytokines, chemokines, hormones and digestive enzymes that play pivotal biological regulatory roles and are very important sources for protein therapeutics. We also identified five G Page 10 of 15 (page number not for citation purposes) Page 10 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/127 BMC Genomics 2006, 7:127 http://www.biomedcentral.com/1471-2164/7/127 http://www.biomedcentral.com/1471-2164/7/127 protein-coupled receptors (GPCRs) among these signa- ture genes, a gene family well established as small mole- cule drug targets. protein-coupled receptors (GPCRs) among these signa- ture genes, a gene family well established as small mole- cule drug targets. Tumor samples and RNA preparation Tumor samples and RNA preparation Tissue samples from a pilot set of 20 breast carcinomas, a small subset of a larger series of 920 unselected early-stage breast cancer patients, for which informed, written con- sent was obtained by the Regional Ethical Committee [38], were analyzed in this study. Samples were fresh fro- zen immediately after surgery and stored at -80°C. All specimens analyzed contained more than 40% tumor cells (of the total number of cells counted). The majority of tumors were invasive ductal carcinomas, T1/T2, N0/N1 and histological grade 2 and 3 (Additional file 6). Total RNA was isolated by phenol-chloroform extraction (TRI- zol reagent, Invitrogen), the quality and integrity of the total RNA was evaluated on the 2100 Bioanalyzer (Agilent Technologies) and the concentration was measured using a NanoDrop spectrophotometer (NanoDrop Technolo- gies). Although we analyzed only 20 tumor biopsies, data were collected using three different microarray platforms; a two-color fluorescent-based cDNA microarray, a 60-mer oligo microarray using two-color fluorescence detection and a 60-mer oligo microarray using chemiluminescence detection. Of 16,611 common genes among these three platforms, 1019, 1054 and 1164 genes, respectively, were identified to be differentially expressed between luminal A and basal-like tumors. Of these, 319 genes were com- mon to all three technologies, which correspond to an overall consistency of 30%. These numbers could prove to be even higher if a more accurate probe match by sequence rather than gene identifiers would be per- formed, as has recently been shown [34]. A few studies have recently been published that aimed to compare vari- ability and consistency between microarray platforms and with different results [35-37]. Our study shows that although there is variability between the platforms, the gene expression profiles emerging from using all three technologies are highly correlated to the biological varia- tion in the data and the same tumor subtype pattern was identified with all three methods. Conclusion h l d We have validated and characterized the two main previ- ously defined clinically relevant subtypes, luminal A and basal-like, in early stage breast carcinomas, using three different DNA microarray platforms. Signature gene pro- files characterizing these two subtypes revealed that dis- tinct molecular mechanisms might have been pre- programmed at an earlier stage in different subtypes of the disease. Our results provide further evidence that these breast tumor subtypes represent biologically distinct dis- ease entities and may require different therapeutic strate- gies. Finally, validated by the gene expression platforms and TaqMan® assay-based real time PCR, the set of 54 pre- dictor genes identified in this study defines a set of highly- validated and potential prognostic molecular markers for these subtypes of breast cancer. Page 11 of 15 (page number not for citation purposes) Agilent whole human genome microarray analysis Agilent whole human genome microarray analysis The Agilent Whole Human Genome Oligo Microarray contains 44,000 60-mer oligonucleotide probes repre- senting 41,000 unique genes and transcripts [43]. Ampli- fication and labeling of 500 ng of total RNA was performed according to the manufacturer's protocol using Cy5 for tumor RNA and Cy3 for the reference RNA (Strat- agene UHR). Hybridization was performed for 16 hrs at 50°C and arrays were scanned on an Agilent DNA micro- array scanner. Images were analyzed and data were extracted using Agilent Feature Extraction Software A.7.5.1. Lowess normalization was performed for within array normalization between the two channels and a lin- ear scaling (geometric mean of each channel signal is set to a value of 1000) was performed for between array nor- malization. Stanford human cDNA microarray analysis Stanford human cDNA microarray analysis The same RNA samples were also analyzed using Stanford Human cDNA microarrays, which contain 42,000 fea- tures representing 24,271 unique cluster IDs (UniGene Build Number 173), manufactured by the Stanford Func- tional Genomic Facility [39]. Amplification was per- Page 11 of 15 (page number not for citation purposes) Page 11 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/127 BMC Genomics 2006, 7:127 gene lists were used as the common genes among all three platforms. formed based on the Van Gelder and Eberwine method [40] using the MessageAmp amplification kit (Ambion) and labeling of 3 µg total RNA per sample was carried out as previously described using incorporation of Cy5 for tumor RNA and Cy3 for reference RNA (Stratagene UHR) [41]. Hybridization at 65°C was performed overnight, the hybridized arrays were scanned on an Agilent DNA micro- array scanner and images analyzed by GenePix Pro v 4.1. The Limma package (R/Bioconductor) [42] was used to perform within array normalization (print-tip loess nor- malization) and between array normalization (median of absolute deviation normalization). Statistical analysis Statistical analyses were performed with the software packages MATLAB® (Mathworks, Natick, MA), R/Biocon- ductor [42] GeneSpring (Agilent Technologies, CA) and Spotfire Functional Genomic (Spotfire, Göteborg, Swe- den). TaqMan® assay-based real-time PCR q y mRNA expression of 85 target genes and 4 endogenous control genes was measured in each of the 20 biopsy spec- imens by real-time PCR using TaqMan® Gene Expression Assays and the ABI PRISM® 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). Four repli- cates were run for each gene for each sample in a 384-well format plate. The probes contain a 6-carboxy-fluorescein phosphoramidite (FAM™ dye) label at the 5' end of the gene and a minor groove binder and non-fluorescent quencher at the 3' end and are designed to hybridize across exon junctions. ~ 4 µg of total RNA from each tumor sample was used to generate cDNA using the ABI High Capacity cDNA Archiving Kit (Applied Biosystems, Foster City, CA) and the real-time PCR reactions were car- ried out following the manufacturer's protocol. TaqMan® Gene Expression Assay IDs are listed in Additional file 6. Among the four measured endogenous control genes (RPS18/PPIA (Alias: cyclophilin A)/GAPDH/PGK1) we chose PPIA for normalization across different genes based on the fact that this gene showed the most relatively con- stant expression in different breast carcinomas (see Addi- tional file 7). Cross-mapping between microarray platforms Cross mapping between microarray platforms All target transcripts of Applied Biosystems Human Genome Survey Microarray were identified by mapping the 60-mer probe sequences to all transcript sequences from both Celera and public databases including Celera hCT, RefSeq NMs, GenBank mRNAs, MGCs, dbESTs, Gen- Bank CDS, Ensembl cDNA; all target transcripts repre- sented on the Stanford cDNA microarray and the Agilent Whole Human Genome Oligo Microarray were identified by the targeted GenBank accession numbers representing the corresponding probes as specified by each manufac- turer. All transcript sequences were then mapped to the Celera-assembled human genome (Build R27) and a tran- script-to-gene-clustering was performed so that each tran- script could be traced to a gene (removing redundancy) (Xiao C. et al, manuscript in preparation). The common target transcrips between Applied Biosystems Human Genome Survey Microarray and the Stanford Human cDNA microarray (17,732 genes) and between the Applied Biosystems Human Genome Survey Microarray and the Agilent Whole Human Genome Oligo Microarray (22,507 genes) respectively, were identified based on their common cluster membership, respectively. Intersect genes (16,611 genes; see Additional file 3) of these two GEO accession The data from three microarray platforms and the Taq- Man gene expression assays have been deposited in Gene Expression Omnibus (GEO 3155) [52]. Profile correlation between microarray and TaqMan® assay-based real-time PCR data Data sets from each microarray platform and TaqMan® Gene Expression assays were normalized sample-wise and gene-wise (z-score transformation) as described above. Pearson's correlation coefficient (R) was calculated between the expression profile for each of the 85 valida- tion target genes across the 20 tumor samples determined by each microarray platform and the expression profile determined by TaqMan® Gene Expression assays. Competing interests Welch-ANOVA analysis coupled with Benjamini and Hochberg False Discovery Rate multiple testing correc- tions were performed using GeneSpring software package to identify the most diferentially expressed genes between the luminal A and basal-like subtypes. p g YW and RRS are employees of Applied Biosystems, CX is an employee of Celera Genomics. Hierarchical clustering Hierarchical clustering dom overlapping p value using the binomial test with all the genes represented by the Applied Biosystems Human Genome Survey Microarray as the reference list [49]. Average-linkage hierarchical clustering analysis and visu- alization was performed using the Cluster and TreeViev programs [44]. When multiple platform data were ana- lyzed together, each data set was first normalized between arrays and between genes independently, and then com- bined for clustering analysis. For the single-color Applied Biosystems microarray platform, gene expression signals were first normalized between arrays to the same median expression level in log2 space, and then normalized by median expression level or by z-score transformation across all samples for each gene. For the two-color array systems (Stanford cDNA microarrays and Agilent oligo arrays), normalization within and between arrays were performed using the log2 ratio of the sample vs. the refer- ence as described earlier, and then normalized by median expression ratio or by z-score transformation across all samples for each gene. For TaqMan® assay-based real-time PCR, -∆Ct = Ct_endogenous control – Ct_gene was calcu- lated as an equivalent of normalized relative gene expres- sion level, and then z-score-transformed across all samples for each gene. The z-score was determined as number of standard deviations of the level of expression of each gene in each sample away from the mean level of expression of that gene across all the samples within the data set of the given platform. http://www.biomedcentral.com/1471-2164/7/127 http://www.biomedcentral.com/1471-2164/7/127 PAM Class prediction was performed by using prediction anal- ysis of microarrays (PAM), a statistical package [45] that applies nearest shrunken centroid analysis and cross-vali- dation to determine a minimal set of predictor genes that achieve optimal prediction accuracy for sample classifica- tion [46]. Pathway analysis y y Pathway analysis was performed using PathArt™ (Jubilant Biosys Ltd., Mahalakshmipuram, Bangalore). PathArt is a curated database of biomolecular interactions with more than 1400 regulatory and signaling pathways. Compared to a few publicly available pathway databases (i.e. Gen- Mapp [50], KEGG [51]), which tend to be heavily enriched in metabolic pathways, the PathArt database emphasizes more on disease specific networks and regula- tory and signaling pathways. The statistical significance of the over-representation of the given pathway within each "signature" gene list was quantified as a similarity p-value (likelihood of a random overlapping) using script SG3b- 1 (BioScripts 2.1, GeneSpring software) based on Fisher's Exact Test. Profile correlation between microarray and TaqMan® assay-based real-time PCR data Authors' contributions TS and YW conceived, designed the study, performed the experiments, analyzed data and wrote the article. CX per- formed cross-platform gene mapping. HJ performed experiments. BN contributed with clinical samples and discussions. RRS and ALBD contributed to conception and design of the study and revising and writing of the article. All authors read and approved the manuscript. Centroid correlation analysis An "intrinsic" gene list consisting of 534 genes repre- sented by 552 clones, was previously selected based on their low variation in expression in successive samples from the same patient's tumor and at the same time, high degree of variation among tumors from different patients [10]. These intrinsic genes have been used to define five subtypes of breast tumors and their core expression cen- troids (i.e., average expression profile of the 534 intrinsic genes) in a data set of 122 breast tissue samples, most of which were locally advanced breast tumors. 526 of the intrinsic genes were mapped to the corresponding genes represented on the Applied Biosystems Human Genome Survey Microarray and 510 were mapped among all three microarray platforms used in this study. Using these mapped genes, we computed the Pearson's correlation coefficient of each sample from this study to each of the five centroids and assigned each sample to the subtype with which it showed the highest correlation. Page 12 of 15 (page number not for citation purposes) BMC Genomics 2006, 7:127 PANTHER™ protein classification system analysis PANTHER™ protein classification system analysis Similar to Gene Ontology™ (GO), PANTHER™ (Protein ANalysis THrough Evolutionary Reationships) Protein Classification System (Applied Biosystems, Foster City, CA) [47] classifies proteins in families/sub-families, molecular functions, biological processes and biological pathways. Compared to GO, the PANTHER™ Protein Classification System provides a more simplified ontol- ogy (vocabulary) of protein function and classifies 25% more proteins than GO [48]. Protein classification over- represented by "signature" genes of the luminal A and the basal subtype were identified and the statistical signifi- cance of the over-representation was quantified by a ran- Page 13 of 15 (page number not for citation purposes) BMC Genomics 2006, 7:127 http://www.biomedcentral.com/1471-2164/7/127 http://www.biomedcentral.com/1471-2164/7/127 http://www.biomedcentral.com/1471-2164/7/127 Additional File 5 5. van't Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, van der KK, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH: Gene expression profiling predicts clinical outcome of breast can- cer. Nature 2002, 415:530-536. TaqMan® Gene Expression assays used in this study. This file contains a gene list of 85 genes with their corresponding TaqMan® Gene Expression Assay IDs, Applied Biosystem Human Genome Survey Microarray Probe IDs, Agilent Human Whole Genome Oligo Microarray Probe IDs, and Stanford Human 42 K cDNA Array SUIDs. Cli k h f fil 6. van de Vijver MJ, He YD, van't Veer LJ, Dai H, Hart AA, Voskuil DW, Schreiber GJ, Peterse JL, Roberts C, Marton MJ, Parrish M, Atsma D, Witteveen A, Glas A, Delahaye L, van V, Bartelink H, Rodenhuis S, Rutgers ET, Friend SH, Bernards R: A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 2002, 347:1999-2009. [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S5.xls] 7. West M, Blanchette C, Dressman H, Huang E, Ishida S, Spang R, Zuzan H, Olson JAJ, Marks JR, Nevins JR: Predicting the clinical status of human breast cancer by using gene expression profiles. Proc Natl Acad Sci U S A 2001, 98:11462-11467. Additional File 4 54-gene set for discrimination between luminal A and basal-like sub- types. This file contains the minimal set of 54 genes that best discrimi- nated luminal A and basal-like tumors by PAM analysis. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S4.xls] 54-gene set for discrimination between luminal A and basal-like sub- types. This file contains the minimal set of 54 genes that best discrimi- nated luminal A and basal-like tumors by PAM analysis. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S4.xls] y 4. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, Van de RM, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein LP, Borresen-Dale AL: Gene expres- sion patterns of breast carcinomas distinguish tumor sub- classes with clinical implications. Proc Natl Acad Sci U S A 2001, 98:10869-10874. References 1. Chang JC, Wooten EC, Tsimelzon A, Hilsenbeck SG, Gutierrez MC, Elledge R, Mohsin S, Osborne CK, Chamness GC, Allred DC, O'Con- nell P: Gene expression profiling for the prediction of thera- peutic response to docetaxel in patients with breast cancer. Lancet 2003, 362:362-369. Additional File 1 The 1210 signature genes represented by 1244 probes that were most differentially expressed between luminal A and basal-like tumors by ANOVA analysis. For each gene, the Applied Biosystems Probe ID, P- value, Fold Change, Gene_Symbol, Gene_Description, GenBank Acces- sion and LocusLink_ID are listed. Click here for file The 1210 signature genes represented by 1244 probes that were most differentially expressed between luminal A and basal-like tumors by ANOVA analysis. For each gene, the Applied Biosystems Probe ID, P- value, Fold Change, Gene_Symbol, Gene_Description, GenBank Acces- sion and LocusLink_ID are listed. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S1.xls] Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S7.eps] [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S1.xls] Acknowledgements Descriptive pathway diagrams for the luminal A and basal-like sub- types. (A) ER signaling pathway is over-represented by the luminal A sig- nature genes. (B) p21-mediated signaling pathway is over-represented by the basal-like signature genes. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S2.eps] Descriptive pathway diagrams for the luminal A and basal-like sub- types. (A) ER signaling pathway is over-represented by the luminal A sig- nature genes. (B) p21-mediated signaling pathway is over-represented by the basal-like signature genes. We are grateful to David Botstein for the initiative to this collaboration. We thank Alexandra Fuller, La-Arni Macalik and Gary Schroth for their technical support and helpful discussions. This work was in part supported by grants from the Norwegian Cancer Society (D99061), the Norwegian Research Council (155218/300) and the SalusAnsvars Award to ALBD. [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S2.eps] Additional File 3 The 16611 common genes mapped among the three microarray plat- forms used in this study. This file contains a gene list of 16611 genes with their corresponding Applied Biosystem Human Genome Survey Microarray Probe IDs, Agilent Human Whole Genome Oligo Microarray Probe IDs and Stanford Human 42 k cDNA array SUIDs. Click here for file 2. Ma XJ, Wang Z, Ryan PD, Isakoff SJ, Barmettler A, Fuller A, Muir B, Mohapatra G, Salunga R, Tuggle JT, Tran Y, Tran D, Tassin A, Amon P, Wang W, Wang W, Enright E, Stecker K, Estepa-Sabal E, Smith B, Younger J, Balis U, Michaelson J, Bhan A, Habin K, Baer TM, Brugge J, Haber DA, Erlander MG, Sgroi DC: A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell 2004, 5:607-616. [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S3.xls] 3. Sotiriou C, Neo SY, McShane LM, Korn EL, Long PM, Jazaeri A, Mar- tiat P, Fox SB, Harris AL, Liu ET: Breast cancer classification and prognosis based on gene expression profiles from a popula- tion-based study. Proc Natl Acad Sci U S A 2003, 100:10393-10398. Additional material Additional File 7 Expression profiles of four tested endogenous control genes in various breast cancer tissues. PPIA (Cyclophilin A) was chosen as the endog- enous control as this gene showed the most relatively constant expression levels (smallest standard deviation and variance) across different breast carcinomas. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S7.eps] Additional File 7 Expression profiles of four tested endogenous control genes in various breast cancer tissues. PPIA (Cyclophilin A) was chosen as the endog- enous control as this gene showed the most relatively constant expression levels (smallest standard deviation and variance) across different breast carcinomas. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-127-S7.eps] Additional File 7 Expression profiles of four tested endogenous control genes in various breast cancer tissues. 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Tumor size (cm); molecular subtype (uc = unclassified); tumor category (tcat) given as T size; nodal status (ncat); histological grade; tumor cell content. Click here for file Tumor characteristics of the 20 samples analyzed in this study. Tumor size (cm); molecular subtype (uc = unclassified); tumor category (tcat) given as T size; nodal status (ncat); histological grade; tumor cell content. Click here for file 8. Perou CM, Sorlie T, Eisen MB, Van de RM, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Bot- stein D: Molecular portraits of human breast tumours. Nature 2000, 406:747-752. 9. Bertucci F, Finetti P, Rougemont J, Charafe-Jauffret E, Cervera N, Tarpin C, Nguyen C, Xerri L, Houlgatte R, Jacquemier J, Viens P, Birn- baum D: Gene expression profiling identifies molecular sub- types of inflammatory breast cancer. Cancer Res 2005, 65:2170-2178. 10. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen H, Pesich R, Geisler S, Demeter J, Perou CM, Lonning PE, Brown PO, Borresen-Dale AL, Botstein D: Repeated observation Page 14 of 15 (page number not for citation purposes) Page 14 of 15 (page number not for citation purposes) BMC Genomics 2006, 7:127 Page 15 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/127 Nishidate T, Katagiri T, Lin ML, Mano Y, Miki Y, Kasumi F, Yoshimoto M, Tsunoda T, Hirata K, Nakamura Y: Genome-wide gene- expression profiles of breast-cancer cells purified with laser microbeam microdissection: identification of genes associ- ated with progression and metastasis. Int J Oncol 2004, 25:797-819. Page 15 of 15 (page number not for citation purposes) Page 15 of 15 (page number not for citation purposes) Page 15 of 15 (page number not for citation purposes)
W2547886876.txt	https://link.springer.com/content/pdf/10.1007%2Fs11101-016-9481-1.pdf	en		Plant defense and herbivore counter-defense: benzoxazinoids and insect herbivores	Phytochemistry reviews	2,016	cc-by	16,712	Phytochem Rev (2016) 15:1127–1151 DOI 10.1007/s11101-016-9481-1 Plant defense and herbivore counter-defense: benzoxazinoids and insect herbivores Felipe C. Wouters . Blair Blanchette . Jonathan Gershenzon . Daniel G. Vassão Received: 10 May 2016 / Accepted: 21 October 2016 / Published online: 5 November 2016 Ó The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Benzoxazinoids are a class of indolederived plant chemical defenses comprising compounds with a 2-hydroxy-2H-1,4-benzoxazin-3(4H)one skeleton and their derivatives. These phytochemicals are widespread in grasses, including important cereal crops such as maize, wheat and rye, as well as a few dicot species, and display a wide range of antifeedant, insecticidal, antimicrobial, and allelopathic activities. Although their overall effects against insect herbivores are frequently reported, much less is known about how their modes of action specifically influence insect physiology. The present review summarizes the biological activities of benzoxazinoids on chewing, piercing-sucking, and root insect herbivores. We show how within-plant distribution modulates the exposure of different herbivore feeding guilds to these defenses, and how benzoxazinoids may act as toxins, feeding deterrents and digestibility-reducing compounds under different conditions. In addition, recent results on the metabolism of benzoxazinoids by insects and their consequences for plant-herbivore interactions are addressed, as well as directions for future research. F. C. Wouters B. Blanchette J. Gershenzon D. G. Vassão (&) Department of Biochemistry, Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany e-mail: vassao@ice.mpg.de Keywords Chemical ecology Detoxification Nutritional indices Metabolism Poaceae Introduction Plants have evolved a diverse repertoire of specialized or ‘‘secondary’’ metabolites in order to alleviate biotic and abiotic stresses. Among these, benzoxazinoids are a group of important defense chemicals widespread in grasses (Poaceae), including economically important crops such as maize, wheat, and rye (but not rice, oat, sorghum, and cultivated barley) (Niemeyer 2009). This class of compounds is also produced by individual species within the dicot families Acanthaceae, Ranunculaceae, Plantaginaceae, and Lamiaceae (Frey et al. 2009; Makowska et al. 2015). These indolederived compounds are regarded as general defense metabolites in plants, being associated with a wide spectrum of direct antifeedant, insecticidal, antimicrobial, and allelopathic activities (Niemeyer 2009), as well as serving to regulate other defense mechanisms (Maag et al. 2015a). Due to their structural diversity, the term benzoxazinoids (BXDs) will be used in this article to refer to both benzoxazinones (glucosides and corresponding aglucones containing a 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one skeleton) and their degradation products, benzoxazolinones. The most common naturally occurring BXD structures, simplified activation and degradation routes, and commonly used acronyms are presented in Fig. 1. 123 1128 Phytochem Rev (2016) 15:1127–1151 Fig. 1 Glucoside hydrolysis of naturally occurring benzoxazinones and degradation to benzoxazolinones via oxo-cyclo/ring-chain tautomerism Benzoxazinones are commonly assumed to be stored as glucosides in vacuoles of undamaged plant cells, and hydrolysis by b-glucosidases increases their reactivity and toxicity (Frey et al. 2009; Niemeyer 2009). BXD-hydrolyzing glucosidases can be found in plastids, cytoplasm, and cell walls (i.e. spatially separated from their glucoside substrates) (Nikus et al. 2001) which upon damage to the plant cell and loss of tissue and cell integrity catalyze the hydrolysis of benzoxazinone glucosides. The resulting unstable benzoxazinone aglucones and their benzoxazolinone degradation products are regarded as the compounds mediating most observed BXD biological activities (Niemeyer 2009; Sicker and Schulz 2002). This compartmentalized system consisting of a stable compound and an activating enzyme resembles other activated two-component defense systems in plants such as glucosinolates and cyanogenic and iridoid glycosides (Morant et al. 2008; Pentzold et al. 2014b). 123 The effects of a variety of BXDs on the survival, growth and feeding behavior of a range of organisms have been studied for many years. Nevertheless, the underlying molecular modes of action and physiological mechanisms responsible for these effects are not completely understood. In addition, the importance of these chemical defenses in a natural context and their modulation by specific characteristics, such as physiology of the target organism and its feeding behavior, are not usually critically evaluated. Given the tissuespecific distribution of BXDs in plants and their activation by glucosidases, the feeding behavior of insect herbivores dictates the amount and the nature of BXDs that they will be exposed to, based on the attacked tissue and the extent of plant cell damage. Finally, even after ingestion of BXDs by the target organism, specialized metabolism of such compounds, as well as other biochemical strategies to avoid toxicity, might play a role on their overall biological effects. Phytochem Rev (2016) 15:1127–1151 Due to the economic and ecological importance of BXDs, many aspects of these compounds have been reviewed, including their biological activities (Macı́as et al. 2009; Niemeyer 2009), organic synthesis (Macı́as et al. 2006; Sicker and Schulz 2002), chemical reactivities (Hashimoto and Shudo 1996; Wouters et al. 2016), and biosynthetic evolution and genetics (Frey et al. 2009; Makowska et al. 2015). The present review aims to categorize the spectrum of biological activities observed for BXDs towards insect herbivores with different feeding behaviors: chewing, piercing-sucking, and root herbivores. These effects are discussed in the ecological context of each interaction, and the contribution of toxicity, digestibility-reduction and antifeedant activities to the overall effects observed on insect herbivores is addressed. Additionally, current knowledge about the metabolism of BXDs in various organisms is summarized. We begin with overviews of BXD biosynthesis, plant distribution and chemical properties to put the biological activities in the proper context. 1129 Biosynthesis and distribution of BXDs in plants The BXD biosynthetic pathway has been mostly established in maize (Frey et al. 2009; Meihls et al. 2013), but other BXD-producing plants have also been investigated (Dick et al. 2012; Schullehner et al. 2008). The general reactions and compartmentalization of BXD biosynthesis are shown in Fig. 2. The formation of BXDs, as well as their genetics and evolution in plants have been comprehensively reviewed (Frey et al. 2009; Gierl and Frey 2001; Makowska et al. 2015). The first committed step of the pathway is catalyzed by BX1, which converts indole-3-glycerol phosphate into indole in the chloroplasts. This enzyme is a homolog of the a-subunit of tryptophan synthase (TSA). In tryptophan synthase, the resulting indole is not released, but rather channeled to the active site of a b subunit (TSB), where it reacts with serine, yielding tryptophan and water. Most likely, the gene encoding BX1 originated from that encoding TSA by Fig. 2 BXD biosynthesis and compartmentalization in plant cell 123 1130 duplication and modification of both function and expression patterns. These changes gave rise to both Bx1 and Igl (indole-3-glycerol phosphate lyase), which mostly produces the free indole released by the plant as a volatile (Gierl and Frey 2001). After this first step, free indole is converted to DIBOA by incorporation of four oxygen atoms. These oxidations are carried out by four cytochrome P450-dependent monooxygenases, BX2-BX5, which are located in the endoplasmic reticulum and are substrate-specific and regioselective for the sequential introduction of oxygen atoms. The DIBOA aglucone thus produced is rendered less reactive in the cytoplasm via glucosylation by the action of the UDP-glucosyltransferases (UGTs) BX8 and BX9, providing a more stable intermediate for further modifications. DIBOA-Glc can be then hydroxylated by the 2-oxoglutarate-dependent dioxygenase BX6, and further O-methylated by the Omethyltransferase BX7, forming DIMBOA-Glc. HDMBOA-Glc can then be formed from DIMBOAGlc via an O-methylation reaction catalyzed by a group of three homologous O-methyltransferases, BX10, BX11, and BX12 (formerly named BX10a, BX10b, and BX10c, respectively, in Meihls et al. 2013). Recently, an additional branch in the BXD pathway has been described: BX13, a 2-oxoglutaratedependent dioxygenase, catalyzes the conversion of DIMBOA-Glc to TRIMBOA-Glc; the latter can be Omethylated by BX7 to form DIM2BOA-Glc, which can be further methylated by the O-methyltransferase BX14 to generate HDM2BOA-Glc (Handrick et al. 2016). The stable BXD glucosides are considered to be transported and stored in the vacuole, while BXDhydrolyzing b-glucosidases are thought to be present in plastids (Frey et al. 2009; Niemeyer 2009; Sicker and Schulz 2002). However, this distribution is not well established and may vary with plant species, tissue and age. Some direct evidence of BXD glucoside distribution was provided by MALDI-MS imaging of metabolites in a maize leaf cross-section, which revealed that DIMBOA-Glc and HMBOA-Glc were localized in cell vacuoles (Korte et al. 2015). The use of antibodies suggests that the subcellular distribution of b-glucosidases varies among plant species and tissues (Nikus et al. 2001). Wheat and rye bglucosidases are mainly localized in cell walls and cytoplasm, while in maize they are mostly found in plastids and proplastids. However, deviations from 123 Phytochem Rev (2016) 15:1127–1151 these general trends have been noted. For instance, Massardo et al. (1994) observed through cell fractionation the presence of BXD b-glucosidases in the vacuole and DIMBOA-Glc in the extravacuolar space of maize parenchyma cells. Upon destruction of the tissue and its accompanying cellular organization caused for example by herbivore damage or pathogen attack, the stable glucosides come into contact with b-glucosidases and are hydrolyzed to reactive aglucones, which are then implicated in BXD toxicity (Cambier et al. 1999). But, in some cases aglucones accumulate. For example, the apoplast of maize leaves contains DIMBOA as well as DIMBOA-Glc and HDMBOA-Glc (Ahmad et al. 2011), while waxes on the surface of maize whorls contain considerable amounts of HDMBOA aglucone (which seems to be stable in the waxy layer), together with DIMBOA and MBOA (Hedin 1993). In roots, BXD aglucones are considered to be actively exuded and diffuse into the soil where they exert their effects on soil microorganisms, root herbivores, and other plants (Belz and Hurle 2005; Pérez and OrmeñoNuñez 1991). The identity of BXDs exuded by roots seems to vary according to growing conditions, sampling, and analytical methods. The aglucones DIBOA and DIMBOA were the main BXDs exuded from roots of hydroponically grown Secale cereale and three Triticum species: T. aestivum, T. durum, and T. spelta (Belz and Hurle 2005). Maize root exudates collected by a trapping system contained the hydroxamic acids DIMBOA and DIBOA, the lactam HMBOA, and the benzoxazolinones MBOA and BOA. In maize hydroponic cultures, however, the glucoside DIMBOA-Glc was additionally detected (Friebe et al. 1998). Exudate extracts obtained by dipping maize roots in dichloromethane contained mainly the unstable derivative HDMBOA (Zhang et al. 2000). However, a method based on liquid extraction surface analysis detected glucosides such as HDMBOA-Glc and DIMBOA-Glc in maize root exudates (Robert et al. 2012). The abundance of BXDs and their proportions vary between plant species and varieties, and also among tissues and developmental stages within plants. For example, the main BXD in rye is DIBOA-Glc (Oikawa et al. 2002), whereas DIMBOA-Glc is the major BXD in aerial parts of wheat and maize (Cambier et al. 2000; Oikawa et al. 2002). In contrast, HDMBOA-Glc is dominant in maize roots, with BXDs being more Phytochem Rev (2016) 15:1127–1151 concentrated in crown roots than in primary and secondary roots (Robert et al. 2012). In maize, BXDs reach the highest concentrations in seedlings that are 10 days-old and decline as the plant grows further (Cambier et al. 2000). Moreover, BXDs are differently allocated in leaves according to their age: DIMBOAGlc was the predominant BXD in young and old maize leaves on plants at growth stages L2 to L4, but at stages L5 to L7 DIBOA-Glc and HMBOA-Glc became the most abundant in older, but not younger leaves (Köhler et al. 2015). The total concentrations of BXDs in different plants can vary with age and biotic stresses, and can reach [0.1 % of maize leaf fresh weight after caterpillar attack (Dafoe et al. 2011, Glauser et al. 2011). HDMBOA-Glc is highly induced in maize after herbivory (Dafoe et al. 2011, Glauser et al. 2011), fungal attack (Oikawa et al. 2004), and in both maize and wheat upon jasmonic acid treatment (Oikawa et al. 2001, 2002). Moreover, young maize leaves display higher inducibility of HDMBOA-Glc and HDM2BOA-Glc upon herbivory than old leaves (Köhler et al. 2015). The induction of these two BXDs is highly localized to the feeding site and their levels remain high for several days (Maag et al. 2016). However, changes in BXD levels away from the feeding area within the same leaf are less pronounced, and no changes are detected in unattacked leaves. Chemical properties and reactivity of BXDs Benzoxazinones can be divided according to their substituent group R1 as lactams (R1 = H), hydroxamic acids (R1 = OH), and N–O-methylated derivatives (R1 = OMe) (Fig. 1). These functional groups and other substituents modulate the stability and reactivity of these compounds and therefore their biological activities. Benzoxazinone glucosides are remarkably stable (Hietala et al. 1960) and require the action of glucosidases for their hydrolysis. The resulting aglucones are cyclic hemiacetals that undergo oxo-cyclo/ring-chain tautomerism via a fast, reversible ring opening reaction (Copaja et al. 1986), and therefore occur as racemic mixtures in solution. However, all known benzoxazinone glucosides produced by plants are (2R)-2-b-Dglucosides (Hartenstein et al. 1993; Hartenstein and Sicker 1994; Kluge et al. 1997; Nagao et al. 1985). 1131 The high activity of BXD b-glucosidases and the instability of aglucones represent a challenge in their extraction and quantitative analysis from natural samples. Once plant material is mechanically disrupted, plant b-glucosidases may quickly hydrolyze benzoxazinone glucosides to aglucones, which spontaneously degrade to benzoxazolinones. The extraction procedure and analytical methods for BXDs have been thoroughly evaluated and compared (Villagrasa et al. 2009), and modern LC–MS protocols are sensitive and accurate. It is important to note, however, that earlier experiments sometimes used colorimetric methods to determine total hydroxamic acids, with no distinction between different structures or even between glucosides and aglucones. Other methods have included calculations of DIMBOA content based on degradation to MBOA, which can be inaccurate due to the non-quantitative nature of this transformation (Woodward et al. 1978) and the fact that HDMBOA also degrades to MBOA. In fact, due to its instability, HDMBOA was likely often missed in chemical analyses for many years, only being considered as a major benzoxazinoid starting in the 1990s (Hedin 1993), although the glucoside was already identified in the 1970s (Hofman et al. 1970). Therefore, when interpreting results in the literature, one must consider possible quantification errors and the fact that certain BXDs were not typically detected. In general, lactams (N–H compounds) are not degraded to benzoxazolinones, whereas hydroxamic acids (N–OH) degrade readily and N–O-methyl derivatives degrade even faster than hydroxamic acids. For example, the half-lives of HDMBOA and DIMBOA aglucones in buffered D2O at pH 5.5 and 24 °C are 1.8 and 25 h, respectively (Maresh et al. 2006). The degradation rates depend on conditions such as pH, temperature and solvent, and structural features such as the nature of the N-substituent group and other substituents on the aromatic ring (Atkinson et al. 1991). Several mechanisms have been proposed to explain this degradation (Bredenberg et al. 1962; Grambow et al. 1986; Maresh et al. 2006; Niemeyer et al. 1982a; Smissman et al. 1972; Wouters et al. 2016). Due to their unique combination of structural features, BXDs are subject to a range of possible reactions that have biological relevance. Upon ring opening, benzoxazolinone aglucones become a-oxoaldehydes, which are potent electrophiles capable of 123 1132 reacting with nucleophilic residues of proteins, such as thiols and amines, and causing enzymatic inhibition (Atkinson et al. 1991; Niemeyer et al. 1982b; Pérez and Niemeyer 1989). The nitrogen atom can also be an electrophilic site upon elimination of the N-substituent group and resulting formation of a nitrenium ion or a reactive o-imidoquinone intermediate (Atkinson et al. 1991; Dixon et al. 2012; Hashimoto et al. 1991; Maresh et al. 2006; Quiroz and Niemeyer 1991). BXD hydroxamic acids possess metal ion chelating properties (Tipton and Buell 1970) that can play a role in Fe(III) uptake (Peth}o 1992a, b, 1993, 2002) and Al(III) resistance by roots (Poschenrieder et al. 2005). Furthermore, benzoxazolinones can interfere with auxin binding in plants (Hasegawa et al. 1992; Hoshisakoda et al. 1994; Venis and Watson 1978), and the products of their metabolism by soil microorganisms are suggested to play a role in allelopathy (Kato-Noguchi et al. 2010; Schulz et al. 2013; Venturelli et al. 2015). Biological effects of BXDs Since their discovery, BXDs have been considered to function in the plant as resistance factors against herbivores, pathogens, and other plants. Many in vitro studies and bioassays have demonstrated that these compounds have inhibitory and toxic effects towards a wide range of target enzymes and organisms, particularly insect herbivores. Most reports focus on important pest species of cereal crops, and include insects from different ecological guilds: caterpillars (chewing herbivores), aphids (piercing-sucking herbivores), and rootworms (root herbivores). These studies are summarized in the following sections and discussed in the ecological context of insect feeding behavior. Moreover, the influence of the toxicity, digestibility-reduction, and antifeedant activities of BXDs on insect physiology is addressed. Allelopathic and antimicrobial activities of BXDs will not be covered in this section as they have already been comprehensively discussed in recent reviews (Macı́as et al. 2009; Niemeyer 2009; Schulz et al. 2013). BXDs have been shown to be present in whole grain cereals, flours, sprouts, bread and beverages derived from rye, wheat, and maize, and might influence human health. Anti-inflammatory, anti-cancer, and anti-microbial activities, as well as stimulatory effects 123 Phytochem Rev (2016) 15:1127–1151 on the central nervous and reproductive systems have been reported for BXDs, mostly using in vitro studies, and the human therapeutic potential of BXDs has been recently reviewed (Adhikari et al. 2015). Effects on chewing herbivores Due to their feeding behavior, chewing herbivores such as lepidopteran larvae disrupt the compartmentalization of BXDs in plant cells during ingestion and are therefore directly exposed to high amounts of BXD aglucones, especially when feeding on leaves from young seedlings where BXD concentrations are highest. Indeed, DIMBOA and BXDs in general have been long known for their toxic and antifeedant activities on caterpillars and for playing an important role in protecting resistant maize lines from herbivore attack (Klun et al. 1967; Reed et al. 1972; Robinson et al. 1978). When evaluating the effects of BXDs on herbivores, it is essential to take into account that most lepidopterans possess an alkaline gut environment (Berenbaum 1980), which facilitates the degradation of hydroxamic acids and N–O-methylated hydroxamic acids into benzoxazolinones, thus leading to biological activities that can be specific to these insects. Furthermore, lepidopteran feeding can also lead to induction of plant BXDs. In the next two sections, we summarize the effects of BXDs on two lepidopteran genera for which considerable data are available in an effort to compare the differences between specialist and generalist feeders in performance, physiology, mode of action and feeding preference, as well as to compare the effects of various BXD structures. Genus Ostrinia The toxicity and deterrence of BXDs towards the European corn borer (ECB, Ostrinia nubilalis) have been extensively investigated due to its economic importance as a pest of maize. When fed in artificial diet, DIMBOA concentrations between 0.05 and 0.5 mg/g (0.24 and 2.37 mM, respectively) and MBOA at concentrations of 0.5–4.0 mg/g (3–24 mM, respectively) both increased mortality and developmental times to pupation in a dosedependent way, but did not affect total weight gain in larvae up to the fifth instar (Campos et al. 1988, 1989). DIMBOA reduced pupal and adult weights in all tested concentrations, and the highest Phytochem Rev (2016) 15:1127–1151 concentration also increased pupal mortality and delayed the emergence of adults. The effects caused by MBOA were similar to those caused by DIMBOA but required higher concentrations, agreeing with the trend that benzoxazolinones are less toxic than benzoxazinones. Further information on the structure–activity relationships of various BXDs fed to O. nubilalis comes from a study employing artificial diets containing 0.5 mM of individual BXDs (Atkinson et al. 1992). Among the natural BXD structures used, DIMBOA and DIBOA showed the highest toxicity, followed by DIM2BOA. The authors suggested that the resulting toxicities are positively linked to the degradation rates of these hydroxamic acids to benzoxazolinones. Indeed, the lactams HMBOA and HBOA, which do not form benzoxazolinones upon degradation, did not inhibit larval growth. Thus degradation of benzoxazinones to benzoxazolinones correlates with increasing toxicity although the benzoxazolinones themselves are less toxic than benzoxazinones as discussed in the previous paragraph. Studies with O. nubilalis have also tracked BXD distribution and excretion dynamics using radioactive 3 H-labeled DIMBOA and MBOA (Campos et al. 1988, 1989). In both cases, the radioactivity was mainly excreted by larvae in the frass and transferred to the pupal case after emergence, suggesting that the adult insect avoids accumulation of BXDs and their metabolites. In fact, a short-term higher level of radioactivity in hemolymph compared to other tissues suggests that these compounds are rapidly transported and excreted. However, the body burden (ratio of radioactivity between body and frass) was constant for all tested concentrations, implying that larvae are not able to increase excretion rate when faced with higher levels of BXDs up to about 2 mM. This is probably consequence of a higher diet consumption, which is consistent with a digestibility-reducing activity of DIMBOA at this concentration. On the other hand, MBOA did not alter diet consumption; thus its effects are due to toxicity and not to antifeedant activity or digestibility reduction. Further experiments evaluating the effects of DIMBOA and MBOA on food consumption and utilization in O. nubilalis also suggest that these compounds have distinct modes of action on insect physiology. Using nutritional indices proposed by Waldbauer (1982), both DIMBOA (0.2 mg/g, 1133 1 mM) and MBOA (3.0 mg/g, 18 mM) reduced weight gain in O. nubilalis in 4-day artificial diet feeding (Houseman et al. 1992). This was not caused by deterrence of feeding, since the consumption index (CI) increased for DIMBOA and remained the same for MBOA treatments. Instead DIMBOA decreased both approximate digestibility (AD) and efficiency of conversion of ingested food into biomass (ECI), but did not change the efficiency of conversion of digested food into biomass (ECD), indicating that DIMBOA affects digestive processes (e.g. inhibiting digestive enzymes), but does not modify the utilization of nutrients after digestion. In agreement, both in vivo and in vitro assays showed that DIMBOA inhibits trypsin and chymotrypsin. Such reduction in digestibility could lead to the observed increase in diet consumption as an attempt by the larvae to compensate for the decrease in food quality. On the other hand, MBOA ingestion did not change AD or ECI, but decreased ECD, suggesting that this compound has an effect on processes occurring after digestion. The effects of O. nubilalis herbivory on maize metabolism are also relevant for the ecology of this interaction. Larval feeding induced the production of HDMBOA-Glc in stems, with a consequent decrease in DIMBOA-Glc (Dafoe et al. 2011). Even though HDMBOA is considered more toxic than DIMBOA, larvae feeding on previously induced stem sections grew more and consumed more plant tissue than on uninduced stems. Feeding on induced stems decreased CI and AD, but increased ECI and ECD, suggesting that, despite being better defended, such tissues are more nutritive than non-induced stems. Indeed, induction by O. nubilalis herbivory increased protein, sucrose, and linoleic acid levels in stems. The authors suggested that high amounts of the auxin 3-indoleacetic acid in O. nubilalis oral secretion and frass could affect plant metabolism and promote the increase in nutritional value of attacked tissue. Given the toxicity and digestibility reducing effects of BXDs on O. nubilalis, it may not be surprising that this insect typically feeds on older maize stems (L11– L13 stage) in which the amounts of HDMBOA-Glc and other BXDs are much lower (3.6 lg/g after 48 h induction) than those found in leaves of young seedlings. In contrast, S. frugiperda larvae, specialists on maize and other grasses which typically feed on maize at the L4 stage, encounter much higher 123 1134 HDMBOA-Glc concentrations (around 30 lg/g constitutively, and 300 lg/g after herbivore induction in young leaves, their preferred food) (Köhler et al. 2015). Screening of maize lines for resistance towards O. nubilalis indicates that in general DIMBOA concentrations of more than 100 lg/g (0.47 mM) in whorl tissues are associated with lower leaf consumption (Barry et al. 1994). Therefore, it has been proposed that O. nubilalis restricts its diet to tissues and plant growth stages that contain lower BXD concentrations in order to avoid their toxicity (Maag et al. 2014). This is consistent with the apparently low capability of this insect to detoxify BXDs, as will be discussed in more detail below. In natural situations, the amount of DIMBOA is not the only factor guiding O. nubilalis feeding behavior. In maize plants, O. nubilalis larvae prefer to feed on immature whorl tissues, despite their higher BXD levels compared to mature tissues (Bergvinson et al. 1995a, 1995b). Such preference was suggested to be a consequence of higher fiber content and cell wall phenolics in mature tissues, which increase leaf toughness and constitute a mechanical defense towards herbivore feeding. Therefore, it was proposed that neonate larvae feed on the younger, more tender whorl leaves in spite of their higher DIMBOA concentration, because these contain higher amounts of protein. As they grow and develop stronger mandibles better able to chew tougher tissues, the larvae move to more mature parts of the plant with lower levels of BXDs. Another study had revealed a similar trend by comparing maize lines with varying DIMBOA concentrations grown under different light conditions. Even though DIMBOA seems to be responsible for high resistance in some genotypes, neonate leaf consumption and survival correlated better with nitrogen content and nutritional value of plants when comparing different light regimes (Manuwoto and Scriber 1985b). For the Asian corn borer (O. furnacalis), which also feeds extensively on maize as well as other grasses, DIMBOA is also a toxin and antifeedant. Choice assays revealed that DIMBOA applied to cabbage leaves showed increasing antifeedant effects up to 0.8 mg/g (3.79 mM) (Yan et al. 1999). Artificial diets with 1 mg/g DIMBOA (4.73 mM) inhibited growth and extended larval developmental time in this species. However, when compared with O. scapulalis, which feeds principally on Artemisia vulgaris and less 123 Phytochem Rev (2016) 15:1127–1151 frequently on maize, O. furnacalis was less affected by BXDs (Kojima et al. 2010). In O. furnacalis, DIMBOA was shown to induce general detoxification reactions including cytochrome P450 and glutathione S-transferase activities (Yan et al. 1995). Acetylcholinesterase was inhibited, while inhibition or induction of general esterases depended on the tissue analyzed. Genus Spodoptera Much DIMBOA research has also been carried out on the larvae of another group of lepidopterans, the genus Spodoptera, which includes the broad generalist S. littoralis, feeding on over 80 families of plants, and the more specialized S. frugiperda, which is mostly restricted to feeding on grasses such as maize. As with the Ostrinia species discussed above, the larvae of the more specialized S. frugiperda perform better on DIMBOA-containing diets than larvae of the generalist S. littoralis. When feeding on DIMBOA at 200 lg/g (0.95 mM, the level found in herbivoryinduced maize plants), the growth of S. frugiperda was similar to that on control diets, but S. littoralis grew significantly less (Glauser et al. 2011). However, on diets containing 40 lg/g DIMBOA (0.19 mM), the level encountered in non-induced maize plants, S. littoralis did not grow differently than when feeding on a control diet, but S. frugiperda grew more quickly. On artificial diets containing 50 and 500 lg/g of the more reactive HDMBOA-Glc, the consumption and preference of S. littoralis and S. frugiperda did not differ from control diets. However, the addition of maize extract to such diets caused deterrence and lower food intake on the high-HDMBOA-Glc diet for both species (Glauser et al. 2011). This was presumably a consequence of the b-glucosidase activity present in the plant extract, causing hydrolysis of HDMBOA-Glc and releasing the highly toxic HDMBOA aglucone, which persists in the diet for around 30 min. On the other hand, an artificial diet containing the benzoxazolinone MBOA at 330 lg/g (2 mM) did not restrict S. frugiperda and S. littoralis growth, but decreased the growth of O. nubilalis (Maag et al. 2014). To gain a more detailed understanding of how S. frugiperda tolerates BXDs, we recently investigated the effects of MBOA on food consumption and utilization by larvae feeding on bean-based artificial Phytochem Rev (2016) 15:1127–1151 1135 diets by measuring the weights of larvae, diet consumed and frass excreted. MBOA at concentrations of 50 and 1000 lg/g (0.3 and 6.06 mM respectively) did not significantly affect larval growth curves (Fig. 3) or the relative growth rate (RGR) after 12 days of feeding (Table 1) compared to feeding on a control diet lacking MBOA. In the high-MBOA treatment, however, we observed a significant decrease in CI and AD. This seems to be compensated by an increase in both ECI and especially in ECD. Taken together, these data suggest S. frugiperda is not affected by MBOA toxicity even at the highest concentration tested, which is higher than the physiological levels encountered in maize seedlings (approximately 2 mM) (Maag et al. 2014). Two hypotheses can be proposed: (1) MBOA exhibits antifeedant activity and decreases CI, but the insect adapts by utilizing its ingested and digested food more efficiently; or (2) MBOA serves as a nutrient for the insect and raises ECD, possibly by increasing nitrogen availability and uptake, with decreased consumption as a response to a richer diet. In any case, MBOA seems to act as a digestibility reducer, either by inhibiting digestive enzymes or interacting with Fig. 3 Growth curves (± SEM) for S. frugiperda larvae fed on artificial diets containing MBOA nutrients in the diet and preventing their digestion. The remarkably high effect on ECD indicates that MBOA acts more critically after the digestion process, suggesting a nutrient role. However, S. frugiperda has been shown to metabolize MBOA via N-glucosylation and to excrete a considerable fraction of it in the frass (Maag et al. 2014). This supports the opposite conclusion, that S. frugiperda recruits detoxification enzymes and expends energy (as UDP-glucose) to facilitate MBOA excretion, thus being subject to a higher metabolic cost that should be reflected in a lower ECD. It is important to note that bioassays performed with minimal diets might overestimate the nutritional value of nitrogen-containing compounds and therefore do not necessarily reflect the importance of MBOA in a natural context. Nevertheless, S. frugiperda seems to have the capacity of feeding on high MBOA diets without negative effects on growth, and the resulting physiological effects are different than those reported for O. nubilalis (Houseman et al. 1992). Although the concentration of MBOA used in this experiment was higher than the concentrations of MBOA usually present in maize, other maize BXDs, such as DIMBOA, DIMBOA-Glc and HDMBOAGlc, are typically present at mM levels in herbivoreinduced plants. Since several BXDs are converted to MBOA in the herbivore gut after hydrolysis (see section below on ‘‘Metabolism of BXDs’’), herbivores such as S. frugiperda could be confronted with MBOA levels similar to those fed in our artificial diets. The feeding preferences of S. frugiperda and S. littoralis on maize plants at different growth stages parallel the performance differences between the two species (Köhler et al. 2015). While the more specialized S. frugiperda preferred to feed on younger leaves in spite of their higher BXD levels and higher degree of induction, feeding of the generalist S. littoralis was distributed over different leaf ages, but especially concentrated on older ones. This indicates that S. littoralis moved more during foraging, possibly to Table 1 Nutritional indices (±SEM) for S. frugiperda larvae fed on artificial diets containing MBOA Treatment N RGR (mg/mg per day) CI (mg/mg per day) AD (%) ECI (%) ECD (%) Control 13 0.1055 ± 0.0080 0.9904 ± 0.0530 43.03 ± 3.11 10.83 ± 0.83 27.14 ± 2.88 MBOA 50 lg/g 15 0.0941 ± 0.0055 0.8337 ± 0.0505 39.63 ± 3.41 12.21 ± 1.27 34.98 ± 5.26 MBOA 1000 lg/g 14 0.0989 ± 0.0042 0.6739 ± 0.0387 27.67 ± 2.83 15.15 ± 0.85* 66.54 ± 10.02** * P \ 0.05, ** P \ 0.01, Tukey’s test for unequal sample sizes 123 1136 avoid locally high concentrations of induced BXDs (Maag et al. 2016). After maize stage L6, however, S. littoralis switched to young leaves, presumably due to the overall decrease in both BXD levels and inducibility as the plant grows older. Moreover, in mutants containing lower BXD levels, the preference of S. frugiperda for young leaves disappeared, and larvae grew less than on high-BXD wild-type plants. This suggests that BXDs differentially influence feeding patterns in S. frugiperda (serving as feeding stimulants or nutrients) as compared to S. littoralis (acting as deterrents and toxins). Food consumption and utilization by another generalist Spodoptera species, the southern armyworm (S. eridania) were compared among maize lines with different DIMBOA levels, which had been bred for resistance towards O. nubilalis (Manuwoto and Scriber 1982). Penultimate instar S. eridania grew less when feeding on high DIMBOA plants, while showing lower ECD and ECI. These larvae displayed higher CI and AD, even though this was apparently not enough to compensate for the high metabolic costs of feeding on tissue with high DIMBOA levels. However, last instar S. eridania larvae grew more on lines with more DIMBOA, possibly because of the induction of detoxification pathways or more efficient food processing caused by previous contact with BXDs. In another study, these authors fed S. eridania on two maize genotypes grown under iron and nitrogen deficiencies, which affect water content, and nitrogen and DIMBOA levels (Manuwoto and Scriber 1985a). Overall, Fe-deficient plants showed higher DIMBOA levels, whereas N-deficient plants had lower DIMBOA levels. Surprisingly, larvae feeding on a highDIMBOA genotype grew more than the group feeding on a low-DIMBOA genotype. Fifth-instar larvae feeding on Fe-deficient plants displayed lower CI and AD, but these were compensated by higher ECD and ECI, resulting in no differences in growth. Nitrogen-deficient plants, however, did not support the development of S. eridania as well as controls, resulting in lower growth, CI, ECD, and ECI, together with high mortality rates, even though these plants contained lower DIMBOA levels compared to ones grown with complete nutrient medium. These results suggest that, even though DIMBOA is important in plant resistance to herbivore attack, other factors such as nitrogen and water content also play a role in determining herbivore performance. Deprivation of 123 Phytochem Rev (2016) 15:1127–1151 nutrients might also affect many other primary and secondary metabolites in plant foliage that impact its nutritional value. In choice assays with another generalist feeding Spodoptera, the beet armyworm (S. exigua), larval feeding was deterred on barley leaves treated with DIMBOA, while this treatment stimulated feeding for S. frugiperda (Rostás 2006). Feeding on artificial diets containing 500 lg/g DIMBOA (2.37 mM) increased mortality and reduced growth in S. exigua in shortterm experiments, and increased developmental time in long-term experiments, but S. frugiperda was not affected. Longer developmental times can affect insect survival in nature making them more vulnerable to predators, parasitoids, and pathogens. However, it is uncertain whether the overall negative effects observed on S. exigua are a consequence of DIMBOA toxicity or antifeedant effects. In summary, benzoxazinones such as DIMBOA seem to be more toxic to caterpillars than benzoxazolinones such as MBOA. The N–O-methyl derivative HDMBOA is suggested to be even more toxic, also towards BXD-resistant species. Experiments with lepidopteran herbivores indicate that Spodoptera spp. are generally more adapted to BXDs than Ostrinia nubilalis. And within the genus Spodoptera, the more specialized grass feeder S. frugiperda seems to be more resistant to the toxic effects of BXDs than S. littoralis, S. eridania, and S. exigua, possibly even benefiting from the presence of BXDs in the diet. Such gradients in BXD resistance are partly explained by the detoxification and metabolic capabilities of each species, as will be discussed in more detail in the following sections. However, insect adaptation to BXD-containing plants might also be influenced by larval feeding behavior and trade-offs between nutritional content and both concentration and induction of chemical defenses (not exclusively BXDs) in plant tissues (McMullen et al. 2009). Effects on aphids Piercing-sucking herbivores from the order Hemiptera, such as aphids, possess modified mouthparts called stylets that are used to pierce through the plant cuticle, epidermis, and mesophyll cells, and feed on the highly nutritious phloem sap (Douglas 2003). Because of this particular feeding behavior, aphids are considered to minimize tissue disruption and Phytochem Rev (2016) 15:1127–1151 consequent activation of glucosylated defenses (Pentzold et al. 2014b). However, the dynamic allocation patterns of BXDs still make them effective defensive compounds towards aphids. Furthermore, apart from their direct toxicity towards aphids, BXDs can trigger callose deposition, serving as a signal to induce another line of plant defense against aphids (Maag et al. 2015a). BXDs have been considered resistance factors of cereals towards several aphid species. Hydroxamic acid levels in cereals were positively correlated with resistance towards Metopolophium dirhodium (Argandoña et al. 1980), Schizaphis graminum (Corcuera et al. 1982), and Sitobion avenae (Bohidar et al. 1986). Moreover, Rhopalosiphum padi displayed higher weight gain and survival when feeding on mutant maize plants with reduced BXD levels compared to wild-type plants (Ahmad et al. 2011). Notably, however, BXD levels in maize were not correlated with resistance towards R. maidis (Bing et al. 1990). The detrimental effects of BXDs on many aphid species have been explored using both artificial diets and plant cultivars with different levels of BXDs. In artificial diets, DIMBOA and MBOA increased mortality in M. dirhodium (Argandoña et al. 1980). In R. padi, MBOA increased reproduction rate in concentrations up to 0.1 mM, but had the opposite effect above this threshold (Hansen 2006). DIBOA increased R. padi mortality in artificial diets, and this was substantiated in a comparison among wild Hordeum species containing different DIBOA levels (Barria et al. 1992). DIMBOA also increased S. graminum mortality in concentrations as low as 1 mM (Argandoña et al. 1981, 1983) and decreased its reproduction rate in sub-lethal concentrations (0.1 mM) (Corcuera et al. 1982), with DIBOA increasing mortality as effectively as DIMBOA (Zuñiga et al. 1983). In choice assays, DIMBOA had antifeedant activity towards S. graminum in artificial diets (Argandoña et al. 1983), whereas R. padi avoided wheat leaves from high-DIMBOA cultivars (Givovich and Niemeyer 1991) and barley leaves treated with DIBOA (Copaja et al. 2006). Additionally, aphid species differ in their susceptibility to BXDs. While DIMBOA up to 2 mM in artificial diets increased the mortality of S. graminum and M. dirhodium, it did not affect R. maidis (Corcuera et al. 1982). Structure–activity relationships comparing the toxicity and antifeedant activity of several BXD 1137 aglucones and analogues towards S. avenae were determined in artificial diets (Escobar et al. 1999). Among the natural compounds tested, the hydroxamic acids, DIMBOA and DIBOA, elicited remarkably higher mortality ([50 % after 89 h) than the lactams, HMBOA and HBOA (\20 %), and the benzoxazolinones (\10 %). Antifeedant activity and toxicity did not follow the same patterns among the tested compounds. For example, MBOA caused low mortality, but was one of the most deterrent compounds. Such discrepancies indicate that the antifeedant and toxic activities of BXDs do not necessarily arise from the same structural features. In another study, DIBOA and DIMBOA were significantly more repellent to R. padi than HBOA and HMBOA were (Bravo et al. 2004). However, in this study BXDs were sprayed on barley leaves, and their allocation and stability during the experiment were not determined. Interestingly, BXD glucosides are also active towards aphids. In S. graminum, DIMBOA and DIMBOA-Glc increased mortality with LD50 values (24 h feeding) of 1.2 mM and 4 mM respectively. Both compounds also decreased reproduction rates at concentrations as low as 0.25 mM and caused appreciable feeding deterrence at 0.5 mM (Corcuera et al. 1985). The glucosides DIMBOA-Glc and HDMBOAGlc increased mortality of M. dirhodium with LD50 values (3 days feeding) of 5.3 mM and 1 mM, respectively, while also decreasing fecundity. Mortality curves similar to fully unfed treatments suggest that these glucosides are also antifeedant in high concentrations (Cambier et al. 2001). However, it is not clear if BXD glucosides display inherent biological activity towards aphids or are activated (hydrolyzed or otherwise metabolized) by their own enzymes after ingestion. The lower performance of aphids on high-BXD containing plants likely derives from a combination of toxic and antifeedant effects. Mortality curves for S. graminum feeding on artificial diets with 8 mM DIMBOA were similar to a non-fed treatment, suggesting that aphids died due to starvation caused by the antifeedant activity of high DIMBOA concentrations rather than toxicity. In order to separate these two effects, aphids were first exposed to diets containing DIMBOA and then transferred to diets without BXDs (Argandoña et al. 1983). Mortality rates followed a biphasic distribution (illustrated in Fig. 4), being highest at intermediate DIMBOA 123 1138 Fig. 4 Representation of detrimental effects on aphids caused by different levels of DIMBOA in diet. Above a certain DIMBOA concentration threshold (dashed line), the antifeedant effect of DIMBOA overcomes its toxicity, decreasing DIMBOA intake and diminishing mortality in aphids. In higher DIMBOA concentrations, the mortality curves matched those of unfed groups concentrations (3–4 mM), due to the toxicity of DIMBOA ingested in the first diet. Mortality decreased at higher DIMBOA concentrations due to lower diet ingestion and DIMBOA uptake, caused by its strong antifeedant effect. A similar experiment comparing DIMBOA and DIMBOA-Glc resulted in highest mortalities at 4 mM and 6 mM respectively (Corcuera et al. 1985). Upon feeding on wheat with different DIMBOA contents, the same biphasic profile was observed for DIMBOA content in bodies of M. dirhodium and S. avenae (Niemeyer et al. 1989), and for both honeydew production and DIMBOA-Glc content in honeydew of R. padi (Givovich et al. 1992), which are parameters reflecting food ingestion. BXDs might increase aphid susceptibility to other plant chemical defenses and insecticides by decreasing their detoxification capabilities. In R. padi, DIMBOA inhibits glutathione-S-transferases and esterases in vivo at dietary concentrations as low as 1.0 and 0.5 mM, respectively (Mukanganyama et al. 2003). UGT activities in S. avenae were lower when the aphids fed on high-DIMBOA wheat cultivars than on low-DIMBOA cultivars, and UGT inhibition by DIMBOA was confirmed in vitro (Leszczynski et al. 1992). The effect of BXDs on aphids depends on their abundance in phloem and other tissues penetrated by these insects on their way to the phloem. In wheat, DIMBOA-Glc was the only BXD hydroxamic acid in 123 Phytochem Rev (2016) 15:1127–1151 phloem sap present at concentrations around 1 mM. Average DIMBOA-Glc concentrations in the phloem did not differ greatly among cultivars with different BXD levels. However, a high variability of DIMBOAGlc levels was found among phloem samples from the same cultivar which may reflect different BXD biosynthesis or transport activity in particular sieve tubes, and suggests that aphids could choose sieve tubes according to their BXD levels (Givovich et al. 1994). Among other tissues, BXD hydroxamic acids were also detected in the mesophyll, but not in the xylem in maize and wheat leaves (Argandoña and Corcuera 1985, Argandoña et al. 1987). Furthermore, the aglucone DIMBOA and the glucosides DIMBOAGlc and HDMBOA-Glc were present in the leaf apoplast in maize (Ahmad et al. 2011) and so might be perceived during aphid stylet penetration. That aphids do encounter BXDs at all is demonstrated not only by their negative effects on aphid performance in the whole plant bioassays as cited above, but also by the presence of these compounds in honeydew. DIMBOA-Glc was detected in honeydew of R. padi (Givovich et al. 1992) and S. avenae (Leszczynski and Dixon 1990) feeding on wheat seedlings, but DIMBOA aglucone and MBOA were low or completely absent. Due to the feeding behavior of aphids, a compound present in the phloem sap can exert both antifeedant and toxic effects, while compounds located in the path followed to reach the phloem are considered to have only antifeedant effects (Cambier et al. 2001) since aphids do not ingest such encountered compounds at a high rate. In this context, the electrical penetration graphic (EPG) technique (Tjallingii 1985) allows the evaluation of individual parameters of overall aphid probing behavior and their modification by BXDs. As the hydroxamic acid content of wheat cultivars increases, S. graminum, R. padi, S. avenae, and M. dirhodium took longer to begin phloem ingestion, and exhibited increased xylem ingestion, possibly to ‘‘dilute’’ ingested BXDs (Givovich and Niemeyer 1991; Givovich et al. 1994). On the other hand, for R. maidis, the time to reach the phloem was not influenced by the plant BXD content, suggesting that this species is insensitive to the antifeedant effect of BXDs while searching for sieve elements. Furthermore, the duration of phloem ingestion did not change according to BXD levels in the plant for any of these five species, implying that BXDs are more critical Phytochem Rev (2016) 15:1127–1151 before the phloem is reached. Surprisingly, DIMBOA and DIMBOA-Glc offered in artificial diets decreased ingestion time for all five aphid species tested, including R. maidis, indicating that both compounds have a similar dose-dependent antifeedant activity on all species in this context. The feeding strategy adopted by R. maidis consists in puncturing fewer cells before reaching the phloem than R. padi, as indicated by EPG comparisons. Therefore, R. maidis minimizes exposure to BXDs in mesophyll cells and avoids their antifeedant effects, although it still suffers from them when BXDs are administered via artificial diets (Givovich and Niemeyer 1995). The influence of BXDs on the feeding behavior of Diuraphis noxia is similar to that on the other species described above (Givovich and Niemeyer 1996; Mayoral et al. 1996). Avoidance of BXDs might be learned by previous experience, as shown by Sitobion fragariae aphids feeding on wheat cultivars with different BXD levels (Ramirez et al. 1999). In a high-BXD cultivar, the time taken by naı̈ve aphids to reach the phloem was higher than in a low-BXD cultivar. However, on a second probing, the time to achieve phloem ingestion did not differ among cultivars. A similar trend was observed for the number of mesophyll cell punctures. Such effects were also observed upon feeding on attacked and non-attacked plants, indicating that no aphidinduced effects in the plant are involved. These data suggest that aphids can adapt their feeding behavior after exposure to BXDs to avoid them in future probes by minimizing mesophyll cell damage. In summary, these data suggest that BXDs protect plants against aphid attacks, acting at different stages from probing to feeding. Before probing has started, aphids can potentially already sense BXD aglucones such as HDMBOA present in leaf surface waxes. Once the epidermis is penetrated, the stylet passes mostly through the apoplast, where BXD glucosides are present. On its way to the phloem, the aphid punctures and probes mesophyll cells, possibly with mechanical disruption of organelles (Brzezina et al. 1986; Hewer et al. 2011). Such a scenario could lead to hydrolysis of BXD glucosides by plant b-glucosidases and expose the aphid to locally high concentrations of antifeedant BXD aglucones. Even if BXD hydrolysis does not occur, the intact glucosides appear to exert antifeedant activity as well. However, it is not clear whether BXD aglucones and glucosides present in the apoplast are perceived by aphid chemoreceptors and provoke 1139 antifeedant responses in this way. Finally, once the aphid reaches a suitable sieve element and starts feeding on the phloem sap, it ingests considerable amounts of BXDs, mostly glucosides. However, the duration of phloem ingestion does not seem to correlate to BXD content in plants, which suggests that BXDs in phloem sap are not concentrated enough to exert antifeedant effects or are masked by other phloem constituents. The antifeedant effects of BXDs towards aphids may not necessarily arise from the same BXDs implicated as antifeedants or toxins against chewing herbivores. Thus, when selecting insect resistant cereal lines, more than one BXD metabolite may be required to cover the full spectrum of insect pests. On the other hand, due to the dependence of aphids on bacterial endosymbionts, the antimicrobial activities of BXDs could also contribute to their overall detrimental effects on aphids. Future investigations on the effects of BXDs on aphids would benefit from taking into account their specialized feeding behavior. Studies using artificial diets offer BXDs as a homogeneous solution and so do not account for their specific allocation to leaves and possible aphid avoidance behavior during probing. Although artificial diet bioassays are useful to assess BXD toxicity, they might overestimate antifeedant effects. Alternatively, EPG studies are useful to determine what features of aphid feeding behavior are associated with BXD avoidance, and how insects respond to them, especially insensitive species such as R. maidis. BXDs may also impact maize defense against aphids by serving as a signal for callose deposition. Maize mutants deficient in BXD production showed lower callose deposition when treated with chitosan, a well-known elicitor of defense responses to fungal pathogens, compared to BXD producing lines (Ahmad et al. 2011). Furthermore, DIMBOA (but not HDMBOA-Glc) induced callose deposition when infiltrated into maize leaves. In another study, callose deposition induced by R. maidis feeding was lower on maize lines with low DIMBOA-Glc (and high HDMBOA-Glc) content, and aphid performance was better, even though HDMBOA-Glc is more toxic to aphids than DIMBOA-Glc when administered in artificial diets (Meihls et al. 2013). Experiments with maize recombinant inbred lines also reported a negative correlation between R. maidis reproduction and DIMBOA content and callose formation 123 1140 (Betsiashvili et al. 2015). However, it is difficult to separate the direct detrimental effects of BXDs on aphids (antifeedant and toxic effects) from indirect effects due to their signaling roles (callose deposition), and further detailed experiments are required to investigate such relations. Both of these types of effects likely play a role in overall plant resistance to insect herbivores, but it is also possible that individual herbivore species are distinctly affected, as observed for R. maidis, which seems to be insensitive to the direct antifeedant effects of BXDs, as discussed above, but is susceptible to the induction of callose. Effects on root herbivores Insect herbivores feeding on belowground plant tissues have had to adjust to the challenges posed by living in the soil and the specific primary and secondary metabolites of roots. Root herbivores are influenced by the antifeedant and toxic activities of plant chemical defenses and exploit root volatiles and exudates for host location and foraging similarly to aboveground herbivores (Erb et al. 2013; Hiltpold et al. 2013). Although progress is being made in this field, the release of BXDs and other secondary metabolites by roots is still not well understood (Baetz and Martinoia 2014; Park et al. 2004). The effect of BXDs on the Western corn rootworm (Diabrotica virgifera virgifera) has been widely studied due to its economic importance as a specialist maize pest. DIMBOA applied to corn roots increased mortality of D. v. virgifera, but it is not clear how DIMBOA was absorbed and possibly metabolized by maize roots and the actual concentrations experienced by the insects. At the highest concentrations applied, larvae came out of the roots before dying, suggesting an antifeedant effect and death by starvation. However, larval death was also observed inside the root, indicating that toxicity also contributes to high mortality. Upon infestation with D. v. virgifera eggs, a high-DIMBOA maize line (1,300 lg/g root fresh weight, 6.16 mM) suffered less damage than a lowDIMBOA line (400 lg/g, 1.90 mM). Insect infestation in the high-DIMBOA line led to a lower adult emergence rate and size when compared to the lowDIMBOA line (Xie et al. 1990). In another experiment, D. v. virgifera feeding on maize lines with root DIMBOA levels ranging from 90 to 250 lg/g (0.43–1.18 mM) did not differ in developmental time 123 Phytochem Rev (2016) 15:1127–1151 and survival, confirming that these values are below a threshold for resistance against this herbivore (Davis et al. 2000). Nevertheless, the positive correlation between root BXD content and resistance towards D. v. virgifera damage was also observed in field experiments with different maize lines (Assabgui et al. 1995a, b). When applied to maize roots, DIMBOA, DIBOA, DIM2BOA, HMBOA, and MBOA were repellent to D. v. virgifera larvae, as observed by choice assays (Xie et al. 1992). Fewer larvae were found inside roots and more stayed on or outside roots when compared to control treatments. BXD treatment also modified host searching behavior, decreasing number of turns and increasing area searched and the locomotion of larvae. A first bioassay-driven fractionation of maize root extracts suggested that MBOA is attractive to D. v. virgifera and could be used as a volatile chemical cue for location of grass hosts in a CO2-rich background (Bjostad and Hibbard 1992). However, in a second study, the authors found CO2 to be the only compound responsible for larval attraction, rather than other components of maize extracts (Bernklau and Bjostad 1998). Furthermore, MBOA applied to maize roots did not show antifeedant activity or toxicity towards D. v. virgifera (Abou-Fakhr et al. 1994). However, recent studies indicate that D. v. virgifera uses BXDs as chemical cues for foraging, despite their toxicity. Maize crown roots were shown to be more nutritious and also to contain higher levels of total and exuded BXDs than primary roots. Nevertheless, D. v. virgifera preferred to feed on crown roots, while feeding by the generalist D. balteata was more distributed between crown, primary, and secondary roots. Accordingly, in no-choice assays, D. v. virgifera larvae gained more weight feeding on crown roots compared to primary roots in both wild-type and lowBXD mutant plants. However, when given the choice between crown and primary roots, D. v. virgifera did not show any preference in low-BXD mutants, suggesting that this insect uses BXDs as chemical cues to locate highly nutritious roots (Robert et al. 2012). The performance of D. v. virgifera was compared to the generalist southern corn rootworm (Diabrotica undecimpunctata howardi) when feeding on a BXDdeficient mutant with low BXD levels and its parental line with high BXD levels (Alouw and Miller 2015). Survival and developmental time were the same when comparing both maize lines. However, D. v. virgifera Phytochem Rev (2016) 15:1127–1151 grew better on the high-BXD line compared to the low-BXD mutant, while no differences were observed in D. u. howardi growth. The better performance of the specialist D. v. virgifera might be related to its ability to exploit BXDs to find nutritious tissues (Robert et al. 2012), while the generalist D. u. howardi is unable to do so. The results from Robert et al. (2012) and Alouw and Miller (2015) suggest that D. v. virgifera is tolerant to BXDs and seem to contradict previous studies showing strong antifeedant and toxic effects. It is possible that by feeding on tissues with high nutritional value, D. v. virgifera overcomes the detrimental effects of BXDs, resulting in better overall performance. On the other hand, the application of pure BXDs onto the root surface used to show repellency in earlier studies does not necessarily reproduce the insect experience in a natural context, considering that their absorption and metabolism by the plant and potential effects of other co-exuded compounds and enzymes are not well-known. Additionally, the earlier studies comparing maize lines only focused on DIMBOA content, not considering other BXDs (such as HDMBOA-Glc) and their distribution among root tissues, which might also play an important role in feeding preference and toxicity against D. v. virgifera. Another specialized root herbivore, the wheat bulb fly (Delia coarctata), showed remarkable attraction to wheat seedling exudates (Rogers and Evans 2013). MBOA attracted larvae in a dose-dependent fashion and might contribute to the activity of the exudate, while DIMBOA elicited a weaker response. Since MBOA is more stable than DIMBOA in the soil (Macı́as et al. 2004), it constitutes a more reliable chemical cue for host location. In soil, BXDs and their derivatives are present as a complex and dynamic mixture whose composition depends on many factors such as temperature, pH, and soil microbiota. Moreover, soil fungi are known to metabolize benzoxazolinones and produce a variety of aminophenols and aminophenoxazinones, as well as their malonylated and acetylated derivatives (Fomsgaard et al. 2004). Such compounds could also possess biologically relevant activities on root herbivores, but this has not yet been investigated in detail. Soil nematodes and microbial pathogens are also exposed to BXD profiles in the soil environment and are subject to their biological activities (Meyer et al. 2009; Zasada et al. 2005). Due to the complexity of the soil matrix, it 1141 is difficult to design bioassays that reflect the BXD concentrations and allocation in the root tissue as perceived by a root herbivore in a natural context. Further studies on BXD degradation and diffusion rates through soil could furnish information to construct more realistic experimental setups to evaluate BXD influence on underground plant protection. Metabolism of BXDs Despite the toxicity, allelopathic and antimicrobial activities of BXDs, some animals, plants, and microorganisms are able to avoid these detrimental effects. Recent reports have increased our understanding about how BXDs are metabolized, absorbed, and excreted by target organisms. Such knowledge provides insight into the coevolution of these plant chemical defenses with plant enemies and the effects of cereal products on human health. Metabolism in insects Several insect species use BXD-containing plants as food, which suggests they have developed resistance strategies such as avoidance, rapid excretion, sequestration, detoxification, or target-site mutation (Després et al. 2007). For example, larvae of M. separata fed on artificial diet containing DIMBOA were found to excrete DIMBOA-Glc, HMBOA-Glc and 1-(2-hydroxy-4-methoxyphenylamino)-1-deoxy-b-glucopyranoside-1,2-carbamate (referred to here as MBOAGlc-carbamate, Fig. 5) in the frass based on NMR analyses (Sasai et al. 2009) in accordance with previously published data (Hofmann et al. 2006; Sicker et al. 2001). Incubation of midgut homogenates Fig. 5 Structures of MBOA metabolites detected in plants and insect frass 123 1142 with DIMBOA and UDP-glucose yielded DIMBOAGlc, indicating a UGT activity. The in vitro DIMBOA glucosylation activity for M. separata was higher than for the non-adapted B. mori. Metabolism of DIMBOA is also observed upon incubation of O. furnacalis larval gut homogenates with UDP-glucose (Kojima et al. 2010). This conversion is not affected by the presence of NADPH or glutathione, and is decreased if the homogenate is treated with heat or proteinases, confirming its enzymatic nature. Assays with larvae of O. scapulalis and O. latipennis, two species known to be less welladapted than O. furnacalis to feed on BXD-containing plants, resulted in lower DIMBOA conversion rates. DIMBOA metabolism by O. furnacalis larval gut homogenates had a pH optimum between 7.2 and 7.8 and was induced after feeding on DIMBOA-containing diet or maize, which was not observed for O. scapulalis (Phuong et al. 2015). However, DIMBOAGlc was not detected, and other products were not identified. Other species that are well adapted to feeding on BXD-containing plants, such as S. frugiperda, have also been suggested to have resistance mechanisms towards these compounds. Both S. frugiperda and S. littoralis larvae feeding on DIMBOA-containing diet were shown to diminish its toxicity by glucosylation reactions, excreting DIMBOA-Glc, HMBOA-Glc, and MBOA-Glc in the frass (Glauser et al. 2011). On the other hand, HDMBOA is considered to be too unstable (Maresh et al. 2006) in the alkaline insect gut to be efficiently conjugated, in agreement with its potent toxic and antifeedant effect even towards the BXD-resistant S. frugiperda. This instability may prevent reglucosylation even if a glucosyltransferase with the appropriate substrate specificity were present. In a screening of many lepidopteran species, glucosylation was shown to be an important process in BXD metabolism. When feeding on maize leaves, larvae of the more resistant species, S. frugiperda, S. littoralis, and S. exigua, excreted DIMBOA-Glc in the frass, while the more susceptible Mamestra brassicae and Helicoverpa armigera did not (Wouters et al. 2014). In vitro assays confirmed that DIMBOA is glucosylated by S. frugiperda larval gut homogenates in the presence of UDP-glucose, suggesting the contribution of UGT enzymes. The insect-derived product, (2S)-DIMBOA-Glc, is an epimer of the original (2R)-DIMBOA-Glc produced by the plant. 123 Phytochem Rev (2016) 15:1127–1151 Such a change in stereochemistry results in the glucoside no longer being a substrate recognized by the plant BXD b-glucosidases, which are still present and active in the gut lumen. Thus this transformation represents a detoxification mechanism since the toxic aglucones can no longer be formed. The benzoxazolinone MBOA is also glucosylated by lepidopteran herbivores. This compound is a product of the spontaneous degradation of DIMBOA and HDMBOA (Fig. 1), two of the most abundant BXDs present in maize leaves, but also shows its own inherent toxicity. Since the alkaline gut of most lepidopteran larvae accelerates DIMBOA and HDMBOA degradation to MBOA, insects are exposed to high levels of this BXD. S. frugiperda and S. littoralis larvae feeding on artificial diets containing MBOA excreted considerable amounts of MBOAGlc, while O. nubilalis larvae were less efficient in this conjugation reaction (Maag et al. 2014). Detailed structural elucidation revealed that the observed MBOA metabolite was 3-b-D-glucopyranosyl-6methoxy-2-benzoxazolinone (MBOA-Glc, Fig. 5) rather than the isomeric MBOA-Glc-carbamate previously characterized as a plant detoxification product (Hofmann et al. 2006; Sicker et al. 2001). To quantify these transformations more directly, we compared the rate of MBOA glucosylation by UDP-glucosyltransferases using in vitro assays of gut homogenates of S. frugiperda and O. nubilalis larvae. S. frugiperda displayed a specific activity for MBOA more than 20-fold higher than O. nubilalis, and no significant differences were observed between O. nubilalis univoltine and bivoltine strains (Fig. 6). Accordingly, the Fig. 6 Specific activity of MBOA glucosylation (±SEM) in gut homogenates of S. frugiperda and O. nubilalis (univoltine and bivoltine strains) (N = 3) Phytochem Rev (2016) 15:1127–1151 tolerance of O. nubilalis to BXD-containing plants appears not to depend on BXD detoxification, but rather on temporal aspects and foraging preferences (Maag et al. 2014). However, it is not known whether UGT activity towards BXDs in O. nubilalis is induced upon exposure to BXDs. The regulation and identity of the UGTs responsible for BXD detoxification in S. frugiperda are currently being investigated. A recent comparison between the performance and transcriptional profiles of S. frugiperda and S. littoralis showed that the total expression of UGT-encoding genes did not change between larvae feeding on artificial diet and on maize leaves (Roy et al. 2016). This supports the hypothesis that the enzymes responsible for BXD glucosylation are constitutively expressed in Spodoptera spp., rather than being induced upon contact with BXDs. Furthermore, S. frugiperda performance on maize was higher than that of S. littoralis, which indicates a greater tolerance to BXDs. To the best of our knowledge there are no detailed studies investigating the metabolism of BXDs in aphids. However, S. avenae reared on wheat over 10 generations possessed increased activities of cytochrome P450 monooxygenases, NADPH cytochrome c reductases, glutathione S-transferases, and esterases, but not catalases (Loayza-Muro et al. 2000). In general, these increases were more pronounced in the low-BXD wheat cultivar tested, presumably due to antifeedant activity and limited BXD ingestion by aphids reared on the high-BXD cultivar. Such upregulation of detoxification enzymes might indicate they are involved in BXD metabolism, but more studies are necessary to confirm this hypothesis. Aphids also have a wide range of enzymes in their saliva (Madhusudhan et al. 1994), which can potentially be secreted during probing and modify BXDs, as has been proposed for phenols (Urbanska et al. 1998). Tolerance towards BXDs has also been suggested for D. v. virgifera. The transcriptional profiles of larvae feeding on a low-BXD maize mutant and on the high-BXD parent line were compared (Miller and Zhao 2015). Differentially expressed genes included a cytochrome P450 and a cathepsin protease. This indicates that D. v. virgifera might detoxify BXDs using P450s, but this hypothesis remains to be tested. Besides detoxification, other strategies such as behavioral avoidance and sequestration might contribute to the resistance towards BXDs observed in 1143 some insect species. For example, the act of snipping the leaf into larger pieces during feeding minimizes plant tissue disruption and, together with an alkaline gut, can inhibit the activation of plant chemical defenses such as BXDs. These mechanisms allow Zygaena filipendulae to limit hydrolysis of cyanogenic glucosides in its host plant and to sequester these compounds for use against predators (Pentzold et al. 2014a). Metabolism in mammals In addition to their importance in assessing the safety and therapeutical uses of BXD derivatives, the metabolism of BXDs in mammals may reveal some common strategies that other animals, including insects, employ to metabolize these compounds. Ingestion of rye bread which naturally contains a mixture of BXDs has revealed some aspects of BXD metabolism in mammals. For example, in rats and pigs, glucoside hydrolysis, hydroxamic acid reduction to the corresponding lactam, and aglucone conjugation to glucuronic acid were important metabolic reactions (Adhikari et al. 2012a, b). In humans, hydroxamic acid reduction and aglucone conjugation with glucuronic acid and sulfate were observed (Adhikari et al. 2013). However, a considerable part of the ingested BXDs was not recovered in the urine and feces in the three studies, indicating that absorption or transformation to unknown metabolites also took place. Conclusions and suggestions for future research Benzoxazinoids constitute a class of activated plant defenses that function against a wide range of insect herbivores and other target organisms. In this review, we have seen that the effects of BXDs on different guilds of insect herbivores are strongly influenced by the specific distributions of the compounds themselves and their activating b-glucosidases. BXD effects are also determined by the unique structural features of these molecules which give them different reactivities, with benzoxazinone N-substitution with either –H, – OH or –OCH3 in particular giving rise to very contrasting aglucone characteristics. Such chemical variation helps to diversify and promote these compounds’ modes of action as toxins, feeding deterrents and digestibility-reducing compounds. Plants can take 123 1144 advantage of such chemical diversity by specifically inducing de novo BXD biosynthesis and regulating their interconversion and accumulation in specific tissues, adapting their defensive strategy to the nature of the attacking insect herbivore. As we have also seen, insect species have varying susceptibility to BXDs not only arising from these bioactivities, but resulting also in part from their ability to metabolize these substances into less harmful derivatives, as well as the distribution and induction of individual BXDs in leaves of different ages and nutritional backgrounds. Herbivore feeding and performance assays on artificial diets are important tools to assess the effects of individual BXDs in a dose-dependent way. However, the stability of these compounds under the bioassay conditions must be considered. Like derivatives of other activated defense compounds, BXD hydrolysis products can react with other diet components (Argandoña et al. 1982) or degrade to other substances such as benzoxazolinones, which might modify the final results. For example, most HDMBOA in an artificial diet degraded within 30 min after HDMBOA-Glc hydrolysis (Glauser et al. 2011) and a similar, albeit slower, decomposition was observed for DIMBOA (Campos et al. 1989). As the degradation of BXD hydroxamic acids and N–O-methyl derivatives to benzoxazolinones is faster at high pH values (Maresh et al. 2006; Niemeyer et al. 1982a), the acidification of diets constitutes an alternative to improve BXD stability during feeding bioassays (Argandoña et al. 1982). Similarly, BXDs applied on plant leaves for bioassays can also degrade or be metabolized quickly, and their persistence should be assessed in such experiments. Additionally, the BXD defense system in plants, like that of other activated defenses, is compartmentalized and depends on temporally and spatially resolved activation by hydrolysis. A bioassay that simulates the tissue-specific distribution of BXDs present in a plant leaf or the gradients of BXDs diffusing through soil would be very valuable, but difficult to design. In the meantime, the limitations of existing experimental bioassay setups must be kept in mind. Studies on the influence of BXDs on food consumption and utilization by insect herbivores are useful to discriminate toxins, digestibility-reduction agents, and antifeedant factors. However, such bioassays should take into account that the concentration and composition of plant BXDs, like that of many 123 Phytochem Rev (2016) 15:1127–1151 other defense compounds, are rapidly and locally induced by the act of herbivory, and in the insect detoxification genes are induced by initial contact with BXDs. Artificial diets provide a more controlled way to assess the effects of single compounds on insect physiology, but have limitations in representing the plant, since induced responses are absent and the nutritional composition of diets is different than the plant, which may modulate toxin activities (Duffey and Stout 1996). In bioassays using specific BXD mutants, the nutritional values and levels of non-BXD metabolites in different lines and plant tissues should also be considered as these may have changed in unplanned ways. For studies on BXD metabolism by insects and other target organisms, future work in determining the structures of metabolites and quantifying their abundance will benefit from ongoing advances in the sensitivity and resolution of modern analytical instruments. In addition, the development of mutant plant lines with low levels of BXDs facilitates the identification of new metabolites by comparative metabolomic approaches (Glauser et al. 2011; Maag et al. 2015b). Nevertheless, feeding single compounds via artificial diets may still be necessary to identify individual metabolites, especially considering the often interlinked metabolism of BXDs, with conversion of hydroxamic acids to lactams, and of multiple benzoxazinones to single benzoxazolinone derivatives. The structural elucidation of metabolites gives rise to hypotheses about the enzymatic pathways involved, which can be confirmed in vitro and characterized on the gene level. The toxicokinetics of BXDs is another important aspect in the description of their biological activities in insects. The absorption, distribution, and excretion of BXDs can be followed with high sensitivity by using radioactive isotopically labelled compounds (Campos et al. 1988, 1989), but these substances are not readily available. Careful studies with unlabeled compounds can also provide important insights on physiological processes and suggest the involvement and location of transporters and detoxification enzymes, and other strategies such as sequestration. Further understanding of the biological effects of BXDs on target organisms and their metabolism requires the convergence of many disciplines, including ecology, evolutionary biology, biochemistry, analytical chemistry, and organic synthesis. The resulting Phytochem Rev (2016) 15:1127–1151 knowledge will undoubtedly provide many new insights on the interactions between BXD-containing plants and other organisms, and can also contribute to the development of new strategies for pest control on BXD-containing crops, such as wheat and maize, that lead to the breeding of new resistant varieties or the synthesis of novel compounds which alter insect BXD processing. 1145 of the experiment, and nutritional indices were calculated according to the following equations (Waldbauer 1982): RGR ¼ Weight gained Time Mean weight CI ¼ Ingested food Time Mean weight AD ¼ Ingested food Feces 100 Ingested food ECI ¼ Weight gained 100 Ingested food Materials and methods Insects Larvae of S. frugiperda (maize strain) were a generous gift from the Department of Entomology of the Max Planck Institute for Chemical Ecology, and were reared on an artificial diet based on white beans (Bergomaz and Boppré 1986), under controlled light and temperature conditions (12:12 h light/dark, 20 °C). Eggs from univoltine and bivoltine strains of O. nubilalis were obtained from the Agroscope Changins (Switzerland) and were reared under the same conditions described above. The O. nubilalis diet was adapted from the literature (Maag et al. 2014), using barley flour instead of wheat germ. Nutritional indices Third instar larvae were individually kept on plastic cups and under the rearing conditions described above for 12 days. MBOA was added to the diets during the cooling step of the preparation process as a solution in ethanol (5 mL for each 100 g diet) or pure ethanol for the control treatment. The diets were poured into Petri dishes, and diet plugs were cut with a cork borer, left for 15 min for ethanol evaporation, and offered to the insects. Diet plugs were replaced daily. Frass and remaining diet in cups were collected daily, freezedried overnight, and weighed in order to quantify ingested food and feces. Larvae were weighed every second day to assess growth. At the end of the experiment and at the end of the life stages studied, larvae were freeze dried and shown to have consistent water content (85 %). All larval weights were calculated as dry mass using water content of each individual at the end of the experiment. Overall weight gain, mean weight, ingested food, and feces were calculated in terms of dry mass over the 12 days ECD ¼ Weight gained 100 Ingested food Feces Enzymatic assays Gut homogenates for in vitro assays were dissected from third and fourth instar larvae of S. frugiperda and O. nubilalis. The caterpillars were dissected in cold 10 mM phosphate buffer (pH 7.0) and the gut tissue was isolated and its contents removed. The rinsed gut tissues were then transferred to a fresh tube and homogenized with 100 lL of 10 mM phosphate buffer (pH 7.0) per gut. Protein concentrations were determined using the method of Bradford (1976). For the in vitro assays, 10 lL of gut suspension were incubated with 75 nmol of MBOA and 150 nmol of UDP-glucose in 100 mM phosphate buffer at pH 7.0 (final assay volume: 50 lL). After an incubation period of 60 min at 30 °C the reaction was stopped by adding 50 lL of MeOH/formic acid (50:50, v/v). The samples were centrifuged at 5000 g for 5 min prior to analysis by HPLC–MS/MS. Aliquots of the gut homogenates were heated at 100 °C for 15 min and used for boiled controls. Chromatographic methods For analytical chromatography procedures, formic acid (0.05 %) in water and acetonitrile were used as mobile phases A and B, respectively, and the column temperature was maintained at 25 °C. The quantitative analysis of MBOA-Glc produced in in vitro assays used an XDB-C18 column (50 9 4.6 mm, 1.8 lm, Agilent Technologies, Boeblingen, Germany) with a flow rate of 1.1 mL min-1 and with the following 123 1146 elution profile: 0–0.5 min, 95 % A; 0.5–6 min, 95–67.5 % A; 6.02–7 min, 100 % B; 7.1–9.5 min, 95 % A. HPLC–MS/MS analyses were performed on an Agilent 1200 HPLC system (Agilent Technologies, Boeblingen, Germany) coupled to an API 3200 triple quadrupole mass spectrometer (Applied Biosystems, Darmstadt, Germany) equipped with a turbospray ion source operating in negative ionization mode. The ion spray voltage was maintained at -4500 V. The turbo gas temperature was 500 °C, nebulizing gas 60 psi, curtain gas 25 psi, heating gas 60 psi and collision gas 5 psi. Multiple reaction monitoring (MRM) was used to monitor the analyte parent ion to product ion conversion with MRM parameters for MBOA-Glc optimized from infusion experiments with a standard (Q1 m/z: 372, Q3 m/z: 164, DP -15 V, EP -4.5 V, CEP -18 V, CE -20 V, CXP -4 V). Both Q1 and Q3 quadrupoles were maintained at unit resolution. Analyst 1.5 software (Applied Biosystems, Darmstadt, Germany) was used for data acquisition and processing. Acknowledgments Open access funding provided by Max Planck Society. The authors gratefully acknowledge the Swiss National Science Foundation (Grant No. 136184) and the Max Planck Society for financial support, the Department of Entomology of the Max Planck Institute for Chemical Ecology for providing S. frugiperda larvae, Noémie Chervet and the Agroscope (Switzerland) for providing O. nubilalis eggs, and Grit Kunert for valuable discussions. 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https://openalex.org/W4310742039	https://ejournal.aissrd.org/index.php/ijess/article/download/99/90	English	null	Social Construction of Young Married Couples in Parenting Children in South Jakarta	International Journal of Education and Social Science	2,022	cc-by-sa	3,512	ISSN : 2686-2239 (online) ____________ ISSN : 2686-2239 (online) ____________ ISSN : 2686-2239 (online) ____________ _ VOL 3 NO 2 OCTOBER 2022 Keywords: Social Construction, Young Married Couples, Parenting SOCIAL CONSTRUCTION OF YOUNG MARRIED COUPLES IN Parenting CHILDREN IN SOUTH JAKARTA Syamsiah, Adisa Cahya Rianti Universitas Nasional Jakarta, Indonesia syamsiahbadruddin0234@gmail.com ISSN : 2686-2239 (online) ISSN 2686 2239 ( li ) _ VOL 3 NO 2 OCTOBER 2022 1Law Number 16 of 2019 concerning Amendments to Law Number 1 of 1974 concerning Marriage. State Gazette of the Republic of Indonesia Year 2019 Number 186, Supplement to the State Gazette of the Republic of Indonesia Number 6401. Central Government. 2 INTRODUCTION From data released by the South Jakarta Religious Court, the application for marriage or marriage made by a couple under the age of has in limit (19 year) on year 2019 as much 53, year 2020 as many as 100, and in 2021 it will return to 53. Here it can be seen that in 2020 applications or marriage dispensations submitted by prospective husbands and wives are quite increasing. Marriage is an important stage in life somebody. Because that, wedding must prepare by ripe from any aspect, for example in terms of mental, economic and other. However, in recent years there have been quite a number of young or early marriages even though the marriage law has been amended 1. This actually makes people think that couples who marry at a young age are still considered a marriage that was carried out too early and without proper preparation. Because marriage at a young age also has an impact on the partner's unpreparedness in family life and in carrying out obligations as well as play a role as person old in nurturing child. Mother those who marry at a young age do not have the readiness to carry out the parenting phase, with limited knowledge or knowledge, limited information and age maturity can be the cause of unpreparedness Mother in nurturing child 2. even though later readiness and people's perseverance old, specifically Mother as teacher first in nurturing will build character quality child. From several research studies that have been conducted, it seems that the quality of parenting is influenced by the quality of marriage or the quality of the relationship between spouses and even the education of the mother/wife 3. quality wedding from couple (husband and wife) is factors that will determine attitudes and behavior child 4. The term early marriage or early marriage is a marriage carried out by someone who has not yet reached adulthood (Koro, 2012: 72). This marriage is often carried out by young men and women who have not yet reached the ideal age to marry or build a household. It can be said that they are not yet established emotionally, financially, and physically or psychologically not ready. The term early marriage or early marriage is a marriage carried out by someone who has not yet reached adulthood (Koro, 2012: 72). 2 Tsunia, N. (2014). Family characteristics, readiness for marriage, wife, and the development of children aged 3-5 years (Thesis). Bogor, Indonesia: Bogor Agricultural Institute. 3 Abstract In married life or household life, children are a gift and gift given by God to complete the happiness of parents. Until now, we still meet couples who get married at a young age. Many of them are actually not ready to have children, thus allowing them to provide inappropriate parenting. The purpose of this study was to determine and understand the social construction of young married couples in South Jakarta in deciding to marry and raise children. This research is a research with qualitative methods using interactive analysis techniques including data reduction, data presentation to draw conclusions. The results of the study indicate that the social construction of young married couples in raising children in South Jakarta is not quite good and well-constructed. There is no readiness of couples to raise children and young married couples in South Jakarta are trying to improve their parenting methods by studying parenting or parenting. 49 49 years (Thesis). Bogor, Indonesia: Bogor Agricultural Institute. 3 Apriliani, FT (2020). The Effect of Young Marriage on Family Resilience. Journal of Social Welfare Vo.07, No. 01, Padjadjaran University. 4C Lai. (2011). Parental marital quality and family environment as predictor of delinquency amongst selected secondary school student in Malaysia. British Journal of Art and Social Science. 5Sunarti, ET (2005). Effect of economic pressure, social support, quality of marriage, parenting and emotional intelligence of children on children's learning achievement Family Nutrition and Media Mass Media Factors / Internet Mass Media Factors / Internet In today 's increasingly sophisticated era, it is very easy for everyone to access whatever they are looking for. If a teenager is not careful, then he can fall into bad influences or promiscuity which begins with seeing, reading, or hearing information that he gets from the internet. factor married by accident y This factor can be said as one result of the use of mass media or the internet. Where by playing on the internet and exploring indecent things that they don't deserve to know, it is important for them (teenagers) to learn and be given socialization about sex education. The occurrence of pregnancy out of wedlock, finally forced them to get married, which aims to clarify the status of the child in the womb. With an accident like this, they are finally forced to get married and are obliged/responsible to behave as husband and wife and become fathers (parents) for their children. So pregnancy that occurs outside of marriage is also one of the factors that create marriage at a young age. With a lot problem from wedding age young which Among them are divorce, quarrels, domestic violence or other findings in previous research, this study also aims to explore the reasons that cause couples to marry at a young age and the readiness and knowledge possessed by young married couples in parenting child. 6Mubasyaroh. (2016). Analysis of the Causes of Early Marriage and Its Impact on the Perpetrators. 7Creswell, J. (2016). Research Design Qualitative & Quantitative Approach ISSN : 2686-2239 (online) VOL 3 NO 2 OCTOBER 2022 Adolescents (boys and girls) who have low education can be the cause of the occurrence of young marriages. The lower the education of adolescents, the higher the risk they have to get married at 6a very young age. Lack of socialization and education from parents can also affect the teenager. INTRODUCTION This marriage is often carried out by young men and women who have not yet reached the ideal age to marry or build a household. It can be said that they are not yet established emotionally, financially, and physically or psychologically not ready. Exist a number of trigger or reason for man and women who marry under the specified age (19 year). factors happening wedding in young age including the following 5: Exist a number of trigger or reason for man and women who marry under the specified age (19 year). factors happening wedding in young age including the following 5: Factor Economy Difficulties in the economic aspect or income and income are one of the factors that trigger marriage at a young age or at an early age, especially for families who experience economic problems will tend to marry off their children at a young age. In this facto case, marriage is considered as the key or a way out for the economic difficulties experienced by the family, so that later the family will experience little economic hardship 4 . Factor Education 1Law Number 16 of 2019 concerning Amendments to Law Number 1 of 1974 concerning Marriage. State Gazette of the Republic of Indonesia Year 2019 Number 186, Supplement to the State Gazette of the Republic of Indonesia Number 6401. Central Government. 2 2 Tsunia, N. (2014). Family characteristics, readiness for marriage, wife, and the development of children aged 3-5 years (Thesis). Bogor, Indonesia: Bogor Agricultural Institute. 3 3 Apriliani, FT (2020). The Effect of Young Marriage on Family Resilience. Journal of Social Welfare Vo.07, No. 01, Padjadjaran University. 4C Lai (2011) Parental marital quality and family environment as predictor of delinquency amongst selected 3 Apriliani, FT (2020). The Effect of Young Marriage on Family Resilience. Journal of Social Welfare Vo.07, No. 01, Padjadjaran University. y y 5Sunarti, ET (2005). Effect of economic pressure, social support, quality of marriage, parenting and emotional intelligence of children on children's learning achievement. Family Nutrition and Media. y y 5Sunarti, ET (2005). Effect of economic pressure, social support, quality of marriage, parenting and emotional intelligence of children on children's learning achievement. Family Nutrition and Media. 50 Parental Factor Parents can also be the cause of children to marry at a young age. However, behind this factor there are several reasons which motivate person old for marry off her son at a young age, namely because parents are worried that their children will fall into promiscuity (adultery) and have bad results; parents wish to extend their relationship by marrying off their relation's children with their children; and the desire to match his child with his brother so that their property or descendants are still in the hands of their family. Parents with low education also greatly influence their children to marry at a young age. The lower the education of parents, the greater the intention or desire person old the for marry off her son in young age even in childhood. So, in this case parents have a very important role in marriage at a young age. Mass Media Factors / Internet ISSN : 2686-2239 (online) _ VOL 3 NO 2 OCTOBER 2022 study that explores a case in depth, and collects complete material or information using a data collection method based on a predetermined time. The case in this study can be in the form of an activity, program, event, and process 8. This type of case study research is in accordance with the method to answer the questions in this study, namely, what are the reasons for couples to marry at a young age, and what strategies or plans they have for parenting. , ( ) Q y Translated by Tjejep Rohindi Rohidi. Jakarta: University of IndonesiaPress. ISSN : 2686-2239 (online) ISSN : 2686-2239 (online) ____________ 8Miles, M.d. (2007). Qualitative Data Analysis. Resource Book About New Methods. RESEARCH METHODS The research location/place chosen in this research is South Jakarta City. The research method used in this study is a qualitative research method. Qualitative research is a type of research that explores and understands the meaning of several individuals or a group that originates from social problems7. This qualitative research is often used in research on people's lives, history, concepts, phenomena, social problems, case studies, and behavior. The type of research used by the researcher in this research is a case study. A case study is a The type of research used by the researcher in this research is a case study. A c 51 51 51 8Miles, M.d. (2007). Qualitative Data Analysis. Resource Book About New Methods. Translated by Tjejep Rohindi Rohidi Jakarta: University of IndonesiaPress ISSN : 2686-2239 (online) __________________________________ VOL 3 NO 2 OCTOBER 2022 YL • Urgent and must get mar- ried right then and there • Married By Iccident LA • It’s been a long time • love/dating since entering high school • Own Will _ VOL 3 NO 2 OCTOBER 2022 Of the various reasons or factors that influence individuals to marry at a young age from the five informants. One of them is because of the religious factor of the informant with the initials TA, according to him marriage is a sacred thing before God and by doing marriage all his affairs become easy and to avoid adultery. However, this is in stark contrast to what is currently experienced after marriage, that taking care of the household, having a good relationship with a partner, and raising children are very difficult things to deal with without any preparation. In this case, Berger sees the problem as a social reality which is the result of the social construction of society towards the individual. social construction into 3 stages, namely 9: a) Externalization Basically, informants who marry at a young age must have the same knowledge and access as they (couples who marry with knowledge) about child rearing and married life. It can become a problem if parenting and the realities of married life are not properly socialized. Socialization related to child care and the reality of domestic life is very necessary, plus this must be socialized by authorized institutions or agencies and has the task of regulating norms and values related to marriage and marriage. This ultimately results in the lack of insight, knowledge and readiness both in raising children or providing household arks by young married couples. g p g y y g p b) Activation object The above statement further makes it clear that many informants who marry at a young age have very little knowledge about child rearing and married life. This indicates that the government or other marriage institutions pay less attention to the situation of young people and especially women who want to get married at a young age. Some of the informants feel lazy or reluctant to find out the right parenting style and how to prepare for marriage properly. In contrast to those who are ready and learn first how to take care of children and prepare everything to face difficult realities. 9Puspitawati, H. (2013). Introduction to Family Studies. Bogor: IPB Press. RESULT AND DISCUSSION In getting married at a young age, of course there are reasons or factors that make someone marry under the age that has been regulated in Law Number 16 of 2019. Various reasons have been obtained from each source or informant for marry in age young. Like which already conveyed by the informant RK (wife) VG (husband) as one of the couples who married young, they deciding want to marry young because existence need Economics and Other from the answer of informant RK who got married because he wanted to advance his trading business, AD informant explained that the strong reason that made him marry his husband was the encouragement of his parents. If informant RK married because of business and informant AD married because of parents, informant TA had other reasons and factors that decided to marry at a young age. Informants on behalf of YL have different reasons and external factors. Because the marriage happened because of an accident. Although the YL informant did not explain during the interview, but as the researcher spent the day at his house, he decided to explain to the researcher that he and his husband got married because of the married by accident factor. Different from the three informants that have been described, there is one informant who decides for marry in age young of course because will they alone and have committed to their partner. the informant is an informant LA Based on the results of the interviews above, it can be seen that there are various reasons and factors that young married couples have. From the explanation above, it can be explained as in the table below: Name Young Married Factors Information RK & VG • Want to build joint venture af- ter high school graduation • Increase economy • Factor economy AD • People's decision old • people factor old TA • Carrying out religious or- ders • Stay away from adultery and slander of the world • Religion influence 8Miles, M.d. (2007). Qualitative Data Analysis. Resource Book About New Methods. Translated by Tjejep Rohindi Rohidi. Jakarta: University of IndonesiaPress. 52 52 CONCLUSION There are various factors that cause young married couples in South Jakarta, including economic factors, parental encouragement factors, religious influence factors, lack of sexual knowledge to self-will factors. Some of these reasons eventually became a factor that made a couple in South Jakarta decide to get married young. From the results of existing interviews, the researchers concluded that when they decided to get married at a young age, couples in South Jakarta did not have readiness in domestic life, one of which was having children. Because if a couple decides to get married at a young age just because they have been dating for too long, because of their parents, or because of the economy, then this will have a bad influence on their domestic life so that it will have an impact on their pattern of parenting. the social construction of young married couples in raising children in South Jakarta has not been well constructed. This is due to the lack of insight, knowledge and socialization obtained by the informants who married at a young age. In fact, to become parents, every couple who gets married at a young age or not really needs readiness, knowledge, and deepening of roles in living household life, including in raising children. REFERENCE S Apriliani, FT (2020). The Effect of Young Marriage on Family Resilience. Journal of Social Welfare Vo.07, No. 01, Padjadjaran University C Lai. (2011). Parental marital quality and family environment as predictor of delinquency amongst selected secondary school student in Malaysia. British Journal of Art and Social Science C Lai. (2011). Parental marital quality and family environment as predictor of delinquency amongst selected secondary school student in Malaysia. British Journal of Art and Social Science Creswell, J. (2016). Research Design Qualitative & Quantitative Approach Miles, M.d. (2007). Qualitative Data Analysis. Resource Book About New Methods. Translated by Tjejep Rohindi Rohidi. Jakarta: University of IndonesiaPress. Miles, M.d. (2007). Qualitative Data Analysis. Resource Book About New Methods. Translated by Tjejep Rohindi Rohidi. Jakarta: University of IndonesiaPress. y j j p y Mubasyaroh. (2016). Analysis of the Causes of Early Marriage and Its Impact on y j j p y Mubasyaroh. (2016). Analysis of the Causes of Early Marriage and Its Impact on the Perpetrators. Puspitawati, H. (2013). Introduction to Family Studies. Bogor: IPB Press. Mubasyaroh. (2016). Analysis of the Causes of Early Marriage and Its Impact on the Perpetrators. Puspitawati, H. (2013). Introduction to Family Studies. Bogor: IPB Press. Puspitawati, H. (2013). Introduction to Family Studies. Bogor: IPB Press. Puspitawati, H. (2013). Introduction to Family Studies. Bogor: IPB Press. Sunarti, ET (2005). Effect of economic pressure, social support, quality of marriage, parenting and emotional intelligence of children on children's learning achievement. Family Nutrition and Media. Sunarti, ET (2005). Effect of economic pressure, social support, quality of marriage, parenting and emotional intelligence of children on children's learning achievement. Family Nutrition and Media. Tsunia, N. (2014). Family characteristics, readiness for marriage, wife, and the development of children aged 3-5 years (Thesis). Bogor, Indonesia: Bogor Agricultural Institute. Tsunia, N. (2014). Family characteristics, readiness for marriage, wife, and the development of children aged 3-5 years (Thesis). Bogor, Indonesia: Bogor Agricultural Institute. ISSN : 2686-2239 (online) about how to deal with the difficulties experienced during the role of a wife or mother. Because there are still many who think that socialization related to child care and prevention of marriage at a young age is very lacking. ISSN 2686 2239 ( li ) ISSN : 2686-2239 (online) __________________________________ VOL 3 NO 2 OCTOBER 2022 about how to deal with the difficulties experienced during the role of a wife or mother. Because there are still many who think that socialization related to child care and prevention of marriage at a young age is very lacking. _ VOL 3 NO 2 OCTOBER 2022 c) Internalization This process or stage of internalization will last a lifetime from an individual through socialization, while he contributes to externalization. Individuals seek to understand the definition of objective reality, but more than that, they also contribute to the construction of shared knowledge. So the individual is an active actor as the shaper, maintainer, and changer of society. To find out the truth of one's thoughts, there is a need for proof. In this internalization process, informants who marry at a young age who already have knowledge and readiness about childcare and in living a married life try or try to provide advice, motivation and input to other informants or young married couples out there who are confused about how to take care of themselves. appropriate for children, how to live married life, as well as 53 53 Legislation g Law Number 16 of 2019 concerning Amendments to Law Number 1 of 1974 concerning Marriage. State Gazette of the Republic of Indonesia Year 2019 Number 186, Supplement to the State Gazette of the Republic of Indonesia Number 6401. Central Government. g Law Number 16 of 2019 concerning Amendments to Law Number 1 of 1974 concerning Marriage. State Gazette of the Republic of Indonesia Year 2019 Number 186, Supplement to the State Gazette of the Republic of Indonesia Number 6401. Central Government. g Law Number 16 of 2019 concerning Amendments to Law Number 1 of 1974 concerning Marriage. State Gazette of the Republic of Indonesia Year 2019 Number 186, Supplement to the State Gazette of the Republic of Indonesia Number 6401. Central Government. 54
https://openalex.org/W4223656814	https://www.nature.com/articles/s41598-022-09717-5.pdf	English	null	Circadian motor activity of non-dominant hand reaches acrophase later than dominant hand	Scientific reports	2,022	cc-by	5,543	Circadian motor activity of non‑dominant hand reaches acrophase later than dominant hand OPEN Vincenzo Natale1, Marco Fabbri2, Monica Martoni3 & Lorenzo Tonetti1 Motor activity during the first half of nocturnal sleep is lateralized to the non-dominant hand. What remains is to determine which account could explain this phenomenon: the more pronounced homeostatic deactivation of the dominant hemisphere or the circadian asymmetry in the hemispheric activation. To better understand the nature of these motor asymmetries, we performed an ecological study assessing the circadian motor activity in 34 evening, 52 intermediate, and 27 morning types. We observed a significant circadian phase delay of the 24-h motor activity pattern of the left hand in comparison to the right hand, regardless of chronotype. Moreover, we replicated higher motor activity in the left hand in comparison to the right hand in late evening that reached statistical significance only in evening and intermediate types. Analysing motor activity around bedtime and wake-up time, we observed a reverse pattern between circadian typologies: evening types showed higher activity in the left hand in comparison to the right hand before bedtime, while morning types showed significantly higher motor activity in the right hand in comparison to the left after wake-up time. Results support the hypothesis of a different circadian phase relationship between the two hemispheres. Since the seventies, it has been documented that during the first half of nocturnal sleep the non-dominant hand is more active in comparison to the dominant one1. This phenomenon has been consistently replicated and appears relatively independent of the NREM/REM (Non-Rapid Eye Movement/Rapid Eye Movement) sleep cycle2–4. However, to date there are no shared explanations for such a phenomenon. We currently know that this particular motor asymmetry can be also documented before sleep onset5,6, suggesting that sleep is not a condition sine qua non. q According to the two-process model of sleep regulation, biological and behavioural circadian variations are driven by two interacting processes: the homeostatic process (S), which increases sleep debt with time spent awake and the circadian process (C), which drives a near 24-h endogenous rhythm7. g y Within this theoretical framework, referring to the S process, it has been suggested that the relative superiority of the non-dominant hand during sleep could derive from a more pronounced homeostatic deactivation of the dominant hemisphere8. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ model, a reversed pattern of motor activity asymmetries between right- and left-handed participants was sup- posed, however, once again, the results did not support this.t p g pp To explain the shift in dominant and non-dominant hand motor activity in the early hours of nocturnal sleep, an alternative hypothesis focussing on the C process has been advanced: the shift could derive from a different circadian phase relationship between the two hemispheres6. This hypothesis was backed up by results obtained analysing diurnal performance oscillation: cognitive performances in verbal tasks (involving predominantly the left hemisphere) are better during the morning, while cognitive performances in spatial tasks (involving predominantly the right hemisphere) are better in the evening14,15. Such a hypothesis agrees also with an under- rated result of the experiment conducted by Kattler and co-authors9: after left-hand somatosensory stimulation, the interhemispheric asymmetry index during the first sleep cycle still shifted to the left hemisphere. Moreover, although both right- and left-hemisphere light stimulations attenuated subjective alertness, only the stimula- tion of the right visual cortex was able to trigger a significant reduction in EEG (electroencephalogram) delta activity16. Much research6,12,13 has shown that the mean motor activity in the left hand is higher in comparison to the right between 10:00 p.m. and 11:00 p.m., as if there was some sort of a key time window. Moreover, in right-handed participants, the right hand reached its acrophase slightly but significantly earlier than the left hand (around 12 min)6. Indeed, such data agree with the hypothesis of the existence of a dual circadian pacemaker in the nervous system, which has been proposed several times over the years based on results deriving from biological experiments performed both in animals17 and humans18.f g To separate the role of S and C processes, different experimental paradigms, such as forced desynchrony or constant routine, are possible19. However, these paradigms pose certain problems in evaluating behavioural variables. For this reason, alternative ecological paradigms have been put forward, such as a chronotype-based paradigm that considers the chronotype as an independent variable20. It has also been suggested that chronotype could constitute a unique tool to access the interplay between the S and C processes under normally entrained day-night conditions21. To further investigate ecologically the role of S and C processes on circadian motor activity asymmetry, we decided to analyse the circadian motor activity of evening, intermediate, and morning types. Circadian motor activity of non‑dominant hand reaches acrophase later than dominant hand OPEN In other words, being more active during wakefulness, the dominant hemisphere could present a greater increase in the sleep debt compared to the non-dominant hemisphere and, therefore, would fall asleep more deeply than the non-dominant one. This hypothesis has been tested with the use-dependent recovery function paradigm. Kattler, Dijk and Borbély9 induced an additional passive movement by vibratory stimuli to the hand for six hours before bedtime and found greater Slow Wave Activity (SWA—a physiological marker of sleep homeostasis10) in the contralateral hemisphere during the first hours of sleep in comparison to the baseline. In the same way, Huber and collaborators11 were able to show that arm immobilization during wakefulness for twelve hours caused a local decrease in SWA in subsequent sleep episodes. In a prospective study, actigraphic data were collected before, during, and after a night without sleep12. However, the greater motor activity of the non-dominant hand late in the evening did not increase after sleep deprivation, as predicted by the use-dependent recovery function model, but, in fact, disappeared. Circadian motor activity of the right and left hand was also studied in a sample of right- and left-handed participants13. According to the use-dependent recovery function 1Department of Psychology “Renzo Canestrari”, University of Bologna, Viale Carlo Berti Pichat 5, 40127 Bologna, Italy. 2Department of Psychology, University of Campania Luigi Vanvitelli, Caserta, Italy. 3Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy. email: vincenzo.natale@ unibo.it \| https://doi.org/10.1038/s41598-022-09717-5 Scientific Reports \| (2022) 12:5748 www.nature.com/scientificreports/ www.nature.com/scientificreports/ It is well known that sleep quantity and sleep quality do not distinguish chronotypes, while they differ in the phase of sleep22: morning types go to bed earlier then intermediate types, and intermediate types go to bed earlier than evening types, while for the wake-up time, the reverse pattern is recognised. Therefore, if circadian motor asymmetries are prevalently driven by a S process, we may expect a similar pattern between chronotypes in the late evening, i.e., a higher level of motor activity in the non-dominant hand in comparison to the dominant hand, regardless of bedtime. By contrast, if circadian motor asymmetries are prevalently driven by a C process, we may expect a different pattern between chronotypes, regardless of sleep condition. In other words, motor asym- metries would always be present around 10 p.m., but they would be less evident in morning types because around that time they are already asleep (masking effect). Conversely, motor asymmetries late in the evening should be more evident in evening types because they usually go to bed later than 10 p.m. In order to better understand the role of sleep condition, we examined motor activity near the phases of wake/sleep and sleep/wake transition, regardless of the specific time of day they occurred. Once again, if circadian motor asymmetries are prevalently driven by the S process, we may expect a similar motor asymmetry late in the evening between the chronotypes. By contrast, if circadian motor asymmetries are prevalently driven by the C process, we may expect a different pattern between the chronotypes with a clearer higher mean motor activity in the left hand in evening types. Results Participants were assigned to one of the three groups: morning types (n = 27, 7 males and 20 females, 23.89%), intermediate types (n = 52, 20 males and 32 females, 46.02%) and evening types (n = 34, 14 males and 20 females, 30.09%). Setting the significance level to p < 0.05, the frequency distribution of circadian typology between males and females was not significantly different (χ2 2 = 1.71, p = 0.42). Moreover, chronotype did not differ by age (F2 110 = 1.56—p = 0.21). g ( 2,110 p ) As expected, morning-types (00:11 a.m. ± 0:56) go to bed significantly earlier than intermediate types (01:04 a.m. ± 1:20), and the latter significantly earlier than evening types (01:49 a.m. ± 1:02) (F2,110 = 14.62—p = 0.00001). Likewise, morning types (08:01 a.m. ± 1:03) wake up in the morning significantly earlier than interme- diate types (08:48 a.m. ± 1:18), and the latter significantly earlier than evening types (10:04 a.m. ± 1:31) (F2,110 = 15.44—p = 0.00001). Actigraphic sleep quantity (TST) and sleep quality (SE%) did not significantly differ by chronotype (respectively: 457 ± 55 min. morning, 432 ± 63 min. intermediate, 450 ± 57 min. even- ing types, F2,110 = 1.74—p = 0.18; 95.0 ± 6.0% morning, 95.5 ± 4.4% intermediate, 95.3 ± 5.3% evening types, F2,110 = 0.08—p = 0.92). 2,110 p Results of the analyses on the motor activity pattern during the 24 h are summarized in Table 1a and shown in Fig. 1. Overall, results confirm a small but significant shift over 24 h. In particular, the mean motor activity of the non-dominant hand is higher at around 10:00 p.m. However, such an effect reaches statistical significance only in evening (t33 = − 2.33—p = 0.03) and intermediate types (t52 = − 3.27—p = 0.002). On the contrary, morn- ing types show a significantly higher mean motor activity in the dominant hand at 10 a.m. (t26 = 2.33—p = 0.03) and 11 a.m. (t26 = − 2.49—p = 0.02).ii 26 p As regards the acrophase, the first factor (hand) was significant (F1,110 = 129.22—p = 0.00001). In particular, the right hand reaches the acrophase significantly earlier (04:42 p.m. ± 0:49) than the left hand (05:02 p.m. ± 0:48). As expected, morning types reach their acrophase (04:01 p.m. ± 2:20) significantly earlier than intermediate types (04:44 p.m. ± 1:24), and the latter earlier than evening types (05:51 p.m. Methods Participants. In order to examine the acti- graphic sleep profile, we considered the following measures: the time the participants went to bed and switched https://doi.org/10.1038/s41598-022-09717-5 Scientific Reports \| (2022) 12:5748 www.nature.com/scientificreports/ off the light (bedtime) and the time the participants last woke up in the morning (wake-up time); total sleep time (TST) (sum, in minutes, of all sleep epochs between sleep onset and sleep end); sleep efficiency percentage (SE%) (the ratio of total sleep time to time in bed multiplied by 100). For each participant, the mean values were calculated over the three nights. off the light (bedtime) and the time the participants last woke up in the morning (wake-up time); total sleep time (TST) (sum, in minutes, of all sleep epochs between sleep onset and sleep end); sleep efficiency percentage (SE%) (the ratio of total sleep time to time in bed multiplied by 100). For each participant, the mean values were calculated over the three nights. Actigraphic motor activity. To evaluate motor activity, actigraphic data were extracted using the version 1.16 of Action 4 software (Ambulatory Monitoring, Inc., Ardsley, NY). The actigraphic recording was divided into 60-min intervals starting from 16:00 h and the hourly mean activity levels over the 24 h were calculated for each participant. Moreover, the MESOR and acrophase were computed using cosinor analyses, which are implemented within the Action 4 software. Cosinor analysis is a statistical technique specifically developed for the study of cyclic functions30. For each participant, the mean values were computed based on 48-h recordings. y y p p p g We also extracted hourly motor activity data considering the hours close to bedtime (i.e., four hours before and four hours after bedtime) and wake-up time (i.e., four hours before and four hours after wake-up time). Statistical analyses. Gender and age differences in chronotype were explored with a chi-squared test and an analysis of variance, respectively.hf y p y The chronotype differences in actigraphic sleep parameters were analysed through a set of analysis of variance with chronotype as independent variable and each actigraphic sleep parameter as dependent variable. y g To analyse the motor activity pattern during the 24 h, a mixed three-way analysis of variance (ANOVA) was performed: hand (two levels: right and left) (within-subjects factor); time of day (24 levels) (within-subjects factor); chronotype (three levels: morning, intermediate, evening type) (between-subjects factor). Methods Participants. yp g g yp j As regards the acrophase and MESOR, we performed a mixed two-way ANOVA: hand (two levels: right and left) (within-subjects factor); chronotype (three levels: morning, intermediate, evening types) (between-subjects factor). To analyse the motor activity pattern during the wake-sleep transition (bedtime), a mixed three-way ANOVA was performed: hand (two levels: right and left) (within-subjects factor); time (8 levels: four hours before and four hours after bedtime) (within-subjects factor); chronotype (three levels: morning, intermediate, evening types) (between-subjects factor). To analyse the motor activity pattern during the sleep–wake transition (wake up time), a mixed three-way ANOVA was performed: hand (two levels: right and left) (within-subjects factor); time (8 levels: four hours before and four hours after wake-up time) (within-subjects factor); chronotype (three levels: morning, intermediate, evening types) (between-subjects factor). Methods Participants. Participants. A sample of 113 healthy university students (41 males, 72 females), mean age = 23.9 ± 4.2, were enrolled as volunteers in the study. After a brief description of the study, participants read and signed a written informed consent form. All participants were all right-handed, as assessed by the Italian version23 of the Edinburgh Handedness Inventory24. Participants were classified as evening, intermediate, or morning types according to the cut-off scores of the Italian version25 of the reduced Morningness-Eveningness Questionnaire (MEQr)26, i.e., 4–10, 11–18, and 19–25, respectively. The MEQr is composed of 5 items taken from the 19-item version of the MEQ27. Since MEQr proved to have good external validity and good discriminant ability between extreme chronotypes, its use has been proposed within the research field28. The MEQr was given at the end of the study so that participants’ behaviour would not be influenced in any way. Actigraphy and procedure. Actigraphic recordings were obtained using the Micro MotionloggerWatch actigraph (Ambulatory Monitoring Inc., Ardsley, NY). Devices were initialized for zero crossing mode to col- lect data in 1-min epochs. Participants wore an actigraph on each wrist for three consecutive nights (excluding Saturday and Sunday) to obtain at least 48 consecutive hours of reliable data. Participants were free to spend their daytime hours and sleep time out of the laboratory. They were also instructed to push the actigraph event marker to signal when they went to bed and woke up in the morning. Each participant provided the written informed consent before being enrolled in this study, carried out during the autumn 2021, that was approved by the Bioethics Committee of the University of Bologna (prot. n. 284786) and carried out in accordance with the Declaration of Helsinki. Actigraphic sleep parameters. To evaluate sleep features, actigraphic data were analysed using Action W-2 (version 2.7) software (Ambulatory Monitoring, Inc., Ardsley, NY). This software identified each epoch as sleep or wakefulness using the mathematical model validated by Cole and co-authors29. According to such model, sleep onset was defined as the first epoch of the first block of 20 min of persistent sleep, while sleep offset as the end of the last sleep episode within the interval of the time spent in bed. Results ± 1:24) (F2,110 = 9.64—p = 0.0001). The interaction between the two factors was not significant. No significant effects were observed on the MESOR. gi gif As regards the motor activity pattern during the wake-sleep transition (sleep onset), results are summarized in Table 1b and shown in Fig. 2. Overall, the results confirmed a higher mean motor activity level in the left hand in comparison to the right hand before sleep. Performing the Tuckey post-hoc test, such a difference is significant only in evening (− 4 h, p = 0.005; − 2 h, p = 0.002; − 1 h, p = 0.01) and intermediate types (− 3 h, p = 0.00006; − 2 h, p = 0.0001). p p Results about the motor activity pattern during the sleep–wake transition (awakening) are summarized in Table 1c and shown in Fig. 3. Post hoc analyses (Tuckey test) only showed a significant difference in morning types with higher mean motor activity in the right hand in comparison to the left (+ 3 h, p = 0.00005). Scientific Reports \| (2022) 12:5748 \| https://doi.org/10.1038/s41598-022-09717-5 www.nature.com/scientificreports/ Table 1. Statistics of the mixed three-way analysis of variance on motor activity pattern during the 24 h (circadian, a), wake-sleep transition (sleep onset, b), and sleep–wake transition (wake-up, c). Results F value Significance (a) Circadian 1 chronotype F2,110 = 0.51 p = 0.60 2 right-left hand F1,110 = 0.34 p = 0.56 3 time of day F23,2530 = 227.84 p = 0.00001 1 × 2 F2,110 = 1.57 p = 0.21 1 × 3 F46,2530 = 6.83 p = 0.00001 2 × 3 F23,2530 = 3.01 p = 0.00001 1 × 2 × 3 F46,2530 = 1.34 p = 0.06 (b) Sleep onset 1 chronotype F2,110 = 2.8 p = 0.06 2 right-left hand F1,110 = 7.0 p = 0.009 3 time F7,770 = 1349 p = 0.00001 1 × 2 F2,110 = 2.0 p = 0.13 1 × 3 F14,770 = 1.7 p = 0.06 2 × 3 F7,770 = 3.5 p = 0.001 1 × 2 × 3 F14,770 = 3.2 p = 0.0001 (c) Wake up 1 chronotype F2,110 = 0.3 p = 0.72 2 right-left hand F1,110 = 0.9 p = 0.33 3 time F7,770 = 1930.0 p = 0.00001 1 × 2 F2,110 = 2.4 p = 0.09 1 × 3 F14,770 = 0.9 p = 0.51 2 × 3 F7,770 = 4.6 p = 0.0001 1 × 2 × 3 F14,770 = 1.8 p = 0.03 F value Significance (a) Circadian 1 chronotype F2,110 = 0.51 p = 0.60 2 right-left hand F1,110 = 0.34 p = 0.56 3 time of day F23,2530 = 227.84 p = 0.00001 1 × 2 F2,110 = 1.57 p = 0.21 1 × 3 F46,2530 = 6.83 p = 0.00001 2 × 3 F23,2530 = 3.01 p = 0.00001 1 × 2 × 3 F46,2530 = 1.34 p = 0.06 (b) Sleep onset 1 chronotype F2,110 = 2.8 p = 0.06 2 right-left hand F1,110 = 7.0 p = 0.009 3 time F7,770 = 1349 p = 0.00001 1 × 2 F2,110 = 2.0 p = 0.13 1 × 3 F14,770 = 1.7 p = 0.06 2 × 3 F7,770 = 3.5 p = 0.001 1 × 2 × 3 F14,770 = 3.2 p = 0.0001 (c) Wake up 1 chronotype F2,110 = 0.3 p = 0.72 2 right-left hand F1,110 = 0.9 p = 0.33 3 time F7,770 = 1930.0 p = 0.00001 1 × 2 F2,110 = 2.4 p = 0.09 1 × 3 F14,770 = 0.9 p = 0.51 2 × 3 F7,770 = 4.6 p = 0.0001 1 × 2 × 3 F14,770 = 1.8 p = 0.03 Table 1. Discussionh The results of actigraphic sleep quality and quantity, and the influence of chronotype on sleep phase agree with the literature22.iit As regards mean motor activity, we confirmed a slight but significant shift (ranging between 15 and 20 min) in the acrophase between the dominant and non-dominant hand late in the evening regardless of chronotype. Looking at mean motor activity over the 24 h, such a phenomenon reaches statistical significance only in evening and intermediate types. It is as if sleep, which occurs much earlier in morning types than the other two circadian types, masks the phenomenon. This interpretation is strengthened by the observation that when we synchronize mean motor activity with wake-sleep transition (sleep onset) such a difference between circadian typologies becomes even more evident. When we synchronize mean motor activity with sleep–wake transition (wake-up) such a difference between circadian typologies becomes specular, i.e., we can see a clearly higher mean motor activity in the dominant hand in morning types but not in evening types. In conclusion, our results seem to indicate that the relative superiority of the non-dominant hand movements late in the evening could derive from a different circadian phase relationship between the two hemispheres, and that sleep differently masks such a phenomenon depending on the sleep phase (chronotype). Within the theoreti- cal framework of the two-process model of sleep regulation, we could speculate that the left hemisphere is more sensitive to the S process, and for this reason “turns off” before the right hemisphere. On the other hand, the right hemisphere could be more sensitive to C processes and continues its activity late in the evening unaffected by the sleep debt accumulated during the day. Furthermore, we could hypothesize that the left hemisphere is in charge of governing S processes, while the right hemisphere is designated to drive the C process. If this is so, we could conclude that chronotype derives from a different hemispheric balance. Morning types could be more sensitive to the left hemisphere (i.e., S process) and for this reason go to bed early. Evening types could be more sensitive to the right hemisphere (i.e., C process) and for this reason do not go to bed when sleep debt reaches a high level. Results Statistics of the mixed three-way analysis of variance on motor activity pattern during the 24 h (circadian, a), wake-sleep transition (sleep onset, b), and sleep–wake transition (wake-up, c). Figure 1. Hourly mean motor activity over the 24 h of the right and left hand in evening, intermediate, and morning types. The white horizontal bar represents the time spent in bed by each chronotype, with the extremes pointing to the bedtime and get-up time. Figure 1. Hourly mean motor activity over the 24 h of the right and left hand in evening, intermediate, and morning types. The white horizontal bar represents the time spent in bed by each chronotype, with the extremes pointing to the bedtime and get-up time. https://doi.org/10.1038/s41598-022-09717-5 Scientific Reports \| (2022) 12:5748 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Hourly mean motor activity, over the time interval from four hours prior to bedtime (− 4, − 3, − 2, − 1) to four hours after bedtime (+ 1, + 2, + 3, + 4), of right and left hand in evening, intermediate, and morning types. Figure 2. Hourly mean motor activity, over the time interval from four hours prior to bedtime (− 4, − 3, − 2, − 1) to four hours after bedtime (+ 1, + 2, + 3, + 4), of right and left hand in evening, intermediate, and morning types. Figure 3. Hourly mean motor activity, over the time interval defined by the four hours before (− 4, − 3, − 2, − 1) and four hours after (+ 1, + 2, + 3, + 4) the get-up time, of the right and left hand in evening, intermediate, and morning types. Figure 3. Hourly mean motor activity, over the time interval defined by the four hours before (− 4, − 3, − 2, − 1) and four hours after (+ 1, + 2, + 3, + 4) the get-up time, of the right and left hand in evening, intermediate, and morning types. https://doi.org/10.1038/s41598-022-09717-5 Scientific Reports \| (2022) 12:5748 \| www.nature.com/scientificreports/ Discussionh Such a conclusion partially agrees with previous research that documented morning-types as having more SWA during the first cycle21 and a steeper slope of Slow Waves when homeostatic sleep pressure is high31 in comparison to evening types. Moreover, such a conclusion also agrees with the observation that morning types show a more marked left-thinking style while evening types are more right-thinkers32. t g y g yp g Our conclusions are prevalently speculative and further studies are needed to understand the origin and significance of circadian motor activity asymmetry. The idea that chronotype may arise from a different com- bination of C and S influences is not new33–35. According to Monk33, the interaction between the two processes contributes to the generation of the wake-sleep behavior pattern based on the needs of the circadian system and environmental demands. The greater role of process C in evening types is compatible with their higher adapt- ability to phase change of sleep in comparison to morning-types, while a lesser role of process C in morning types might explain why they are more affected by changes in the sleep–wake cycle22.h f The strength of this study is certainly its ecology: we studied the participants while they continued to carry out their daily activities outside of the laboratory. At the same time, this is also its principal limitation because the ecological approach should always suggest caution in separating the role of S and C processes20. Furthermore, among the limitations the lack of the assessment of potential gender differences, due to the relatively small size of the sample, can be quoted. Such limitation could be overcome by future studies on samples larger in size. Data availabilityh The data underlying this article cannot be shared publicly for the privacy of individuals that participated in study. 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The assessment and analysis of handedness: The Edinburgh inventory. Neuropsychologia 9, 97–113 (1971). ihh 25. Natale, V., Esposito, M. J., Martoni, M. & Fabbri, M. Validity of the reduced version of the Morningness–eveningness questionn Sleep Biol. Rhythms. 4, 72–74 (2006). 6. Adan, A. & Almirall, H. Horne and Ostberg morningness–eveningness questionnaire: A reduced version. Personal. Individ. Dif 12, 241–253 (1991). Ö 7. Horne, J. A. & Östberg, O. A self-assessment questionnaire to determine morningness–eveningness in human circadian rhythms Int. J. Chronobiol 4, 97–110 (1976). 28. Di Milia, L., Adan, A., Natale, V. & Randler, C. Reviewing the psychometric properties of contemporary circadian typology meas- ures. Chronobiol. Int. 30, 1261–1271 (2013).i ures. Chronobiol. Int. 30, 1261–1271 (2013). 9. Cole, R. J., Kripke, D. Author contributions V.N. designed the study, collected the data, performed the statistical analyses, and wrote a first draft of the manuscript. M.F., M.M., and L.T. collected the data, reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript. © The Author(s) 2022 Additional information Correspondence and requests for materials should be addressed to V.N. Correspondence and requests for materials should be addressed to V.N. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W2945054721	https://revistas.pucsp.br/index.php/cognitiofilosofia/article/download/40877/27839	English	null	All is not Vanity: William James versus Ernest Renan	Cognitio	2,019	cc-by	9,081	1 Richard Shusterman at the beginning of his paper, 2012, p. 433-454, gives some detail of the Strenuous mood concept from a conceptual point of view. It omits, however, a fundamental text from James to understand this notion, namely: The energies of Man. This omission can be saved by reading the article by Patrick K. Dooley, 2001, p. 18-28. For an approach to the cultural field where it emerges see SPEZIALE, 1980-1981, and COLELLA, 2016. On the subject of virility see: TOWNSEND, 1996. On the economic- energetic language of Neurasthenia and its relation to moral vigor see: LUTZ, 1991. http://dx.doi.org/10.23925/2316-5278.2018v19i2p242-257 http://dx.doi.org/10.23925/2316-5278.2018v19i2p242-257 All is not Vanity: William James versus Ernest Renan Nem tudo é vaidade: William James versus Ernest Renan José Jatuff Universidad Nacional de La Rioja – Argentina josejatuffdj@hotmail.com José Jatuff Universidad Nacional de La Rioja – Argentina josejatuffdj@hotmail.com Abstract: In James’ work, there is an explicit reaction against Renan’s insincerity and vanity as the dominant moral tone. The way in which James judges Renan in particular, and the Latin spirit in general, is related to an early identification with the German spirit through his Protestant background. Within this framework, we will see that through Carlyle’s figure, James opposes the objective moral of work to Renan’s interior gnostic sensitivity. Since there exists an overt link between Carlyle and Calvinism, the component of Protestant ethics in James’ proposal becomes manifest. Consequently, the purpose of this paper is to show that strenuous mood, as a characteristic of courage and manhood, has a Protestant tone. Keywords: Carlyle. Ethics. Protestantism. Renan. Strenuous mood. Resumo: Na obra de James, há uma reação explícita contra a falsidade e a vaidade como o tom moral dominante. O modo como James julga Renan em particular, e o espírito latino em geral, está relacionado a uma identificação inicial com o espírito germânico através de um contexto protestante. Dentro dessa estrutura, nós veremos que por meio da figura de Carlyle, James opõe-se à moral objetiva da obra para com a sensibilidade gnóstica interior de Renan. Visto que há uma conexão óbvia entre Carlyle e o Calvinismo, o componente da ética protestante na proposta de James torna-se manifesta. Consequentemente, o propósito deste artigo é mostrar que o humor extenuante, como uma característica de coragem e virilidade, possui um tom protestante. Palavras-chave: Carlyle. Ética. Humor extenuante. Protestantismo. Renan. 2 JAMES, 1920, p. 326. 3 Idem, p. 326. 1 Introduction Our purpose is to reveal through different papers the various components that constitute the concept of strenuous mood,1 which is a key to understanding the Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 242 242 All is not Vanity: William James versus Ernest Renan heroic aspect of William James’s ethics. In Europe in the 19th century, different intellectuals opposed the already well-established cultural tendencies that focus on the group, a set of reflections tending to highlight individuals and exceptional attitudes giving shape to what today is known as the heroic Victorian criticism of mass society. In the United States, the reflection about men and exceptional attitudes takes its own shape towards the end of this century, mainly influenced by the energetic language of the entropy theory and by a Darwinian biological model with touches of Zola’s naturalism. Reflections on vigor, struggle, commitment, manhood and strong attitudes are common in the United States and seem to respond to a need to deal with the mediocrity and relaxation of the civilized bourgeois life of the time. By using his skill as a psychologist, James takes part in this debate without abandoning moral exhortation. Hereafter, some of the components of strenuous mood will be highlighted, evidencing it as a key concept of James’s ethical proposal by showing his moral opposition to Ernest Renan and the strategic way in which he uses Thomas Carlyle to present his own position in which objective vigor opposes futile sentimentality. 2 JAMES, 1920, p. 326. 2 The path of the objective attitude In his essay on ethics written one year earlier, he says that infinite demand leads to “strenuous mood” penetrated by a “tragic urgency.” Throughout history, he adds, we find on the one hand antagonism between Puritanism, the energy attitude, and the ethics of the infinite and, on the other, the careless temper, the genial moods, the ethics of prudence, and the satisfaction of the merely finite.4 The attitude of irony and persiflage as well as the attitude of careless and easy going temper are opposed, in different ways, to the ethical mood that defines James’s thinking and personality. In his Manuscript Lectures (1880-1881), he states: “Religion means: not everything is vanity,”5 and about twenty years later, in conference two of The Varieties of Religious Experience, he explains this idea when he analyzes human beings’ attitude towards the universe as a whole: renouncement. James knows them, yet, maintains that moral will acquires its highest performance when linked to a religious ideal that demands sacrifice. In his essay on ethics written one year earlier, he says that infinite demand leads to “strenuous mood” penetrated by a “tragic urgency.” Throughout history, he adds, we find on the one hand antagonism between Puritanism, the energy attitude, and the ethics of the infinite and, on the other, the careless temper, the genial moods, the ethics of prudence, and the satisfaction of the merely finite.4 The attitude of irony and persiflage as well as the attitude of careless and easy going temper are opposed, in different ways, to the ethical mood that defines James’s thinking and personality. 4 JAMES, 2009, p. 252. 5 JAMES, 1988, p. 177. 6 JAMES, 1917, p. 36. 7 Idem, p. 39. 8 Ibid., p. 36. 2 The path of the objective attitude In a letter to Salter W. M. from Florence on October 6, 1892— at the time James was spending five months with his family in Europe—he says about Renan’s death: “So wizard Renan is no longer among us!”2 In relation to the great French intellectual, in different sections of James’ writings we come across a complex treatment of recognition and criticism: a considerable respect for his artistic qualities, but a strong rejection of his moral attitude. What James finds harmful in Renan—and also partly in the Latin spirit—defines his own seriousness. The queer thing was that he so slowly worked his way to his natural mental attitude of irony and persiflage, on a basis of moral and religious material. He levitated at last to his true level of superficiality, emancipating himself from layer after layer of the inhibitions into which he was born, and finally using the old moral and religious vocabulary to produce merely musical and poetic effects. That moral and religious ideals, seriously taken, involve certain refusals and renunciations of freedom, Renan seemed at last entirely to forget. On the whole, his sweetness and mere literary coquetry leave a displeasing impression, and the only way to handle him is not to take him heavily or seriously. The worst is, he was a prig in his ideals […].3 The attitude of “irony” and “persiflage” opposes the moral and religious ideals that always involve “prohibition” and “renouncement.” It is noteworthy that, although these may be the dominant notes of some traditional religions, it is not so easy to assert the same of morality. Complete systems in moral matters do not presuppose these traits, or otherwise propose an economy of pleasures rather than Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 243 Cognitio: Revista de Filosofia renouncement. James knows them, yet, maintains that moral will acquires its highest performance when linked to a religious ideal that demands sacrifice. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 9 Ibid., p. 38. 10 Ibid., p. 38. 11 About this point and the James-Santayana relation see: DEL CASTILLO, 2011. 12 JAMES, 1987, p. 327. 2 The path of the objective attitude Give ourselves up, according to the hour, to confidence, to skepticism, to optimism, to irony, and we may be sure that at certain moments at least we shall be with the truth […]. Good-humor is a philosophic state of mind; it seems to say to Nature that we take her no more seriously than she takes us. I maintain that one should always talk of philosophy with a smile. We owe it to the Eternal to be virtuous; but we have the right to add to this tribute our irony as a sort of personal reprisal. In this way we return to the right quarter jest for jest; we play the trick that has been played on us. Saint Augustine’s phrase: Lord, if we are deceived, it is by thee! remains a fine one, well suited to our modern feeling. Only we wish the Eternal to know that if we accept the fraud, we accept it knowingly and willingly. We are resigned in advance to losing the interest on our investments of virtue, but we wish not to appear ridiculous by having counted on them too securely.9 wise. In utrumque paratus, then. Be ready for anything—that perhaps is wisdom. Give ourselves up, according to the hour, to confidence, to skepticism, to optimism, to irony, and we may be sure that at certain moments at least we shall be with the truth […]. Good-humor is a philosophic state of mind; it seems to say to Nature that we take her no more seriously than she takes us. I maintain that one should always talk of philosophy with a smile. We owe it to the Eternal to be virtuous; but we have the right to add to this tribute our irony as a sort of personal reprisal. In this way we return to the right quarter jest for jest; we play the trick that has been played on us. Saint Augustine’s phrase: Lord, if we are deceived, it is by thee! remains a fine one, well suited to our modern feeling. Only we wish the Eternal to know that if we accept the fraud, we accept it knowingly and willingly. 2 The path of the objective attitude In his Manuscript Lectures (1880-1881), he states: “Religion means: not everything is vanity,”5 and about twenty years later, in conference two of The Varieties of Religious Experience, he explains this idea when he analyzes human beings’ attitude towards the universe as a whole: This sense of the world’s presence, appealing as it does to our peculiar individual temperament, makes us either strenuous or careless, devout or blasphemous, gloomy or exultant, about life at large; and our reaction, involuntary and inarticulate and often half unconscious as it is, is the completest of all our answers to the question, “What is the character of this universe in which we dwell?6 From this perspective, religion is defined by a type of attitude that makes human beings become “solemn, serious, and tender […] If glad, it must not grin or snicker; if sad, it must not scream or curse.”7 The opposite of this seriousness and solemnity towards the whole world that is qualified as religious is exemplified by the attitude, who cares? All is vanity. Two authors are relevant here: Voltaire and Renan. In his old age, the former confesses to a friend: “I get a hundred pike-thrusts, I return two hundred, and I laugh. […] I can look upon the world as a farce even when it becomes as tragic as it sometimes does, all comes out even at the end of the day, and all comes out still more even when all the days are over.”8 But the one who defines vanity as an attitude more clearly is Renan, who asserts: There are many chances that the world may be nothing but a fairy pantomime of which no God has care. We must therefore arrange ourselves so that on neither hypothesis we shall be completely wrong. We must listen to the superior voices, but in such a way that if the second hypothesis were true we should not have been too completely duped. If in effect the world be not a serious thing, it is the dogmatic people who will be the shallow ones, and the worldly minded whom the theologians now call frivolous will be those who are really Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 244 244 All is not Vanity: William James versus Ernest Renan wise. In utrumque paratus, then. Be ready for anything—that perhaps is wisdom. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 […] to collaborate in the fraud which lies at the base of things; the finest function of genius is to be the accomplice of God, to connive at the eternal policy, to assist in spreading the traps and nets, to help to deceive individuals for the good of the aggregate, to be the instrument of this grand illusion by 11 About this point and the James-Santayana relation see: DEL CASTILLO, 2011. 12 JAMES, 1987, p. 327. 2 The path of the objective attitude 2018 245 Cognitio: Revista de Filosofia preaching virtue to men, while he knows all the time that they shall draw no profit from it.13 In this theology, God’s place is occupied by a deity, a mixture of genius of the species and nature in the cold and modern sense of the term, but which, as we see in the second dialogue, has another end in addition to perpetuating itself as a wheel. In James’s words, “The savant, the philosopher, is what, through all her sidereal systems, Nature is seeking to form.”14 Philosophy is the end of creation, but it is extremely difficult and rare. When it appears, it should occupy the center of the historical scene; however, this is not so. The philosopher used to live off princes’ crumbs and now lives off the world’s crumbs. But it is good for this to happen. It is right for the superficial and selfish spirits of the time to occupy a central spot. By dedicating themselves to consuming world’s banality, they unconsciously collaborate to making the philosopher—who opposes them both in taste and lifestyle—remain locked in his library, where he should be. In the third dialogue, we find an apocalyptic view of the current course of history and terror of the Commune. The only positive aspect that allows us to enjoy Renan, James says, is the image of a “League of Sages” (league of savants) who will keep the world in order through fear and may be called gods. At the end, it seems that the intellectual oligarchy and the commoners shake hands in terror: “Fear, mistrust of time and the persuasive force of what is good, seem to be ingrained in the bones of most of the present generation of Frenchmen […].”15 The other texts of the volume, James says, have the same tone, the same virtues, and the same flaws than these, and their only quality is sincerity, which in this case, is not favorable. Renan even boasts of being a stranger in his time, someone who has no hope (sans espérance)16 and who can stand at a futile distance. “The true atheist is a frivolous man” is one of his most quoted statements. 13 Idem, p. 329. 14 Ibid., p. 329. 15 Ibid., p. 330. 16 Ibid., p. 331. 17 Idem, p. 331. 2 The path of the objective attitude We are resigned in advance to losing the interest on our investments of virtue, but we wish not to appear ridiculous by having counted on them too securely.9 James concludes that religion attacks such “chaffing” talk from Renan, favoring “gravity” over “pertness;” “it says hush to all vain chatter and smart wit”.10 As Santayana said, for his former teacher, the first thing to be erased is the cynical attitude, regardless of whether it is true or false.11 James concludes that religion attacks such “chaffing” talk from Renan, favoring “gravity” over “pertness;” “it says hush to all vain chatter and smart wit”.10 As Santayana said, for his former teacher, the first thing to be erased is the cynical attitude, regardless of whether it is true or false.11 The last Renan is the one that James rejects the most. Therefore, his review of the Dialogues et Fragments Philosophiques (1876) reveals his rejection of Renan’s cynical and arrogant attitude as well as his own position. Anyone who believes that France still remains robust and fertile, he tells us, only has to read this work to realize that it is nothing else than a monument to mental ruin, dandyism and insincerity. The dialogues are simply “priggishness rampant, an indescribable unmanliness of tone compounded of a sort of histrionically sentimental self-conceit, and a nerveless and boneless fear of what will become of the universe if l’homme vulgaire is allowed to go on.”12 The notion of God is replaced by a cold-blooded destiny, and none of the characters in the dialogues fully represent his thought, but just different parts of his brain remaining skeptical in the end. In the first dialogue, he states that even though the universe is a fatal mechanism, it is also true that there is a final purpose in which we all collaborate by pursuing our own goals, and that the task of the great intellectual who understands the mechanism is […] to collaborate in the fraud which lies at the base of things; the finest function of genius is to be the accomplice of God, to connive at the eternal policy, to assist in spreading the traps and nets, to help to deceive individuals for the good of the aggregate, to be the instrument of this grand illusion by Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 2 The path of the objective attitude But already in his ‘Antichrist,’ published after the Commune, he spoke of the summit of wisdom being the persuasion that at bottom all is vanity; and if this book be really half trifling, he would seem practically to have espoused that persuasion—in other words, to have become a frivolous man, or, according to his own definition, an atheist. Indeed, if one were to seek a single phrase which should define the essence of religion, it would be the phrase: all is not vanity. The solace and anæsthetic which lies in the conclusion of Ecclesiastes is good for many of us; but M. Renan’s ostentatious pretension to an exquisite sort of religious virtue has debarred him from the right to enjoy its comforts.17 246 246 All is not Vanity: William James versus Ernest Renan Although decomposition or decay ruin Renan’s mind, to James the worst thing is the immense pedantry of the person who witnesses the worst scene from a distance—becoming an accomplice of its suspected artisan—with a sardonic smile. And all this is based on an aristocratic sensitivity! “This cannot be the best of men. The political or spiritual hero will always be the one who, when others crumbled, stood firm till a new order built itself around him […].”18 The review clearly demands a little more vigor when faced with crisis: “[…] must not take it hard if we insist on a little more courage in him when the wind begins to blow.”19 However, vanity, cynicism and derision are part of the wide gap between Renan and James. The question of virility and vigor is gravitating here, or in other words, the difference is expressed in these terms. The signifiers used are quite clear. On the one hand, we have the “feminine cowardice” of the person who “butterflies” a “pink optimism”, and on the other, that who forgets his “emotions” and “complaints” “and gets to work as a man.”20 We have noticed two sets of well-defined features both in his Letters and in his works. On the one hand, there is the Puritan, energetic, courageous and vigorous attitude; solemn, serious, tender and bravely masculine. On the other hand, there is the feminine cowardice and insincerity of the cynical attitude that in relaxation yields to vanity. These two opposing columns cannot be defined clearly since the features mentioned above are not always present. 20 What divided James from Santayana is complex. It is linked to the ideal of life that each one holds and, for that reason, not to be treated here, but we find between that which divides them something that is of the utmost importance to enrich the already mentioned signifiers. “Santayana introduces himself to James’s class at Harvard in 1883 and his first impression is that James considers him too weak for philosophy. The first thing he asks is, Are you sure you’re interested in philosophy? Do you really want to dedicate yourself to philosophy? […] Santayana must have had a hard time. “I still have the broken gesture […] I must have seemed weak and not promising,” he told to James’s secretary, Daniel Cory. “Santayana,” commented Cory, “always suspected that this professor of ‘brave masculinity’ perceived some deficiency in him and from that very moment he did not cease to feel uncomfortable and not wanted.” It seems that Santayana (reader of Petrarch and lover of the Classics in general, asking from this perspective about the “good,” “excellent” and “beautiful” human existence—Latin in a Protestant world—with another emotional and sexual structure) does not fit in the virility of the strenuous mood. See DEL CASTILLO, 2011, p. 294. 18 Ibid., p. 332. 19 Ibid., p. 330. Italics added by the author. 19 Ibid., p. 330. Italics added by the author. See ELIAS, 2001. 2 The path of the objective attitude However, it is quite clear that the general framework of the opposition between vain, affected sentimentality and frank, committed vigor responds to the 19th century debate between the Latin and Germanic spirit.21 Although in James’s work, such a debate does not appear, we find, however, many comments and additions that place him on the German side. With admiration he comments, for example, on the mighty powerful construction of German morality in which life dramas hardly ever have any effect. In his 1882 journey, he states that Americans only lack the German abdominal depth of temperament. We have seen how Puritan ethics is considered vigorous and we know that in relation to Renan’s death, James speaks about his vanity, superficiality 21 21 See ELIAS, 2001. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 247 247 Cognitio: Revista de Filosofia and cynicism as well as the French intellectuals’ of his time who have generally fallen into a “bitter decay.” He says the same about Florence in October, the year of Renan’s death (1892). In a letter, he writes that “Germany is good but Switzerland is better”, so good—he continues—that it is indescribable; their healthiness is beyond words: “the roads, the mountains, the customs, the institutions, the people.” Not a breath of art, poetry, esthetics, morbidness, or suggestions! It is all there, solid meat and drink for the sick body and soul, ready to be turned to, and do you infallible good when the nervous and gas-lit side of life has had too much play. What a see-saw life is, between the elemental things and the others! We must have both; but aspiration for aspiration, I think that of the over-cultured and exquisite person for the insipidity of health is the more pathetic. After the suggestiveness, decay and over- refinement of Florence this winter, I shall be hungry enough for the eternal elements to be had in Schweiz.22 Though he inclines towards the ascetic Protestant lifestyle of those countries, Florence annoys him with its note of exuberance and sensuality he considers “decadent.” To understand James’s rejection of Florence, we have to assume and understand his personal preference for Protestant solemnity, purity, and sanity, or else his appraisals sound absolutely arbitrary. In another letter written in October, he says that although Florence is “delicious,” he has an “organic protest” against certain things there. 22 JAMES, 1920, p. 328. Italics added by the author. 23 Idem, p. 330. Italics added by the author. 24 Ibid., p. 342. Italics added by the author. 25 Ibid., p. 342. Italics added by the author. 26 CUCHE, 1992, p. 12. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 2 The path of the objective attitude He feels that the air is lacking outside as if he were breathing in a closed environment. “The general debility; which pervades all ways and institutions, the worn-out faces, etc., etc.”23 In March 1893, he wrote to Henry—with whom he argues all the time on the opposite views they both have on these places—that his tourist gaze allows him to enjoy Italy while among those who have been living there for a while, “the sweet decay breathed in for six months has produced a sort of physiological craving for a change to robuster air.” He keeps saying that although Italy has fulfilled its role very well, there are times when “the Florentine debility becomes really hateful to one” and ends up congratulating Henry for choosing the strongest milieu.24 In June of the same year while in the United States, he complains: “There is a strange thinness and femininity hovering all over America, so different from the stoutness and masculinity of land and air and everything in Switzerland and England […].”25 We have emphasized these appreciations between the German and Latin cultures because their characterizations partly respond to the 19th century debate on Culture versus Civilization. In the French vocabulary of the 18th century, the concepts of ‘culture’ and ‘civilization’ “belong to the same semantic field and reflect the same fundamental conceptions,”26 although with reference to the individual and the collective Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 248 248 All is not Vanity: William James versus Ernest Renan respectively. The concept of civilization becomes dominant in France because the philosophers of Enlightment support a government based on knowledge and reason. The term Kultur appears in Germany in the 17th as the transposition of the French term, although it evolves in a very different way and quickly becomes a sign of distinction and opposition to the “French civilization.” French manners were a model for the German court, and many bourgeois intellectuals opposed the so- called “spiritual” values to “courtly” values. 27 Idem, p. 15. 28 Ibid., p 15. 29 JAMES, 2009, p. 208. 30 Ibid., p. 208. 31 Ibid., p 211. 32 Ibid., p 212. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 2 The path of the objective attitude 2018 249 Cognitio: Revista de Filosofia our performances and our violations of duty are for a common purpose, the attainment of subjective knowledge and feeling, and that the deepening of these is the chief end of our lives,— and at what point on the downward slope are we to stop?33 our performances and our violations of duty are for a common purpose, the attainment of subjective knowledge and feeling, and that the deepening of these is the chief end of our lives,— and at what point on the downward slope are we to stop?33 In practical terms, this leads to sentimentality or unlimited sensuality, a current that leads one to seek to experience everything, James concludes. To his judgement, we find nothing else in the romantic school of Paris. Both Renan and Zola, though with different sensitivities, cultivate an aesthetic that has nothing to say when the hour of restlessness comes: “[…] under the pages of both there sounds incessantly the hoarse bass of vanitas vanitatum, omnia vanitas, which the reader may hear, whenever he will, between the lines.”34 Zola is attributed his own, but to our purpose, what he says about Renan is quite revealing: He “[…] plays the coquette between the craven unmanliness of his Philosophic Dialogues and the butterfly optimism of his Souvenirs de Jeunesse.”35 Renan’s insincere and unmanly attitude seems to incarnate, at least in part, the decadence attributed to the Latin spirit by Germanic criticism, and although it is evident that, in general terms, James’s character and ethical proposal are contrary to this attitude, in the text The dilemma of Determinism, his virile proposal becomes concrete in the figure of Thomas Carlyle. In a very illuminating essay—somewhat forgotten nowadays—Josiah Royce (1855-1916) points, among other things, to what he considers the foundation of James’s moral proposal, where what James calls the philosophy of objective behavior is roughly clear. This proposal—that is, as “one awakens from some feverish dream, full of bad lights and noises, to find one’s self bathed in the sacred coolness and quiet of the air of the night”36—has, according to Royce’s description, three main characteristics: first, the need to escape from sloth: “And sloth at any level of our development remains one of the most treacherous and mortal enemies of moral will;” secondly, the need to “avoid the dangers in which Hamlet’s type souls fall a prey. 33 Ibid., p 212. 34 Ibid., p. 214. 35 Ibid., p. 214. 36 Ibid., p. 218. 37 ROYCE, 1912, p. 29. 2 The path of the objective attitude There are two words that will allow to define this opposition of the two systems of values: everything that originates in the authentic and contributes to the intellectual and spiritual enrichment will be considered as belonging to the culture; On the other hand, everything that is nothing more than bright appearance, lightness, superficial refinement, belongs to civilization, just as depth opposes superficiality.27 Understood in these terms, culture marks the eighteenth-century German intellectual bourgeoisie whose sincerity opposes the French refinement of the court. In the 19th century, it becomes the stamp of the German nation: “sincerity, depth, spirituality, will be considered, from this moment, as specifically German.”28 Is this not close to what James says about Switzerland and Germany, the centers of the Protestant world? Does James not claim some sort of sincerity, depth and spirituality against Renan’s cynical refinement? In The dilemma of Determinism, James tells us that it is not easy to escape from pessimism, and that one of the ways to do so is to explain all evil as apparent: “[…] bleach the devil, disinfect the universe.”29 This subjectivist attitude, which is also known as Gnosticism, states that evil is not really evil, but an opportunity for our sensitivity to acquire a deeper knowledge of the world. Perhaps the last purpose of the world is the enrichment of our subjective consciousness, and for this to happen, there must be contrasts. According to this perspective: “Life is a long feast of the tree of knowledge,”30 and it would be better in this case, James asserts, to adopt a dramatic point of view: “[…] to treat the whole as a great play without end that the spirit of Universe, in the attempt to realize its own content, tries and represents eternally for itself.”31 Let us remember the magic pantomime we have just mentioned. This attitude, although it has every right to exist and presents itself as an alternative to pessimism, leads to “the most corrupt curiosity:”32 Once dismissed the notion that certain duties are good in themselves, and that we are here to do them, no matter how we feel about them; once we consecrate the opposite notion that Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 2 The path of the objective attitude That is, they discourage the spirit that reflectively divides the inner self and that leaves it divided […] the divided self is indeed, unless it can heal its deadly wound, by fitting action, a lost soul;” and thirdly, he emphasizes courage, not exterior courage, but that which “fits us to meet our true spiritual enemies—the courage that arises anew from despair and that undertakes, despite tribulations, to overcome the world—such courage is one of the central treasures of the moral life.”37 Although one may wonder whether the characteristics Royce lists are indeed the most important from James’s ethics, they are certainly well defined features that show the connection between James and Carlyle. In The Dilemma of Determinism, he opposes Romantic subjectivism and Renan’s feminine cowardice to the objective attitude he adheres to by using Carlyle’s thought as an example in a lecture given to Harvard Divinity Students, published in the Unitarian Review of September 1884. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 250 250 All is not Vanity: William James versus Ernest Renan However, the assumption that behavior deserves our ultimate acknowledgement instead of sensitivity, can be found in an intense letter from June 8th, 1866.38 There, James tries to encourage his friend Thomas W. Ward who suffered from melancholy, “your melancholy tone about yourself.” In this letter, we find his concern about the relation between human beings’ worldview and their mental and moral state, some thoughts about the oscillation of consciousness towards optimism and pessimism, and ideas that advance concepts of the sick and healthy soul.39 What is relevant in this case is that concerns such as melancholy, mood changes and the “fragmentary condition” of the cosmos—which caught the attention of philosophical tradition, and therefore, received varied answers—appear in James as a demand linked to willpower in an attempt to respond to such concerns almost twenty years before the cited essay: I think we ought to be independent of our moods, look on them as external, for they come to us unbidden, and feel if possible neither elated nor depressed, but keep our eyes upon our work and, if we have done the best we could in that given condition, be satisfied.40 Young James seems already convinced of what he will assert much later: that the only escape from the depth of vanitas vanitatum, omnia vanitas is practical. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 38 JAMES, 1920, p. 78. 39 Ibid., p. 76. 40 Ibid., p. 78. 41 JAMES, 2009, p. 215. 42 Ibid., p. 215. 43 Ibid., p. 215. 44 BORGES, 1956, p. 22. 45 JAMES, 2009, p. 215. 2 The path of the objective attitude This is the most important concept Carlyle taught us, James says in the essay: “Hang your sensibilities! Stop your sniveling complaints, and your equally sniveling raptures! Leave off your general emotional tomfoolery, and get to WORK like men!”41 By paying attention to behavior and not to sensitivity, we break with subjectivism. The key lies in the tasks to be fulfilled and the changes to promote regardless of the emotions they entail: “No matter how we succeed in doing these outward duties […] No matter how we feel; if we are only faithful in the outward act and refuse to do wrong, the world will in so far be safe, and we quit of our debt toward it.”42 The philosophy of objective behavior is also characterized as “chaste and sane and strong.”43 In the preliminary study of his translation of Carlyle’s work on heroes, Borges (1899-1986) tells us: “No one has felt like him that this world is unreal (unreal like nightmares, and atrocious). From this general phantasm, rescues one thing, work, not its result, well understood, is mere vanity, mere image, but its execution.”44 In The Dilemma of Determinism, James claims: “[…] our responsibility ends with the performance of that duty, and the burden of the rest we may lay on higher powers.”45 38 JAMES, 1920, p. 78. 39 Ibid., p. 76. 40 Ibid., p. 78. 41 JAMES, 2009, p. 215. 42 Ibid., p. 215. 43 Ibid., p. 215. 44 BORGES, 1956, p. 22. 45 JAMES, 2009, p. 215. 251 Cognitio: Revista de Filosofia Carlyle’s figure is recurrent in James’s work, who adheres to the heroic vision of history,46 but as one of the constituents of strenuous mood opposed to Renan’s banal refinement. From these links we can see two related elements. On the one hand, there is a large dose of ascetic Protestant work, and on the other, part of the typical characteristics attributed to the Germanic spirit. Carlyle himself “[…] in 1870 acclaimed the victory of the ‘patient, noble, profound, solid and pious Germany’ on the ‘boastful, vainglorious, gesticulating, quarrelsome, restless, hypersensitive France’.”47 Within this general inclination toward the Germanic, James seems to accept the Protestant asceticism of work as his own, breaking from the subjective sentimental drift. But what is the nature of that asceticism? What role does it play in Carlyle’s intellectual proposal? 46 See JAMES, 2009, p. 293-301. 47 BORGES, 1956, p. 11. 48 WEBER, 1991, p. 41. 49 Idem, p. 42. 50 Ibid., p. 42. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 50 Ibid., p. 42. 51 Ibid., p. 61. 52 Ibid., p. 75. 2 The path of the objective attitude Max Weber (1864-1920) claims that Catholicism—as in Classical Antiquity—lacks an expression such as the German word “profession” (beruf) whose religious connotation is clear when used in its full meaning. Weber states that this word’s specific meaning appears for the first time in the Lutheran translation of the Bible. Although its meaning is not faithful to the original version, it belongs to the “spirit of the one who translated it.”48 Where it said, “persevere in your work,” we find, “persevere in your profession”.49 The adoption of this current meaning was not to be expected in Protestants’ everyday language, whereas in the past, no signs of it appeared in the Protestant sacred or profane literature, except for a German mystic who exerted a great influence on Luther: In any case, what was absolutely new was that the most honorable content of one’s own moral behavior consisted precisely in conscience of duty. In the performance of professional work in the world. That was the inescapable sequel of the sacred sense, so to speak, of the work and what led to the ethical-religious concept of profession: concept that translates the dogma extended to all the Protestant creeds, opposite to the interpretation that the ethics of the Catholicism divulged of the evangelical norms in praecepta and consilia that preaches that the only way to live in the life that satisfies God is to accept— not the overcoming of earthly morality through the mediation of monastic asceticism but—the observation in the world of the duties that are imposed to each one for the position in life and that therefore comes to become for him in profession.50 Although the Reformation cannot be understood without Luther’s strong personality and mind, without Calvinism his reformist work would not have lasted. However, Weber states that continuity occurred in the rupture. Therefore, regardless of how superficial the research might be, what becomes explicit is that Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 252 252 All is not Vanity: William James versus Ernest Renan Calvinist religious and work life are fundamentally different from Catholics’ and Lutherans’. The point of disagreement lies in the weight of belief in the absolute power of providence, since eternity has already decided who will be saved and who will not. Within Calvinism, the idea that our destiny depends on the decision of an absolutely free God is preponderantly present. 2 The path of the objective attitude Ultimately, there is a creature who is separated from Him by an insurmountable abyss and deserves eternal death unless He decides otherwise: Calvinist religious and work life are fundamentally different from Catholics’ and Lutherans’. The point of disagreement lies in the weight of belief in the absolute power of providence, since eternity has already decided who will be saved and who will not. Within Calvinism, the idea that our destiny depends on the decision of an absolutely free God is preponderantly present. Ultimately, there is a creature who is separated from Him by an insurmountable abyss and deserves eternal death unless He decides otherwise: In his pathetic cruelty, this doctrine had to have above all a special consequence for the spirit of the generation that surrendered to the enormous coherence. The sensation of an unheard of inner solitude on the part of the isolated individual. In the question of eternal salvation, the most decisive of questions for a person of the Reformation era, the human being was condemned to walk alone in his street to fulfill a destiny determined from eternity.51 The inner emotional movements, and sometimes, the despair caused by the uncertainty of being a sinner and not knowing whether one is doomed to the eternal flames or deserves paradise, no longer possess the “magical” mechanisms of liberation available in the grace of the Catholic sacraments. Confession, for example, cuts the inner undercurrent of guilt and uncertainty with the certainty of God’s forgiveness. Repentance and contrition, atonement, hope of grace, and certainty of forgiveness offer a discharge of tension for the believer’s life and destiny. At the psychological level, we find the compelling need to verify certitudo salutis. In this respect, Calvinists lack the old balm and develop another device to answer the question about their salvation: doubts about whether they are saved or not are taken as lack of faith. With iron faith, they are forced to believe that they are saved as long as they can show concrete actions. Webber shows two different moods in Luther and Calvin, one more focused on the inner self and another on action. In both, we can find: […] deep differences in the conditions necessary for salvation, valid for the classification of all practical religiosity at all. The virtuous believer can be sure of his state of grace, whether he is a receiver of a tool of divine Power. 2 The path of the objective attitude In the first case his religious life will be inclined towards an emotional mystical culture, in the second, to an ascetic activity. Luther was closer to the first type, the second belonged to Calvinism.52 […] deep differences in the conditions necessary for salvation, valid for the classification of all practical religiosity at all. The virtuous believer can be sure of his state of grace, whether he is a receiver of a tool of divine Power. In the first case his religious life will be inclined towards an emotional mystical culture, in the second, to an ascetic activity. Luther was closer to the first type, the second belonged to Calvinism.52 […] deep differences in the conditions necessary for salvation, valid for the classification of all practical religiosity at all. The virtuous believer can be sure of his state of grace, whether he is a receiver of a tool of divine Power. In the first case his religious life will be inclined towards an emotional mystical culture, in the second, to an ascetic activity. Luther was closer to the first type, the second belonged to Calvinism.52 From the moment Calvin considers that emotional evidence—however sublime it may be—is capricious and does not count, faith has to be credited for its objective results to serve as a reliable basis for certitudo salutis. Subjective drift is opposed to rational and methodical life. Compliance with the coldness of the profession becomes the technical means, not to buy beatitude—or to deserve it—but to free oneself from anguish. Execution demanded at the point of perfection is then a Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 253 Cognitio: Revista de Filosofia mechanism that silences or represses—according to the frame of appropriation we choose—the existential anxiety of the ultimate end of life itself: “To put it in modern terms, Puritan—asceticism like all rational ascesis—impelled the human being to maintain his ‘constant motivations’ over ‘affections.’ Especially those motivations that this same ascesis had led him “to exercise.”53 mechanism that silences or represses—according to the frame of appropriation we choose—the existential anxiety of the ultimate end of life itself: “To put it in modern terms, Puritan—asceticism like all rational ascesis—impelled the human being to maintain his ‘constant motivations’ over ‘affections.’ Especially those motivations that this same ascesis had led him “to exercise.”53 Therefore, two paths can ultimately be taken, the subjective or the objective one. 2 The path of the objective attitude American Methodism, for example, whose exalted ecstasies in the conversion crisis are taken as a certainty of holiness, took the subjective path. The objective path, on the other hand, has to do with abandoning the instability typical of all emotional certainty by adopting a mundane-professional life with the shape of a rational ascetic of work fulfillment, which in psychological terms bridges the trust of being saved. In negative terms, professional duty erases the anxiety of not knowing if we are doomed. This excerpt taken from Weber’s classic work accounts for both the origin and the relation between ethics and work in the Protestant world. William James’ religious sensitivity, his Protestant background, and his early advice to overcome melancholy through duty show us an easily identifiable mood. The Protestant attitude of duty at work appears in the article The Dilemma of Determinism expressed in Carlyle’s Calvinism. p y Finally, it is convenient to give a brief account of this component so that all relations may be properly established. At the beginning of his work on heroes Carlyle says: For, as I take it. Universal History, the history of what man has accomplished in this world, is at bottom the History of the Great Men who have worked here. They were the leaders of men, these great ones; the modellers, patterns, and in a wide sense creators, of whatsoever the general mass of men contrived to do or to attain, all things that we see standing accomplished in the world are properly the outer material result, the practical realization and embodiment, of thoughts that dwelt in the Great Men sent into the world: the soul of the whole world’s history, it may justly be considered, were the history of these. Too clearly it is a topic we shall do no justice to in this place!54 Charles F. Harrold (1897-1948) asserts that Carlyle’s social writings find inspiration in the hope of a “rebirth of the heroic and the spirit of obedience and loyalty.”55 Both doctrines of predestination possess a similar logic. Whereas most people are considered dark and insignificant, there are some chosen ones—the great men, the heroes—whose origin is transcendent and who must be followed and obeyed. Obedience is a virtue in so far as it is the obedience to the hero, destined to guide by nature. 53 Ibid., p. 75. 54 CARLYLE, 1956, p. 3. 55 HARROLD, 1936, p. 480. 2 The path of the objective attitude This is clearly a revival of the idea of the chosen for salvation where Calvinist fatalism of the transcendent operates in a non-dogmatic way. At the same time, we find a strong belief in a natural moral order. “What is Nature […] Why Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 254 254 All is not Vanity: William James versus Ernest Renan do not I name it god?”56 In turn, speculative questions receive a strong rejection in this vision of the world. Questions such as: How can the moral order of the world, with the density of the eternal, be frustrated by the work of the insignificant and finite human being? receive as a response aversion and condemnation, as if they were something pathological or evil. The writer possesses much of the Calvinist hostility to theological mysteries. Expressed in non-dogmatic terms, he feels hostility towards speculation: “True knowledge for Carlyle was never a logical or demonstrative knowledge”, but a knowledge that arises from the “infiltration into the consciousness of nature and the origin of the laws by which humanity is governed.”57 Together with this conception of the Calvinist and anti-enlightened source, we find that the true life, which is morally valuable, is not the inner life, full of research and questions, but the life of objective action, work, struggle, self- denial, and even martyrdom. Carlyle states: “Difficulty, self-denial, martyrdom, death are the seductions that act in the heart of man.”58 The illogical ways of the Foreman (Taskmaster) who, far from the world, irrationally decides about people’s lives, and demands, in turn, strict obedience had Milton say: “I can go down to hell; but such a God will never have my respect.”59 However, as Weber cautions, such a doctrine has proved to be a surprising stimulus for activity and self-forgetfulness. The belief in a ‘chosen’, pre-determined outcome of human activity, and in man’s obligation to ‘assist’ in that outcome, is a belief which, despite its intellectual difficulties, has stirred the energy of millions. Carlyle’s social gospel of work and self-denial reflects the Calvinist disdain for ‘torturing anxiety’, as seen in Methodism, for example, with its eye forever turned on its own navel. It is labor, therefore, rather than emotional satisfaction or intellectual inquiry, which becomes the heart of Carlyle’s social ideal. 56 CARLYLE, 1896, p. 171. 57 FROUDE, 1877, p. 52. 58 CARLYLE, 1956, p. 81. 59 APUD WEBER, 1991, p. 59. 60 Harrold, Charles Frederick. óp. cit., p. 482. Italics added by the author. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 60 Harrold, Charles Frederick. óp. cit., p. 482. Italics added by the author. 2 The path of the objective attitude Yet, it is labor in a strictly Calvinistic interpretation: labor not for the individual but for ‘the divine’, for the whole. The end would be ‘the moralization of all life’ with worldly callings exalted as the means of spiritual expression, and sometimes, unfortunately, worldly progress employed as the measure of spiritual success. For Carlyle, such labor would become a form of asceticism in reality with no claim of reward, “all work being essentially worship, a sort of sacrament”.60 g y p In his essay, James contrasts the interior-subjectivist current—with all the characteristics we have reviewed—with Carlyle’s objective attitude and his emphasis on duty and work. The passage from one sensitivity to another can be clearly seen. However, James says very little about the source, nature, reach and meaning of this morality which urges us to abandon sentimental drift and “work like men.” This is awkward even in the light of the extensive and conscientious commentary received by the subjectivist or gnostic attitude. Having used Carlyle to exemplify objective morality involves a certain set of outcomes. We hope to have made the necessary connections to prove the Protestant component of strenuous mood. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 255 Cognitio: Revista de Filosofia 3 Final Words In order to fight against melancholy, young James advises us to stand firm in the accomplishment of a task beyond our feelings. Many of his value judgments can be understood from his work and the letters he wrote. He opposes Renan’s banal and cynical refinement to Carlyle’s seriousness and virility. To Florence’s general decadence, he contrasts Switzerland’s sanity and sanctity. I hope to have proven that, in general terms, this is value distribution. In the same direction and moving towards the specific—taking into account Royce’s words—the courage of strenuous mood has a component of Calvinist work ethic, which though evident in Carlyle, does play a role in James as well. This component is found throughout James’s work. However, if this statement is too difficult to show, it is expected that at least it remains clear that what is established in The Dilemma of Determinism to the very well reconstructed gnostic and subjectivist attitude—which had led to the ruin of the French of the time—is opposed by the objective attitude about which he says very little. We have stressed that this attitude has much of the Protestant work ethic so influential among people of Calvinist descent. Work, which at the time of the Reformation was the right way to deal with the desperate uncertainty of salvation, is now, at the end of the Century’s crisis the device that faces the onslaught of vanitas vanitatum, omnia vanitas. References BORGES, Jorge Luis. Estudio preliminary. In: Carlyle y Emerson: De los heroes. Hombres representativos. Buenos Aires: W. M. Jackson, Inc., 1956. BORGES, Jorge Luis. Estudio preliminary. In: Carlyle y Emerson: De los heroes. Hombres representativos. Buenos Aires: W. M. Jackson, Inc., 1956. CARLYLE, Thomas. Sartor Resartus. Boston: ed. A. MacMechan, 1896. _____. De los héroes. Translated by Miguel C. Perales. Buenos Aires: W. M. Jackson, Inc., 1956. COLELLA, E. Paul. The geography of strenuousness: America in William James’s narrative of moral energy. Transactions of the Charles S. Peirce Society. v. 52, n. 1, p. 93-113, Winter 2016. CUCHE, Denys. La noción de cultura en las ciencias sociales. Buenos Aires: Nueva Visión, 1992. DEL CASTILLO, Ramon. Estetas y profetas: equívocos de Santayana y James. In: Miradas transatlánticas: Intercambios culturales entre Estados Unidos y Europa. Madrid: Fundamentos, 2011. DOOLEY, Patrick K. The strenuous mood: William James ‘Energies in Men’ and Jack London’s ‘The Sea-Wolf’. American Literary Realism. v. 34, n. 1, p. 18-28, Jun. 2001. ELIAS, Norbert. El proceso de la civilización: Investigaciones sociogenéticas y psicogenéticas. Translated by Ramón García Cotarelo. México: Fondo de Cultura Económica, 2001. FROUDE, J. A. Calvinism, Short Studies on Great Subjects. New York: 2nd Ser, 1877. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 256 All is not Vanity: William James versus Ernest Renan HARROLD, Charles Frederick. The nature of Carlyle’s Calvinism. Studies in Philology. v. 33, n. 3, p. 475- 486, Jul. 1936. JAMES, William. The varieties of religious experience. New York: Longmans, Green, and Co., 1917. _____. The letters of William James. v.1. Edited by JAMES, Henry. Boston: The Atlantic Monthly Press, 1920. _____. Essays, Comments, and Reviews. Cambridge: Harvard University Press, 1987. _____. Manuscript Lectures. Cambridge: Harvard University Press, 1988. _____. La voluntad de creer. Translated by Ramon Vilà Vernis. Barcelona: Marbot, 2009. LUTZ, Tom. American Nervousness, 1903: an anecdotal history. Ithaca: Cornell University Press, 1991. ROYCE, Josiah. William James, and other essays on the philosophy of life. New York: The Macmillan company, 1912. SHUSTERMAN, Richard. Thought in the strenuous mood: pragmatism as a philosophy of feeling. New Literary History. v. 43, n. 2, p. 433-454, Mar 2012. SPEZIALE, Marcia Jean. Oliver Wendell Holmes Jr., William James, Theodore Roosevelt, and the strenuous life. Heinonline. v. 13, Conn. L. Rev, p. 663-681, 1980- 1981. TOWNSEND, Kim. Manhood at Harvard. New York: Norton, 1996. WEBER, Max. Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 References La ética protestante y el espíritu del capitalismo. Translated by José Chávez Martínez. México: Premia Editora, 1991. Data de recebimento: 12-02-2018 Data de aprovação: 18-11-2018 Data de recebimento: 12-02-2018 d Data de recebimento: 12-02-2018 Data de aprovação: 18-11-2018 Cognitio, São Paulo, v. 19, n. 2, p. 242-257, jul./dez. 2018 257
W2949650858.txt	https://www.frontiersin.org/articles/10.3389/fenvs.2019.00089/pdf	en		Does Protection of Marine Areas Safeguard Coral Reefs From Human-Source Pollution?	Frontiers in environmental science	2,019	cc-by	3,337	BRIEF RESEARCH REPORT published: 14 June 2019 doi: 10.3389/fenvs.2019.00089 Does Protection of Marine Areas Safeguard Coral Reefs From Human-Source Pollution? Alphaxand Kaimba 1 , Santie de Villiers 2,3 and Sammy Wambua 1,3* 1 Department of Biological Sciences (DBS), Pwani University, Kilifi, Kenya, 2 Department of Biochemistry & Biotechnology (DBB), Pwani University, Kilifi, Kenya, 3 Pwani University Bioscience Research Centre (PUBReC), Pwani University, Kilifi, Kenya Edited by: Xiaoshou Liu, Ocean University of China, China Reviewed by: Chun-hua Li, Chinese Research Academy of Environmental Sciences, China Wenzhe Xu, Tianjin University of Science and Technology, China *Correspondence: Sammy Wambua sammywambua@gmail.com Specialty section: This article was submitted to Marine Pollution, a section of the journal Frontiers in Environmental Science Received: 12 February 2019 Accepted: 27 May 2019 Published: 14 June 2019 Citation: Kaimba A, de Villiers S and Wambua S (2019) Does Protection of Marine Areas Safeguard Coral Reefs From Human-Source Pollution? Front. Environ. Sci. 7:89. doi: 10.3389/fenvs.2019.00089 Marine biodiversity is under increasing threat as the area covered by corals diminishes under pressure from climate change and human activities, most of which lead to marine pollution. In Kenya, marine protected areas (MPAs) are the key strategy used to protect coral reefs and biodiversity. However, MPAs’ effectiveness to prevent pollution of the reefs has not been specifically assessed. We determined if the levels of surrogates of human-source pollution, i.e., E. coli and nutrient concentrations on Kenyan coral reefs, varied with increasing levels of marine protection at the Kilifi creek (least protection), Malindi Reserve (moderate protection), and Kuruwitu Conservancy (strictest protection). The most probable number (MPN) of E. coli was estimated by serial dilution while nitrate and orthophosphate concentrations were determined spectrophotometrically. As protection increased from “least,” to “moderate” and “strictest,” E. coli concentrations (MPN/100 mL) decreased from 29, to 16 and undetectable, while mean orthophosphate concentrations increased from 0.326, to 0.422 and 0.524 mg/L, respectively. Mean nitrate concentrations, on the other hand, showed no trend with protection. These results suggest the potential of marine protection to mitigate coral reef pollution, especially from microbes. They also point to the possibility that multiple sources of pollution exist on which marine protection may have little or no effect. Significantly, this pilot study points to the need for improved study design to definitively determine the role MPAs may play in protecting against pollution. Keywords: MPA (marine protected area), E. coli—Escherichia coli, nutrients (nitrogen and phosphorus), coral reefs, pollution INTRODUCTION The Western Indian Ocean (WIO) hosts the second largest coral reef biodiversity globally (Obura, 2012) and holds potential wealth for the region due to the significant economic role that oceans play to support jobs, tourism, and fisheries. Surprisingly, the blue economy of the WIO region remains underdeveloped. The oceans’ economic value depends on healthy coral reefs (Obura et al., 2017), which cover only 0.1% of the ocean floor but host at least 25% of all marine biodiversity (Hoegh-guldberg et al., 2017), provide goods and services such as seafood, recreational possibilities, coastal protection, and aesthetic and cultural benefits (Moberg and Folke, 1999). However, coral reef ecosystems are increasingly under threat mainly from climatic changes as evidenced by mass coral bleaching Frontiers in Environmental Science \| www.frontiersin.org 1 June 2019 \| Volume 7 \| Article 89 Kaimba et al. Marine Protected Areas and Human-Source Pollution (Wiedenmann et al., 2013), compromise resilience, promote algal dominance over corals (Hall et al., 2018) and suppress calcification (Kinsey and Davies, 1979). and/or death following episodes of elevated sea surface temperatures (Hughes et al., 2017). The situation is aggravated by overfishing and pollution (Hughes et al., 2003) from human activities on the coast zone leading to a decrease in the abundance of coral reef species. While corals may recover from bleaching episodes (Hughes et al., 2003; West and Salm, 2003), nutrient influx, especially of nitrate and phosphate, is thought to increase susceptibility to bleaching (Wiedenmann et al., 2013), compromising this resilience (Hall et al., 2018). The scale of human impacts is, therefore, bound to intensify over time as temperatures are expected to keep rising, while the human population also increases on coastal zones attracted by the goods and services of the marine ecosystems (Neumann et al., 2015). Over the past 50 years, Kenya has mitigated anthropogenic impacts on its coastal and marine resources, through MPAs as the primary marine ecosystems management strategy (Samoilys and Obura, 2011). Two classes of MPAs, differing in the level of protection, were established and enforced by the Kenya Wildlife Services (KWS): marine parks’ “no-take” areas protected from all forms of consumptive utilization, and marine reserves which are contiguous to parks acting as buffers. Here, artisanal, but not commercial fishing is allowed (Tuda and Omar, 2012). Owing to the success of MPAs, local communities are increasingly establishing and enforcing conservation areas termed as “locally managed marine areas” (LMMAs) which adopt the “no-take” protection management plan, like marine parks. Most LMMAs are administered with financial and/or technical support from non-governmental organizations and/or the government. The primary goal for the implementation of MPAs and LMMAs is biodiversity conservation and the regulation of fisheries. These efforts have generally been successful, especially in increased and improved fish populations (Ransom and Mangi, 2010; Samoilys and Obura, 2011). While these strategies seem effective to curb the direct impacts of human-related pressures such as overfishing and fishery-related damages, it is necessary to interrogate their effectiveness on subtler effects that may result from spatially removed urbanization, tourism development, agriculture, and industrialization. Globally, these activities are major drivers of marine pollution (Shahidul Islam and Tanaka, 2004) posing health risks to fishery products, consumers and recreational users on the coasts (Kilinc and Besler, 2014), as well as leading to the death of corals and other reef organisms (Hipsey et al., 2008). We conducted a pilot study to assess whether area-based marine protection may have the potential to curb humansource pollution. In coral reefs under differing levels of protection, we measured concentrations of surrogates for human-source pollution including, E. coli, a conventional indicator for fecal pollution and the subsequent public health risk from pathogenic microorganisms (Fremaux et al., 2009). We also measured nitrate and phosphate as they are known to be increased, in marine environments, by human activities (Jickells, 1998; Wiedenmann et al., 2013; Sayadi et al., 2016). Furthermore, nitrate and phosphate are important for the optimum physiological functioning of coral symbionts and in determining coral reef resilience (Rosset et al., 2017) whereby their enrichment is thought to increase susceptibility to bleaching Frontiers in Environmental Science \| www.frontiersin.org MATERIALS AND METHODS Study Sites Sampling was done between March and April 2018 during the long rains season at three sites on the northern coast of Kenya: (1) Kuruwitu Conservancy, an LMMA administered “notake” area since 2006, had the “strictest” protection safeguarded from all forms of consumptive utilization and human activities, save for research and tourism; (2) Malindi Marine Reserve, established in 1968 (Lambo and Ormond, 2006), was considered to be under “moderate” protection—the reserve is contiguous to Malindi Marine Park where it acts as a buffer to the “notake” area. Here, artisanal fishing as well as research and tourism is allowed, but commercial forms of utilizations are prohibited (Tuda and Omar, 2012); and (3) Kilifi creek is not protected hence was designated the “least protected”—it is open to fishing, recreation, and experiences effluents from surrounding hotels and residential houses. Sampling Duplicate samples of 1L seawater each were collected in the morning between 08:00 and 12:00 at 1–2 meters depth at low tide within 15–20 cm of the dominant species of coral (Acropora spp.) 500–2,000 m from the shore. The samples were then transported on ice to the laboratory for processing within 2 h of sampling. Laboratory Methods Nitrate concentration was determined with ultraviolet spectrophotometry at 275 nm. Phosphate concentration was derived from orthophosphate colorimetric measurement at 690 nm using the Stannous Chloride method (APHA et al., 1999). Concentrations of viable E. coli cells were estimated by a three-tube most probable number (MPN) method as described by Vincy et al. (2015). RESULTS E. coli concentrations decreased with increased levels of protection. At Kuruwitu, the site with the strictest protection, E. coli was not detected (<3 MPN/100 mL), whilst Malindi (moderate protection) recorded 16 MPN/100 mL and Kilifi (no protection) 29 MPN/100 mL. Between-site differences of E. coli density were not statistically significant (Figure 1). Orthophosphate mean concentrations differed significantly among sites, increasing with the degree of marine protection (Table 1): the highest concentration, 0.524 mg/L, was observed at Kuruwitu followed by Malindi (0.422 mg/L) and Kilifi (0.326 mg/L) (P < 0.01). Nitrate mean concentrations also differed significantly among sites but did not correlate with the level of protection. Kuruwitu registered 0.566 mg/L, Kilifi 0.188, and Malindi, 1.402 (P < 0.01). 2 June 2019 \| Volume 7 \| Article 89 Kaimba et al. Marine Protected Areas and Human-Source Pollution fertilizers, as well as commercial farms. The conservancy administration reported of having had to seek the intervention of National Environmental Management Agency (NEMA) to stop residents from discharging swimming pools wastewaters directly into the ocean during the initiation phase of the LMMA. This suggests that the level of marine protection may also explain the variations in E. coli concentrations observed. Agriculture-related activities are the main contributors of both orthophosphates and nitrate in rural settings (Coulter et al., 2004). We expected Malindi (where the Sabaki river meets the ocean) to contain higher mean nutrient concentrations than Kuruwitu since the Sabaki river runs through a catchment area with farming activities. However, the opposite was observed. Run-off from Kuruwitu lawns and commercial farms may explain the elevated orthophosphate and nitrate concentrations compared to Malindi where little or no farming occurs in the immediate vicinity of the sampling site. Furthermore, the Sabaki river has been shown to have the lowest discharge in the ocean during the north-eastern to south-eastern Monsoon transition period (March–April), which was when this study sampled. Most river sediment was deposited along the beach and intertidal area, and not in the neritic zone where this study sampled (Munyao et al., 2003). These observations support our results, assuming that nutrients are deposited in a similar manner as sediments. There are no universal E. coli and nutrients references for tropical coral reefs but in comparison to local standards elsewhere, the levels observed in this study are of public and environmental health concern. Although the E. coli concentration was lower than the threshold allowable for bathing beaches in parts of Europe (Baudart et al., 2009), it may have been much higher at the shore close to the point where the pollutants entered the sea as bacterial numbers would decrease as they moved toward the open ocean due to tidal dilution and the bactericidal activity of seawater (Vaccaro et al., 1950). The nutrient concentrations assessed were also high, consistent with eutrophication (Okuku et al., 2011) or may have been the result of resuspended sediments (which accumulate nutrients at higher concentrations than seawater) following disturbances caused by rains and/or sampling activities. This finding is alarming and needs to be confirmed by a long-term follow-up study with supplementary data on the possible impact of the nutrient elevations on reef biodiversity. This pilot study indicated that human activities around the Kenyan Coastal zone likely increase pollution that may impact coral reef health. An exact cause for the observed variations may not be conclusively specified yet, but the likelihood of marine protection in mitigating microbial pollution is suggested. This study, therefore, confirms the need to research the topic, and informs on critical areas of consideration in designing an effective study to definitively determine the role, if any, of MPAs in curbing marine pollution. FIGURE 1 \| E. coli concentrations (MPN/100 mL) in water sampled at three different coral reef sites. Error bars indicate 95% confidence intervals. TABLE 1 \| Nutrient mean concentrations for water samples collected from the coral reef sites. Nutrient Kuruwitu Malindi Kilifi P 0.523 ± 0.018 0.421 ± 0.001 0.325 ± 0.004 <0.01 0.565 ± 0.007 0.187 ± 0.001 1.401 ± 0.004 <0.01 ORTHOPHOSPHATE (mg/L ± SD) NITRATE (mg/L ± SD) Between-sites comparisons tested by ANOVA. DISCUSSION The coral reef sites studied differed in both surrogates of humansource pollution investigated. As expected, E. coli concentrations decreased as the level of protection increased. Conversely, mean orthophosphate concentrations increased with increasing protection while mean nutrient concentration did not correlate with protection. These observations can be explained by the wide and differing range of human activities taking place around each study site, the level of marine protection and the proximity of each sampling site to a town. In urban settings, wastewater and solid waste disposal is the principal cause of microbial pollution and nutrient loading (particularly nitrate) in the ocean (Wakida and Lerner, 2005; Okuku et al., 2011). The Kilifi site, which registered the highest E. coli and nitrate concentrations is a creek dividing two business centers, Mnarani and Kilifi towns, which together constitute the capital of Kilifi County. The creek is surrounded by resorts and residential houses leaking and/or disposing of wastewater into the ocean, and the beach is frequented by both foreign and local residents and holiday makers for recreation. In contrast, Malindi Marine Park and Kuruwitu Conservancy have limited, if any, exposure to urban pollution due to their distance from towns and levels of protection; the Malindi site (moderate protection) is about 10 km from the town, and Kuruwitu (highest protection) about 23 km from Kilifi town. The Kuruwitu site shares a beach with several affluent residential homes with swimming pools, lush lawns utilizing Frontiers in Environmental Science \| www.frontiersin.org DATA AVAILABILITY The datasets generated for this study are available on request to the corresponding author. 3 June 2019 \| Volume 7 \| Article 89 Kaimba et al. Marine Protected Areas and Human-Source Pollution AUTHOR CONTRIBUTIONS SW performed data analysis. AK wrote the first draft of the manuscript. 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https://openalex.org/W2585776394	https://europepmc.org/articles/pmc5390278?pdf=render	English	null	Inkjet printing-based volumetric display projecting multiple full-colour 2D patterns	Scientific reports	2,017	cc-by	6,802	Inkjet printing-based volumetric display projecting multiple full- colour 2D patterns received: 30 January 2017 accepted: 14 March 2017 Published: 13 April 2017 Ryuji Hirayama1,2, Tomotaka Suzuki1, Tomoyoshi Shimobaba1, Atsushi Shiraki3, Makoto Naruse4, Hirotaka Nakayama5, Takashi Kakue1 & Tomoyoshi Ito1 Ryuji Hirayama1,2, Tomotaka Suzuki1, Tomoyoshi Shimobaba1, Atsushi Shiraki3, Makoto Naruse4, Hirotaka Nakayama5, Takashi Kakue1 & Tomoyoshi Ito1 In this study, a method to construct a full-colour volumetric display is presented using a commercially available inkjet printer. Photoreactive luminescence materials are minutely and automatically printed as the volume elements, and volumetric displays are constructed with high resolution using easy-to- fabricate means that exploit inkjet printing technologies. The results experimentally demonstrate the first prototype of an inkjet printing-based volumetric display composed of multiple layers of transparent films that yield a full-colour three-dimensional (3D) image. Moreover, we propose a design algorithm with 3D structures that provide multiple different 2D full-colour patterns when viewed from different directions and experimentally demonstrate prototypes. It is considered that these types of 3D volumetric structures and their fabrication methods based on widely deployed existing printing technologies can be utilised as novel information display devices and systems, including digital signage, media art, entertainment and security. Volumetric displays directly render a three-dimensional (3D) image onto the true volume physical space1,2. Each volume element (voxel) of a 3D object is physically present at the required location and thus a natural visual perception is afforded from the surrounding. A variety of volumetric displays are intensively studied to achieve next-generation human–computer interaction and other applications3–12. A potential use of volumetric displays includes 3D visualization without the need to wear devices such as special glasses, which is highly relevant in medical applications, architectural design, advertising and entertainment. pp g g In previous studies, an algorithm was proposed for the design of 3D structures, which projects multiple 2D patterns in different directions13–15. As shown in Fig. 1a, a 3D glass structure designed by the algorithm pro- vides multiple 2D images independently to each of the viewpoints. As opposed to conventional 3D structures demonstrated in previous studies16,17, the algorithm in the present study provides grayscale images and projection directions that can be configured more flexibly. In addition, a multi-colour volumetric display based on the 3D arrangement of photoreactive luminescence materials was developed18. A volumetric display that projected three multi-colour 2D patterns was demonstrated by arranging two types of quantum dots that emitted red and green lights. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Graduate School of Engineering, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. 2Research Fellow of the Japan Society for the Promotion of Science, 5-3-1 Kojimachi, Chiyoda-ku, Tokyo 102-0083, Japan. 3Institute of Management and Information Technologies, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. 4Network System Research Institute, National Institute of Information and Communications Technology, 4-2-1 Nukui-kita, Koganei, Tokyo 184-8795, Japan. 5Center for Computational Astrophysics, National Astronomical Observatory of Japan, 2-21-1 Osawa, Mitaka, Tokyo 181-8588, Japan. Correspondence and requests for materials should be addressed to R.H. (email: hirayama@chiba-u.jp) Scientific Reports \| 7:46511 \| DOI: 10.1038/srep46511 Inkjet printing-based volumetric display projecting multiple full- colour 2D patterns b Inkjet printer Printed 2D sheet Excitation light Full-colour 3D image c Inkjet-printing-based volumetric display Multiple full-colour 2D patterns Inkjet-printing-based volumetric display a Pattern A Pattern B Pattern C Pattern D 3D glass crystal b a c Figure 1. Concept of the study. (a) 3D glass structure projecting four monochromatic patterns in different directions. (b) Volume construction method enabling a high resolution. (c) Inkjet printing-based volumetric display projecting multiple full-colour 2D patterns. b 16 18 17 19 20 11 13 12 14 15 6 8 7 9 10 1 3 2 4 5 10 mm Y Z X 12.5 mm pacers 20 films 35 mm 35 mm V irradiation a d c θ θ = -30° 0° +30° Figure 2. Prototype of the volumetric display based on inkjet printing. (a) Overview under natural light. (b) Cross-sectional images printed on the films. (c) Original 3D image represented with computer graphics from three different viewpoints. (d) Photographs of the volumetric display when excited by ultraviolet light. b 16 18 17 19 20 11 13 12 14 15 6 8 7 9 10 1 3 2 4 5 10 mm Y Z X 12.5 mm Spacers 20 films 35 mm 35 mm UV irradiation a d c θ θ = -30° 0° +30° Figure 2. Prototype of the volumetric display based on inkjet printing. (a) Overview under natural light. (b) Cross-sectional images printed on the films. (c) Original 3D image represented with computer graphics from three different viewpoints. (d) Photographs of the volumetric display when excited by ultraviolet light. +30° Y Z X 12.5 mm Spacers 20 films 35 mm 35 mm UV irradiation a θ c 10 mm d c d a Figure 2. Prototype of the volumetric display based on inkjet printing. (a) Overview under natural light. (b) Cross-sectional images printed on the films. (c) Original 3D image represented with computer graphics from three different viewpoints. (d) Photographs of the volumetric display when excited by ultraviolet light. is composed of several layers of transparent films, and 2D patterns of fluorescent ink are printed on each of the films. The printed fluorescent materials arranged in a 3D layout are excited by external light irradiation (e.g. ultraviolet light), each excited fluorescent material, on returning to the ground state, emits light, which together form a 3D image. Inkjet printing-based volumetric display projecting multiple full- colour 2D patterns The optical excitation of the voxels in the volumetric display resolves the issue of occlusion as opposed to conventional methods that necessitate electrical wiring, which significantly degrades the quality of 3D images. i However, all the manufacturing processes related to the aforementioned optically excited volumetric dis- play were performed manually, i.e., the voxels were manually cut into a cubic shape and manually assembled to construct the volume. Accordingly, the volumetric display device reported in a previous study18 comprised only 8 × 8 × 8 voxels each with 5 mm side-lengths. This leads to difficulties in increasing the display resolution and representing full-colour images. In this study, a manufacturing method is proposed with a high resolution volumetric display based on inkjet printing technology, which enables the minute and automatic location of photoreactive luminescence materials on appropriate places. Figure 1b shows the concept of the proposed method. The proposed volumetric display 1Graduate School of Engineering, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. 2Research Fellow of the Japan Society for the Promotion of Science, 5-3-1 Kojimachi, Chiyoda-ku, Tokyo 102-0083, Japan. 3Institute of Management and Information Technologies, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. 4Network System Research Institute, National Institute of Information and Communications Technology, 4-2-1 Nukui-kita, Koganei, Tokyo 184-8795, Japan. 5Center for Computational Astrophysics, National Astronomical Observatory of Japan, 2-21-1 Osawa, Mitaka, Tokyo 181-8588, Japan. Correspondence and requests for materials should be addressed to R.H. (email: hirayama@chiba-u.jp) Scientific Reports \| 7:46511 \| DOI: 10.1038/srep46511 1 www.nature.com/scientificreports/ b a Inkjet printer Printed 2D sheet Excitation light Full-colour 3D image c Pattern A Pattern B Pattern C Pattern D Inkjet-printing-based volumetric display Multiple full-colour 2D patterns 3D glass crystal Figure 1. Concept of the study. (a) 3D glass structure projecting four monochromatic patterns in different directions. (b) Volume construction method enabling a high resolution. (c) Inkjet printing-based volumetric display projecting multiple full-colour 2D patterns. b a Inkjet printer Printed 2D sheet Excitation light Full-colour 3D image c Pattern A Pattern B Pattern C Pattern D Inkjet-printing-based volumetric display Multiple full-colour 2D patterns 3D glass crystal Figure 1. Concept of the study. (a) 3D glass structure projecting four monochromatic patterns in different directions. (b) Volume construction method enabling a high resolution. (c) Inkjet printing-based volumetric display projecting multiple full-colour 2D patterns. Inkjet printing-based volumetric display projecting multiple full- colour 2D patterns In this method, a massive amount of voxels and a full-colour representation can be achieved at a low cost using the widely available inkjet printers by adjusting the ratio of three fluorescent inks emitting primary colours (red, green and blue). g Furthermore, an algorithm proposed in a previous study13 is extended to design a 3D structure that projected multiple full-colour 2D patterns, as shown in Fig. 1c. As described below, the study succeeded in experimentally demonstrating 3D structures projecting three and four full-colour patterns. Each of the patterns can only be reconstructed from a designated viewpoint according to the proposed volumetric display method based on inkjet printing. Results F ll l Full-colour 3D image representation. First, a prototype of the inkjet printing-based volumetric display is presented to demonstrate its ability to represent an arbitrary full-colour 3D image. Figure 2a shows the overview of the prototype. Fluorescent inks (SO-KEN Inc., Trick Print Ink) are used as photoreactive luminescence materi- als that mainly comprise europium complex (red), β-quinophthalone (green) and coumarin dyes (blue). An inkjet printer (SO-KEN Inc., ‘TPW-105PB’) prints the inks on 0.1 mm thick polyester transparent films (Folex imaging Inc., ‘BG-32’) with a maximum resolution of 5,760 × 1,440 dpi. The rendering space of the 3D image corresponds to 35 mm × 35 mm × 12.5 mm. Twenty layers of the printed films are stacked at 0.5 mm intervals.ill i In addition, 3D figures of flowers and butterflies comprising 51,767 full-colour points are used as a source for the 3D objects. Figure 2b shows twenty cross-sectional images of the 3D objects. Each layer includes 300 × 300 pixels and is printed on the films. In order to excite the printed fluorescent ink, the ultraviolet light Scientific Reports \| 7:46511 \| DOI: 10.1038/srep46511 2 www.nature.com/scientificreports/ a b c d Pattern A Pattern A Pattern C Pattern C Pattern B Pattern B 16 18 17 19 20 11 13 12 14 15 6 8 7 9 10 1 3 2 4 5 16 18 17 19 20 11 13 12 14 15 6 8 7 9 10 1 3 2 4 5 a b c d Pattern A Pattern A Pattern C Pattern C Pattern B Pattern B 16 18 17 19 20 11 13 12 14 15 6 8 7 9 10 1 3 2 4 5 16 18 17 19 20 11 13 12 14 15 6 8 7 9 10 1 3 2 4 5 Figure 3. Prototypes of the volumetric display projecting three full-colour patterns. (a) Cross-sectional images printed on the films. (b) Original images recorded on the volume. (c) Projected patterns from different viewpoints of the 3D structure in the simulation. (d) Projected patterns of the volumetric display. The photographs used as the original images can be found at http://free-photos.gatag.net/?s=201304180900 (pattern A), https://unsplash.com/photos/HQqIOc8oYro (patterns B) and https://unsplash.com/photos/clkYWlgOHIQ (pattern C). All of them are licensed under the Creative Commons Public domain (https://creativecommons. org/publicdomain/zero/1.0/deed.en). b c d Pattern C Pattern C Pattern B Pattern B Pattern C Pattern C d Pattern B Pattern B d a Figure 3. Results F ll l Prototypes of the volumetric display projecting three full-colour patterns. (a) Cross-sectional images printed on the films. (b) Original images recorded on the volume. (c) Projected patterns from different viewpoints of the 3D structure in the simulation. (d) Projected patterns of the volumetric display. The photographs used as the original images can be found at http://free-photos.gatag.net/?s=201304180900 (pattern A), https://unsplash.com/photos/HQqIOc8oYro (patterns B) and https://unsplash.com/photos/clkYWlgOHIQ (pattern C). All of them are licensed under the Creative Commons Public domain (https://creativecommons. org/publicdomain/zero/1.0/deed.en). peaked at 365 nm (AS ONE, LUV-4) is irradiated on the volumetric display in the perpendicular direction. Figure 2c shows the 3D objects rendered by a computer simulation viewed from different perspectives. Figure 2d presents images of an experimentally fabricated volumetric display obtained from different viewpoints. In par- ticular, θ in Fig. 2c and d depicts a horizontal angle between the perpendicular to the films and the viewing direc- tion. The highest quality of the 3D image is obtained when it is viewed in a direction perpendicular to the films (θ = 0°). Although the images obtained from the diagonal directions (θ = ±30°) are blurred, it is confirmed that the volumetric display appropriately represents motion parallax. 3D structure projecting multiple full-colour 2D patterns. Next, a 3D structure projecting three dif- ferent full-colour patterns in different directions is designed, and hereafter the patterns are referred to as patterns A, B and C. The concept of the algorithm presented in a previous study13 is extended, and the extended algorithm is used to determine the voxel values of the full-colour 3D structure (please refer to the Method section for details). The original patterns comprised 512 × 512 pixels with three channels (red, green and blue), and each of the channels can represent 256 gradations. As shown in Fig. 1c, pattern B is set such that it is projected in the direction perpendicular to the films. The projection directions of the other two patterns (A and C) correspond to ±30° around the Y-axis. Figure 3a shows twenty cross-sectional images of the designed 3D structure when three full-colour images, as shown in Fig. 3b (right: A, middle: B and left: C), are used as the original patterns.i t Simulations are performed to confirm the successful projection of original 2D patterns from the designed 3D structure. Scientific Reports \| 7:46511 \| DOI: 10.1038/srep46511 Results F ll l b c Pattern A Pattern A Pattern C Pattern C Pattern B Pattern B Pattern D Pattern D X Y Z Pattern A Pattern B Pattern C Pattern D a b a Figure 4. Prototype of the volumetric display projecting four full-colour patterns. (a) Scheme of the prototype that indicates the projection directions of the patterns. (b) Projected patterns from different viewpoints of the 3D structure in the simulation. (c) Projected patterns of the volumetric display. The photographs used as the original images can be found at http://free-photos.gatag.net/?s=201304180900 (pattern A), https://unsplash.com/photos/HQqIOc8oYro (patterns B), https://unsplash.com/photos/clkYWlgOHIQ (pattern C) and https://www.pexels.com/photo/clown-fish-swimming-128756/ (pattern D). All of them are licensed under the Creative Commons Public domain (https://creativecommons.org/publicdomain/zero/1.0/ deed.en). UV Camera Printed film NVIS NUV a c b NUV = 0 5 10 15 20 25 NVIS = 0 5 10 15 20 25 Figure 5. Evaluation of an effect based on a number of films. (a) Experimental setup. Captured images based on the number of clear films placed (b) between the printed film and the ultraviolet source and (c) between the printed film and the camera. UV Camera Printed film NVIS NUV a UV Camera Printed film NVIS NUV a c b NUV = 0 5 10 15 20 25 NVIS = 0 5 10 15 20 25 Figure 5. Evaluation of an effect based on a number of films. (a) Experimental setup. Captured images based on the number of clear films placed (b) between the printed film and the ultraviolet source and (c) between the printed film and the camera. a Figure 5. Evaluation of an effect based on a number of films. (a) Experimental setup. Captured images based on the number of clear films placed (b) between the printed film and the ultraviolet source and (c) between the printed film and the camera. projected patterns when compared with that in Fig. 3c. However, four different patterns are recognised from each viewpoint. The images in Fig. 4c correspond to the images of the experimentally fabricated volumetric display observed from different viewpoints. Although the images are not clear when compared with that of the simula- tions, the four different patterns can be successfully observed. Experimental evaluation. The quality of the 3D images of the proposed volumetric display partially depends on the number of the films since the transparency of the films used in practice is not perfect. Scientific Reports \| 7:46511 \| DOI: 10.1038/srep46511 Results F ll l It should be noted that the simulation did not involve the effects of the light absorption by the films or inks since the primary objective involved confirming the validity of the design method. Figure 3c shows the three different patterns projected from the designed volumetric display. Deteriorations caused by other patterns (cross talk) are shown. However, the projected patterns are recognised as the original patterns, as shown in Fig. 3b. It is confirmed that the proposed algorithm13 is extended to full-colour representations. i p p g p Twenty films with printed cross-sectional images are stacked to create a prototype of the volumetric display. The images in Fig. 3d correspond to the images of the volumetric display observed from different viewpoints. In order to decrease the effects due to photo absorption of the ultraviolet excitation light by the films (for details please refer to the experimental evaluation section), the volumetric display is irradiated by two ultraviolet lights placed on the top and the bottom of the display. The observed patterns are not as clear as the ones predicted in the simulations. Nevertheless, the three full-colour patterns are recognised from each viewpoint (see Supplementary Video S1).hi The projection axes can actually be configured freely. As shown in Fig. 4a, a prototype of the volumetric display projecting four patterns is created to experimentally demonstrate such a characteristic. In the experi- ment, the Z-axis is rotated ±20° around both the X- and Y-axes to derive the projection axes. Figure 4b shows the simulation results of the projected patterns from the display. Degradation is observed in the contrast of the Scientific Reports \| 7:46511 \| DOI: 10.1038/srep46511 3 www.nature.com/scientificreports/ ure.com/scientificreports/ X Y Z Pattern A Pattern B Pattern C Pattern D a b c Pattern A Pattern A Pattern C Pattern C Pattern B Pattern B Pattern D Pattern D Figure 4. Prototype of the volumetric display projecting four full-colour patterns. (a) Scheme of the prototype that indicates the projection directions of the patterns. (b) Projected patterns from different viewpoints of the 3D structure in the simulation. (c) Projected patterns of the volumetric display. The photographs used as the original images can be found at http://free-photos.gatag.net/?s=201304180900 (pattern A), https://unsplash.com/photos/HQqIOc8oYro (patterns B), https://unsplash.com/photos/clkYWlgOHIQ (pattern C) and https://www.pexels.com/photo/clown-fish-swimming-128756/ (pattern D). All of them are licensed under the Creative Commons Public domain (https://creativecommons.org/publicdomain/zero/1.0/ deed.en). Discussion Th d This section discusses the resolution of the proposed volumetric display. As described in the demonstration of a full-colour 3D image representation, the inkjet printer used in this study can print 2D patterns of fluorescent inks at an in-plane high resolution (a maximum of 5,760 × 1,440 dpi). However, the depth resolution (out-of-plane resolution) is only approximately 42 dpi, as determined by the film thickness (0.1 mm) and intervals (0.5 mm) between the films. Here, the stacking interval of 0.5 mm was empirically determined based on three factors, which are depth resolution, depth size and the number of films. The stacking interval is minimized in order to increase depth resolution. However, a trade-off exists between depth resolution and depth size since the number of the films in the current study is only twenty. For instance, in the case where the twenty films are stacked without interval to achieve higher depth resolution, the depth size of the resultant display is only 2.0 mm (20 × 0.1 mm), which insufficient to generate a volumetric image. Future work to improve display resolution by increasing the number of films is planned.i i p With respect to these films, increased transparency is indispensable to avoid deterioration of the image with the increase of the films (as quantitatively evaluated previously). For example, a heat-curable resin polydimethyl- siloxane (PDMS) is used in various optical applications18–20 due to its superior transparency. The transmittance of the PDMS exceeds 90% when the optical path length corresponds to 10 mm; thus, the transmittance of a 0.1 mm thick, PDMS film could exceed 99%. At this time, to the best of our knowledge, thin PDMS films, onto which inkjet printers can print fluorescent inks, are not commercially available. We consider the development of such PDMS films as a topic for future research that will afford an interesting comparison with the current system. Furthermore, the combination of PDMS and fluorescent materials in 3D printing represents another potential approach21 and an interesting future research topic.h The 3D object data used in this study are sparsely distributed within the volume. In particular, greater than 98% of the total voxels have “zero” values (represented in perfect black in Fig. 2b and transparent (no-ink) in the experiments). This indicates that the florescent inks were not printed in a majority of the region that the voxels exist. Discussion Th d Therefore, we assumed that the influence of the fluorescent ink absorption was not significant in the cur- rent system whereas the transmittance of the films was analysed. However, it is expected that increasing depth resolution and displaying high-density printed objects may potentially decrease transmission efficiencies and lead to image deterioration due to self-absorption by the inks. Our future work will need to consider technologies to overcome these effects, such as the development of a design method that can effectively correct transmission issues by modulating the 3D layout and the amount of ink while maintaining the quality of resultant images for human perception. In terms of the safety of the proposed volumetric display, it is important to consider potential hazards associ- ated with the use of ultraviolet light. In this study, we employed fluorescent inks excited by ultraviolet light since they are accessible and compatible with commercially-available inkjet printers. Specifically, the current system is physically grounded in the frequency down-conversion (from shorter wavelength to longer wavelength) of fluorescent inks. In this case, protection of the observers’ eyes from harmful ultraviolet wavelengths or stray radiation must be carefully considered. We used UV cut-off filters while observing the images throughout the experiments, and strongly recommend that adequate means of protection be implemented in the use of this technology. Conversely, frequency up-conversion (from longer wavelength to shorter wavelength) technologies have been studied in the field of nanophotonics22,23. Although these frequency up-conversion techniques still require substantial performance improvements (e.g. operating wavelengths, conversion efficiencies) in order to be feasibly applied to our proposed method, they suggest that the current system may potentially be operated using infrared light, which is not hazardous, instead of ultraviolet light. Next, the projected full-colour patterns of the 3D structure designed by the extended algorithm are discussed. As shown in Figs 3 and 4, it is confirmed that the algorithm can be successfully adapted to the full-colour images. However, certain background noises (cross talks) were observed in the computer simulations. It is considered that such a background noise can be suppressed by introducing an iteration algorithm proposed in a previous study15 or by other optimisation methods. The iteration method demonstrated in the literature15 found that the iteration between 3 steps (recording, projecting and updating) may effectively reduce the cross talk between different images. Results F ll l This result is attributed to the diffusion and the refraction of the emitted visible light at the film surfaces. The aforementioned findings indicate that the transparency of the mother materials for inkjet printing radically affects the quality of the volumetric display. This is an important result that can be explored in detail in future studies. Scientific Reports \| 7:46511 \| DOI: 10.1038/srep46511 Results F ll l This type of number-of-layer dependency of the image quality is evaluated by performing an experiment based on the setup, as schematically shown in Fig. 5a. A single layer on which a 2D pattern comprising red, green and blue circles is printed and is sandwiched by multiple layers of transparent films (namely, films without printed patterns). Specifically, NUV and NVIS denote the numbers of transparent films placed between the pattern-printed film and the ultraviolet light source and between the pattern-printed film and the camera, respectively. The increase in NUV and NVIS is unavoidable to realise a high resolution volumetric display in depth direction. However, the increase in NUV and NVIS further attenuates the ultraviolet light required to excite the florescent ink and the colour visible light emitted, respectively. Figure 5b shows the captured images when NVIS = 0 and NUV = 0, 5, …, 25. It should be noted that the camera was focused on the printed film. It is confirmed that the increase in NUV led to a significant decrease in the bright- ness and contrast of the image. This result is attributed to the transmittance of a film in the ultraviolet region (365 nm) that corresponds to 82%. When NUV = 20 and NUV = 25 the ultraviolet light for excitation is attenuated to 2% and less than 1%, respectively.hh y The images shown in Fig. 5c depict the captured images when NUV = 0 and NVis = 0, 5, …, 25. The decrease in the brightness and contrast of the image is lower than those of the results shown in Fig. 5b. This is because the transmittance of a film in the visible region is higher (about 90%) than that in the ultraviolet region. In contrast, it Scientific Reports \| 7:46511 \| DOI: 10.1038/srep46511 4 www.nature.com/scientificreports/ is confirmed that the increase inNVIS leads to the deterioration of the images with respect to sharpness. This result is attributed to the diffusion and the refraction of the emitted visible light at the film surfaces. The aforementioned findings indicate that the transparency of the mother materials for inkjet printing radically affects the quality of the volumetric display. This is an important result that can be explored in detail in future studies. is confirmed that the increase inNVIS leads to the deterioration of the images with respect to sharpness. Discussion Th d This algorithm can also be applied when the projected images are in full-colour.h h The projected patterns of the experimentally fabricated devices deteriorated when compared with the simula- tion results. It is presumed that the deterioration is caused by the degradation of 2D patterns printed on the film with respect to the contrast (brightness) as well as the blurring effects, as observed in Fig. 5c. The degradation of the contrast of the patterns is attributed to the absorption of the emitted light by the films. Thus, the contrast can be decreased by using highly transparent materials as films. The blurring is minimised by increasing the display resolution in the depth direction, and this will also be examined in detail in a future study. In addition to the image quality improvement, the aim of the study involved realising a dynamic volumetric display system for practical applications of the proposed algorithm. In a previous study, an optically addressing method was proposed based on photochromic materials, i.e., photoreactive materials with unique characteristics of a reversible colour transformation24. The volumetric display is developed using this method, and the algorithm experimentally demonstrated in this study is applied on the display. In summary, in this study, a manufacturing method of a high resolution volumetric display based on inkjet printing is proposed and experimentally demonstrated. The inkjet printer enables the minute and automatic loca- tion of photoreactive materials (that are used as the voxels of the 3D images) at appropriate places. Moreover, an Scientific Reports \| 7:46511 \| DOI: 10.1038/srep46511 5 www.nature.com/scientificreports/ V(x, y, z) b a c(uc, vc) b(ub, vb) a(ua, va) a’ (ua, va) PC PB PA PA V(x, y, z) c(uc, vc) b(ub, vb) a(ua, va) a’ (ua, va) V(x, y, z) V(x, y, z) Figure 6. Schematic of the algorithm. (a) Voxel values are determined by multiplying the pixel values of the original patterns. (b) Projected pattern is given by the summation of the voxel values. V(x, y, z) a c(uc, vc) b(ub, vb) a(ua, va) PC PB PA c(uc, vc) b(ub, vb) a(ua, va) V(x, y, z) b a’ (ua, va) PA V(x, y, z) a’ (ua, va) V(x, y, z) b a Figure 6. Schematic of the algorithm. (a) Voxel values are determined by multiplying the pixel values of the original patterns. (b) Projected pattern is given by the summation of the voxel values. Methods l Perpendicular lines PA, PB and PC are drawn from the voxel to the patterns A, B and C, respectively. These lines are referred to as projection axes of the patterns.h j 3. The voxel value V(x, y, z) is calculated as shown in Eq. (1) wherein a(ua, va), b(ub, vb) and c(uc, vc) corre- spond to the pixel values of the original patterns A, B and C at the intersections with each projection axis. Each pixel consists of red, green and blue components as follows: 3. The voxel value V(x, y, z) is calculated as shown in Eq. (1) wherein a(ua, va), b(ub, vb) and c(uc, vc) corre- spond to the pixel values of the original patterns A, B and C at the intersections with each projection axis. Each pixel consists of red, green and blue components as follows: = × × . V x y z a u v b u v c u v ( , , ) ( , ) ( , ) ( , ) (1) a a b b c c (1) Next, the projected patterns of the 3D structure comprising the voxels determined by Eq. (1) are considered. It is assumed that the pixel values of the projected patterns are given by summations of the voxel values along their projection axes, as shown in Fig. 6b. For example, a′(ua, va) corresponds to the pixel value of the projected pattern A and is given by Eq. (2) as follows: Next, the projected patterns of the 3D structure comprising the voxels determined by Eq. (1) are considered. It is assumed that the pixel values of the projected patterns are given by summations of the voxel values along their projection axes, as shown in Fig. 6b. For example, a′(ua, va) corresponds to the pixel value of the projected pattern A and is given by Eq. (2) as follows: ∑ ′ = . a u v V x y z ( , ) ( , , ) (2) a a PA (2) In addition, a(ua, va) is constant along the projection axes PA, and thus a′(ua, va) can be represented as Eq. (3) as follows: ∑ ′ = ⋅ . Discussion Th d algorithm to design 3D structures is developed to project multiple full-colour patterns, and volumetric displays based on the proposed method are experimentally demonstrated. Quantitative analysis of the observed images is performed to clarify important future research agenda. Methods l Volume construction. The quantum yield of red, green and blue inks corresponds to 0.43, 0.85 and 0.89. Furthermore, 0.5 mm thick acrylic plates are used as spacers and placed between the twenty films.h i A method to create the twenty cross-sectional images of a 3D object is described, as shown in Fig. 2b. The object data a created with computer graphics software and is composed of 3D position coordinates (x, y and z) and bright information (R, G, B). It is assumed that the twenty films are placed on z = Z1, … Z20, and a point of the 3D object is at (Ox, Oy, Oz). When Zn < Oz < Zn+1, the point is printed at (Ox, Oy) of the film placed at z = Zn. All the points are printed on the films in the same manner. Algorithm. The algorithm to create the cross-sectional images of the 3D structure that exhibits multiple 2D patterns is described. It is based on a previous study13 and is incremented to correspond to the full-colour rep- resentation. The algorithm can design a 3D structure that exhibits an arbitrary number of patterns (there is a trade-off between the number of the patterns and their image quality). Nevertheless, for the purpose of simplicity, the case wherein the number of patterns corresponds to three is considered, as shown in Fig. 6a. Specifically, V(x, y, z) is a voxel value of the 3D structure that indicates the amount of inks at (x, y, z). In the study, the full-colour images are treated; thus, each of the voxel values comprise three colour components, namely red, green and blue that correspond to VR(x, y, z), VG(x, y, z) and VB(x, y, z), respectively. The voxel value V(x, y, z) can be determined as follows: The voxel value V(x, y, z) can be determined as follows: 1. Each of the original patterns is set up on the direction in which it is required to be projected. 1. Each of the original patterns is set up on the direction in which it is required to be projected. g p p q p j 2. Perpendicular lines PA, PB and PC are drawn from the voxel to the patterns A, B and C, respective lines are referred to as projection axes of the patterns.h g j 2. References & Prasad, P. N. Light upconverting core–shell nanostructures: nanophotonic contro emerging applications. Chem. Soc. Rev. 44, 1680–1713 (2015). g g pp 4. Hirayama, R. et al. Optical addressing of multi-colour photochromic material mixture for volumetric display. Sci. Rep. 6, 31543 (2016). Author Contributions R.H., Tomotaka S., M.N. and T.I. directed the project. Tomoyoshi S. suggested the basic concept of this study. H.N., A.S. and T.I. suggested the algorithm demonstrated in this study. R.H., Tomotaka S. and T.I. designed and performed the experiments. R.H. Tomotaka S., N.M. and T.K. analysed the data. All authors contributed the discussions and reviewed the manuscript. Methods l a u v a u v b u v c u v ( , ) ( , ) ( , ) ( , ) (3) a a a a P b b c c A (3) Scientific Reports \| 7:46511 \| DOI: 10.1038/srep46511 6 www.nature.com/scientificreports/ As a result, the projected pattern is given by multiplying the original pattern and a background noise, which corresponds to interference from the other two patterns. The component of the original pattern in Eq. (3) tends to be more dominant than that of the background noise when 2D images are generally used as the original patterns. Therefore, the projected patterns are recognised as the original patterns used to determine the voxel values. The same trend is observed for the pixels of patterns B and C; therefore, three patterns are recognised from the 3D structure. Acknowledgementsh Acknowledgements This study was partially supported by the Japan Society for the Promotion of Science Grant-in-Aid No. 16J30007 and No 25240015 and the Core-to-Core Program A Advanced Research Networks g This study was partially supported by the Japan Society for the Promotion of Science Grant-in-Aid No. 16J30007 nd No. 25240015 and the Core-to-Core Program, A. Advanced Research Networks. References References 1. Blundell, B. G. & Schwarz, A. J. The classification of volumetric display systems: characteristics and predictability of the image space. IEEE Trans. Vis. Comput. Graphics 8, 66–75 (2002). G Th d l d l h l Ad O h ( ) p p 2. Geng, J. Three-dimensional display technologies. Adv. Opt. Photonics 5, 456–535 (2013). gh y g p 3. Sullivan, A. DepthCube solid-state 3D volumetric display. Proc. SPIE 5291, 279–284 (2004). 4. Parker, M. Lumarca. Proc. ACM SIGGRAPH Asia 2009 Art Gallery & Emerging Technologies: Adaptation 77, Yokohama, J (2009). 5. Barnum, P. 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Hirayama, R. et al. 3-D crystal exhibiting multiple 2-D images with directivity. Proc. ACM SIGGRAPH Asia 2015 Posters 1, Kobe, Japan (2015). 15. Hirayama, R. et al. Image quality improvement for a 3D structure exhibiting multiple 2D patterns and its implementation. Opt. Exp. 24, 7319–7327 (2016). ofstadter, D. R. Gödel, Escher, Bach: an Eternal Golden Braid. (Basic Books, New York, 1979). 16. Hofstadter, D. R. Gödel, Escher, Bach: an Eternal Golden Braid. (Basic Books, New York, 1979). 17. Mitra, N. J. & Pauly, M. Shadow Art. ACM T. Graph. 28, 156 (2009). 17. Mitra, N. J. & Pauly, M. Shadow Art. ACM T. Graph. 28, 156 (2009). Mitra, N. J. & Pauly, M. Shadow Art. ACM T. Graph. 28, 156 (2009) 18. Hirayama, R. et al. Design, implementation and characterisation of a quantum-dot-based volumetric display. Sci. Rep. 5, (2015). 9. Wang, Y., Bish, S., Tunnell, J. W. & Zhang, X. MEMS scanner enabled real-time depth sensitive hyperspectral imaging of biologica tissue. Opt. 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Sel. Topics Quantum Electron. 15, 1380–1386 (2009). 23. Chen, G., Ågren, H., Ohulchanskyy, T. Y. & Prasad, P. N. Light upconverting core–shell nanostructures: nanophotonic control for (2009). 23. Chen, G., Ågren, H., Ohulchanskyy, T. Y. & Prasad, P. N. Light upconverting core–shell nanostructures: nanophotonic control for emerging applications Chem Soc Rev 44 1680 1713 (2015) 23. Chen, G., Ågren, H., Ohulchanskyy, T. Y. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing Interests: The authors declare no competing financial interests. Competing Interests: The authors declare no competing financial interests. How to cite this article: Hirayama, R. et al. Inkjet printing-based volumetric display projecting multiple full- colour 2D patterns. Sci. Rep. 7, 46511; doi: 10.1038/srep46511 (2017). How to cite this article: Hirayama, R. et al. Inkjet printing-based volumetric display projecting multiple full colour 2D patterns. Sci. Rep. 7, 46511; doi: 10.1038/srep46511 (2017). How to cite this article: Hirayama, R. et al. Inkjet printing-based volumetric display projecting multiple full- colour 2D patterns. Sci. Rep. 7, 46511; doi: 10.1038/srep46511 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. 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W2601812130.txt	https://zenodo.org/record/2023689/files/article.pdf	fr		Sur la fragilité des aciers doux cémentés	Revue de métallurgie	1,905	public-domain	3,292	SUR LA FRAGILITÉ DES ACIERS DOUX CÉMENTÉS PAR M. J. LECARME Lorsqu’on cémente un acier doux, on constate que le métal est devenu moins résistant au choc et que la fragilité augmente avec la profondeur de la cémentation. On a attribué ju s q u ’ici la cause de cette fragilité au traitement thermique que subissent les pièces pendant la cémentation, dont l'effet se mani feste principalement par l’augmentation plus ou moins grande des polyèdres de ferrite. L ’expérience m'ayant montré, au contraire, que l’action de la chaleur était secondaire, j ’ai entrepris une série d’essais dont je vais résumer ici les plus inté ressants et qui démontrent : 1° Que le traitement thermique qui accompagne la cémentation a pour effet de détruire en partie les qualités que l’acier doux avait acquises au laminage, forgeage, etc., mais sans le rendre cassant; 2° Que la fragilité due à la cémentation est produite par une transformation d’ordre chimique du noyau doux, et qui a lieu en même temps que la cémentation superficielle de l'acier ; 3° Que cette transformation donne aux aciers doux de diverses provenances une fragilité très variable bien que ces aciers soient d'une composition chimique très voisine et aient des propriétés mécaniques analogues. Voici d ’abord quelques faits typiques q u ’il me semble intéressant de signaler. J ’ai réussi à plier à bloc des fers à grains qui avaient subi un recuit de 1.000° pen dant 125 jours en les réchauffant à 900° et les trempant dans l'eau à 10°; tous les échantillons ne m ’ont pas donné d'aussi bons résultats, mais ils ont toujours accusé une flexion très notable. Or, les mêmes échantillons cémentés à 1 mm. de profondeur, puis la couche cémentée une fois supprimée par un usinage quelcon que, le barreau a été trempé comme précédemment : l’essai à la presse n ' a donné aucune flexion, et l’échantillon a cassé net. Je ne cite cette expérience qu'à titre d'exemple, car le recuit prolongé des barres a été fait dans des conditions qui ne se présentent pas en pratique ; en particulier, le contact des gaz du four n ’a pas été complètement évité et la température n ’a pas été constante. Un autre fait qui me paraît très net est le suivant : il est arrivé que parmi les pièces soumises à la cémentation, les témoins d’acier doux qui sont placés dans les caisses à côté des pièces et destinés à contrôler la profondeur obtenue n 'o nt pas été enduits de cément par erreur ; ils ont alors subi le même traitement th er mique que les pièces et que les autres témoins, mais sans être cémentés superfi ciellement. Après trempe, ils ont été tous cassés au b alancier ; or, ceux qui n ’é- M. J. LECARME 517 taient pas cémentés se sont pliés fortement, les autres se brisant après une légère flexion. Par conséquent il faut en conclure que la fragilité du centre d’une pièce cémentée est liée à la cémentation superficielle et q u ’elle ne dépend pas seulement du traitement thermique comme on le croyait jusqu’ici. Je pourrais citer encore bien d ’autres faits qui se produisent fréquemment dans une usine où l'on fait beaucoup de cémentation et où l’on est appelé à traiter journellement des aciers de toutes provenances. Mais comme une partie des phénomènes observés se trou vent reproduits dans les expériences que je vais décrire, il me paraît inutile d’in sister ici. Pour rechercher la cause de la fragilité des aciers doux cémentés, j ’ai choisi parmi les aciers du commerce deux qualités courantes et donnant des résultats très différents lorsqu’on leur fait subir un traitement identique. Voici les constantes moyennes de ces aciers : 1° — Acier B : Composition chimique 0/0 Constantes mécaniques R = 37 kg. A 0/0 = 35 C = 0,100 p = 0,031 lu = 0,300 Si = 0,750 2° — Acier C T de Firminy. Constantes mécaniques Composition chimique 0/0 C = 0,090 P = 0,065 H= 46,8 kg. A 0/0 = 29,8 Mn = 0,623 Si =0,152 E = 35,6 Σ = 0,67 Ces aciers étant cémentés à diverses profondeurs puis trempés à 800° à Peau, donnent des résultats très différents : l'acier CT, pour 1 mm. de cémentation fléchit fortement tandis que l ’acier B casse net. Il faut donner à l’acier CT une profondeur de cémentation allant jusqu'à 3 mm. pour obtenir une fragilité com parable. Il en résulte que l’acier B est beaucoup plus sensible que l’acier CT pour un même traitement, puisqu’il exagère notablement l’effet de fragilité. Dans les expériences qui vont suivre, j'ai cherché à augmenter encore ce phénomène en maintenant la température de cémentation à 1.000°. J ’ai opéré sur des barres rondes de 20 mm. de diamètre et de 20 cm. de lon gueur. Les aciers ont été divisés en quatre groupes que j ’appellerai A, B, C, D. Chaque groupe contient dix barrettes de 20 cm. de longueur, numérotées de 1 à 10. Les éprouvettes portant le même numéro dans chaque série ont subi le même traitement thermique. Les séries A, B, C, D, correspondent à un même traitement chimique. La série A correspond à un traitement thermique seul, c’est-à-dire que les éprouvettes ont été chauffées dans une matière inactive et à l’abri des gaz. Elles ont été placées à cet effet dans des tubes en fer hermétiquement clos et remplis de terre réfractaire Muller, qui n ’altère pas la surface du métal et empêche toute oxydation. 518 REVUE DE MÉTALLURGIE La série B correspond à un traitement thermique seul, mais en un milieu carburé, très peu actif, constitué par du charbon de cornues finement pulvérisé et qui ne cémente pas quand il est pur. La série G correspond à un traitement thermique et chimique, les pièces étant entourées de charbon de cornue comme précédemment, mais enduites d’un cément au carbone énergique. La série D comprend un traitement analogue au précédent, mais le charbon de cornue étant remplacé par du charbon de bois fin, préalablement calciné pour éviter tout dégagement gazeux. En plus, les derniers numéros (9 et 10) de chaque série ont été enduits d’un anticément, c’est-à-dire d’un corps qui empêche la cémentation et qui permet ainsi, par suite de la cémentation partielle d ’une pièce mécanique, de conserver une plus grande souplesse à la partie non cémentée. C’est précisément à cause de ce dernier point que j’ai cru intéressant de rechercher l’action des anticéments sur le centre des aciers. Les pièces ainsi préparées ont été mises au four pendant le temps indiqué : le chauffage étant terminé, les tubes ont été retirés du four et abandonnés au re froidissement spontané. Les tubes étant alors ouverts, les éprouvettes ont été brossées pour enlever tous les dépôts, le métal s’est trouvé intact pour toutes les barrettes. Chaque barrette a été sciée d’abord en deux parties, puis sur chacune d’elles une rondelle de 15 mm. a été prélevée pour l’examen micrographique. Après numérotage des rondelles, chaque lot a été divisé en deux groupes, dont l’un a subi une trempe à partir de 800° dans un liquide à 10° trempant énergi quement. Voici les divers résultats obtenus : Acier B naturel. — Plie à bloc sans criques. Le même trempé à 800°, plie à bloc sans criques. Expérience A : À 2. — A plié jusqu’à former un angle de 20°(presque à bloc) puis un choc un peu trop violent du balancier l’a brisé. La cassure montre un grain fin et soyeux. Si la pression avait été constante, l’éprouvette ne se serait pas brisée. A 4. — A plié à bloc sans criques. A 6. — A plié jusqu’à former en angle de 30° puis un choc du balancier l’a brisé. A 8. — S’est plié à bloc sans criques. A 10. — S’est plié à bloc sans criques. L'acier C T a pu, dans les mêmes conditions, et pour tous les cas être plié à bloc sans former de criques. Les essais de traction ont donné, avec l’acier C T : 1° Métal naturel : B = 46kg,8 ; A = 29,8 : 2° Métal trempé à 800° : R z= 54kg,2 ; A = 13,8 ; 3° Métal ayant subi le traitement A 4 : R = 53ke,2 ; A = z 16,8. 519 M. J. LECARME Tableau résumant les essais effectués sur l’acier B Traitement thermique seul Terre réfractaire D C B A Traitement ent thermique therm ique d. Traitement thermique Traitem dans cornue avec C . de bois dans cornue cément. et cément. 1reheure n on trempé . 1. essai de flexion inutile » » 1000° trempé à 800°. 2. plie presque à bloc (20°) plie à angle droit plieàpeine et casse casse net 2 heures non trempé . 3. — 1000° »trempé à 800°. 4. plie à bloc casse net plie et casse avaut casse net l’angle droit 3 heures non trempé . 5. — 1000° trempé à 800°. 6. plie jusqu’à (30°) casse net plie de 20° et casse casse net lOheures non trempé . 7. — 1000° trempé à 800°. 8. plie à bloc casse net plie à peine etcasse casse net lOheures non trempé . 9. — avec anticément 1000° trempé à 800°. 10. plie à bloc plie à angle droit _ — sans casser Section 1. Naturelle........................... 2. Naturelle trempée à 800° . . 3. Recuite 2 h. terre et trempée. 3. Recuite 2 h. cornue et trempée. 5. Cémentée 2 h. non trempée . 6. Cémentée 2 h. et trempée . . 7. Centre d’une barre cémentée 2 h. puis trempée . . . . 8. Éprouvette cémentée 10 h. et trempée............................ Limite élastique deCharge rupture Allongement Striction S-s s totale par mm2 totale par mm* total 0/0 Diamètre éprouvette Longueur de mesure Tableau des essais de traction : Acier C. T. F irminy. mm. 10 mm2 mm. kg. 78 72 2,800 — — 3,900 — — 3,700 — — 3,180 — — 2,800 — — 4,500 — — 4,450 4,080 — — — — — — — — — kg. kg. 35,6 3,680 49,6 4,260 47,1 4,180 40,5 4,220 35,6 4,400 57,3 4,500 56,6 5,200 51,9 4,080 kg. 46,8 54,2 53,2 53,7 56,0 57,3 66,2 51,9 21,5 10,0 12,0 10,0 5,5 0,0 29,8 13,8 16,8 13,8 7,6 0,0 9,0 12,5 0,0 0,0 0,67 0,64 0,57 0,56 0,07 0,0 0,27 » Micrographie acier B . (Photo 1). 1° Métal naturel. — G = 160 d. — L ’aspect de ce métal est normal, la perlite étant rassemblée entre les grains de ferrite comme dans tous les aciers doux. (Photo 2). 2° Métal trempé à 800°. — La trempe a été effectuée dès que la température a atteint 800°, sans la dépasser et sans la maintenir plus de quel ques secondes pour éviter le recuit. Les polyèdres de ferrite ont conservé leurs contours qui se sont accentués par la formation d ’une mince couche de troostite qui apparaît en noir sous l’action du réactif A que nous a indiqué M. le Chatelier : les parties blanches sont formées de ferrite et de martensite. 520 REVUE DE MÉTALLURGIE (Photo 3). 3° Métal recuit. — Les micrographies montrent que l'effet du recuit s’est traduit par une augmentation des éléments, qui ont conservé leur as pect primitif et leurs formes. Ce qui est à noter ici est l accroissement d’abord très rapide des polyèdres, qui paraissent conserver ensuite les dimensions qu’ils ont acquises pendant la première heure de chauffage. L’examen des micrographies correspondant aux essais A 1, A 3, A 5, A 7, montre que les changements d as pect sont de peu d’importance : ils sont du même ordre que les diverses parties d’un même échantillon. Leur aspect est celui de la photo 3 (A 7). Mais la trempe agit alors en modifiant complètement la texture correspon- Fig. 1. — Acier B naturel. G= 160. dant à la photo 2. Ainsi la photo 4 montre un mélange de martensite encore unie aux plages perlitiques du métal re cu it, et aucune ressemblance n ’existe plus entre le métal recuit 1 h. et 4 h. par exemple. Ceci semble démontrer que l’effet du recuit seul n ’est pas décelé complètement par la micrographie, mais que la trempe rend la transformation plus sensible, du moins par l’emploi des méthodes d’attaque employées. (Photo 4). — Cet effet paraît montrer très nettement l’action effective des anticéments sur le métal. Ainsi la photo 5 montre le résultat obtenu par un recuit identique à celui de l’échantillon A 7 (photo 3) mais en présence d’un anticément, et trempé à 800°. (Photo 5). — Malheureusement il paraît difficile de déterminer la nature des constituants ainsi obtenus. En particulier la fig. 5 montre des plages blanches im portantes qui paraissent formées de ferrite bordée elle même d ’un filet de troostite qui enveloppe les parties noires de constitution assez confuse. Il ne semble pas y M. J. LECARME 521 avoir un rapport bien net entre cette texture et la non fragilité de l’acier. J e pour- Fig. 2. —Acier B trempé à 800°. G= 160. Fig. 3. — Acier B recuit 10h. à 1000°. G= 160. suis des essais dans cette voie sur divers aciers, car chaque sorte d’acier doux donne un résultat différent. 67 522 REVUE DE MÉTALLURGIE Expérience B. Essais mécaniques : B 2. — S ’est courbé à angle droit. Fig. 4. —Acier B recuit 1 h. à 1000° et trempé à 800°. G = 160. Fig. 5. — Acier B recuit 10 h. à 1000° eu présence d’un auticément et trempé à 800°. G =160. B 4. — A fléchi un peu moins, puis a casse. B 6. — A fléchi de 20° puis a cassé. M. J. LECARME 523 B 8. — A accusé une légère flexion et a cassé net. B 10. — S’est courbé à angle droit sans casser. Cette expérience montre que les barres ont acquis une plus grande fragilité que celles de l'expérience A. Les cassures ont fait apercevoir une légère couche durcie, mais de faible teneur en C. La lime l’entame facilement. Ceci ne se pro duit pas avec du graphite pur; mais ici le charbon de cornue employé contenait, outre les gaz qu’il a pu absorber, des corps étrangers, notamment du soufre. Il y a donc eu cémentation, qui, bien que très faible, a produit immédiatement une fra gilité très supérieure à celle que donne le simple recuit. La micrographie n ’in- Fig. 6. — Acier B. Cémeuté 10 h. et trempé à 800°. G= 160. dique rien de bien net. Les éléments ont conservé les mêmes dimensions que par le simple recuit, et les parties noires sont devenues plus intenses par le même mode d’attaque. Le bord extérieur montre une légère carburation se traduisant par une teinte jaune sur l’échantillon, alors que la cémentation ordinaire donne une teinte noire. L’acier C T dans ces conditions, donne une flexion beaucoup plus accentuée comme dans le cas précédent. L’essai de traction a donné pour le cas B 4 : R = 53,7 ; A = 13,8. Expérience C. — Les résultats sont à peu près les mêmes que ceux obtenus dans le traitement D. Les micrographies sont semblables, je n ’insisterai donc pas. Expérience D. — Les essais de flexion donnent dans tous les cas une rupture brusque dès que la limite d’élasticité de la couche durcie est dépassée. L’acier G T fléchit avant de casser : voici le résultat de traction : Traitement D 3 : R = 56 kg. A = 7,6 D 4 : R = 5 7 kg,3 A = 0. 524 REVUE DE MÉTALLURGIE En taillant l’éprouvette dans le même métal cémenté suivant le traitement D 3, de façon à ne conserver que le centre, puis en la trempant suivant D 4, on obtient : R = 66kg,2 A = 12,5 Σ = 0,27. Ce résultat paraît très intéressant, et je poursuis une étude comparative des divers aciers dans cet ordre d’idées. La micrographie 6 correspondant au traitement D 8 montre bien la modifi cation profonde qui s’est produite : la texture martensitique apparaît nettement et semblerait indiquer que le carbone a pénétré ju sq u ’au centre du métal. Malheu reusement, l ’analyse chimique démontre le contraire, du moins en employant les méthodes ordinairement usitées. Pour expliquer ce fait, je suppose que le carbone se combine peu à peu à l’azote qui se dissout très facilement au rouge dans le fer, et qui échappe ensuite à l’attaque malgré toute les précautions prises pour éviter les pertes. De fait, on trouve moins de C combiné dans le centre d ’un acier doux cémenté que dans le métal primitif. Ceci m’a amené à étudier plus particulièrement la forme sous laquelle se trouve le C dans les aciers. Ce travail n ’est pas encore assez complet pour en tirer actuellement des conclusions, mais je puis dire, dès maintenant, que l ’hypothèse de Fremy semble se vérifier sur tous les aciers, même sur ceux que l’on obtient au four électrique. Tous les essais de cémentation que j ’ai pu faire sur les divers aciers que nous avons en magasin donnent des résultats conformes à l ’expérience que j ’ai citée relativement à l’acier B. Mais la fragilité obtenue est variable avec les aciers. Le point de transformation de l'acier doux se trouve déplacé d’une ma nière plus ou moins g r a n d e ; il peut y avoir un écart de 300° pour les divers aciers doux. Ceci nous a permis de les classer en différents groupes, et ce phéno mène peut donner lieu à une méthode très précise pour reconnaître en deux heures la provenance d’un acier. Je réunis actuellement les documents qui pour raient amener la relation qui existe entre leur composition chimique et le dépla cement de leur point de transformation principal. Ce qui est intéressant à noter au point de vue industriel, c’est qu’il est pos sible de supprimer d’une façon presque complète la fragilité de l'acier doux ainsi obtenu. Il suffit pour cela de connaître exactement la nature d e là transformation qui s’est effectuée au centre du métal, ce qui s ’obtient assez facilement par compa raison avec des échantillons classés, et en tenant compte des divers facteurs pouvant dépendre de la cémentation. Une fois ces données connues, il suffit de faire subir un recuit et une trempe correspondant au nouveau métal qu’on a ob tenu et qui dans la plupart des cas est complètement différent du métal primitif. On peut alors obtenir des pièces qui se courbent à angle droit bien qu’étant cé mentées à des profondeurs allant ju s q u ’à 3 mm. et plus : la texture a perdu alors son aspect cristallin et est devenue fibreuse. Conclusions. — On peut tirer des essais précédents ainsi que de ceux que M. J. LECARME 525 j ’ai pu faire sur des aciers de plusieurs marques différentes, les conclusions sui vantes : 1° La fragilité des aciers doux cémentés ne provient pasdu recuit q u i accompagne la cémentation ; 2° La cémentation superficielle au carbone est accompagnée d’une transfor mation chimique du centre du métal qui paraît homogène pour toute la masse. Le nouveau métal produit, traité par la méthode ordinaire, c’est-à-dire simple ment trempé vers 800°. devient fragile par cette opération ; 3° Par un traitement thermique et une trempe appropriés, il est possible de supprimer en partie la fragilité sans nuire à la dureté de la couche superficielle. On obtient alors au centre de la pièce une texture fibreuse, sauf pour les aciers qui se trouvent sur la limite de deux groupes et auxquels il est très difficile de rendre les qualités qu’ils possédaient primitivement s’ils sont cémentés assez profondément.
W2057808808.txt	http://www.scirp.org/journal/PaperDownload.aspx?paperID=43663	en		Simulations of Colliding Uniform Density H2 Clouds	International journal of astronomy and astrophysics	2,014	cc-by	14,073	International Journal of Astronomy and Astrophysics, 2014, 4, 192-220 Published Online March 2014 in SciRes. http://www.scirp.org/journal/ijaa http://dx.doi.org/10.4236/ijaa.2014.41018 Simulations of Colliding Uniform Density H2 Clouds Guillermo Arreaga-García1, Jaime Klapp2,3, Julio Saucedo Morales1 1 Departamento de Investigación en Física, Universidad de Sonora, Hermosillo, Mexico Instituto Nacional de Investigaciones Nucleares, Ocoyoacac, Mexico 3 Departamento de Matemáticas, Cinvestav del Instituto Politécnico Nacional, México D.F., Mexico Email: garreaga@cifus.uson.mx, jaime.klapp@inin.gob.mx, jsaucedo@cifus.uson.mx 2 Received 18 December 2013; revised 18 January 2014; accepted 25 January 2014 Copyright © 2014 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Abstract In this paper we present a set of numerical simulations designed to study the interaction process of HII molecular clouds. For the initial conditions we assume head-on and oblique collisions of binary identical clouds placed adjacent to one another, with their surfaces just in contact. The colliding initial clouds are uniform density molecular gas spheres with rigid body rotation. The cloud initial conditions are chosen to favor its gravitational collapse as an isolated system. To study the effect of the self-gravity of the cloud in the collision process, we consider several models in which the approaching speed of the colliding clouds increases from zero up to several times the initial sound speed of the barotropic gas. We present the outcome of these collision models for several values of the impact parameter b, which depends on the initial radius of the cloud. We have explored the parameter space of the approaching velocity Vapp of the colliding clouds for configurations that may result in seeds for the formation of more complex systems. Such systems are expected to include filaments and gas clumps, where the star formation process is still possible despite the occurrence of the collision. We show hereby that collisions may have a major and favorable influence on the star formation process. Keywords Star Formation; Cloud Collisions; Protostellar; Binaries 1. Introduction Protostellar cloud collisions are expected to occur in a great variety of astrophysical phenomena, for example, as a consequence of collisions between gas-rich galaxies; or due to the presence of strong tidal forces within a single giant cloud. For instance, [1] identified mechanisms they suggested could initiate the formation of agglomerations How to cite this paper: Arreaga-García, G., et al. (2014) Simulations of Colliding Uniform Density H2 Clouds. International Journal of Astronomy and Astrophysics, 4, 192-220. http://dx.doi.org/10.4236/ijaa.2014.41018 G. Arreaga-García et al. of matter within the cloud, due to the expansion of regions leading to a compression in other regions of the same cloud. He presented observational evidence of gas compression at the interface of two colliding molecular clouds, and proposed that collisions may be an alternative mechanism to explain the formation of massive stars in galaxies. Indeed, collisions between neutral molecular clouds are frequently mentioned as a possible mechanism for initiating the star formation process. For instance, based on observed carbon molecular lines, [2] suggested that the formation of the stars in the IRAS04000 + 5052 cluster was induced by the collision of two molecular clouds. The theoretical study of collisions has a long history. It was started in the early 80 s, but even nowadays the academic interest in astrophysical collisions has never fallen off completely, see for instance [3] [4]. In the old numerical simulations of colliding gas clouds that were done with particle based codes, the clouds partially penetrated each other; and in the case of collisions with high Mach numbers, they passed completely one to another, see [5]. These non-physical results were the consequence of the neglect of artificial viscosity that was later used by [6]. In simulations based on grid techniques, there were also important limitations, for example, those considering only two spatial dimensions or neglecting self-gravity. In spite of this, the complexity of the phenomenon was uncovered many years ago. For instance, [7] studied head-on collisions with several two clouds combinations, in which one of them represented a giant molecular cloud, and the second one was smaller and denser than the former. They also considered several cases according to the internal density structure of the clouds: some were uniform while others clumpy. In their two dimensional hydrodynamical calculations, the effect of the self-gravity of the gas was unfortunately not taken into account. Besides, [8] performed two dimensional collision simulations between atomic hydrogen clouds using the AMR (Adaptive Mesh Refinement) numerical technique. In their hydrodynamic equations, they included heating and cooling functions as well as a magnetic pressure term. They considered homogeneous clouds with cylindrical symmetry and surface perturbations, and showed that a filament forms at the interface of the clouds and that the gas flow outwards from the edge of the filament. They estimated that a numerical simulation based on particles with a similar resolution should include at least one million particles. Unfortunately, again their simulations did not include self gravity. Additionally, [9] also considered collisions between giant spherical clouds within the framework of the Smoothed Particle Hydrodynamics (SPH) Lagrangian technique. The mass of each cloud was 2222M⊙ with a radius of 10 pc. Their collision models with clouds at a temperature of 20 K and with an approaching speed of 10 km∙s−1 produced fragments with a size of 0.1 pc and with a peak density number of 104 cm−3. The conclusion of their calculation was that cloud collisions certainly stimulate the formation of clumpy structures. A drawback of this study is that the authors only used 4096 particles to model such a huge cloud. There are non-spherical collision models reported in the literature as well, for instance [10] considered two identical, parallel gas slabs, semi-infinite in extent so that each slab could be considered two dimensional, in such a way that the collision takes place in only one spatial dimension. Although the papers mentioned above are only a representative sample of the vast amount of existing literature, in view of them, we believe it is timely to devote this paper to make new numerical simulations of the collision process, taking advantage of both new capabilities of modern computers and improved numerical techniques. We now present numerical high resolution three-dimensional (3D) hydrodynamical simulations of spherical cloud collisions, including the self-gravity of the gas. As mentioned earlier, we restrict ourselves to considering only binary collisions between identical clouds. Our main objective in this paper is to explore the parameter space of the collision models; we have proposed several approaching velocities, simply as sample values chosen only on the basis of the different outcomes that each simulation produces. We also emphasize that along this project we are always interested in studying the effects of the collision on a cloud which is already collapsing. Another subject deserving further study, is the case of clouds starting from hydrodynamical equilibrium. This was already considered by [4] [11], who simulated cloud collisions between two clouds initially in hydrodynamical equilibrium (since each cloud was modeled as Bonnor-Ebert sphere). Collisions between dissimilar clouds has also considered by [12]. An important result of our simulations, is that we have explored the possible approaching velocities of the colliding clouds (for an initial cloud with well defined physical characteristics, see Section 2). Because of this, we now know the range of approaching velocities that allow an initial binary colliding clouds system to remain as a binary system capable of undergoing further gravitational collapse and eventually 193 G. Arreaga-García et al. produce a multiple protostellar system. It is in this sense that our results have to be compared with those of [13], that obtained that it is more likely that a colliding system will in general result in disruption and dispersal of the cloud involved with no chance of forming proto stars. It must be emphasized that this work hereby devoted to study collisions with clouds with a clear initial tendency to a gravitational collapse prior to collision, has enabled us to uncover subtle collision effects about the formation and fragmentation of the collapsing clumps. To have observed these effects have made an important difference in the results derived from the simulations when we compare with those results reported by [4] [11] [12]. Besides, during the phase that comes after the collision before the density reaches the critical density ρ ~ 10−18 gr∙cm−3, shearing and Kelvin-Helmholtz instabilities are observed. Fragmentation has been observed in somefilaments, which is a direct consequence of the occurrence of the collision. The integral properties of the resulting gas clumps are calculated with the purpose of estimating if they are likely to collapse, virialize or disperse. The initial conditions for the isolated cloud are such that it will collapse in the absence of a collision as its thermal and rotational energy ratios with respect to the gravitational energy, have been chosen to have initially the numerical values α0 = 0.26 and β0 = 0.16, respectively. However, when we consider the translational kinetic energy that comes from the approaching velocity Vapp of the clouds, the collision system is unbound for Vapp greater than about Mach three. As a result of the collision process a large fraction of the translational kinetic energy is transformed into heat that is radiated away because the clouds and the shock front that forms in the interphase between the clouds are isothermal. This produces are duction of the global value of α + β for all models. For the head-on models we found that they all get a final configuration near equilibrium even for very high Vapp. This is not the case for the oblique models, which have a final value near equilibrium for Vapp less than about Mach three and all b values. The final α + β values get away from equilibrium for Vapp greater than Mach eight and high b values. Hence we only get very high dissipation, and thus the possibility of fragmentation for initially unbound systems for head-on or near head-on collisions. The observation that collisions favor molecular cloud fragmentation is a very important result in the area of star formation, because each dense gas knot formed along the filament in the central core of the colliding cloud, could eventually form a proto-star. The outline of the paper is as follows. In Section 2 we describe the characteristics of the particle distribution that represents the initial cloud, which will be involved in all the subsequent collisions. In Section 3 we show the geometry of the collision models and the parameter values chosen for the Gadget2 code. In Section 4, we describe the most important features of the time evolution of our simulations by means of two-dimensional (2D) isodensity plots, despite the fact that the collision events are fully 3D. In Section 5 we discuss the relevance of our results in view of those reported by previous works and finally we make some concluding remarks. 2. The Initial Cloud We consider a spherical cloud with radius R0 = 3.0138 × 1018 cm = 0.97 pc = 2.0038 × 105 AU and mass M0 = 2.21M⊙. The average density of this cloud is ρ0 = 1.3024 × 10−21 gr/cm3, from which we can estimate the time required by a test particle to reach the center of the cloud from the outermost regions, known as the free fall time of the cloud and given by 3π 5.8 × 1013 sec = 1.846Myr . = 32G ρ0 τ ffc = (1) Our initial cloud can be considered as a typical prestellar cloud, though of a very small size such that it can be called a clump according to [14]. It is relatively easy to observe these small clouds as isolated systems in low mass star forming regions such as Taurus [15]. If these clouds have also existed within more dense cloud environments such as the Orioncluster, they would have had multiple dynamical encounters. In the collision events more frequently observed, the involved clouds are very likely to be typical giant molecular clouds with radius ~ 40 pc and mass ~ 105 M⊙. We have set five million SPH particles to generate the initial cloud by a traditional Monte Carlo scheme, in which the particles are randomly located in the volume space. Let u, v and w be random uniform variables taking real values within the interval [0,1]; then according to the fundamental probability conservation law for a system with spherical symmetry, we have that 4πr 2 ρ ( r ) dr sin θ dθ dϕ = dudvdw . By means of integration we obtain that the spherical coordinates ( r , θ , ϕ ) of the particles are related to the uniform random variables by the 194 G. Arreaga-García et al. following equations: u= 4π M0 ∫0 ρ ( r ′)r ′ dr ′, r 2 1 − cos (θ ) 1 θ sin (θ ′ ) dθ ′, = 2 2 ∫0 1 φ φ dφ ′, = w = 2π 2π ∫0 = v (2) where M0 is the total mass contained in the cloud of radius R0; it must be noticed that the first relation should be numerically integrated to obtain r once that u has taken an allowed uniform random value. The mathematical function f(r) in which we are interested to model the cloud’s radial distribution of matter was first introduced by [16], and later on studied by [17] as a building block of an analytical model to study the collapse of clouds. This radial density function is given by:  Rc ρ ( r ) = ρc   r 2 + R2 c  η   ,   (3) where ρc, Rc and η are three free parameters that fix the shape of the radial density profile. As it was mentioned by [16], the main characteristic of this Plummer-like density function is its radial behavior: constant at first and then rapidly falling off with radius. Due to the density behavior, this mathematical model captures quite well the observed fact that prestellar clouds are mainly formed by a strongly centrally condensed low mass core, which is surrounded by a gas envelope steeply declining in density, see [15]. The Plummer parameters are Rc = 3.0 × 1018 cm and ρc = 1.3025 × 10−21 gr∙cm−3. The initial radius R0 is slightly larger than Rc, because for this paper we are mainly interested in studying the collision of uniform density clouds, see Figure 1. The cutting density ρc is very near the average density ρ0. In an article to come, we will study collisions between centrally condensed clouds, in which Rc < R0. This future study promise to be interesting, as [18] [19] have already shown that the extension of the envelope with respect to the core in a centrally condensed cloud, is also an important dynamical factor playing a crucial role in the fate of the cloud’s collapse. Additionally, we consider that the cloud is in counterclockwise rigid body rotation around the Z axis; therefore the initial velocity of the i-th SPH particle is given according to vi =Ω0 xri =Ω0 ( − yi , xi , 0 ) , where Ω0 is the constant magnitude of the initial angular velocity. For the cloud considered in this paper, the initial sound speed and angular velocity have the following values: c0 = 16600 cms −1 , = Ω 0 2.5969 × 10−15 s −1 . (4) As it is quite common in the literature of gravitational cloud collapse [20] [21], the dynamical properties of the initial distribution of particles modeling the cloud are usually characterized by means of the thermal and rotational energy ratios with respect to the gravitational energy, denoted by α and β, respectively. The ρ0 and Ω0 values have been calculated for the energy ratios to initially have the following numerical values: α = Etherm = 0.26055, Egrav β = Erot 0.16134. = Egrav (5) We recall that these selected values of α and β are known to favor the occurrence of collapse in the cloud. As done in our previous papers, we also implement a density perturbation on the initial particle distribution, such that at the end of the evolution of the cloud as an isolated system, it might result in the formation of binary systems. This perturbation is applied to the mass of each particle mi according to: = mi m0 (1 + a cos ( mφi ) ) , 195 (6) G. Arreaga-García et al. Figure 1. Upper left panel: Radial density profile for the distribution of SPH particles modeling the initial uniform cloud, as measured from the first Gadget2 snapshot. Upper right panel: Ratio of the hydrodynamical (due to the pressure gradient only) to the gravitational acceleration for several times of the first stage of the cloud’s evolution. Lower left panel: Velocity field of an XZ view of a collision scenario where the velocity of cloud’s particles is seen to point outwards during the initial evolution stage. Lower right panel: The time evolution of the peak density obtained for the collapse of the cloud as an isolated system. where m0 is the unperturbed mass of the simulation particle, the perturbation amplitude is set to a = 0.1 and the mode is fixed to m = 2. We have found that this mass perturbation is almost irrelevant in the scenario of the cloud collision models. The characteristics and physical properties of the clouds expressed in Equations (5) and (6) are quite common and we refer the reader to [20] [21] for reviews of the most important results of collapse calculations which have given place to great conceptual advances in the state of the art. Let us now say something about the way in which we account for the thermodynamics of the gas. Most authors have used an ideal equation of state. As the observed star forming regions basically consist of molecular hydrogen clouds at 10 K with an average density of 1 × 10−20 gr∙cm−3, the ideal equation of state is a good approximation. However, once that gravity has produced a substantial contraction of the cloud, the gas begins to heat. In order to take into account this increase in temperature, we use the barotropic equation of state proposed by [22]. Thus in this paper we carry out all our simulations using the following equation of state:    γ −1  = p c  1 +   ,    crit   2 0 (7) where for the molecular hydrogen gas the ratio of specificheats γ = 5 3 because we only consider translational degrees of freedom of the hydrogen molecule. For the critical density we consider only one single value of crit = 5.0 × 10−14 gr∙cm−3. However, a word of warning should be made. By comparing the results of our previous works in [23] [24] with those of [25] for the collapse of an isolated and rigidly rotating cloud with a uniform density profile, we have concluded the barotropic equation of state in general behaves quite well and that it captures all the essential thermo dynamical phases of the collapse. But this does not mean that the same comparison would always be correct for more complex initial conditions or for collision models as those we are considering in this paper. 196 G. Arreaga-García et al. Despite these facts and that we know that it is indispensable to include all the detailed physics of the thermal transition in order to achieve the correct results, be it in a collision or not, we carry out the present simulations with the barotropic approximation, because we know that there are other computational and physical factors that could have a stronger influence on the outcome of a simulation. The collision clouds considered for this work have approaching velocities of up to about thirty times the sound speed, see Section 3 and Table 1. For these high velocities a shock is formed in the interface between the clouds and the artificial viscosity used for the calculations transform kinetic into thermal energy that increases the gas temperature. However, as we will now show, the cooling time τ cool is both much shorter than the local sound crossing time, and the free fall time, hence the isothermal condition can be used for the shock region. For an initial density of the clouds of = 0 1.3024 × 10−21 gr∙cm−3, its particle number density is −3 = nH = 542 cm , where mp is the proton mass and μ the mean molecular weight of the gas, which 0 ( µ mp ) we take to be 2.4. For a strong adiabatic shock, from the Rankine-Hugoniot jump condition, the density in the thin dissipative region is about four times the pre-shock density, and so we expect the post-shock number density to be about n ps = 2170 cm−3. We can calculate the cooling time from the expression [26] τ cool = 2.4 × 105 yrs. nH (8) Using our estimate for the post shock region we obtain that τ cool = 442 yrs . On the other hand, for our clouds the local sound crossing time = τ cross R= 28.285 Myrs and the free fall time τ ffc = 5.2 Myrs . We can then 0 c0 assume that the shock remains isothermal. 3. Collision Models and Computational Considerations In this paper we use the Gadget 2 code which implements the SPH technique. We use five million simulation particles to model each colliding cloud, as we explain below. 3.1. The Collision Geometry The simulations considered in this paper include both head-on and oblique collisions, see Figure 2. For the head-oncollisions, the initial position in rectangular coordinates of the center of mass (CM) of each of the colliding clouds is located at ( x, = y, z ) X= ( 0, − R0 , R0 ) , respectively. We recall the ( 0, R0 , R0 ) and X CM= 2 CM 1 reader that R0 is the initial radius of the cloud. The average velocity of the center of mass of the clouds points along the Y axis and are VCM= ( 0, −Vave , 0 ) 1 and VCM 2 = ( 0, Vave , 0 ) . We define the approaching speed Vapp (with respect to the center of mass of the collidTable 1. The collision models. Model b/Rₒ Vapp/cₒ log10(ρmax/ρₒ) tmax/tffc αinitial βinitial (α + β)initial αfinal βfinal (α + β)final HO-1 0 2.46 10 5.27 0.19 0.21 0.4 0.093 0.443 0.536 HO-2 0 4.92 5.37 4.32 0.19 0.5 0.69 0.082 0.565 0.647 HO-3 0 9.84 4.31 3.65 0.19 1.6 1.85 0.062 0.554 0.616 HONR-4 0 9.84 5.99 3.45 0.19 1.5 1.73 0.061 0.589 0.65 HO-5 0 30.78 11.14 3.22 0.19 15 15.24 0.077 0.475 0.552 M1 0.5 2.52 5.42 5.47 0.19 0.22 0.41 0.094 0.425 0.519 M2 1 2.74 6.26 5.51 0.19 0.24 0.43 0.107 0.414 0.521 M21 1 8.58 3.05 4.38 0.19 1.32 1.52 0.091 0.564 0.655 M3 −1 4.58 4.57 5.32 0.19 0.46 0.65 0.097 0.589 0.686 M31 −1 8.58 9.65 4.86 0.19 1.32 1.52 0.105 0.91 1.015 M4 −2 2.74 3.95 5.5 0.2 0.25 0.46 0.13 0.431 0.561 M41 −2 4.58 4.01 5.5 0.2 0.49 0.69 0.127 0.581 0.708 M42 −2 8.58 5.43 5.5 0.2 2.1 2.3 0.134 1.586 1.72 197 G. Arreaga-García et al. Figure 2. The initial geometry of the colliding clouds for the head-on collision (left panel), and for the oblique collisions with a positive impact parameter b (right panel). ing binary system) of the clouds by means of Vapp = 2 Vave. For the oblique collision case, the colliding clouds are initially displaced (with an impact parameter b along the X-axis) transversely to the collision symmetry axis (the Y-axis). For these cases, the center of mass of the clouds are initially placed at the coordinates $ X CM 1 = ( b 2, R0 , R0 ) and X CM 2 = ( − b 2, − R0 , R0 ) , see Figure 2. The approaching velocity is defined in the same way as was done for the head-on collision case. To consider a significant sample of models, we choose severalimpact parameters and approaching velocities. For instance, the impact parameter takes the values b = 0, R0/2, R0 and 2R0. It should be noticed that this range of values is well motivated physically, as it follows from an statistical cloud collision study in the context of interacting galaxies [27]. 3.2. The Collision Approaching Velocity With the purpose of studying the influence of the self-gravity of the cloud in the outcome of the collisions, we define several models in which we change the cloud’s displacement speed Vave. Therefore, according to the collision model under consideration, each colliding cloud can individually reach different stages of gravitational collapse before the collision takes place. It has been known since the first molecular line studies that most molecular clouds are observed to have a hierarchical structure, which consist of small gas condensations in supersonic random motion embedded in a larger and more diffuse parent cloud. According with the classification scheme of [14], such small gas clumps are observed to have a size between 0.3 and 3 pc and a velocity in the 0.3 - 2 km∙s−1 range. For instance, let usmention two cloud examples reported by [28]: the cloud OMC1within the Orion Complex, which has a velocity dispersion of 2.5 km∙s−1, size of 1.2 pc and a mass of 1000M⊙; the clouds B7 and B19 observed in the Taurus complex have a velocity dispersion of 0.9 km∙s−1, a similar size of 1.0pc, but with a mass of 140M⊙, much smaller than for the OMC1 cloud. These data for molecular cloud condensations have common features with the clouds modeled in this manuscript. Bergin [14] also reported that the typical sound speed for this kind of cloud (with a temperature around 10 K), is approximately $c0 = 0.2 km∙s−1. Then, for such random movements the ratio v/c0 of the velocity of the cloud to the sound speed, would be in the range Mach v/c0 = (0.3/0.2 - 2/0.2) = (0.15 - 10). Now, if we want to consider a collision between two of this kind of gas clumps, then the approaching velocity (also known as the relative or impact velocity of the clouds) of the colliding clouds will at least be 2v/c0, which gives us an approaching velocity Vapp in the range Mach (0.30 - 20). In order to be consistent with the observational data we have just described, the approaching velocity formost of our models has been chosen in the range Mach (0 - 10). Besides, we have included model HO-5 with an approaching velocity of Mach 30 just for the sake of comparison with [4] that modeled cloud collisions with Mach 25 and 35 and those of [29] that also modeled cloud collisions with velocities in the range Mach (25 - 40). In Table 1 we summarize all the collision models considered in this paper. The entries of the data in Table 1 are as follows. Column one shows the label chosen to identify the model. Column two indicates the impact parameter b of the collision models in terms of R0; it should be noticed the appearance of a sign before the magni- 198 G. Arreaga-García et al. tude of b, whose meaning will be explained in detail below in Section 4.4, but in advance we say that it is related to the orientation of the colliding clouds along the X-axis. Column three indicates the approaching velocity of the colliding clouds, expressed in terms of the initial sound speed of the cloud, whose numerical value is given in Equation (4). Columns four and five show the peak density and maximum evolution time reached in our runs following the colliding process. We recall that we normalize density and time with the average density = 1.3 × 10−21 gr∙cm−3, and with the free fall time τ ffc = 1.846 Myr, respectively. 0 For each individual cloud α = 0.26055 and β = 0.16143, see Equation (5), which gives that α + β = 0.42693. For the initial collision systems we show in columns six, seven and eight the global values of α, β and α + β and in columns nine, ten and eleven the same quantities for the last computed model for each of the initial configurations. The values for the last six columns are calculated for the global collision system and includes the kinetic translational energy that after the collisions contribute to the α and β values of the resulting system. From the column eight of Table 1 we notice that only models HO-1, M1, M2 and M4 are bound, that is only for systems with Vapp/c0 less than about three. In Table 2 we sum up the main result of the collision models. 3.3. The Evolution Code We carry out the time evolution of the initial distribution of particles with the fully parallel Gadget2 code, which is described in detail by [30]. Gadget2 is based on the tree-PM method for computing the gravitational forces and on the standard SPH method for solving the Euler equations of hydrodynamics. Gadget2 incorporates the following standard features: (i) each particle i has its own smoothing length hi; (ii) the particles are also allowed to have individual gravitational softening lengths εi, whose values are adjusted such that for every time step εihi is of order unity. Gadget2 fixes the value of εi for each time-step using the minimum value of the smoothing length of all particles, that is, if hmin = min(hi) for i = 1, 2... N, then εi = hmin. The Gadget2 code has an implementation of a Monaghan-Balsara form for the artificial viscosity [31] [32]. The strength of the viscosity is regulated by setting the parameter αν = 0.75 and βν = ( 3 2 ) αν , see Equation (14) in [30]. We have fixed the Courant factor to 0.1. 3.4. Resolution According to [33], the resolution requirement for avoiding the growth of numerical instabilities is expressed in terms of the Jeans wavelength λJ , which is given by λJ = πc 2 , G Table 2. The main outcome of the collision models. Model Main outcome HO-1 binary + bridge HO-2 weak binary + strong filament HO-3 filament + single cloud HONR-4 longitudinal filament HO-5 long transverse filament M1 binary + bridge M2 binary + bridge M21 almost binary + bridge M3 triple system M31 weak binary + strong filament M4 binary + bridge M41 binary + almost bridge M42 unbounded binary? 199 (9) G. Arreaga-García et al. where G is Newton’s universal gravitation constant, c is the instantaneous sound speed and ρ is the local density. To obtain a more useful form for a particle based code, the Jeans wavelength λJ is written in terms of the spherical Jeans mass MJ, which is defined by 5 4  λJ  π2 π   = = MJ 3  2  6 3 c2 G3 ρ . (10) Nowadays it is well known that the Jeans requirement l < λJ 4 (where l is a characteristic length scale of the grid for a mesh based code) is a necessary condition to avoid the occurrence of artificial fragmentation. For particle based codes, [34] showed that there is also a mass limit resolution criterion which needs to be fulfilled besides that of [33]. They showed that an SPH code produces correct results involving self-gravity as long as the minimum resolvable mass is always less than the Jeans mass MJ. If we now approximate the instantaneous sound speed by c = p ρ , then according to Equation (7), we have 5 π2 = MJ 6 3 γ −1   ρ  2 1 + γ   .  G 3   ρcrit   c03 (11) Following [34], the smallest mass that an SPH calculation can resolve is mr = M J ( 2 N neigh ) , where Nneigh is the number of neighboring particles included in the SPH kernel. For our collision models to comply with the Jeans requirement, the mass particle mp must be such that mp/mr < 1. The mass of the simulation particles is m p = M t N p = 2 ∗ M 0 N p , where Mt is the total mass contained in the simulation box; the total number of particles Np is 10 million for all the models in this paper. To verify that the Jeans stability condition is satisfied we will use the collision model HO-1, which is the one that reached the highest maximum density of all models, see Table 1. For this model we followed its collapse −3 until a peak density of = 1.30 × 10−11 gr∙cm . The average particle mass (after the mass perturbation given max −7 by Equation (6)) is = mp M 0 = N p 4.47 × 10 M ⊙ . For all models our assumed initial sound speed is given by Equation (4), and hence from Equation (11) the minimum Jeans mass for model HO-1 is given by ( M I ) HO −1 = 0.06 M ⊙ , from which we obtain m= 6.4 × 10−5 M ⊙ . Thus, for model HO-1 we obtain the ratio m p mr = 0.07 , and the Jeans resolution requirer ment is quite easily satisfied. For the rest of the collision models, the minimum Jeans mass is expected to be greater than for model HO-1 because their maximum density is less than for model HO-1. It is then clear that for all models the Jeans length criterion is satisfied as well. 4. Results In order to show the main results of our simulations, we present iso-density plots for a slice of matter parallel to the XY plane. The procedure to illustrate the results of these 3D phenomena with 2Diso-density plots is as follows. We first locate the SPH particle with maximum density in the entire volume space of a simulation. The z-coordinate of that particle, say zmax, determines the height of the thin slice of material. The width of the slice is determined such that about 10,000 SPH particles enter into the slice, which is centered around the zmax coordinate. Once the SPH particles defining the slice have been selected, we set a color scale related to the iso-density curves: yellow colors indicate areas with higher densities, whereas blue those with lower densities and green and orange correspond to intermediate density regions. It should be noted that there is no relation between the density colors associated with different panels even in the same plot. At the bottom of each iso-density panel, we include two numerical values to illustrate the different stages of the evolution process: the left value is the peak density ρ(t) at time t; and the right one the actual time t normalized with one of the two free fall times: τ ffc for the cloud and τ ff for the collision, as we explain below. In fact, there is a further time scale that can be defined for the collision system, which is expected to be slower than for the isolated cloud because the spatial extension of the new system has doubled, and its average density is smaller than ρ0 , and therefore its associated τ ff must be longer than τ ffc . The new time scale is given by τ ff = 1.64 × 1014 sec = 5.22 Myr . 200 (12) G. Arreaga-García et al. It should be clear that the times given in Equations (1) and (12) are only time estimates and are useful just as normalizing factors for our evolution plots. The evolution of the uniform density cloud model has been reproduced by many groups worldwide using different codes based both on grids and particles, see for instance [20] [21] and references there in. We also successfully reproduced the uniform model results in [23] and [24], from which we established there liability of our calculations for evolving the cloud with the Gadget2 code, as we followed the collapse process until peak densities of the order of 3.0 × 10−11 gr∙cm−3. Now, in this paper we use the same Gadget2 code to evolve the initial conditions for a bigger but still rotating cloud and to follow the process of the collisions as well. 4.1. The Collapse of the Initial Cloud We describe in this Section the temporal evolution of the initial cloud as an isolated system which evolves under the influence of its own gravitational force. Numerical simulations performed so far have proved that an isolated rotating cloud contracts itself on an almost flat configuration in more or less the free fall time τ ffc of dynamical evolution. For the present work, the initial model is constructed using the Monte Carlo method described in Section 2 that generates an initial density and velocity distributions which produces a peculiar behavior, namely, that the cloud starts its evolution with an spatial expansion of the particles so that the peak density decreases by up to two orders of magnitude before increasing again. Let us describe this phenomenon. In the upper right panel of Figure 1 we show for several early evolution times the absolute value of the ratio of the hydrodynamic to the gravitational radial acceleration as a function of the radius of the cloud. These accelerations have been calculated by dividing the cloud into a number of spherical shells and averaging the radial accelerations within each shell. The hydrodynamic acceleration which is due to the pressure gradient, is clearly dominant in the innermost and outermost regions of the cloud, and produces an initial expansion of the cloud, see the lower right panel of Figure 1. Soon after the start of the calculation the cloud adjust itself so that the hydrodynamical to gravitational acceleration ratio approaches the equilibrium value of 1.0 in the innermost region, see the top-right panel of Figure 1, and the collapse of the whole cloud begins. The ratio tends to 1.0 in the center because the very central region cannot collapse. The outermost region that has very little mass maintains a ratio closer but greater than 1.0, which means that it very slowly keeps expanding, while the rest of the cloud collapses. This is partly due to the fact that near the boundary of the system the interpolating kernel introduces small errors, which makes the boundary pressure to be lower than it should be. This is known as the density deficiency problem and there are some methods for correcting it, see for example [35] [36], but for the problem at hand this effect does not introduce significant errors in the calculations. The handling of the density deficiency problem has not been incorporated into the Gadget2 code. Despite this behavior, the cloud clearly shows a tendency to collapse onto itself as expected. There is a time when the cloud truly begins its collapse as its peak density increases with time, first slowly but then very rapidly at the final stages of the collapse, as can be appreciated in the lower right panel of Figure 1. As expected on the basis of our previous publications, despite that the cloud of this paper is slightly different in its behavior, the isolated cloud described in Section 2 collapses in such a way that the core begin to lengthen while forming a well-defined filament that is surrounded by a halo, still having cylindrical symmetry, as can be seen in the last panel of Figure 3. It was important in reaching this result to have included the mass perturbation given in Equation (6), although as mentioned earlier, this perturbation seems to play no role at all in the collisions. It is interesting to notice that under the influence of self-gravity alone (with no collision), it takes quite a , i.e., t 5.6945 long time for the cloud to collapse until densities of 5.0 × 10−15 gr∙cm−3 = = τ ffc 1.8 Myr . As we will now see, with collisions, those densities are reached in a much shorter evolution time. 4.2. The Collision Process The approaching velocities Vapp of our collision models varies from Mach 2.46 to 30.78 (see Table 1) and the clouds are initially almost in contact. The systems are bound only for Vapp less than about Mach three, the remaining models are unbound but most become bound or nearly reach equilibrium as a result of the collision. Then just after the start of the simulations, the clouds experience a collision and the first effect is that they are compressed and a shock front is formed that propagates to the two clouds. The artificial viscosity transforms ki- 201 G. Arreaga-García et al. Figure 3. Isodensity plot illustrating the gravitational collapse process of the isolated and rotating cloud which we shall collide. We recall that = τ ffc 5.821 × 1013 sec = 1.846 Myr. netic energy into heat, as was described in the last paragraph of Section 2, that is radiated away in a very short timescale and we can assume that the shock and the clouds remain isothermal. During this process the kinetic energy and heat lost by the system makes it possible to approach equilibrium thereby increasing the fragmentation probability. During the collision process a slab of material is formed along the shock front. The clouds then expand but eventually collapse as will be later described. The shocked slab which is formed as a result of the supersonic collision is susceptible of having various instabilities, which has been studied by [37] for the linear regime and by [38] for the non-linear case. For the present work the relevant ones are the shearing and gravitational instabilities. The shearing instability dominates at low density while the gravitational one takes over for densities above the critical density  η  λ  T  (13) = shear 7.86132 × 10−22    NTSI    gr∙cm−3 pc pc    K  where η is the amplitude of the perturbation, λNTSI the length of the unstable mode and T the temperature [39]. Among the shearing instabilities the main ones are the non-linear thin shell instabilities (NTSI) and the Kelvin-Helmholtz (KH) instability. The NTSI instability is expected to occur in the shocked slab just after the cloud collision, where non-linearbending and breathing modes could also be present. 4.3. The Head-On Collisions In order to study the collision process when the approaching speed increases, we have considered five head-on 202 G. Arreaga-García et al. collision models: in the five models H0-1, H0-2, H0-3 andH0-5, the rotating clouds are approaching each other; while for model HONR-4 the colliding clouds have not been endowed with initial rotation. The same approaching velocities were chosen for models H0-3 and HONR-4 in order to study the effect of cloud rotation on the collisions. In the head-on collision models, two identical clouds are placed in contact barely touching each other. Each cloud shares some of its particles which are closer to the edge of the neighboring cloud. Due to gravity and the approaching velocity the clouds collide producing a compression first, that is followed by an expansion. During this phase a contact layer forms between the clouds that thickens as the approaching speed of the clouds increases. A shock wave propagates into the clouds producing a collection of filaments and clumps that can be seen in the top left panel of all the evolution plots. The expansion of the clouds is eventually stopped by gravity and the collapse phase begins. Just after the collision, a filament or slab of colliding particles quickly forms emphasizing the interface of the clouds. This is the phase where the slab may experience the NTSI and KH instabilities. From our numerical reλNTSI 0.1 = pc,η 2λNTSI and T = 10 K, and so the sults we estimate that the parameters in Equation (13) are= NTSI and KH instability are suppressed when the density goes above = 1.57 × 10−18 gr∙cm−3. crit In the next three head-on collision models the clouds approach each other with increasing velocities. In model H0-1, we have Vapp = 2.46c0 ; we show the evolution of this model in Figure 4. Model H0-2 has an approaching speed of Vapp = 4.92c0 and its evolution can be seen in Figure 5. For Vapp = 9.84c0 we have two cases, model HO-3 whose iso-density plot is shown in Figure 6, and the initially non-rotating model HONR-4, shown in Figure 7. Finally, the highest approaching speed we have considered is Vapp = 30.78c0 , which corresponds to model HO-5 whose isodensity plot is presented in Figure 8. We notice that as we increase the approaching velocity of the clouds, the bridge that forms in the interface Figure 4. Isodensity plot for model H0-1. 203 G. Arreaga-García et al. Figure 5. Isodensity plot for model H0-2. Figure 6. Isodensity plot for model H0-3. 204 G. Arreaga-García et al. Figure 7. Isodensity plot for model H0NR-4. Figure 8. Isodensity plot for model H0-5. 205 G. Arreaga-García et al. becomes larger and thicker while the effect of the cloud’s self-gravity is less important. This indicates that many particles from the clouds are flowing very quickly to the bridge, to the point that the two initial clouds cannot be distinguished anymore in model HO-3 after a time of 1.29τ ff . For model HO-3 the final effect of the collision is to replace the two initial colliding clouds by a single cloud that more rapidly collapses towards a long filament. It is interesting to notice the bending of the bridge in all these models; this bending is due to the individual rotation of the clouds. In model H0NR-4 all the matter collides in the interface of the clouds forming a longitudinal filament as is shown in Figure 7. For the purpose of comparing the result of our 3D simulations with Fig. 38 of [8], in Figure 9, we zoom the filament that results from model H0NR-4. The density contours and the velocityfield in this figure suggest the appearance of bending and KH instabilities. During the initial phase after the collision, the cloud’s peak density wiggles in time, as can be observed for model H0NR-4 in the top panels of Figure 10. These wiggles are a consequence of the density shock developed after the clouds collision in the mid plane. For model H0-3 we have also found the presence of density wiggles although of a smaller amplitude, see the bottom panels of Figure 10. To understand the effect of an even higher approaching velocity, we have included model H0-5 with an approaching speed of 30.78 times the sound speed. As expected the density shock is stronger and in Figure 8 we sketch its evolution. Again we observe the formation of a strong filament in the interface of the clouds that rapidly expands as a strong flow of particles reaches the filament ends. However, the expansion eventually stops and the filament bounces back to begin the expansion in the transversal direction, along the Y-axis. This behavior has been previously observed by [4]. The density wiggles are shown in the fourth panel of Figure 10. For the sake of looking for other kinds of instabilities in model H0-5, we have also included some plots with velocity distributions in Figure 11. For the collision systems considered the initial and final values of α, β and α + β are shown in Table 1. For the initial head-on configurations, α + β increases from 0.404 for model HO-1 to 15.247 for model HO-5, which is a very high number and a consequence of the high Vapp value of Mach 30.78. In all head-on models a large fraction of the translational kinetic energy is transformed into heat and is radiated away. This is the reason why the Final α + β values reach the equilibrium value of 0.5. 4.3. Oblique Collisions The oblique collisions are characterized by an impact parameter b which depends on the initial radius R0 of the cloud. The model with the smallest b has b = R0 2 , while that with the largest b has b = 2.0R0. The sign of b is important because it determines the initial orientation of the colliding clouds as we now explain. For instance, for model M1 with the smallest b, we have placed the first cloud such that its center of mass in rectangular coordinates is X CM 1 = ( R0 4, R0 , R0 ) , while the center of mass of the second cloud is Figure 9. Zoom in of the filament of model H0NR-4 as it is shown in the last panel of Figure 7. We show isodensity curves (top panel) and the velocity field (bottom panel). 206 G. Arreaga-García et al. Figure 10. Time evolution of the peak density in the midplane of the collision for the XZ plane (continuous line), and for the entire collision volume (dashed line), for model H0NR-4 (top-left panel and zoomed in top-right panel), model H0-3 (bottom left panel), and model $H0-5$ (bottom right panel). Figure 11. Velocity distribution in the XY plane for model H0-5 for t = 0.69τ ff and = 1.55 × 10−20 gr cm-3 (top panel), and = t 1.13τ ff= max 1.022 × 10−16 gr∙cm−3, which cormax responds to the last frame of the isodensity plot shown in Figure 8 (bottom panel). X CM 2 = ( − R0 4, − R0 , R0 ) . We consider this orientation to correspond to a positive impact parameter b as labeled in the second column of Table 1. In this configuration the clouds are moved along the X-axis in the same sense than their rotational motion. For the opposite case, when b < 0, the clouds are again displaced along the X-axis, but in the opposite direction to its rotation. As expected, the result of the collision for model M1 is quite similar to that obtained for the head-on low approaching velocity collisions. Indeed, the outcome of this model is very similar to that obtained for model HO-1, 207 G. Arreaga-García et al. because the approaching speed of these two models is almost equal, see Figure 12. It is unnecessary to calculate more models with increasing Vapp with this impact parameter because we would expect that the results are very similar to those obtained for the head-on collision models. Let us now consider models M2 and M21, where the impact parameter has been increased to b = R0, again with the positive orientation. The approaching velocity for the later is almost three times that of the former, with velocities given numerically by Vapp = 2.74c0 and Vapp = 8.52c0 , respectively. As expected, in these two models the size of the bridge of particles inthe colliding interface is very sensitive to the approaching speed value. Whereas in model M2, it is always possible to distinguish the form of the two colliding clouds, in model M21, it is clearly seen that the participating clouds tend to merge, while the bridge becomes larger and thicker, see Figure 13 and Figure 14, respectively. In model M21, the lack of time for self-gravity to act on the clouds is evident. On the other hand, the results for models M2, HO-1 and M1 are very similar because self-gravity had enough time to act on all of them prior to the collision. It is instructive to compare the outcome of models HO-3 and M21, whose approaching velocities are very similar. In both models the bridges have a similar size, although their orientations are quite different as a consequence of the impact parameter of model M21, see again Figure 6 and Figure 14 for a comparison. For models M3 and M31 we have not increased the magnitude of the impact parameter, but we have changed its sign as well approaching speeds. The rectangular coordinates of the center of mass for the colliding clouds are now X CM 1 = ( − R0 2, R0 , R0 ) and X CM 2 = ( R0 2, R0 , R0 ) , respectively. That is, we now have b = −R0, see Table 1. The magnitude of the approaching speed for these models is Vapp = 4.58c0 and Vapp = 8.52c0 , respectively. This orientation will also be used for the next models. One of the most interesting evolutions is produced by model M3, as can be seen in Figure 15. In this case, the bridge starts forming in an inclined orientation and it ends up almost vertical along the colliding symmetry axis (the Y-axis); besides, it is much thicker and smaller Figure 12. Isodensity plot for model M1. 208 G. Arreaga-García et al. Figure 13. Isodensity plot for model M2. Figure 14. Isodensity plot for model M21. 209 G. Arreaga-García et al. Figure 15. Isodensity plot for model M3. than in all the previous cases. We will describe in Section 4.5 the final evolution. As usual, the next model M31, has the same initial orientation than model M3, but we have almost doubled its approaching speed. The outcome of model M31 is quite different to that of model M3, as can be seen in Figure 16. For the last models labeled M4 and M41, we further increase the impact parameter b up to the value of −2.0R0. We observe that depending on the magnitude of the approaching speed, the bridge of particles in the interface of the colliding clouds can remain and act as a gravitational link between the clouds, as can be seen in Figure 17 and Figure 18; or when this approaching velocity is sufficiently high, the clouds move next to each other and the bridge disappears, see Figure 19. It turns out to be very interesting to analyze the initial and final α, β and α + β values for the oblique models, see This is not the case for the oblique models, which have a final near equilibrium value for Vapp less than about Mach three and all b values. The final α + β values do not reach the equilibrium for Vapp greater than eight and high b values. Hence we only get very high dissipation for head-on or near head-on collision. 4.4. Physical Properties In this section we describe the integral properties of the clumps or fragments found in some of our simulations; properties which characterize the physical state of the resulting clumps, among others, the energy ratios α and β already defined in Equation (5). We first proceed to find the center of the clump which is given by the location of the particle with the highest density in the region where the clump is located. With the clump center determined, xcenter, we then find all the particles which have a density above (or equal to) some minimum density value ρmin and that, at the same time, are located within a given maximum radius rmax from the clump center. With this set of Ns particles we can estimate the integral properties of the clump. 210 G. Arreaga-García et al. Figure 16. Isodensity plot for model M31. Figure 17. Isodensity plot for model M4. 211 G. Arreaga-García et al. Figure 18. Isodensity plot for model M41. Figure 19. Isodensity plot for model M42. 212 G. Arreaga-García et al. We use the smoothing kernel for calculating the density of particle i by means of= ρi ρ= ( ri ) mW1 ( ri , h ) , where W1 ( ri , h ) is the spline kernel given in Equation (A1) of [40]. For the gravitational potential, we use another kernel such that Φ i =Φ ( ri ) =G ( m h )W2 ( ri h ) , where the kernel W2 is now given in Equation (A3) of the same reference. The softening length h appearing in these two kernels, sets the neighborhood on the point r outside of which no particle can exert influence on r, that is, for r > h both kernels vanish: W1 = 0 and W2 = 0. We use several values for h, with the purpose that the number of neighbor particles for any point (or particle) be near 50. The next step is to make a sum over all the set of Ns particles, from which we obtain the density and the gravitational potential for every particle i = 1 N s due to the presence of all other particles j ≠ i. We keep Ns to be around 100,000 particles. Then we approximate the thermal energy of the clump, by calculating the sum over all the Ns particles, that is Etherm = ∑ i =s1 N 3 Pi ( ρ ) mi , 2 ρi (14) where P is the pressure associated with particle i with densityρi by means of the equation of state given in Equation (7). In a similar way, the approximate potential energy is N 1 E pot = ∑ i =S1 mi Φi . 2 (15) Although a bit more complicated, the rotational energy of the set of Ns particles is calculated with respect to the Z-axis of the located clump, as follows. Let xi and vi be the position and velocity of particle i. Then the coordinates of those particles in the clump with respect to the clump center are u= xi − xcenter . The azimuthal angle i φ associated with the rotation of particles with respect to the Z-axis can be calculated by taking the ratio of the projection of coordinates of the particle with the unitary vectors i = (1,0,0) and j = (0,1,0), that is we can write = φi arctan ( ui ⋅ j ui ⋅ i ) . Therefore the angle φ also depends on the particle i, that is, φi . Then the rotational energy can be estimated by taking the projection of the velocity along the unitary azimuthal vector eφi = − sin (φ ) i + cos (φ ) j , that is = Erot ∑ i =1 2 mi ( vi ⋅ eφ ) . Ns 1 2 i (16) We report in Table 3 the results obtained by the application of the preceding calculation procedure to the lastsnapshot obtained in each simulation. It must be noted that the numerical results for integral properties unfortunately depend on the values chosen for two cutting parameters, ρmin and rmax, therefore we inevitably commit certain ambiguity in defining the clump boundaries. For instance, the minimum density is fixed arbitrarily. We will next describe the entries of Table 3 as follows (see the next page). In column one, we repeat the label of the model as many times as needed to account for all the identified clumps in the model. In column two we indicate the number of particles (Ns) entering into the set of particles that approximate the physical state of the clump with respect to the energy calculation. In columns three, four and five we show the Cartesian coordinates of the center of every identified clump, expressed in terms of the side length of the simulation box, 2R0. Later on, in column six we show the ratio of the minimum density to the initial central density of the cloud, which is the lowest value that a particle entering in the set can indeed have. In column seven we indicate the maximum radius in units of 2R0. In column eight we show the softening length h in units of R0. In columns nine and ten we present the energy ratios α and β as previously defined, and finally in the last column we add α + β. In Table 4 we show for some models the peak velocity in sound speed units (Mach number) and the maximum acceleration normalized with as = 3.64 × 109 cm∙s−2. For almost all models the maximum velocity is about Mach 10 and since quite large maximum accelerations are reached, this makes the computational time step to get very small. Because of this in some models the evolution cannot be followed to very high density. For model HO-1 the first and second fragments have α = 0.19, 0.23, β = 0.12, 0.13, and α + β = 0.315, 0.366, respectively. The fragments have already collapsed by ten orders of magnitude in density, and the low α + β values suggest that they will continue to do so, inhibiting any further fragmentation. The β values are very low because the collision is head-on. The original clouds have β = 0.16134 and as a result of the collision is reduced to about 0.12 - 0.13. The central filament rotates as a consequence of the initial rotation of the clouds, it contains 213 G. Arreaga-García et al. Table 3. Physical properties of the resulting protostellar clumps. Model Np x/(2Rₒ) y/(2Rₒ) z/(2Rₒ) h/Rₒ α β (α + β) HO-1 88631 0 0.186 0.498 log10(ρmin/ρₒ) Rmax/(2Rₒ) 3.5 0.2 0.003 0.192 0.122 0.315 HO-1 113829 −0.04 −0.15 0.479 3.5 0.2 0.003 0.234 0.131 0.366 M1 116227 −0.097 −0.168 0.498 3 0.2 0.016 0.609 0.271 0.88 M1 115889 0.095 0.183 0.499 3 0.2 0.016 0.619 0.22 0.839 M2 194996 0.22 0.195 0.495 2.5 0.15 0.004 0.108 0.433 0.541 M2 130620 −0.2 −0.15 0.499 3 0.15 0.007 0.109 0.392 0.501 M3 157861 0.05 0.031 0.461 3 0.1 0.002 0.198 0.165 0.364 M3 127926 0.161 −0.2 0.495 2.35 0.1 0.004 0.442 1.573 2.01 M3 139838 −0.24 −0.04 0.46 2 0.1 0.004 0.462 1.289 1.751 M4 120585 0.46 −0.23 0.498 2.65 0.15 0.0024 0.21 0.288 0.498 M4 120618 −0.49 0.23 0.536 2.65 0.15 0.0024 0.21 0.316 0.531 M41 116129 0.51 −0.016 0.499 2.6 0.25 0.002 0.2 0.614 0.823 M41 116336 −0.52 0.05 0.497 2.6 0.25 0.002 0.21 0.674 0.887 Table 4. Peak values for the velocity (normalized with the sound speed) and the acceleration (normalized with as = 3.64 × 109 cm∙s−2 obtained for the last snapshot of some representative runs. Model vmax/c0 amax/as HO-1 14.25 159.6 HO-2 11.15 105.5 HO-3 12.37 77.4 M1 11.18 83.7 M2 11.85 72.9 M3 12.31 25.2 M4 11.95 63.7 the highest density regions and fragments into several clumps. For the oblique collision model M1, the impact parameter b/R0 = 1/2 and the approaching velocity is Vapp = 2.52 co, see Figure 12. The fragments in the last panel of this figure are well defined, one is binary (top-right) and the other one single (lower-left). The fragments have shifted from the original head-on collision alignment because of the impact parameter b, but also as a result of the rotation of the clouds which is counterclockwise. The α + β values of the two fragments are 0.86 and 0.90, respectively. These numbers are well above the virial value and the system at the last computed time step has only been able to increase its density by about five orders of magnitude. Both fragments need to lose a significant amount of angular momentum for the collapse to proceed at a faster rate. The central filament is well defined, its maximum density is similar to that of the fragments and at the last computed model shows signs of fragmentation. From Figure 20 we observe that there is a flow of particles going in all directions, as can also be seen in Figure 21 where we sketch the 3D bridge structure. Model M2has some similarities to model H0-1, compare the last frames of Figure 4 and Figure 13. The fragments for model M2 are more detached from the filament, which is due to the combination of the initial angular velocity of the clouds and its initial condition that corresponds to an oblique collision with an impact parameter b/R0 = 1/2. For the HO-1 case the fragments are less detached from the filament because we only have the contribution from the initial angular velocity of the clouds. The central filament is dissolved, and it can only survive if the impact parameter b/R0 is low (<1/2). Model M2 is very interesting because as we shall see, it produced two filament-like fragments that appear to be subfragmenting. In Section 3.4 we showed that the Jeans length criterion is satisfied for all models. Despite 214 G. Arreaga-García et al. Figure 20. Velocity field for the last snapshot obtained for models M1 (top panel) and M3 (bottom panel). Figure 21. Dot plot for the last snapshot obtained for model H0-1 when t = 1.863655τff and log10(ρmax/ρ0) = 10.93. In this plot each dot represents an SPH particle and only thoseparticles having log10(ρ/ρ0) > 2.5 have entered, which are 800,566, that is, only 8 percent of the total number of particles. this, in order to ensure that the result of model M2 is not affected by artificial fragmentation, we simulated again this model with 15 million particles, this represents 50% more than for the rest of the models and basically obtained the same result. In Figure 22 we show an amplification of the resulting system of the resimulated model M2 for the last computed time step. We refer to the two fragments as top-right and lower-left. The filament-like structure of the two fragments is produced by the initial compression of the clouds that generates a density maximum along a line parallel to the collision shock front and at the center of the clouds. This is an initial condition very different from the standard m = 2 perturbation, see Equation (6). If clouds do not experience a strong direct collision, that is, if the impact parameter b/R0 > 2, or the separation of the clouds is much greater than their radius, the induced perturbation is a mixture of an m = 2 and m = 3 perturbation, but if the collision is strong and b < 1 the perturbation is filament-like. An amplification of the top-right and lower-left fragments of Figure 22 shows that they have sub fragmented into several clumps. For the lower-left fragment α = 0.110, β = 0.392, and α + β = 0.501, that is, the system composed by this fragment has virialized, the collapse has stopped completely and the newly formed fragments are expected to become stars after they reach higher densities. For the top-right fragment α = 0.108, β = 0.433, α + β = 0.541, and the system is also close to virial equilibrium. Model M3 for which b/R0 = −1 and Vapp = 4.58c0 evolved into two single fragments and the filament decayed into a central fragment see Figure 15. For the central fragment, from Table 4, α + β = 0.36, and so we expected that it will continue collapsing to much higher densities. The outer two fragments have α + β = 2.016 and 1.752, respectively, and in particular, very high rotation energies, so for them to be able to collapse it is required that they lose large amounts of angular momentum. The velocity field for this model is shown in Figure 20, from where we can appreciate that many particles are orbiting around the system in circular orbits but not really col- 215 G. Arreaga-García et al. Figure 22. We show here an amplification of the last snapshot obtained for a resimulation of model M2 which should be compared with the last panel of Figure 13 (top panel), and of this panel a further amplification of the top-right filament (middle panel), and of the lower-left filament (bottom panel). lapsing. The small b and quite high Vapp produce the very high rotational velocities in the outer fragments. We now describe model M4 that has b/R0 = −2.0 and Vapp = 2.74c0. The two outer fragments are single, well defined, and have α + β = 0.4986 and 0.53105, respectively, which is very close to the virial value of 0.5. The fragments require to lose thermal and rotational energy to be able to reaching higher densities. The central filament quickly disappears because of the high b value. Model M41 with b/R0 = −2.0 and Vapp = 4.58c0 has a similar revolution to model M4 which has the same b but lower Vapp. The α value of the fragments is about 0.21 but the β value is above 0.60 which makes α + β take values above 0.82 and so the fragments need to get rid of some angular momentum to be able to collapse. The higher angular moment is generated during the collision as a result of the higher Vapp. The velocity and acceleration particle distributions for some of the models are shown in Figure 23. In this figure f means the fraction of particles in the simulation which have a velocity (left panel) and an acceleration (right panel) less than the number indicated in the horizontal-axis, respectively. We show in Table 4 the maximum values for velocities and accelerations obtained for the last snapshot of some simulations. 5. Discussion In this paper we carried out a fully 3D set of numerical hydrodynamical simulations within the framework of the SPH technique, aimed to follow the collision process of two small rotating uniform density clouds. The initial conditions for the isolated cloud are such that it will collapse in the absence of a collision. However, when we consider the translational kinetic energy that comes from the approaching velocity Vapp of the clouds, the collision system is unbound for Vapp greater than about three, that is, (α + β)initial > 0.5, see Table 1. 216 G. Arreaga-García et al. Figure 23. Fraction of particles f having less than or equal to the peak velocities as indicated in the X-axis (top panel); the peak accelerations expressed in terms of as = 3.64 × 10−9 cm/sec2 (right panel). As a result of the collision process a large fraction of the translational kinetic energy is transformed into heat that is radiated away because the clouds and the shock front that forms in the inter phase between the clouds are isothermal. This produces a reduction of the global value of α + β for all models. For the head-on models we found that they all get a final configuration near equilibrium even for very high Vapp. This is not the case for the oblique models, which have a final value near equilibrium for Vapp less than about Mach three and all b values. The final α + β values get away from equilibrium for Vapp greater than eight and high b values. Hence we only get very high dissipation, and thus the possibility of fragmentation for initially unbound systems for head-on or near head-on collision. When the cloud collides, the gravitational collapse speeds upin some regions and we do observe fragmentation in some of the models, see for example Figure 13 and Figure 22. This result is different from that of [13] that found that most supersonic head-on cloud collisions will probably do not result in fragmentation, unless the interstellar gas can cool much faster than it is normally assumed. They concluded that most cloud collisions produce a disruption or dispersion of the clouds involved. In this work we have observed that the result of simulation depends mainly on two physical factors that compete to exert more influence on the collision process. These factors are self-gravity and the flowing of particles from the colliding interface. The occurrence and extension of these effects are regulated by the value of the approaching velocity of the colliding clouds. The two cloud collision forms a shock front, the clouds are first compressed but then expand. In all simulations the shock front produces a collection of filaments and clumps, and the bending and KH instabilities are observed for densities lower than about 10−18 gr∙cm−3, i.e. before the collapse is truly underway. With no collision or with a very slow approaching velocity, self-gravity dominates the evolution of both clouds. In these cases, the main effect of self-gravity on the system is the decreasing size of each of the clouds. The collision system quickly reaches a configuration in an advanced state of gravitational collapse consisting of two gas clumps (the cores of the clouds) linked by a bridge of matter. In some cases the clumps or fragments have sub-fragmented into an elongated filamentary structure that is very close to virial equilibrium. Besides that, there is always a sharing region of particles between the clouds that gives place to the formation of a bridge of gas, even when no collision occurs. On the other hand, when a collision takes place with a significant approaching velocity, self-gravity plays a minor role and the fate of the system is entirely determined by the strong flow of particles in the interface of the clouds. In these cases, the main effect on the system is to replace the two initial collapsing clouds by a single cloud that is rapidly shocked to give place to an elongated and irregular filamentary structure formed in the shock region. This filamentary structure increases its size very rapidly as it is continuously fed by in falling material coming from the original clouds. The second important parameter of the collision process is the impact parameter b. For instance, it should be noted that for models with the same approaching velocity and with the same magnitude of the impact parameter 217 G. Arreaga-García et al. b, but with a different sign of b, we have obtained different outcomes. We have tried to consider several parameters in order to make a representative study, but as the space parameter is very large, it cannot be covered in a single work. It is interesting to note that the particle with the highest density in the simulations was not always found in the bridge of matter, but there were cases where it was located in the remnants of the clouds; remnants that hopefully will end up as protostellar cores. For model H0-1, in Figure 21, we show in a dot plot the 3D structure of the bridge. Each dot represent an SPH particle and we have only included those that have a density greater than a certain value, which is given in the bottom of the figure. It can be appreciated that the bridge is highly irregular as many particles flow very fast in all directions, and are continuously ejected from the collision region. In view of our simulations, the future of the bridge is as yet unclear; but it is likely that most of the gas is destined to remain in this filamentary structure as the collapse progress further. Although many clumps of matter get formed near the principal bridge, these clumps are very thin and small, then it could be the case that the diameter of these clumps are in general greater than the Jeans length. This is another reason why the bridge would not be an appropriate site for protostellar formation until it cools further. As described in Section 3.4, the Jeans mass requirement is a necessary condition for avoiding the occurrence of artificial fragmentation. As far as we know, the only way to ensure the correctness of a simulation is by making a convergence analysis in which the total number of particles increases systematically. Although this analysis is beyond the present manuscript, we have been careful to resimulate at least model M2 with 15 million particles, that is, 50% more than for the rest of the models. In fact, in Figure 22 we show an amplification of the last snapshot computed in this resimulation of model M2. We have pleasantly found convergence. The filament-like structure of the two fragments is produced by the initial compression of the clouds that generates a density maximum along a line parallel to the collision shock front and at the center of the clouds. This is an initial condition very different from the standard m = 2 perturbation, see Equation (6). If clouds do not experience a strong direct collision, that is, if the impact parameter b/R0 > 2, or the separation of the clouds is much greater than their radius, the induced perturbation is a mixture of an m = 2 and 3 perturbation, but if the collision is strong and b/R0 < 1 the perturbation is filament-like. Besides, we note that the filament developed in the central region of each clump, show a clear tendency to fragment by forming small knots along the filament. This behavior contrasts with the fate of the filament developed for the initial cloud when it evolved as an isolated system, which shows no tendency to fragment, as can be appreciated in the last panel of Figure 3. We conclude that the fragmentation of the filaments in model M2 is a consequence of the collision. 6. Concluding Remarks It was [40] who predicted that the infall of gas colliding into the galactic disk could have a huge influence on the star formation process, and at the same time, to inherit a very peculiar physical structure to the interstellar gas. It is already well known that recent observations show some elongated structures in the Orion Molecular Cloud, which appear to be common in the observed interstellar medium. In this manuscript we have been able to capture and show some of the essential features of the collision process. For instance we have obtained irregular systems of filaments and clumps of neutral gas, which could be somehow compared with those already observed. We also investigated what are the physical properties of the resulting proto stars in this collision scheme, which hopefully could be compared with observations. Moreover, we claim to have observed some kind of fragmentation in the filaments of model M2, which is a direct consequence of the occurrence of the collision. This result can be considered as an evidence that collisions may have an important influence on the star formation process. Calculations have been performed by colliding two identical polytropes (with their surfaces just in contact including self-gravity and viscosity) with the purpose to test the conservation of energy in codes based on the SPH technique [41]. 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https://openalex.org/W2068101487	https://europepmc.org/articles/pmc4486412?pdf=render	English	null	The Thebesian valve and coronary sinus in cardiac magnetic resonance	Journal of interventional cardiac electrophysiology	2,015	cc-by	3,919	J Interv Card Electrophysiol (2015) 43:197–203 DOI 10.1007/s10840-015-9994-3 J Interv Card Electrophysiol (2015) 43:197–203 DOI 10.1007/s10840-015-9994-3 Abstract Purpose There is no complex research exploring usefulness of cardiac magnetic resonance in the evaluation of the coro- nary sinus including Thebesian valve, which can be useful before selected electrophysiology procedures. Conclusions In most of the cases, it is possible to visualize and measure the coronary sinus using cardiac magnetic reso- nance with SSFP sequence. In selected cases, it is necessary to perform additional dedicated short sequences. Thebesian valve was visualized in almost 50 % of patients. Methods One hundred twenty-two patients aged 49.2±17.2 (42 women) were included in the study; 4 of them were ex- cluded. A steady-state free-precession (SSFP) sequence was the basis of the visualization and analysis of the coronary sinus as well as Thebesian valve. In selected cases, dedicated coro- nary sinus sequences were created. All data were evaluated by experienced cardiac magnetic resonance investigators. Keywords Coronary sinus . Thebesian valve . Cardiac magnetic resonance . CRT Results We were able to visualize the coronary sinus by using basic SSFP sequence in all patients, however in four cases in suboptimal quality. Average length of the coronary sinus was 39.73±16.9 mm, average diameter was 9.81±9.3 mm, and average angle of the entrance of the coronary sinus into the The Thebesian valve and coronary sinus in cardiac magnetic resonance Rafal Mlynarski & Agnieszka Mlynarska & Maciej Haberka & Krzysztof S. Golba & Maciej Sosnowski Received: 20 January 2015 /Accepted: 6 March 2015 /Published online: 12 April 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com right atrium was 111.37±13.8°. The Thebesian valve as the gate of the coronary sinus was found in 56 cases (45.9 %). In 21 patients (17.2 % of all), the valve was porous or almost totally covered the coronary sinus ostium, which can poten- tially create problems during CS cannulation. Conclusions In most of the cases, it is possible to visualize and measure the coronary sinus using cardiac magnetic reso- nance with SSFP sequence. In selected cases, it is necessary to perform additional dedicated short sequences. Thebesian valve was visualized in almost 50 % of patients. right atrium was 111.37±13.8°. The Thebesian valve as the gate of the coronary sinus was found in 56 cases (45.9 %). In 21 patients (17.2 % of all), the valve was porous or almost totally covered the coronary sinus ostium, which can poten- tially create problems during CS cannulation. 1 Introduction The in vivo anatomy of the coronary sinus including an anal- ysis of Thebesian valve as well as target veins has been de- scribed in many papers [1–3]. The Thebesian valve is a caudal remnant of the embryonic sinoatrial valve. It is usually a semi- circular fold of membrane in the right atrium at the orifice of the coronary sinus. It is on the posterior, inferior surface of the heart, medial to the inferior vena cava opening [1, 4]. Its role in normal physiology is not known, but some experts believe that it may prevent the regurgitation of blood into the sinus during the contraction of the atrium [5]. Unfortunately, expe- rience from clinical practice suggests that the valve may pose difficulties during cannulation of the coronary sinus during cardiac resynchronization therapy (CRT). Therefore, studies on the anatomy of the Thebesian valve have potential practical implications [6, 7]. Visualization of the coronary venous sys- tem is an important element of cardiac resynchronization ther- apy as well as during ablation or even the delivery of stem cells into the heart [8]. The most common method of in vivo visualization to date is cardiac computed tomography (CT) in R. Mlynarski (): A. Mlynarska: K. S. Golba Department of Electrocardiology, Upper Silesian Heart Centre, Ziolowa 45/47, 40-635 Katowice, Poland e-mail: Rafal_Mlynarski@mp.pl R. Mlynarski (): A. Mlynarska: K. S. Golba Department of Electrocardiology, Upper Silesian Heart Centre, Ziolowa 45/47, 40-635 Katowice, Poland e-mail: Rafal_Mlynarski@mp.pl A. Mlynarska Department of Internal Nursing, School of Health Sciences in Katowice, Medical University of Silesia, Katowice, Poland M. Haberka Department of Cardiology, School of Health Sciences in Katowice, Medical University of Silesia, Katowice, Poland M. Haberka K. S. Golba Department of Electrocardiology and Heart Failure, School of Health Sciences in Katowice, Medical University of Silesia, Katowice, Poland K. S. Golba Department of Electrocardiology and Heart Failure, School of Health Sciences in Katowice, Medical University of Silesia, Katowice, Poland M. Sosnowski Unit for Noninvasive Cardiovascular Diagnostics, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland J Interv Card Electrophysiol (2015) 43:197–203 198 Table 1 Main indications for CMR in the patients included Purpose No. of cases Percentage Cardiomyopathy 49 40.2 Arrhythmogenic right ventricular dysplasia (ARVD) 17 13.9 Myocarditis 16 13.1 Ejection fraction evaluation 10 8.2 Overall 10 8.2 Cardiac neoplasm 9 7.4 Myocardial scar/viability 4 3.3 Sarcoidosis 3 2.5 Pulmonary hypertension 2 1.6 Anatomical abnormalities 2 1.6 Table 1 Main indications for CMR in the patients included the consensus of experts [9]. A special scheme for the visual- ization of veins dedicated for cardiac resynchronization in cardiac CT was also presented by our team [10]. However, cardiac computed tomography has some serious disadvan- tages. Most of the patients who are qualified for CRT implan- tation are patients with advanced heart failure, for whom the administration of β-blockers during the examination is con- traindicated. Many potential patients have also suffered renal failure, and therefore, the administration of a contrast agent is contraindicated. There is an alternative—it is cardiac magnetic resonance (CMR). In this examination, it is possible to visualize the func- tion of the heart as well as its anatomy in a noninvasive way using the next generation of CMR devices. An exact analysis of the ejection fraction in some patients for whom suboptimal images in the echo examination were obtained is recommend- ed before qualification for an implantable cardioverter defibril- lator (ICD). Using tagging function in some patients qualified for cardiac resynchronization may be also helpful in finding those who might respond well to the therapy. However, it is unclear whether it is possible to visualize the coronary venous system and Thebesian valve in CMR without creating special sequences to analyze the raw data. All magnetic resonance examinations were performed by using a GE Optima MR450w 1.5T with GEM Suite with a dedicated cardiac coil GE body 3D small cardiac. The GEM is an integrated system that combines high-density RF surface coils with software that permits high-quality images to be obtained. M. Haberka A highly homogeneous magnet with a 50×50×50- cm field of view to cover more anatomy is used in this equip- ment. Image acquisition parameters were as follows: slice thickness was 6–8 mm with a 1-mm interslice gap and field of view was typical 40×40 and matrix 192×192. Post- processing of images was performed by using a dedicated console. The purposes of the study were to evaluate the usefulness of CMR in the evaluation of the coronary sinus including Thebesian valve, to design a methodology for fast coronary venous system visualization, and to evaluate the quality of CMR images. Imaging protocol includes a steady-state free-precession cine imaging (SSFP; FIESTA/45) sequence which uses the T2 steady-state contrast mechanism for the evaluation of the cardiac anatomy, acquired in different standardized planes, including axial, 2-, 3-, and 4-chamber, and short-axis oblique planes covering the right atrium. These sequences provide images of fluid-filled structures in a very short time and were the basis of the visualization and analysis of the coronary sinus as well as Thebesian valve. More detailed assessment of the coronary venous vasculature was performed if there were dif- ficulties with visualization of the coronary sinus ostium; in such cases, dedicated additional sequences were created. 3 Results however in four cases in suboptimal quality. Using this se- quence, the average length of the coronary sinus that was visualized was 39.73±16.9 mm. The average diameter of the coronary sinus ostium was 9.81±9.3 mm. Average diameter of the ostium in patients with a valve present was 11.9±17.9 and without Thebesian valve was 8.9±0.4; p=0.429 NS. The average angle of the entrance of the coronary sinus into the right atrium was 111.37±13.8°. We were able to visualize the proximal parts of posterolateral veins in 66 (54.1 %) patients. The hemodynamic measurements of the patients included that were obtained using cardiac magnetic resonance are presented in Table 3. The hemodynamic measurements of the patients included that were obtained using cardiac magnetic resonance are presented in Table 3. We were able to visualize the coronary sinus by using steady-state free-precession (SSFP) scans in all patients, Table 2 Proposed nomenclature for the types of Thebesian valves adapted from cardiac computed tomography [1] Type of Thebesian valve Description A1 The semilunar membrane is visible from the atrium wall. The membrane covers less than 50 % of CS ostium A2 The semilunar membrane is visible from the atrium wall. The membrane covers more than 50 % of CS ostium B1 The semilunar membrane is visible from the interatrial septum. The membrane covers less than 50 % of CS ostium B2 The semilunar membrane is visible from the interatrial septum. The membrane covers more than 50 % of CS ostium C Thebesian valve is built from two separate parts. There is a gap between parts D The almost whole CS ostium is covered by a membrane The quality of the visualization of the coronary sinus in most cases (109 cases; 89.2 %) was acceptable to perform a clinical analysis (mean score obtained was 4, range 3–5). In 24 (19.7 %) patients, we obtained optimal quality (score 5) of visualization. 2 Methods One hundred twenty-two patients aged 49.2±17.2 (42 wom- en) were included in this trial. Four of them were excluded due to significant anatomical anomalies of the heart found in the exam. Indications for an examination using cardiac magnetic resonance were typical, and they are presented in Table 1. Patients were excluded if they had impaired renal excretory function (calculated glomerular filtration rate below 30 mL/ min/1.73 m) except during dialysis. Pregnant women or lac- tating women as well as patients with decompensated conges- tive heart failure who were unable to lie flat during an MRI or with a known allergy to gadolinium-based contrast agents were also excluded. All data were evaluated by two CMR investigators. An arbitrary scale of the quality of the images of the coronary venous system, which was adapted from the cardiac computed tomography scale of visualization, was introduced and used [10]. The highest grade on the scale was 5 and the lowest was 1, and the final score was the consensus of investigators. Typical exclusion criteria for an MRI scan were the following: An example of anatomy of the coronary sinus with visible Thebesian valve in CMR/fast imaging employing steady-state acquisition (FIESTA) is presented in Fig. 1. The nomenclature for the Thebesian valve was based on the classifications of the Thebesian valve in cardiac computed tomography that were proposed and published by the authors [1]. This nomenclature is presented in Table 2. – Cardiac pacemaker or implantable defibrillator implanted (acceptable if device and leads carry an MRI safety mark) – Any other ferromagnetic implants that are not acceptable for cardiac MRI – Any other implanted device (e.g., insulin pump, drug in- fusion device) J Interv Card Electrophysiol (2015) 43:197–203 199 Fig. 1 Example of the anatomy of the coronary sinus with a visible Thebesian valve in CMR/FIESTA (type C—Thebesian valve is built from two separate parts; there is a gap between parts). RA right atrium, LV left ventricle, CS coronary sinus Fig. 1 Example of the anatomy of the coronary sinus with a visible Thebesian valve in CMR/FIESTA (type C—Thebesian valve is built from two separate parts; there is a gap between parts). RA right atrium, LV left ventricle, CS coronary sinus 3 Results Analysis for the patients with quality score 1– Table 2 Proposed nomenclature for the types of Thebesian valves adapted from cardiac computed tomography [1] Table 3 Average values of the main cardiac function parameters for the patients included Average value ±SD Ejection fraction (%) 47.56 ±14.9 Stroke volume (mL) 71.74 ±22.3 End diastolic volume (mL) 168.73 ±82.2 End systolic volume (mL) 97.42 ±74.5 Peak filling rate (mL) 324.18 ±125.0 Peak ejection rate (mL) 384.76 ±138.4 Cardiac output (L/min) 4.58 ±1.5 Table 3 Average values of the main cardiac function parameters for the patients included J Interv Card Electrophysiol (2015) 43:197–203 200 54.1 9 7.4 0.8 1.6 9.8 17.2 0 10 20 30 40 50 60 No valve Valve A1 Valve A2 Valve B1 Valve B2 Valve C Valve D Presence of Thebesian valve in % Fig. 2 Graphic distribution of different types of Thebesian valves 54.1 9 7.4 0.8 1.6 9.8 17.2 0 10 20 30 40 50 60 No valve Valve A1 Valve A2 Valve B1 Valve B2 Valve C Valve D Presence of Thebesian valve in % Fig. 2 Graphic distribution of different types of Thebesian valves Presence of Thebesian valve in % Presence of Thebesian valve in % case in which the Thebesian valve was found, we were able to describe its clinical characteristics. Examples of Thebesian valves are presented in Fig. 3. If visualization SSFP was not acceptable diagnostically, an additional visualization sequence of the coronary sinus ostium to the right atrium was performed. An example of such a sequence is presented in Fig. 4. 4 Discussion The anatomy of the coronary sinus was well recognized in vivo in cardiac computed tomography [1, 10, 11]. The pop- ularity of cardiac magnetic resonance has increased, and this examination has some advantages, especially for patients for whom no contrast agent can be used and those who cannot be exposed to radiation. It is important to remember that the visualization of vessels is not the strongest aspect of CMR. A comparison of cardiac computed tomography from our im- age library with CMR from this research is presented in Fig. 5. It can be seen that the images are comparable. In our CT image 2 (10.8 %) made it difficult to draw any conclusions. Average quality of visualization was 3.55±0.9. The Thebesian valve as the gate of coronary sinus was found in the 56 cases (45.9 % included patients). Graphic dis- tribution of different types of Thebesian valves is presented in Fig. 2. The most frequent variant (21 cases; 17.2 % of all included) was D in which a significant part of the sinus ostium is covered by the valve. What is most important is that in each The Thebesian valve as the gate of coronary sinus was found in the 56 cases (45.9 % included patients). Graphic dis- tribution of different types of Thebesian valves is presented in Fig. 2. The most frequent variant (21 cases; 17.2 % of all included) was D in which a significant part of the sinus ostium is covered by the valve. What is most important is that in each Fig. 3 Examples of various types of Thebesian valves in CMR/FIESTA. RA right atrium, LV left ventricle, CS coronary sinus Examples of various types of Thebesian valves in CMR/FIESTA. RA right atrium, LV left ventricle, CS coronary sinus J Interv Card Electrophysiol (2015) 43:197–203 201 Fig. 4 Additional sequence of the visualization of the coronary sinus ostium to the right atrium if the basic visualization (FIESTA) was not acceptable diagnostically. RA right atrium, CS coronary sinus Fig. 4 Additional sequence of the visualization of the coronary sinus ostium to the right atrium if the basic visualization (FIESTA) was not acceptable diagnostically. References 1. Mlynarski, R., Mlynarska, A., Tendera, M., & Sosnowski, M. (2011). Coronary sinus ostium: the key structure in the heart’s anatomy from the electrophysiologist’s point of view. Heart and Vessels, 26, 449– 456. 2. Silver, M. A., & Rowley, N. E. (1988). The functional anatomy of the human coronary sinus. American Heart Journal, 115, 1080–1084. We agree that whole-heart 3D reconstructions are more useful to recognize the anatomy of the coronary venous sys- tem; however, in order to add clinical value, an analysis of the Thebesian valve and of its types (if present) should be always performed. 3. Pejković, B., Krajnc, I., Anderhuber, F., & Kosutić, D. (2008). Anatomical variations of the coronary sinus ostium area of the human heart. Journal of International Medical Research, 36, 314–321. 4. Katti, K., & Patil, N. P. (2012). The Thebesian valve: gatekeeper to the coronary sinus. Clinical Anatomy, 25, 379–385. 5. Kautzner, J. (2009). Thebesian valve: the guard dog of the coronary sinus? Europace, 11, 1136–1137. 4 Discussion RA right atrium, CS coronary sinus of the visualization of the coronary sinus ostium to the right atrium if the basic visualization (FIESTA) was not acceptable m CS coronary sinus database of the coronary venous system, there is 46∼55 % visualization of the Thebesian valve [1]. Those results are almost equal with the results presented in this study (45.9 %). coronary veins despite the fact that a few papers have been published. When we prepared the methodology for this paper, one of the aims was to simplify and check whether it is pos- sible to analyze images retrospectively. As a result, we first tried to use the sequence that is always performed during After analyzing the literature on the subject, we are aware that there is not one common standard for the visualization of Fig. 5 Graphic comparison of the visualization of the coronary sinus ostium in computed tomography as well as CMR. RA right atrium, LV left ventricle, CS coronary sinus Fig. 5 Graphic comparison of the visualization of the coronary sinus ostium in computed tomography as well as CMR. RA right atrium, LV left ventricle, CS coronary sinus ualization of the coronary sinus ostium in computed tomography as well as CMR. RA right atrium, LV left ventricle, Fig. 5 Graphic comparison of the visualization of the coronary sinus ostium in computed tomography as well as CMR. RA right atrium, LV left ventricle, CS coronary sinus J Interv Card Electrophysiol (2015) 43:197–203 202 cardiac magnetic resonance—FIESTA. The FIESTA se- quence uses a T2 steady-state contrast mechanism to provide high SNR images with a strong signal from fluid-filled tissues while suppressing background tissue in order to produce a contrast and anatomic detail of small structures. In addition, the ultrashort repetition time (RT) and echo time (TE) permit extremely short acquisition times—shorter than fast spin echo (FSE)—and the images can be post-processed using MIP, vol- ume rendering, or 3D navigator techniques. half of the patients, which may provide a way to recognize problems for CRT implantation and other procedures. Financial No financial support Conflict of interest There are no conflicts of interest for any of the authors. Chiribiri et al. [13] examined 31 participants in CMR using whole-heart imaging and intravascular contrast agents, and Younger et al. [12] used 3D reconstructions along with stan- dard orthogonal planes in all of the coronary sinuses and great cardiac veins with relations to the mitral valve and LCx. Both authors visualized the coronary sinus in all of the participants. The authors concluded that cardiovascular magnetic reso- nance might provide important information for the selection of candidates for these procedures. Similarly, Ibrahim et al. reported possibility of visualization of the coronary sinus and great cardiac, posterior interventricular, and anterior inter- ventricular veins in 100 % of patients by both MRI and MDCT [14]. Detection of the posterior vein of the left ventri- cle and the left marginal vein by MRI was reported as 97 and 81 %, respectively. Authors’ contributions All authors read and approved the final manuscript. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. 6 Limitations of study We agree that visualization of the coronary sinus and Thebesian valve may not be recommended for selection of patient for CRT due to limited data actually available. How- ever, the decision of a surgeon to implant a CRT may be affected by these additional imaging data. In our opinion, car- diac magnetic resonance may create a road to visualize Thebesian valve and to visualize potential obstruction of the coronary sinus ostium. The authors believe that imaging data are mostly created to support surgeons in the decision-making process and when choosing the most optimal equipment. An- other technical disadvantage of cardiac magnetic resonance is the lack of the possibility to perform another reconstruction after finishing the examination (except 3D whole-heart imag- ing if available). As was mentioned earlier, our paper is not the first that describes the visualization of coronary veins using CMR. However, none of the papers cited below analyzed the Thebesian valve. Younger et al. in 2009 examined 31 cardiac CMR studies in 3D reconstructions with gadolinium [12]. The authors confirmed that the cardiac venous system was visual- ized in all of the patients. They also visualized the anterior lateral and posterior veins. They documented myocardial in- farction on late gadolinium enhancement (LGE) images in five patients. The authors concluded that the coronary venous anatomy can be reliably demonstrated using a comprehensive CMR protocol and a standard extracellular contrast agent. Like us, they think that a combination of coronary venous anatomy imaging, assessment of ventricular function, and LGE may be useful in the management of patients with LV dysfunction who are being considered for CRT. Financial No financial support 5 Conclusions 6. Kuroda, M., Takahashi, T., Mita, N., Kagaya, S., Miyoshi, S., & Saito, S. (2013). Difficult cannulation of the coronary sinus due to a large Thebesian valve. Anesthesia and Analgesia, 116, 563–566. In most cases, it is possible to visualize and measure the cor- onary sinus using cardiac magnetic resonance with SSFP se- quence. In selected cases, it is necessary to perform additional, dedicated short sequences. Thebesian valve was visualized in 7. Cao, M., Chang, P., Garon, B., & Shinbane, J. S. (2013). Cardiac resynchronization therapy: double cannulation approach to coronary venous lead placement via a prominent Thebesian valve. Pacing and Clinical Electrophysiology, 36, e70–e73. J Interv Card Electrophysiol (2015) 43:197–203 203 12. Younger, J. F., Plein, S., Crean, A., Ball, S. G., & Greenwood, J. P. (2009). Visualization of coronary venous anatomy by cardiovascular magnetic resonance. Journal of Cardiovascular Magnetic Resonance, 11, 26. 8. Randhawa, A., Saini, A., Aggarwal, A., Rohit, M. K., & Sahni, D. (2013). Variance in coronary venous anatomy: a critical determinant in optimal candidate selection for cardiac resynchronization therapy. Pacing and Clinical Electrophysiology, 2013, 94–102. 9. Taylor, A. J., Cerqueira, M., Hodgson, J. M., et al. (2010). ACCF/SCCT/ACR/AHA/ASE/ASNC/NASCI/SCAI/SCMR 2010 appropriate use criteria for cardiac computed tomography. Journal of Cardiovascular Computed Tomography, 4, 407.e1–33. 13. Chiribiri, A., Kelle, S., & Köhler, U. (2008). Magnetic resonance cardiac vein imaging: relation to mitral valve annulus and left cir- cumflex coronary artery. JACC. Cardiovascular Imaging, 1, 729– 738. 10. Mlynarski, R., Sosnowski, M., Wlodyka, A., Kargul, W., & Tendera, M. (2009). A user-friendly method of cardiac venous system visual- ization in 64-slice computed tomography. Pacing and Clinical Electrophysiology, 32, 323–329. 14. Ibrahim, W. G., El Khouli, R. H., Abd-Elmoniem, K. Z., Matta, J. R., McAreavey, D., & Gharib, A. M. (2014). Optimization of free- breathing whole-heart 3-dimensional cardiac magnetic resonance im- aging at 3 tesla to identify coronary vein anatomy and to compare with multidetector computed tomography. Journal of Computer Assisted Tomography, 38(6), 941–948. 11. Genc, B., Solak, A., Sahin, N., Gur, S., Kalaycioglu, S., & Ozturk, V. (2013). Assessment of the coronary venous system by using cardiac CT. Diagnostic and Interventional Radiology, 19, 286–293.
https://openalex.org/W4286274224	https://ela.kpi.ua/bitstream/123456789/52402/1/259068-Article%20Text-600807-1-10-20220707.pdf	English	null	Accuracy assessment of marker recognition using ultra wide angle camera	Technology audit and production reserves	2,022	cc-by	4,656	1. Introduction to the no need to use additional equipment, its comfortable ergonomics, wide range of provided features, and variety of tools for software development for this device [3]. Despite all the advantages, Microsoft HoloLens 2 has limitations. Both design flaws of device and drawbacks of AR tech nology itself make the Microsoft HoloLens 2 ineffective to be applicable in certain scenarios. The modern state of augmented reality (AR) technology allows it usage in various fields of human activity, including medicine. AR makes it possible to interact with virtual objects in the real world. In this way, AR technology is more attractive compare to virtual reality when used in applications for medical visualization [1]. Tracking method used in Microsoft HoloLens 2 is inside- out tracking. Only those cameras and sensors that are built into the device itself provide information on the orienta tion of the device in the real world. In order to provide update of spatial relationships of virtual and real objects, such method needs information from the tracking of fidu cial markers attached in the real scene. Fiducial markers provide unambiguous identification of object position and pose in three-dimensional space [4, 5]. The most widely used cases of AR technology in medi cine are the visualization of the patient’s state indicators during examination and the visualization of surgical in strument positions during surgery. For mentioned cases, the output can be provided into medical card helmet, interactive MRI scan, and any other device with AR technology on board. Conventional smart phone, tablet, and even computers with plugged camera can be used as AR devices to superimpose virtual objects onto physical objects in real space. However, the most convenient in case of visualization for medical purposes is usage of special helmets or head mounted displays. They do not take up hands and the imposition of information on the user’s field of view (FoV) allows users to immerse fully in the process of interaction with augmented objects [2, 3]. According to the manufacturer information, the mono scopic camera of Microsoft HoloLens 2 covers 52 degrees [6], while the FoV of user, excluding the rotation of the eyes and peripheral vision, is 114 degrees [7]. In practice, us age of mentioned monoscopic camera results in a small area beyond which interaction with virtual objects are not possible. INFORMATION TECHNOLOGIES INFORMATION TECHNOLOGIES UDC 004.932:616-073.756.8 DOI: 10.15587/2706-5448.2022.259068 Article type «Reports on Research Projects» UDC 004.932:616-073.756.8 DOI: 10.15587/2706-5448.2022.259068 Article type «Reports on Research Projects» UDC 004.932:616-073.756.8 DOI: 10.15587/2706-5448.2022.259068 Article type «Reports on Research Projects» How to cite Alkhimova, S., Davydovych, I. (2022). Accuracy assessment of marker recognition using ultra wide angle camera. Technology Audit and Production Reserves, 3 (2 (65)), 6–10. doi: http://doi.org/10.15587/2706-5448.2022.259068 Alkhimova, S., Davydovych, I. (2022). Accuracy assessment of marker recognition using ultra wide angle camera. Technology Audit and Production Reserves, 3 (2 (65)), 6–10. doi: http://doi.org/10.15587/2706-5448.2022.259068 ACCURACY ASSESSMENT OF MARKER RECOGNITION USING ULTRA WIDE ANGLE CAMERA Head mounted displays may provide an attractive alternative to traditional surgery naviga tion systems because allow users to stand at the first point of view and interact with objects in their surroundings naturally. Thus, the object of research in this study is recognition accuracy of fiducial markers in zones where ultra- wide angle camera distort the most. This is motivated by the need to increase user workspace for interaction with markers compare to the workspace provided with such popular augmented reality device as Microsoft HoloLens 2. In this study, the recognition accuracy is evaluated using ArUco square markers with taking into account different marker sizes and their positions in the camera view space. The marker positions include the center of the camera view space as well as such zones where lenses distort the most as top left, top right, bottom left, and bottom right corners. Obtained results show that recognition accuracy is good enough to be applicable for surgical navigation and In this study, the recognition accuracy is evaluated using ArUco square markers with taking into account different marker sizes and their positions in the camera view space. The marker positions include the center of the camera view space as well as such zones where lenses distort the most as top left, top right, bottom left, and bottom right corners. Obtained results show that recognition accuracy is good enough to be applicable for surgical navigation and failures referred to the distortion occurs are available in less than 0.2 % of all cases. This gives a possibility to increase workspace for interaction with markers compare to the Microsoft HoloLens 2. At the same time, the workspace for interaction could not reach the actual view space of the camera since recognition fails in cases where marker’s body is partially visible in the captured image (i. e., marker position is at the image boundaries). Keywords: augmented reality, marker recognition, ArUco fiducial markers, recognition accuracy, surgical navi gation, ultra-wide angle camera. Received date: 30.04.2022 Accepted date: 11.06.2022 Published date: 16.06.2022 © The Author(s) 2022 This is an open access article under the Creative Commons CC BY license How to cite Alkhimova, S., Davydovych, I. (2022). Accuracy assessment of marker recognition using ultra wide angle camera. Technology Audit and Production Reserves, 3 (2 (65)), 6–10. doi: http://doi.org/10.15587/2706-5448.2022.259068 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 ACCURACY ASSESSMENT OF MARKER RECOGNITION USING ULTRA WIDE ANGLE CAMERA Svitlana Alkhimova, Illia Davydovych Modern devices that support augmented reality technology are widely used in various fields of human activity, including medicine. Head mounted displays may provide an attractive alternative to traditional surgery naviga tion systems because allow users to stand at the first point of view and interact with objects in their surroundings naturally. Thus, the object of research in this study is recognition accuracy of fiducial markers in zones where ultra- wide angle camera distort the most. This is motivated by the need to increase user workspace for interaction with markers compare to the workspace provided with such popular augmented reality device as Microsoft HoloLens 2. h d h l d k h k d Modern devices that support augmented reality technology are widely used in various fields of human activity, including medicine. Head mounted displays may provide an attractive alternative to traditional surgery naviga tion systems because allow users to stand at the first point of view and interact with objects in their surroundings naturally. Thus, the object of research in this study is recognition accuracy of fiducial markers in zones where ultra- wide angle camera distort the most. This is motivated by the need to increase user workspace for interaction with markers compare to the workspace provided with such popular augmented reality device as Microsoft HoloLens 2. In this study, the recognition accuracy is evaluated using ArUco square markers with taking into account different marker sizes and their positions in the camera view space. The marker positions include the center of the camera view space as well as such zones where lenses distort the most as top left, top right, bottom left, and bottom right corners. Obtained results show that recognition accuracy is good enough to be applicable for surgical navigation and failures referred to the distortion occurs are available in less than 0.2 % of all cases. This gives a possibility to increase workspace for interaction with markers compare to the Microsoft HoloLens 2. At the same time, the workspace for interaction could not reach the actual view space of the camera since recognition fails in cases where marker’s body is partially visible in the captured image (i. e., marker position is at the image boundaries). Keywords: augmented reality marker recognition ArUco fiducial markers recognition accuracy surgical navi including medicine. 2. Research methodology In this study, binary square fiducial markers were used as visual markers to be recognized. The main advantage of such markers is that camera pose can be obtained with only one marker usage [13]. Moreover, binary square fiducial markers are widely used in medical research and practice, as they are simple, highly reliable, and multifunctional solution [14–16]. Of the factors mentioned above, usability of such aug mented reality-based system as Microsoft HoloLens 2 will benefit from an increased FoV and achieved a good enough level of recognition accuracy of fiducial markers. Ultra-wide angle camera with high resolution can be a solution to increase FoV. Such a camera has a lens whose focal length is shorter than 24 mm in full-frame equivalent FoV. Depending on the shape and location of the lens group, the angle of view can range from 52 to 180 degrees (Fig. 1). ArUco library is an open source popular library for detection of square fiducial markers. ArUco library is an open source popular library for detection of square fiducial markers [17]. ArUco library was chosen as it has high accuracy of recognition on all three axes of the Cartesian plane and faster than similar tools for markers detection. This choice also provides compliance with the hardware capabilities of AR devices (i. e., most of them are not equipped with any sensors other than cameras). At the same time, usage of ultra-wide angle cameras pro vides images that usually suffer from distortions. Distortion of the geometry and proportions of the objects at the image can affect the accuracy of recognition [10, 11]. However, these defects can be corrected. The most common way to get rid of distortions is to calibrate the internal parameters of the camera with the help of calibration boards [12]. The disadvantages are that calibration should be performed in dividually for each camera, requires additional time and availability of appropriate software. The ArUco library includes several dictionaries contain ing sets of tokens with different numbers of bits. Having a smaller bit size helps to identify markers better for cases when marker size in image is too small, but a larger bit size can help to get position in three-dimensional space more accurately [13]. Therefore, the object of research in this study is recogni tion accuracy of fiducial markers in AR-based system ap plicable for surgical navigation. INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES have a lower resolution of the light-sensitive matrix compared to monoscopic camera. This leads to obtaining of a lower level of recognition accuracy of fiducial markers [8, 9]. 1. Introduction Presence of two pairs of stereoscopic cameras on the glasses sides could solve the issue with a small FoV. However, stereoscopic cameras of Microsoft HoloLens 2 The most commercially successful AR device is the Microsoft HoloLens 2 (Microsoft Corporation, USA) due TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 6 ISSN 2664-9969 INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES ISSN 2664-9969 For experiments A and B where the same bit size of markers were used, better recognition accuracy (by 5.4 % on average) was observed for markers with larger physical sizes. For experiments B and C where the same physical size of markers were used, better recognition accuracy (by 6.2 % on average) was observed for markers with smaller bit sizes. It can be explained as captured images of markers with different physical sizes have different resolution in case the markers have the same position and at a fixed distance from the camera. This, in its turn, could lead to loss of information on some marker’s details or ele ments of inner binary matrix may not be clear in the captured image. In these experiments, the ultra-wide angle camera was represented by the 8MP ultra-wide angle camera of Xiao mi Redmi Note 8 Pro (Xiaomi Inc., China). The camera properties are: f/2.2 aperture, 13 mm focal length (FoV is 120 degrees), 1/4.0″ diagonal light-sensitive matrix, 1.12 μm pixel size. Distance from the camera position to the markers position was restricted to 70 cm. This value was taken from [18]. It is based on the assumption that interaction with the object to which the markers are applied is constrained by anthropometric and biomechanical limits (i. e., the maximal distance an average individual is able to reach forward from shoulder to fingertip). The analysis of the images with failed recognition showed that most of the failures occurred because of lighting is sues, i. e., due to insufficient illumination of the scene or presence of glare from lighting on the marker surface. The other significant part of failures was referred to the cases where only part of the fiducial marker was visible in the image. As any specific preprocessing for distortion correction was not applied to the images extracted from the video files, few recognition failures were referred to the distortion occurs (i. e., less than 0.2 % of all cases). To find out failed recognition cases due to the distortion presence, correction algorithm was applied to the input images copied beforehand. The OpenCV module was utilized to provide radial and tangential correction. Coefficients of radial and tangential were determined from the calibration data obtained before the experiments. INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES Difference between the initial recognition rate and the rated obtained on the corrected data was considered as a measure of failed cases referred the distortion occurs. In each experiment, video data were collected from scenes with markers placed in the FoV of the camera in five diffe rent positions (i. e., in the center, top left, top right, bottom left, and bottom right corners of the camera view space). The position of the camera was fixed for all experiments. In each experiment, video data were collected from scenes with markers placed in the FoV of the camera in five diffe rent positions (i. e., in the center, top left, top right, bottom left, and bottom right corners of the camera view space). The position of the camera was fixed for all experiments. Since the current study is interested in analyzing a re cognition accuracy of fiducial markers in AR-based system applicable for surgical navigation, interaction with individual components in such system is an important aspect for its operator. Therefore, operator interacted with markers and rotated them in any direction during the video record ing process. Video record lasted about 2 minutes for each marker position. In total, 15 videos were recorded. Since the current study is interested in analyzing a re cognition accuracy of fiducial markers in AR-based system applicable for surgical navigation, interaction with individual components in such system is an important aspect for its operator. Therefore, operator interacted with markers and rotated them in any direction during the video record ing process. Video record lasted about 2 minutes for each marker position. In total, 15 videos were recorded. The marker recognition was implemented using Python programming language (version 3.9.1), OpenCV open-source computer vision library (version 4.5.5), and cv2.aruco mo dule [13]. The program input was images previously extracted from the video files. In case of success recognition, the pro gram saved the coordinates of marker’s corners and center to be used to confirm marker recognition. The processed images also were saved at the program output. Images with failed marker recognition were marked and saved separately. In order for the marker to be successfully recognized, it should be positioned on white-light background or have white border around the marker to enable detection. 2. Research methodology Different physical sizes and number of bits of markers were used in this study to assess their possible impact on the recognition accuracy (Fig. 2). Experiments were conducted with markers that have 4×4 bits and 3×3 cm physical sizes (Experiment A), 4×4 bits and 5×5 cm physi cal sizes (Experiment B), 7×7 bits and 5×5 cm physical sizes (Experiment C). The aim of this research is to analyze a recognition accuracy of fiducial markers in zones where ultra-wide- angle camera distort the most with taking into account different marker sizes. a b Fig. 1. Comparison of captures images by wide angle and ultra-wide angle camera: a – example of an image from monoscopic camera of Microsoft HoloLens 2 (FoV is 52 degrees); b – example of an image from 8MP camera of Xiaomi Redmi Note 8 Pro (FoV is 120 degrees) Fig. 1. Comparison of captures images by wide angle and ultra-wide angle camera: a – example of an image from monoscopic camera of Microsoft HoloLens 2 (FoV is 52 degrees); b – example of an image from 8MP camera of Xiaomi Redmi Note 8 Pro (FoV is 120 degrees) a b c Fig. 2. Markers used in this study: a – marker with 4×4 bits and 3×3 cm physical sizes (Experiment A); b – marker with 4×4 bits and 5×5 cm physical sizes (Experiment B); c – marker with 7×7 bits and 5×5 cm physical sizes (Experiment C) Fig. 2. Markers used in this study: a – marker with 4×4 bits and 3×3 cm physical sizes (Experiment A); b – marker with 4×4 bits and 5×5 cm physical sizes (Experiment B); c – marker with 7×7 bits and 5×5 cm physical sizes (Experiment C) used in this study: a – marker with 4×4 bits and 3×3 cm physical sizes (Experiment A); b – marker with 4×4 bits and 5×5 cm physical sizes (Experiment B); c – marker with 7×7 bits and 5×5 cm physical sizes (Experiment C) Fig. 2. Markers used in this study: a – marker with 4×4 bits and 3×3 cm physical sizes (Experiment A); b – marker wi physical sizes (Experiment B); c – marker with 7×7 bits and 5×5 cm physical sizes (Experiment C TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 7 INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES In addition, the white elements of inner binary matrix of the marker should have good contrast respect to the black frame and the marker’s body should be fully visible in the image. Among the mentioned issues related to markers recogni tion failures, the cases caused due to insufficient illumina tion can be solved through applying algorithms of exposure compensation and the cases caused due to distortion occurs can be solved through calibration procedure with further applying of distortion correction algorithms. As for the cases caused due to the image contained only part of the marker, possibility to increase the recognition rate is com plicated by the lack of built-in methods to process markers partially visible in the image. For this reason, the workspace for interaction with markers is smaller than the actual view space provided by the ultra-wide angle camera (i. e., by 15.85 % smaller for markers that have 3×3 cm physi cal sizes and by 25.96 % smaller for markers that have 5×5 cm physical sizes in our experiments). In order for the marker to be successfully recognized, it should be positioned on white-light background or have white border around the marker to enable detection. In addition, the white elements of inner binary matrix of the marker should have good contrast respect to the black frame and the marker’s body should be fully visible in the image. A h i d i l d k i 3. Research results and discussion The recognition accuracy was calculated as the frac tion of the number of images with recognized marker to the total number of images with certain marker position in given experiment. Table 1 lists the recognition accuracy of the markers by experiments. In all three experiments, the used algo rithm successfully recognized most of markers in images captured by ultra-wide angle camera. Table 1 Table 1 Markers recognition accuracy by experiments, % Position Experiment A Experiment B Experiment C center 91.8 93.1 89.4 up right 71.7 74.0 72.0 up left 82.4 89.8 84.6 down right 75.0 89.8 78.1 down left 84.1 85.4 76.9 Markers recognition accuracy by experiments, % TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 References 1. Vassallo, R., Rankin, A., Chen, E. C., Peters, T. M. (2017, March). Hologram stability evaluation for Microsoft HoloLens. Medical Imaging 2017: Image Perception, Observer Performance, and Technology Assessment, 10136, 295–300. doi: http://doi.org/ 10.1117/12.2255831 1. Vassallo, R., Rankin, A., Chen, E. C., Peters, T. M. (2017, March). Hologram stability evaluation for Microsoft HoloLens. Medical Imaging 2017: Image Perception, Observer Performance, and Technology Assessment, 10136, 295–300. doi: http://doi.org/ 10.1117/12.2255831 2. Eckert, M., Volmerg, J. S., Friedrich, C. M. (2019). Augmented Reality in Medicine: Systematic and Bibliographic Review. JMIR mHealth and uHealth, 7 (4), e10967. doi: http://doi.org/ 10.2196/10967 3. Moro, C., Phelps, C., Redmond, P., Stromberga, Z. (2020). HoloLens and mobile augmented reality in medical and health science education: A randomised controlled trial. British Journal of Educational Technology, 52 (2), 680–694. doi: http://doi.org/ 10.1111/bjet.13049 Despite its contributions, this study has some limitations to be considered. First, let’s use data collected under the same lighting conditions. Poor lighting conditions affect the result of the marker recognition. Especially, it happens when final texture or geometry of marker in the image gets corrupted. Second, this study has taken into account only 2D ArUco markers. Usage of 3D cube with an ArUco marker attached to each side of the cube can affect the recognition accuracy as angle between the camera plane and the marker plane does not exceed 45 degrees. Finally, our study did not compare results of marker recognition from different commercial or open-source software, which can be applied for that. Only OpenCV library was used. 4. Olson, E. (2011). AprilTag: A robust and flexible visual fi ducial system. 2011 IEEE international conference on robo tics and automation, 3400–3407. doi: http://doi.org/10.1109/ icra.2011.5979561 5. Romero-Ramirez, F. J., Mu oz-Salinas, R., Medina-Carnicer, R. (2018). Speeded up detection of squared fiducial markers. Image and Vision Computing, 76, 38–47. doi: http://doi.org/10.1016/ j.imavis.2018.05.004 6. Microsoft: Learn about HoloLens 2 features and review technical specs. Available at: https://www.microsoft.com/en-us/hololens/ hardware Last accessed: 20.05.2022 7. Howard, I. P., Rogers, B. J. (1995). Binocular vision and stereopsis. Oxford psychology series No. 29. Oxford University Press, 736. doi: https://doi.org/10.1093/acprof:oso/9780195084764.001.0001 In the future, it is necessary to further examine recogni tion accuracy at the image boundaries and find a way to expand the workspace for interaction with fiducial markers close to the maximum possible. 8. Brand, M., Wulff, L. A., Hamdani, Y., Sch ppstuhl, T. (2020). References Accuracy of Marker Tracking on an Optical See-Through Head Mounted Display. Annals of Scientific Society for Assembly, Handl ing and Industrial Robotics. Vieweg, Berlin, Heidelberg: Springer, 21–31. doi: http://doi.org/10.1007/978-3-662-61755-7_3 Markers recognition accuracy by experiments, % Since usage of ultra-wide angle camera aims to solve difficulties related to limited workspace in AR-based system, distribution of failed recognition cases where the fiducial marker left the workspace (i. e., partially visible in the image) was analyzed separately (Fig. 3). Regardless of the physical sizes and number of bits of markers, the recognition accuracy in the center of image is on average higher than in the corners. The recognition accuracy in the corners relative to the center of image degrades by 13.5 %, 8.35 %, and 11.5 % on average in Experiment A, B, and C, respectively. The results of this analysis revealed that despite the limited workspace, interaction with marker placed in the corners of the camera view space leads to cases where only part of the fiducial marker was visible in the image. On average, the mentioned factor reached 20.47 % of the total number of all unrecognized cases. TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 8 8 ISSN 2664-9969 INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES INFORMATION TECHNOLOGIES Fig. 3. Distribution of failed recognition cases where only part of the fiducial marker was visible in the image: a – Experiment A; b – Experiment B; c – Experiment C a b c a Fig. 3. Distribution of failed recognition cases where only part of the fiducial marker was visible in the image: a – Experiment A; b – Experiment B; c – Experiment C There was a noticeable increase in the percentage of failed recognition cases where the fiducial marker crossed the right side of workspace and was partially visible in the image. Right-side cases occurred more than 2.15 times often than lift-side cases. It can be explained by biome chanical impact during the interaction with marker, as operator was right-handed person. Since a movement to the left side by the right hand would be farther than reaching to the right side, it was more easer to cross the right side of workspace for the right-handed operator. TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 4. Conclusions 9. Thabit, A., Niessen, W. J., Wolvius, E. B., van Walsum, T. (2022). Evaluation of marker tracking using mono and stereo vision in Microsoft HoloLens for surgical navigation. Medical Imaging 2022: Image-Guided Procedures, Robotic Interventions, and Mo deling, 12034, 253–262. doi: http://doi.org/10.1117/12.2607262 In this study, we evaluated recognition accuracy of fiducial markers in zones where ultra-wide angle came ra distort the most with taking into account different marker sizes. 10. Zhao, H., Ying, X., Shi, Y., Tong, X., Wen, J., Zha, H. (2020). RDCFace: radial distortion correction for face recognition. Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 7721–7730. doi: http://doi.org/10.1109/ cvpr42600.2020.00774 It is possible to conclude that the proposed usage of ultra-wide-angle camera is feasible for fiducial markers recognition in AR-based system applicable for surgical navigation. The accuracy that could be achieved in dis tortion zones of ultra-wide angle camera is sufficient in case where marker’s body is fully visible in the captured image. The recognition failures referred to the distortion occurs were less than 0.2 % of all cases. However, issues of failed recognition of makers partially visible in the image reduce the workspace for interaction with fiducial markers compare to the actual view space provided by the ultra-wide angle camera. 11. Miki, D., Abe, S., Chen, S., Demachi, K. (2020). Robust human motion recognition from wide-angle images for video surveillance in nuclear power plants. Mechanical Engineering Journal, 7 (3), 19–00533–19–00533. doi: http://doi.org/10.1299/mej.19-00533 12. Remondino, F., Fraser, C. (2006). Digital camera calibration methods: considerations and comparisons. International Archives of the Photogrammetry, Remote Sensing and Spatial Informa tion Sciences, 36 (5), 266–272. doi: https://doi.org/10.3929/ ethz-b-000158067 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 9 9 Corresponding author TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES ISSN 2664-9969 Recognition, 47 (6), 2280–2292. doi: http://doi.org/10.1016/ j.patcog.2014.01.005 Recognition, 47 (6), 2280–2292. doi: http://doi.org/10.1016/ j.patcog.2014.01.005 13. OpenCV: Tutorials for contrib modules. Detection of ArUco Markers. Available at: https://docs.opencv.org/4.x/d5/dae/tuto rial_aruco_detection.html Last accessed: 20.05.2022 18. Looker, J., Garvey, T. (2015). Reaching for Holograms: Assess ing the Ergonomics of the Microsoft™ Hololens™ 3D Gesture Known as the «Air Tap». Proceedings from International Design Congress. Gwangju: KSDS, 504–511. 14. Liu, X., Plishker, W., Shekhar, R. (2021). Hybrid electromag netic-ArUco tracking of laparoscopic ultrasound transducer in laparoscopic video. Journal of Medical Imaging, 8 (1). doi: http:// doi.org/10.1117/1.jmi.8.1.015001 15. O dal, P., Heczko, D., Vysock , A., Mlotek, J., Nov k, P., Virgala, I. et. al. (2020). Improved Pose Estimation of Aruco Tags Using a Novel 3D Placement Strategy. Sensors, 20 (17), 4825. doi: http://doi.org/10.3390/s20174825 Svitlana Alkhimova, PhD, Department of Biomedical Cyberne Svitlana Alkhimova, PhD, Department of Biomedical Cyberne tics, National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Kyiv, Ukraine, ORCID: http://orcid.org/0000- *Svitlana Alkhimova, PhD, Department of Biomedical Cyberne tics, National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Kyiv, Ukraine, ORCID: http://orcid.org/0000- 0002-9749-7388, e-mail: alkhimova.svitlana@lll.kpi.ua tics, National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Kyiv, Ukraine, ORCID: http://orcid.org/0000- 0002-9749-7388, e-mail: alkhimova.svitlana@lll.kpi.ua 16. Luzon, J. A., Stimec, B. V., Bakka, A. O., Edwin, B., Ignjatovic, D. (2020). Value of the surgeon’s sightline on hologram registra tion and targeting in mixed reality. International Journal of Computer Assisted Radiology and Surgery, 15 (12), 2027–2039. doi: http://doi.org/10.1007/s11548-020-02263-3 Illia Davydovych, Department of Biomedical Cybernetics, National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Kyiv, Ukraine, ORCID: https://orcid.org/0000-0001- 9987-8267 17. Garrido-Jurado, S., Mu oz-Salinas, R., Madrid-Cuevas, F. J., Mar n-Jim nez, M. J. (2014). Automatic generation and detec tion of highly reliable fiducial markers under occlusion. Pattern TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 10
https://openalex.org/W2059295122	https://link.springer.com/content/pdf/10.1007%2Fs13679-012-0020-0.pdf	English	null	Nutrition Labeling to Prevent Obesity: Reviewing the Evidence from Europe	Current obesity reports	2,012	cc-by	5,890	Nutrition Labeling to Prevent Obesity: Reviewing the Evidence from Europe Stefan Storcksdieck genannt Bonsmann & Josephine M. Wills Published online: 26 June 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Published online: 26 June 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com and Obesity related health issues (2007–2013)”, the Euro- pean Commission reports 30–70 % of EU adults to be overweight and 10–30 % obese [1]. As obesity is a condi- tion of multifactorial origin, preventative measures need to consider various dimensions including individual, societal, economic, and environmental aspects. In this context, the “Strategy” identified consumer information as one of four priority areas for which it aims to provide guidance for action to EU Member States [1]. The 2004 Global Strategy on Diet, Physical Activity and Health by the World Health Organization listed nutrition labeling as an important means to meet the consumers’ requirement for “accurate, standard- ized and comprehensible information on the content of food items in order to make healthy choices” [2]. Likewise, the Organization for Economic Co-operation and Development (OECD) views nutrition labeling as “a main tool for pre- venting increasing rates of obesity and unhealthy diets in OECD countries” [3]. European public health professionals and other stakeholders appear to agree that (mandatory) nutrition labeling is one of the more important policy options for obesity prevention, but food and health educa- tion were also considered relevant [4]. This is supported by research showing that consumers report that they value the on-pack provision of nutrition information [5]. Abstract Overweight and obesity are major public health problems in the European Union (EU). Providing nutrition information on foods and menus is considered a relevant means to guide consumers toward more healthful food choices, in part characterized by adequate energy intakes to achieve and maintain a healthy body weight. Various formats of back-of-pack and front-of-pack nutrition labeling can cur- rently be found across the EU, with varying levels of penetra- tion. Experimental studies show that consumers are reasonably able to understand and use the different systems to identify more healthful food products from given choice sets. However, European studies assessing the impact of nu- trition labeling on actual dietary intake are scarce, and no real- life evidence exists linking nutrition label use with measured changes in body weight. Curr Obes Rep (2012) 1:134–140 DOI 10.1007/s13679-012-0020-0 Curr Obes Rep (2012) 1:134–140 DOI 10.1007/s13679-012-0020-0 ECONOMY AND ENVIRONMENT (A DREWNOWSKI, SECTION EDITOR) Nutrition Labeling to Prevent Obesity: Reviewing the Evidence from Europe This review summarizes how Euro- pean consumers respond to nutrition labels when shopping for food or eating out of home, considering evidence published between 2007 and mid-March 2012. Keywords Nutrition labeling . Obesity prevention . Consumer behavior . Public policy Keywords Nutrition labeling . Obesity prevention . Consumer behavior . Public policy S. Storcksdieck genannt Bonsmann (*): J. M. Wills European Food Information Council (EUFIC), Tassel House, Rue Paul Emile Janson 6, 1000 Brussels, Belgium e-mail: stefan.storcksdieck@eufic.org J. M. Wills e-mail: jo.wills@eufic.org Introduction Although a new mandatory nutrition labeling legislation was adopted in December 2011 [6], any existing European studies into the role nutrition labeling could play in helping people choose healthful, balanced diets, were carried out on the backdrop of voluntary nutrition labeling. Only in the presence of a nutrition claim did nutrition labeling become mandatory in the EU, as laid down in Directive 90/496/EC of 1990 [7] and Directive 2000/13/EC of 2000 [8]. Depend- ing on the claim, either the “big 4” (energy, protein, carbo- hydrate, and fat) or the “big 8” (“big 4” plus sugar, saturated fat, fiber, and sodium) had to be stated. An audit of the Overweight and obesity are widespread in the European Union (EU). In its 2010 Implementation progress report about the “Strategy for Europe on Nutrition, Overweight Curr Obes Rep (2012) 1:134–140 135 (over half in Poland) thought incorrectly children needed more calories than an adult man [13]. penetration of nutrition information on food and drink labels in the EU plus Turkey, carried out in 2008/2009, showed that the basic nutrition table was present (back-of-pack) on 84 % of over 37,000 products from five predefined food and drink categories (sweet biscuits, breakfast cereals, carbonated soft drinks, chilled fresh ready meals, yogurts) [9]. Penetration was lowest in Slovenia at 68 % and highest in the United Kingdom (UK) and Ireland at 97 %. There was an uneven split between “big 8” and “big 4” across countries, with the UK most often providing the “big 8” (94 % of all products audited), and Turkey least often (19 %). The average split was 49 % “big 8” and 34 % “big 4” (not summing up to 84 % due to rounding errors). In their systematic review, Campos et al. [14] highlight that a number of studies have shown consumers to be struggling with quantitative nutrition label information. This was espe- cially true for certain patient groups (diabetics, chronic kidney disease), older adults, adolescents, infrequent label users, and those with lower education levels, but appeared to be amena- ble to change through educational efforts targeted at label knowledge and understanding. The need for simplicity was demonstrated by van Kleef et al. [15] who tested different front-of-pack energy sign- posts with consumer focus groups in the UK, Germany, The Netherlands, and France. Introduction Among the formats–from basic calorie labels to complex schemes including daily reference values and information about how much physi- cal activity would balance out the stated calories–the sim- plest format was liked best. The more complex the scheme became, the less it was liked, and this effect was substantially more pronounced in German respondents compared to the other three countries. Dietary energy intake and physical activity are the most immediate contributors to energy balance. In case of sus- tained positive energy balance (ie, when more calories are being consumed than expended), overweight and obesity ensue. As energy (usually given in kJ and kcal per 100 g [mL]) is commonly one of the core information items in nutrition labeling, the above figures give an idea of the availability of dietary energy labeling on prepackaged food and drink products in Europe. Much less effort has gone into nutrition labeling outside the supermarket setting. In early 2006, McDonald’s introduced Guideline Daily Amount (GDA) labeling (energy, protein, fat, carbohydrates, and salt) on product packages in Italy in conjunction with the Olympic Winter Games held in Turin, followed by a rollout across Europe and the rest of the world in the months thereafter [10]. As concerns governmental action, the most prominent example appears to be the UK, where the national Department of Health in early 2011 initiated a Public Health Responsibility Deal [11]. Amongst others, the deal includes a pledge for out-of-home calorie labeling, “asking catering businesses, who sell food in out of home settings, to provide calorie information for customers on menus or menu boards, to help people make healthier choices” [11]. By early March 2012, 45 business partners had signed the pledge, 38 of them having submitted concrete delivery plans. First moni- toring results were announced for April 2012, to be followed by annual reports every April thereafter. To the best of our knowledge, research causally linking nutrition labeling with total energy intake over time, and corresponding changes in body weight, is lacking. A large part of the research into nutrition labeling stems from North America (the United States in particular), with comparative- ly little evidence from Europe [14]. The studies discussed below have looked at the potential of nutrition labeling to guide Europeans toward more healthful diets, mainly charac- terized by products lower in fat, saturated fat, sugar, or salt. Introduction As most of these key nutrients also provide calories, reduced dietary intakes thereof may be considered a proxy for lower energy intakes. Potential Impact of Nutrition Labeling on Food Purchases Modeling Approaches Evidence from Retail Stores In a nationally representative survey carried out with 11,781 respondents from France, Germany, Poland, Sweden, Hun- gary, and the UK, two thirds of shoppers were observed looking at the front of food and drink packages in the supermarket, with less than 15 % looking elsewhere on the pack [13]. Whereas many people tend to say they use nutrition information regularly when shopping for food [12, 22–24], only a relatively small number (16.8 %) was actually observed doing so [25••]. French consumers showed the lowest objectively measured use at 8.8 %, con- trasted by UK consumers with 27.0 %. Similarly, the UK Food Standards Agency reported lower actual use than claimed by respondents [23]. Understanding and use of nutrition labels are positively correlated with female gender, higher income, better nutrition knowledge, and a general interest in healthy eating [14, 24, 25••, 26, 27]. As with Tesco, the UK retailer Sainsbury’s reported some beneficial effects on product sales in some catego- ries from a nutritional perspective after the introduction of traffic light signposting on their own brand ranges (cited in [24]). However, when Sacks et al. [31•] studied sales data of ready meals and sandwiches from a UK retailer employing traffic light labeling, they found no consistent nutritionally desirable effects related to the introduction of the labeling scheme. In other words, consumers did not purchase more of the products nutritionally better for them in the presence of traffic light labeling. The authors con- cluded that larger studies of longer duration would be required before traffic light labeling could be considered a promising public health intervention. Across surveys, calories and fat are stated fairly consis- tently as the most important nutrition information looked for, with the (back-of-pack) nutrition table being the main source [14, 24, 25••]. However, it was observed that few consumers look at the back of the pack [25••]. To help consumers access nutrition information more quickly, vari- ous front-of-pack schemes such as GDA and traffic light labeling have emerged in the EU over the past years, and used on a voluntary basis, yet with unclear outcomes [28]. The traffic light scheme, developed by the UK Food Stand- ards Agency, depicts whether a food is high (red), medium (amber), or low (green) in key nutrients (eg, fat, saturates, sugar, and salt, and usually includes calories per portion [without color coding]). Modeling Approaches Modeling Approaches Various studies [16–21] have modeled the potential impact of nutrition labels on dietary intakes or obesity in Europe. As an example, Sassi et al. [21] estimated that a mandatory nutrition labeling system for food sold in stores would decrease obesity rates in Europe by 2.5 % compared to a baseline situation where no such labeling existed. The model labeling scheme would inform consumers about nutrient content and portion size, supported by retailers posting explanations on how to read the labels and about the benefits of a healthy diet. Indi- vidual counseling by a combination of physicians and dieti- tians came out as most effective among the interventions assessed, with a population decrease in obesity prevalence of 6.5 %. Whereas the mandatory nutrition labeling intervention Regardless of these efforts to make energy (calorie) in- formation ubiquitous, the main question is whether people use this information when shopping for food or eating out, and with what outcome. Moreover, it is fair to assume that consumers need to know their energy requirements to make appropriate dietary choices based on the energy information provided. Two pan-European surveys [12, 13] indicated that while a majority of consumers know experts recommend to consume less calories, they were less certain about daily energy requirements for the average female (2000 kcal) and male (2,500 kcal). Knowledge about differences in calorie needs for men versus women and younger versus older adults was reasonably good, but over a third of respondents 136 Curr Obes Rep (2012) 1:134–140 resulted in an estimated 15 million disability-adjusted life years (DALYs) averted, the individual counseling approach computed to a reduction of 50 million DALYs. default signal lower energy products, they guide consumers toward products with a lower content of certain calorific nutrients. Therefore, using more of these products instead of counterparts not eligible for the logo could be seen as a way of reducing energy intake. Modeling approaches are helpful to estimate what changes in consumers’ food choices (read: dietary intakes) are required to bring about significant public health effects. However, they provide little, if any, information about consumers’ actual use of nutrition labels, its deter- minants, and how label use could be improved to result in healthier food choices. Modeling Approaches GDA labeling appears to be the most widespread front- of-pack system, with an average EU penetration of 25 %, ranging from 63 % in the UK down to 2 % in Turkey [9]. As reported by Grunert and Wills [24], the UK retailer Tesco found that after introduction of GDA labeling, sales of some more healthful products went up whereas sales of compara- ble products with a less favorable nutrient profile went down. However, a number of methodological issues were pointed out, thus limiting the overall relevance of the data. Within the EU 7th Framework Programme-funded project FLABEL (Food Labeling to Advance Better Education for Life) [29], a scientific assessment of the impact of introduc- ing GDA labeling on product sales was attempted, again based on Tesco sales data [30]. Unfortunately, the analysis revealed that price changes at the same time as the GDA labeling introduction masked any potential effects the new labeling system may have had on sales. What is Required to Make Nutrition Labeling More Helpful and Relevant to European Consumers? In general, the influence of nutrition labeling on food pur- chasing decisions is weak, especially when compared to other factors such as taste, price, use by-date, brand, conve- nience, and family preferences [22–24, 30, 41]. However, addressing a few barriers identified by FLABEL and other researchers could help optimize nutrition label use and thus its impact on dietary intakes. Posting nutrition information–in the format of star ratings for most healthful choices–at the point-of-purchase in two Belgian university canteens failed to significantly improve meal choices by customers [36]. Better objective nutrition knowledge, stronger health and weight control motives, and a greater openness to change meal choices at baseline de- termined the best results. However, the authors pointed out that a generically more healthful meal supply might have made the intervention more effective. While nutrition labels are already widely available [9], complete penetration on food and drinks products is consid- ered helpful [30, 31•, 32, 33, 34•, 35–39]. Consistent label format and positioning emerged as important factors for easy and quick access [39, 40•]. A previous representative survey involving six European countries showed that con- sumers can use different labeling systems similarly success- fully to identify the most healthful option out of a choice of three ready meals/pizzas [25••]. This and other research [22, 23, 26] suggest it does not matter so much which system is used on product packages, as long as it is presented in the same format and place on all products. The provision of multiple systems should be avoided as it may cause consumer confusion and frustration [23, 42]. Consistency, especially if supported by promotional and education campaigns, should aid familiarity with the nutrition labeling system, which in turn may enhance actual use [43]. In a Dutch cinema setting, Vermeer et al. [37] labeled soft drinks with portion size and GDA information (experimen- tal setting) or just the volume in milliliters (control) on two consecutive evenings and compared customers’ portion decisions. Neither portion information nor GDA labeling led to significant changes compared to the control condition, yet the relatively small sample size of 101 subjects may have resulted in insufficient statistical power to detect mean- ingful differences. Experimental labels were noticed by 68.8 % of participants and control labels by 49.8 %, the difference again not being statistically significant. Vyth et al. Evidence from Retail Stores The GDA labeling system was developed in a collaborative effort by the UK government, consumer organizations, and the food industry, and shows the amount of key nutrients and energy per portion of a food or beverage, and what percentage that portion contributes to a person’s daily guideline amount of those nutrients or energy. Health logos such as the Choices logo or the Swed- ish Keyhole are a type of scheme that requires foods to fulfill certain nutrient profiling criteria before being eligible to carry the logo on pack. While these logos do not provide information about the energy content of foods and do not by Vyth et al. [32] aimed to study consumers’ use of the Choices logo in nine Dutch supermarkets and observed that shoppers who claimed to be logo users had purchased significantly more products bearing the Choices logo. Self-reported logo use was positively associated with sev- eral food choice motives including health, weight control, and product information. The authors qualified that 1) overall response rate was low, 2) not all eligible products carried the Choices logo (manufacturers voluntarily choose to join the scheme), and 3) observations were based on single shopping occasions, which may not reflect habitual food purchasing behavior. Consumer preferences for a specific nutrition label for- mat seem to differ by country [15, 27, 33]. However, re- gardless of whether consumers said or were objectively found to look at, like, understand, or use nutrition labeling systems, none of the above studies allows any conclusion as Curr Obes Rep (2012) 1:134–140 137 products, with 12 control cafeterias over a period of 3 weeks showed no impact of the intervention. to whether consumers’ purchasing decisions were in any way influenced by the existence of such information. Find- ings from a study carried out in Germany and Belgium suggest that nutrition labeling has little impact on consum- ers’ buying decisions [33]. The relatively higher use and understanding of nutrition labeling systems by UK consum- ers indicates, however, that “intensive public debate on nutrition and labeling issues can indeed affect people’s thinking and behaviour” [25••]. This is supported by find- ings from Möser et al. [33] showing that GDA labeling was preferred in Belgium while traffic lights were preferred in Germany. Evidence from Out-Of-Home Eating Settings Frequent out-of-home eating is associated with higher intakes of total energy and energy from fat [34•], and a higher risk of overweight and obesity [35]. Therefore, pro- viding guidance to consumers toward making healthier food choices when eating out may be considered appropriate. Our literature search yielded four European studies published since 2007 assessing the impact of nutrition labeling on dietary intake in out-of-home eating settings [36–39]. What is Required to Make Nutrition Labeling More Helpful and Relevant to European Consumers? Evidence from Retail Stores Both countries have a similar availability of GDA labels [9], but in Germany consumer organizations, health professional associations, and insurance companies strongly supported the introduction of traffic lights, and the topic was widely covered in the media. Qualitative research commissioned by the UK Food Standards Agency [39] assessed the impact on consumers of posting calorie information in catering outlets. Dimen- sions considered were visibility of the calorie label, its location, format, and availability, as well as consumer understanding and usage, including impact on food choices. Standing out from other information was shown to be important, and this was aided by sufficient label size, the use of a distinct color, and possibly a consistent label format and location–findings that are in line with FLA- BEL data reported by Bialkova and van Trijp [40•]. Ad- vertising the existence of calorie labeling increased consumer awareness, and respondents stated that provi- sion on all food items would help them to accurately judge calorie contents of full meals [39]. Unfortunately, at the time of this assessment, calorie labeling in catering outlets had been very new, which is why actual use was report- edly low. Evidence from Out-Of-Home Eating Settings Beyond the Nutrition Label Low income and lack of time may be major barriers to buying more basic and healthful foods; providing more information– in the form of nutrition labeling–will increase neither of these two [14, 24, 46]. Furthermore, nutrition labels are more likely to be read by those who have an interest in healthy eating, show better nutrition knowledge, and thus may display health- ier eating patterns already [14, 25••]. In this context, findings from FLABEL [30] and others [32] indicate that expanding a given food/drink category by adding more healthful products can improve overall healthfulness of actual choice by the consumer. Nutrition labels, especially health logos, are seen as a potential driver for product reformulation in an attempt to meet eligibility criteria [17, 26, 30, 47]. Furthermore, Barreiro-Hurlé et al. [48] noted that clear and truthful nutrition and health claims may reach out to those who are less likely to read nutrition labels, such as people with lower nutrition knowledge or more hedonic lifestyles. What is Required to Make Nutrition Labeling More Helpful and Relevant to European Consumers? [38] tested whether introducing the nutrient profile-based Choices logo on products in Dutch worksite cafeterias improved customers’ food purchase decisions from a nutritional point of view. A comparison of food sales (sandwiches, soups, salads, snacks, and fruits) in 13 inter- vention cafeterias, where the logo was placed on eligible Consumers’ attention and motivation remain major bar- riers to using nutrition labels [25••, 44•], thus limiting any potential impact on health. Eye-tracking research measuring how long consumers look at nutrition labels indicated a time 138 Curr Obes Rep (2012) 1:134–140 span of 25–100 ms regardless of the system used [30]. This period is far too short for any conscious processing of the information. However, the presence of a health logo can slightly improve attention to the nutrition label [40•], which may be considered relevant within the (fairly) common condition of shopping under time pressure. attention to nutrition labels in real life. This lack of attention is partly driven by a lack of motivation, but the grander scheme suggests that price, taste, convenience, and shop- ping habits are simply far more important than nutrition information when making food purchasing decisions. Shop- ping under time pressure–a common phenomenon among today’s consumers–further impedes nutrition label use for healthy food shopping. Actively seeking out nutrition information requires a certain level of motivation. Such motivation could derive from the presence of a diet-related disease (eg, type 2 diabetes, hypertension), which would make nutrition infor- mation more personally relevant. Research shows that con- sumers with a health goal in mind are more likely to pay attention to and use nutrition labels [32, 44•, 45]. The opposite was observed when consumers followed their own preferences or were given a hedonic goal. The new EU food information regulation, which makes nutrition labeling mandatory, provides an opportunity for monitoring the impact of this policy on public health. How- ever, simply providing such information will not be enough to justify expectations for a (positive) change in people’s dietary habits. Instructive educational campaigns are re- quired that raise awareness, understanding, and the motiva- tion to use nutrition labels, taking into account the diverse needs of the European consumers. Authors’ Note After several years of negotiation, the European Commis- sion in December 2011 made nutrition labeling mandatory on food and drink products. With a few exemptions, manu- facturers must disclose information on the package about energy and six nutrients; total fat, saturated fat (saturates), carbohydrates, sugars, protein, and salt–in this order, and expressed per 100 g (mL) of product [6]. This information should be presented in the same field of vision, usually on the back of the pack, and may in addition be expressed on a per portion basis. Manufacturers who already provided nu- trition information in the past must comply with the new regulation by December 2014, whereas those who have yet to introduce nutrition labeling on their products are given until December 2016. Front-of-pack labeling remains voluntary under the new regulation, yet if information is repeated on the front of the pack, specific rules apply. Front-of-pack information can be the content of energy alone or in combination with fat, saturates, sugar, and salt. Energy can be presented per 100 g (mL) alone or additionally expressed per portion. The new regulation maintains the requirement to display energy in both kilojoules (kJ) and kilocalories (kcal) (there are 4.2 kJ in each kcal). When this information is declared for a portion or unit (eg, amount per biscuit), the size of a portion/unit must also be indicated, in conjunction with the number of portions or units contained in the package. References 15. van Kleef E, van Trijp H, Paeps F, Fernández-Celemín L. Consumer preferences for front-of-pack calories labeling. Public Health Nutr. 2008;11(2):203–13. Papers of particular interest, published recently, have been highlighted as: 16. Hieke S, Wilczynski P. Colour Me In - an empirical study on consumer responses to the traffic light signposting system in nutrition labeling. Public Health Nutr. 2012;15(5):773–782. • Of importance •• Of major importance 17. Roodenburg AJC, Schlatmann A, Dötsch-Klerk M, et al. Potential Effects of Nutrient Profiles on Nutrient Intakes in the Netherlands, Greece, Spain, USA, Israel, China and South-Africa. PLoS One. 2011;6(2):e14721. 1. European Commission. Strategy for Europe on nutrition, over- weight and obesity related health issues – Implementation Progress Report 2010. Brussels: European Commission; 2010. 18. Balcombe K, Fraser I, Salvatore Di F. Traffic lights and food choice: a choice experiment examining the relationship between nutritional food labels and price. Food Policy. 2010;35(3):211– 20. 2. World Health Organization (WHO). Global strategy on diet, phys- ical activity and health. Geneva: WHO; 2004. 3. Organisation for Economic Co-operation and Development (OECD). Promoting sustainable consumption – good practices in OECD countries. Paris: OECD Publishing; 2008. 19. Temme EH, van der Voet H, Roodenburg AJ, et al. Impact of foods with health logo on saturated fat, sodium and sugar intake of young Dutch adults. 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EC (2011). References Regulation (EU) No 1169/2011 of the European Par- liament and of the Council of 25 October 2011 on the provision of food information to co nsumers, amending Regulations (EC) No 1924/2006 and (EC) No 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/ EEC, Council Directive 90/496/EEC, Commission Directive 1999/ 10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) No 608/2004. Official Journal of the European Union L 304, 22.11.2011, p. 18–63. g 22. Food Safety Authority of Ireland. A research study into consumers’ attitudes to food labeling. Dublin: Food Safety Authority of Ireland; 2009. 23. Malam S, Clegg S, Kirwan S, McGinigal S. Comprehension and use of UK nutrition signpost labeling schemes. London: Food Standards Agency; 2009. 24. Grunert KG, Wills JM. 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Conclusions Nutrition labeling is considered a relevant component of pub- lic health policies attempting to stem the obesity epidemic in Europe. Scientific evidence to prove its actual use by consum- ers and the resulting impact on dietary energy intake, body weight, and health remains largely absent. Allison [49••] rightfully stated that “[i]f we are to understand the value of any macro-environmental manipulation intended to reduce obesity levels, we must eventually measure body weight, fat, or obesity levels”. While consumers like to see nutrition information on food and drink packages and appear able to use any labeling scheme to choose more healthful options out of a limited choice set under experimental conditions, they pay little Acknowledgments The European Food Information Council receives some funding from companies in the European Food and Drink industry. However, no companies were consulted in the drafting of this review, and there are no conflicts of interest. 139 Curr Obes Rep (2012) 1:134–140 consumer research. Webinar released on 18 February 2011. Available at http://www.eufic.org/webinars/consumerresearch/index.html. Accessed 6 March 2012. Disclosure No potential conflicts of interest relevant to this article were reported. 13. Wills JM, Grunert KG. Pan-European consumer research on in- store behaviour, understanding and use of nutrition information on food labels, and nutrition knowledge – Results from the European Study. Webinar released on 5 November 2008. Available at http:// www.eufic.org/webinars/eufic/paneuropeanlabelresearch/europe/ index.htm. Accessed 6 March 2012. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. 14. 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https://openalex.org/W3215423329	https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/article/download/4100/3012	English	null	Building the Resilient Indonesia’s Education in Pandemic Era: Lessons from Taiwan and the United States	Jurnal kependidikan	2,021	cc-by-sa	4,392	Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Building the Resilient Indonesia’s Education in Pandemic Era : Lessons from Taiwan and the United States Lalu Suprawesta1, Amja Manullang2, Mohammad Ainul Maruf3* 1Graduate Institute of Injury Prevention and Control, College of Public Health, Taipei Medical University, Taipei, Taiwan 1Department of Sport and Health Education, Faculty of Sport Science and Public Health, Universitas Pendidikan Mandalika, West Nusa Tenggara, Indonesia 2International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 3Faculty of Public Health, University of Muhammadiyah Jakarta 3Global Health and Health Security, College of Public Health, Taipei Medical University, Taipei, Taiwan Corresponding Author. Email: arvin.ainul@umj.ac.id Lalu Suprawesta1, Amja Manullang2, Mohammad Ainul Maruf3 1Graduate Institute of Injury Prevention and Control, College of Public Health, Taipei Medical University, Taipei, Taiwan 1Department of Sport and Health Education, Faculty of Sport Science and Public Health, Universitas Pendidikan Mandalika, West Nusa Tenggara, Indonesia 2International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 3Faculty of Public Health, University of Muhammadiyah Jakarta 3Global Health and Health Security, College of Public Health, Taipei Medical University, Taipei, Taiwan *Corresponding Author. Email: arvin.ainul@umj.ac.id Abstract: The aim of the study is to analyze the measures from Taiwan and the United States that responded to the Covid-19 in high education institutions. The method that was conducted in this study is qualitative with a narrative review as a data technique. Data collected from reviewed the relevant literature that meets inclusion criteria as the primary object. The descriptive qualitative analysis technique was used. The result of this study concludes that some measures from Taiwan and United States can be matched according to the characteristic in Indonesia. These two countries provide several lessons in tackling the Covid-19 problem in the education sector by preventing the transmission since the beginning, taking mitigation steps to reduce the speed of the spread of the virus, applying clear regulations and guidelines in various fields of life including education, and have a sense of sensitivity to relaxed and tightened of regulations. Article History Received: 23-08-2021 2 Revised: 24-10-2021 Accepted: 22-11-2021 Published:.11-12-2021 2017 Key Words: Education, Higher Education Institutions, Lessons Learned, Covid-19. How to Cite: Suprawesta, L., Manullang, A., & Maruf, M. (2021). Building the Resilient Indonesia’s Education in Pandemic Era: Lessons from Taiwan and the United States. Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran, 7(4), 778-784. doi:https://doi.org/10.33394/jk.v7i4.4100 https://doi.org/10.33394/jk.v7i4.4100 This is an open-access article under the CC-BY-SA License. https://doi.org/10.33394/jk.v7i4.4100 Introduction According to the CDC, higher education institutions, together with state and local health officials, may determine whether and how these considerations are applied while adapting to the unique needs and circumstances. The realization of the project must be guided by the practicable, acceptable, and adaptable needs of each school member and the community. In addition, educators can play a significant role in preventing in-school transmission through physical distancing and mask use. In response to the outbreak of Covid-19 in Indonesia, Minister of Education and Culture Nadiem Makarim issued Circular Letter Number 4 of 2020 on March 24, 2020. This established letter informed Indonesian educators of emergency-specific policies regarding the implementation of educational practices during the spread of the Covid-19. Through this circular, the Minister of Education and Culture sets forth the Minister's 2020 Policy for Home-Based Learning. In November 2020, Directorate General of Higher Education (DIKTI) released a circular letter Number 6 of 2020 informing that universities students starting in January 2021 can be held face-to-face and online (hybrid learning). Students' exams, like the national exams and equivalency exams, were all abolished in February 2021, according to circular letter number 1 of 2021 issued by the Minister of Education. In Taiwan, the Ministry of Education created general guidelines for college campuses to ensure the safety of students and faculty. There were detailed guidelines for the creation of a task force at each university, as well as school-based risk screening that is based on the student's travel history, job, contacts, and clusters. The following safety measures were also outlined: measures on self-management of health, and a process for reporting suspected cases. And policies on school closings and make-up classes were also made (Cheng, Wang, Shen, & Chang, 2020). A variety of mitigation measures were implemented in some states in the United States in August 2020, including increased physical spacing in classrooms, the requirement for students to wear face masks in class and other common areas, as well as adjustments to dining to reduce overcrowding (Walke, Honein, & Redfield, 2020). Lastly, all classes were moved to an online format, which has proven to be extremely successful. During the period of online classes, the university focused on facilitating access to testing, expanding contact tracing, isolation, and quarantine operations, and implementing screening tests for asymptomatic people, as well as enhancing the data systems to support these measures (Fox, Bailey, Seamon, & Miranda, 2021). Introduction This narrative review article discusses how education institutions in Taiwan and the United States responded to the Covid-19 pandemic then we discussed the kind of measures done in both contries that can be followed in Indonesia. Introduction Society is now more aware of the field of public health and the role of public health professionals in addressing the pandemic. Public health has long been known to play a key role in population health (Brisolara & Smith, 2020), especially in prevention strategies. The significance of this expertise allowed some countries in Asia, such as Taiwan, South Korea, and Singapore, to fight the coronavirus more successfully (Bauchner & Sharfstein, 2020). At the time of this update, the Covid-19 pandemic continues to have a terrible impact on lives worldwide. As of August 2021, there have been 200.566.338 cases and 4.624.642 deaths in the world (Worlemeter,2021). In Indonesia, 3.440.396 confirmed cases had occurred, resulting in 95.723 deaths. There are 9.9 percent of cases with 0.5% of deaths among school- aged children (6-18 years old), and 25.1% of cases with 2.8% of deaths among those aged 19- 30 years old (Satuan Petugas Penanganan Covid-19, 2021). l d d k As health and safety concerns remain unclear, there is considerable pressure on schools to find a way to educate every student in all segments while causing the least amount of disruption (Hale et al., 2021). So, a quick response from higher education leaders is \|778 Copyright © 2021, Authors Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 urgently needed for effective mitigation strategies. According to the CDC, higher education institutions, together with state and local health officials, may determine whether and how these considerations are applied while adapting to the unique needs and circumstances. The realization of the project must be guided by the practicable, acceptable, and adaptable needs of each school member and the community. In addition, educators can play a significant role in preventing in-school transmission through physical distancing and mask use. urgently needed for effective mitigation strategies. Jurnal Kependidikan Vol. 7, No. 4 (December 2021) Lesson from Taiwan In general, compare to Indonesia, Taiwan have taken some measures that affect the resilience in education program in pandemic era. The lessons from Taiwan including 1) Developing the robust national public healthcare network, 2) Taking fast response before the first cases reported, 3) Developing the guidelines in preventing pandemic spread by involving all sectors and practicing in various conditions that may be impacted by pandemic, 4) Constructing an integrated educational program for pandemic condition, and 5) Taking some measures that sufficient to prevent and contain viral spread by all colleges and universities meber depending on the incidence and prevalence. Taiwan has procedures in place to prevent any future epidemics, based on lessons learned during the 2003 SARS outbreak. A robust national public health network, complete universal healthcare for all people, a thriving medical research and pharmaceutical sector, and improved infection control methods are all strengths of the country. Since the epidemic, Taiwan has started a long-term investment in expanded capacity at the Centers for Disease Control (CDC), hospitals, and infectious disease laboratories. The Ministry of Health and Welfare (MOHW) directed the Taiwan CDC, which is responsible for communicable disease prevention, surveillance, investigation, and control, has made significant efforts to improve its disease control capacity by strengthening the regulatory power of the Communicable Disease Control Act (Ministry of Justice, 2020). ( y ) The Taiwanese government's response to Covid-19 is to take steps of speed, vigilance, and decisiveness. When the first reports of a new virus in China emerged on December 31, 2019, Taiwan began screening arrivals from Wuhan, imposing home quarantine on individuals with fever and possible respiratory symptoms. The Taiwan CDC created the Central Epidemic Command Center (CECC) on January 20, 2020, in response to the rising severity of the epidemic in China and neighboring regions, with the minister of the MOHW as the main commander and high-rank officials from other ministries as the core members. The leadership of the CECC and the Taiwan CDC are critical in combating the Covid-19 epidemic. Personal hygiene policies, preventative actions in schools and businesses, the identification and isolation of suspected and proven cases, the expropriation of mask makers, and mask rationing all necessitate efficient public-private engagement and partnerships. (Control, 2020; Han, Chiou, McKee, & Legido-Quigley, 2020; Yeh & Cheng, 2020). ) The Taiwanese CDC released a set of guidelines outlining preventive actions to be taken by various actors in various situations. Research Method The method that was conducted in this study is qualitative with a narrative review as a data technique. Data collected from reviewed the relevant literature that meets inclusion criteria as the primary object. We searched for information and news regarding higher education's response to the Covid-19 pandemic in Taiwan and the US from Google Scholar, PubMed databases, and ministry of education websites. The sources were limited to English and Indonesian languages by the time frame between January 2020 and August 2021. The search terms used were "Taiwan", "US", "higher education response", "college", "university campus", "Covid-19 and students", "Covid-19 and higher education", and "Covid-19 response". The data were analyzed in descriptively qualitative. Jurnal Kependidikan Vol. 7, No. 4 (December 2021) Jurnal Kependidikan Vol. 7, No. 4 (December 2021) Copyright © 2021, Authors Copyright © 2021, Authors \|779 Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/ind Jurnal Kependidikan Vol. 7, No. 4 (December 2021) Lesson from Taiwan The Ministry of Education has declared campus closures for both public and private institutions. The CECC stated on March 20, 2020 that all instructors, students, and staff in high school and lower will be prohibited from going abroad. Many colleges and universities have also freely implemented online classrooms and teleconferencing. Schools and other educational institutions have returned to normal as the pandemic has decreased since late May. The Taiwanese Ministry of Education created broad rules for college campuses to ensure the safety of students and employees [Ministry of Education, 2020]. The guide describes the formation of working groups at each university and the risk assessment at the school based on travel history, occupation, contacts and groups; self-management of sanitary and quarantine measures, general sanitation measures (including the use of masks indoors); ventilation and sanitation principles, School assembly regulations, suspected case reporting procedures and policy formulation. There are some consequences and impacts of the pandemic for college students in Taiwan. The construction of an integrated educational program for health professionals, Jurnal Kependidikan Vol. 7, No. 4 (December 2021) \|780 Copyright © 2021, Authors Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 lifestyle modifications and college students' health condition, and the learning efficacy of physical classroom and online learning for dentistry education during the Covid-19 pandemic are among the latest research. Health professionals’ workers may increase their learning opportunities through the hospital wide-courses, continued placement of test questions and learning files on digital learning platforms, placement of journal highlights in cloud folders and using the digital learning platform on mobile phones accessible outside the hospital (Chiu et al., 2021). The lifestyles change of college students were found by decreased frequency of daily activities and had fewer opportunities for socialization and interaction with peers and faculty under the new procedures created by the pandemic of Covid-19 [Chen et.al., 2019]. Dental education may adopt the combination of physical classroom and online course as the future trend since the dental students tended to have online class learning was better than physical classroom but more feel convenience to have physical classroom examination than online examination (Yu-Fong Chang, Wang, Lin, Cheng, & Chiang, 2021). Lesson from Taiwan Taiwan have successfully done some measures such as active campus-based screening and access control; school-based travel, occupant, contact, cluster (TOCC) screening and quarantine protocol; student and faculty quarantine as needed; mobilization of administrative and health center staff; regulation of dorms and cafeteria; and reinforcement of personal hygiene, environmental sanitation, and indoor air ventilation. These measures will be sufficient to prevent and contain viral spread on campus depending on the incidence and prevalence of disease in a school’s location (Cheng et al., 2020). Jurnal Kependidikan Vol. 7, No. 4 (December 2021) Lesson from the United States Covid-19 was first detected in the US in January and February 2020 among travelers from Hubei Province, China, and their family contacts (Jernigan & CDC Covid Response Team, 2020). Since then, the US CDC has taken several measures to prevent local transmission from occurring. However, there were certain factors that make the spread of the virus difficult to avoid such as the continued viral importation from other countries, the participation at mass and gathering events, the introduction of the virus into potentially amplification-prone facilities, and the obstacles in virus detention, and the other cryptic transmission (Schuchat & CDC COVID Response Team, 2020). Until now, there were at least 4 waves of Covid-19 transmission in the US (Wilson, 2021). Even in the US, Covid-19 pandemic has had a detrimental impact on higher education, including dental education, medical education, and other field of education (Ahmed, Allaf, & Elghazaly, 2020; Ferrel & Ryan, 2020; Iyer, Aziz, & Ojcius, 2020; Rose, 2020). The reopening of college and universities in the fall of 2020 presented new problems and risk for transmission on campuses and neighboring communities. The risk of severe health outcomes might be lower among college students. However, the faculty, university staff and family of college students at home and in the communities may be at a significantly higher risk of severe illness and death if infected (Walke et al., 2020). g The US CDC anticipated existing risks and problems by issuing guidelines with the tagline “plan, prepare, and response” for colleges and universities. Those guidelines include the guiding principles and mitigation strategies for reopening the institution, when to quarantine on campus, testing for Covid-19, case investigation and contact tracing, guidance for shared or congregate housing, guidance of using transportation, and also how to cope with stress for student, administrators, faculty and staff (Center for Disease Control and Prevention, 2020). The guidelines are also accompanied by easy-to-understand posters and videos which can be accessed on their websites. Jurnal Kependidikan Vol. 7, No. 4 (December 2021) Jurnal Kependidikan Vol. 7, No. 4 (December 2021) \|781 Copyright © 2021, Authors Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. Conclusion Indonesia has its own characteristics that cannot simply be matched with the situation in Taiwan and the United States. However, there are several lessons that can be learned from the two countries in tackling the COVID-19 problem in the education sector. First, it is very important to prevent transmission from the beginning. Second, when local transmission cannot be overcome, it is necessary to take mitigation steps to reduce the speed of the spread of the virus. Third, there needs to be clear regulations and guidelines to be applied in various fields of life, including education. Fourth, those regulations need to be disseminated and accompanied by law enforcement. Fifth, there needs to be a sense of sensitivity to know when regulations can be relaxed and when they can be tightened. When planning to reopen a school or campus, the considerations used must be based on a rational approach. Indeed, young people are not categorized as vulnerable groups. However, their mobility can endanger vulnerable groups. Moreover, in Indonesia, usually, young people live with their families at home. Under one roof there can be three generations, grandparents, parents, and children. Even in implementing distance learning from home, it is necessary to pay attention to whether the student has supporting devices, such as laptops, and internet access for instance, in their homes. The gap between rural and urban areas, between the rich and the poor, also needs special attention, both from the central and local governments. Lesson from the United States 778-784 In dealing with the potential decline in the number of public health workers (ASPH Policy Brief, 2008; Leider, Coronado, Beck, & Harper, 2018), several public health schools and programs had contributed to the public health response to Covid 19 by providing applied practice experiences for students (Burns, Strickland, & Horney, 2021; Council on Education for Public Health, 2020). They keep running the fieldwork initiative in the midst of challenges such as travel restrictions, university closure, working on-site restrictions, and others (Centers for Disease Control Prevention, 2021). This showed that public health students can take part in efforts to mitigate the devastating effects of the pandemic. p g g p Distance learning education also plays an important role during this pandemic in the US. Fortunately, the US has a long history of developing distance learning (Cook & Dupras, 2004). There are several key players from the US who develop Massive Open Online Courses (MOOCs), such as edX, Pluralsight, Udacity, Udemy, Simplilearn, Edmodo, LinkedIn, NovoEd, Skillshare, and Khan Academy. It puts the US as the leading MOOC platform on this planet. Moreover, up to 80% of colleges in the US offer their courses online (Wotto, 2020). However, some argued that distance learning cannot simply replace traditional education especially if schools and students do not prepare it properly (Christakis, 2020). References Ahmed, H., Allaf, M., & Elghazaly, H. (2020). COVID-19 and medical education. The Lancet Infectious Diseases, 20(7), 777-778. ASPH Policy Brief. (2008). Confronting the Public Health Workforce Crisis. Retrieved August 23, 2021, from ASPH http://www.nevadapublichealthfoundation.org/wp- content/themes/nphf/userfiles/WorkforceShortage2010Final.pdf Bauchner, H., & Sharfstein, J. (2020). A Bold Response to the COVID-19 Pandemic: Medical Students, National Service, and Public Health. Jama, 323(18), 1790-1791. doi:10.1001/jama.2020.6166 j Brisolara, K. F., & Smith, D. G. (2020). Preparing Students for a More Public Health-Aware Market in Response to COVID-19. Prev Chronic Dis, 17, E56. doi:10.5888/pcd17.200251 Burns, K. F., Strickland, C. J., & Horney, J. A. (2021). Public health student respo COVID-19. Journal of Community Health, 46(2), 298-303. Center for Disease Control and Prevention. (2020). College & Universities: Plan, Prepare, and Respond. Retrieved August 23, 2021, from The US CDC https://www.cdc.gov/coronavirus/2019-ncov/community/colleges- universities/index.html Centers for Disease Control Prevention. (2021). Considerations for institutions of higher education. Cheng, S. Y., Wang, C. J., Shen, A. C., & Chang, S. C. (2020). How to Safely Reopen Colleges and Universities During COVID-19: Experiences From Taiwan. Ann Intern Med, 173(8), 638-641. doi:10.7326/m20-2927 Chiu, T. F., Chu, D., Huang, S. J., Chang, M., Liu, Y., & Lee, J. J. (2021). Facing the Coronavirus Pandemic: An Integrated Continuing Education Program in Taiwan. Int J Environ Res Public Health, 18(5). doi:10.3390/ijerph18052417 j p Christakis, D. A. (2020). School reopening—the pandemic issue that is not getting its due. JAMA pediatrics, 174(10), 928-928. Control, T. C. f. D. (2020). Prevention and Control of COVID-19 in Taiwan. Retrieved from https://www.cdc.gov.tw/En/Category/Page/0vq8rsAob_9HCi5GQ5jH1Q Cook, D. A., & Dupras, D. M. (2004). A practical guide to developing effective web-based learning. Journal of general internal medicine, 19(6), 698-707. Council on Education for Public Health. (2020). COVID-19 Related Updates: Practice Experience. Retrieved August 23, 2021, from Council on Education for Public Health https://ceph.org/covid19/ Ferrel, M. N., & Ryan, J. J. (2020). The impact of COVID-19 on medical education. Cureus, 12(3). Fox, M. D., Bailey, D. C., Seamon, M. D., & Miranda, M. L. (2021). Response to a COVID- 19 Outbreak on a University Campus - Indiana, August 2020. MMWR Morb Mortal Wkly Rep, 70(4), 118-122. doi:10.15585/mmwr.mm7004a3 Government of Taiwan, 2021. Communicable Disease Control Act. Ministry of Justice Laws & Regulations Database of The Republic of China. Retrieved August 02, 2021 from https://law.moj.gov.tw/ENG/LawClass/LawAll.aspx?pcode=L0050001 Hale, T., Angrist, N., Goldszmidt, R., Kira, B., Petherick, A., Phillips, T., . . . Tatlow, H. (2021). Recommendation This pandemic should be a point where improvements are carried out on an ongoing basis to avoid the impact of the next pandemic in the future. Educational institutions themselves in which there are teachers/faculty members, students and education managers, need to see this as an opportunity to improve the quality of education as a whole. As the main actors of the education sector, they must be able to adapt to changes and implement innovations that are appropriate to the context in which they are located. It is also possible to see learning from other regions or other countries while still paying attention to local wisdom. Jurnal Kependidikan Vol. 7, No. 4 (December 2021) Jurnal Kependidikan Vol. 7, No. 4 (December 2021) Copyright © 2021, Authors \|782 Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran https://e-journal.undikma.ac.id/index.php/jurnalkependidikan/index Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Email: jklppm@undikma.ac.id Vol. 7, No. 4 : December 2021 E-ISSN: 2442-7667 pp. 778-784 Jurnal Kependidikan: Jurnal Kependidikan Vol. 7, No. 4 (December 2021) References A global panel database of pandemic policies (Oxford COVID-19 Government Response Tracker). 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https://openalex.org/W4322493739	https://ueaeprints.uea.ac.uk/id/eprint/91649/1/mra.01273_22.pdf	English	null	Draft Genome Sequence of a Preterm Infant-Derived Isolate of Candida parapsilosis	Microbiology resource announcements	2,023	cc-by	1,390	Steve A. James,a Andrea Telatin,a David Baker,a Rhiannon Evans,a Paul Clarke,b,c Lindsay J. Hall,a,d Simon R. Cardinga,c Steve A. James,a Andrea Telatin,a David Baker,a Rhiannon Evans,a Paul Clarke,b,c Lind Steve A. James,a Andrea Telatin,a David Baker,a Rhiannon Evans,a Paul Clarke,b,c Lindsay J. Hall,a,d aGut Microbes and Health Research Programme, Quadram Institute Bioscience, Norwich, United Kingdom bNeonatal Intensive Care Unit, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, United Kingdom cNorwich Medical School, University of East Anglia, Norwich, United Kingdom dZiel–Institute for Food and Health, Technical University of Munich, Freising, Germany ABSTRACT Candida parapsilosis is a human fungal pathogen of increasing incidence and causes invasive candidiasis, notably in preterm or low-birthweight neonates. Here, we present the genome sequence of C. parapsilosis NCYC 4289, a fecal isolate from a preterm male infant. C andida parapsilosis is a dimorphic ascomycete yeast belonging to the Lodderomyces clade, a large monophyletic group of species, which includes a number of important human pathogens (e.g., Candida albicans) (1). Although often found in the human gut (2, 3), C. parapsilosis is primarily a skin commensal and is present in the hospital environment (4), especially the neonatal intensive care unit (NICU). With a capacity to form persistent bioﬁlms, it can spread by horizontal transmission and is regarded as a signiﬁcant neonatal pathogen, with low-birthweight preterm neonates at particular risk of infection (5). Here, we combined short- and long-read sequencing to obtain the genome sequence of C. parapsilosis NCYC 4289, a feces-derived isolate from a 3115-week-old premature male infant delivered by cae- sarean section. A fecal homogenate was prepared in sterile phosphate-buffered saline (PBS) and cultured on Sabouraud dextrose (SD) agar plates containing penicillin (25 U/mL) and streptomycin (25 U/mL) at 30°C. Species identity, from single colonies, was determined by PCR ampliﬁcation and Sanger sequencing of the ribosomal DNA internal transcribed spacer (ITS) region of the ribosomal DNA locus using primers ITS1F (6) and ITS4 (7). The ITS sequence of strain NCYC 4289 (GenBank accession number ERZ15609610) is 100% identical to that of the C. parapsilosis type strain CBS 604 (GenBank accession number AY391843). Downloaded from https://journals.asm.org/journal/mra on 22 March 2023 by 139.222.123.80. For short- and long-read sequencing, total genomic DNA was extracted from a stationary- phase SD culture (MasterPure yeast DNA puriﬁcation kit; Cambio), and cells were treated with Zymolyase (0.25 mg/mL) to aid cell wall disruption with an additional proteinase K treatment step included prior to DNA precipitation. GENOME SEQUENCES Draft Genome Sequence of a Preterm Infant-Derived Isolate of Candida parapsilosis Steve A. James,a Andrea Telatin,a David Baker,a Rhiannon Evans,a Paul Clarke,b,c Lindsay J. Hall,a,d Simon R. Cardinga,c REFERENCES Thomson NM, Gilroy R, Grifﬁth L, Adriaenssens EM, Stanley R, Charles IG, Elumogo N, Wain J, Prakash R, Meader E, Mather AE, Webber MA, Dervisevic S, Page AJ, O’Grady J. 2021. CoronaHiT: high-throughput sequencing of SARS-CoV- 2 genomes. Genome Med 13:21. https://doi.org/10.1186/s13073-021-00839-5. 1. Lachance M-A, Boekhout T, Scorzetti G, Fell JW, Kurtzman CP. 2011. Candida Berkhout (1923), p 987–1278. In Kurtzman CP, Fell JW, Boekhout T (ed), The yeasts; a taxonomic study, 5th ed, vol 2. Elsevier, Amsterdam, Netherlands. Thomson NM, Gilroy R, Grifﬁth L, Adriaenssens EM, Stanley R, Charles IG, g Page AJ, O’Grady J. 2021. CoronaHiT: high-throughput sequencing of SARS-CoV- Page AJ, O’Grady J. 2021. CoronaHiT: high-throughput sequencing of SARS-CoV 2 genomes. Genome Med 13:21. https://doi.org/10.1186/s13073-021-00839-5. g , y g g p q g 2 genomes. Genome Med 13:21. https://doi.org/10.1186/s13073-021-00839-5. nomes. Genome Med 13:21. https://doi.org/10.1186/s13073-021-0083 2. Strati F, Di Paola M, Stefanini I, Albanese D, Rizzetto L, Lionetti P, Calabro A, Jousson O, Donati C, Cavalieri D, De Filippo C. 2016. Age and gender affect the composition of fungal population of the human gastrointestinal tract. Front Microbiol 7:1227. https://doi.org/10.3389/fmicb.2016.01227. 9. Telatin A, Fariselli P, Birolo G. 2021. SeqFu: a suite of utilities for the robust and reproducible manipulation of sequence ﬁles. Bioengineering (Basel) 8:59. https://doi.org/10.3390/bioengineering8050059. 3. Schei K, Avershina E, Oien T, Rudi K, Follestad T, Salamati S, Odegard RA. 2017. Early gut mycobiota and mother-offspring transfer. Microbiome 5: 107. https://doi.org/10.1186/s40168-017-0319-x. 10. Chen SF, Zhou YQ, Chen YR, Gu J. 2018. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890. https://doi.org/10.1093/bioinformatics/ bty560. g 4. Sabino R, Sampaio P, Carneiro C, Rosado L, Pais C. 2011. Isolates from hos- pital environments are the most virulent of the Candida parapsilosis com- plex. BMC Microbiol 11:180. https://doi.org/10.1186/1471-2180-11-180. y 11. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error- prone reads using repeat graphs. Nat Biotechnol 37:540–546. https://doi .org/10.1038/s41587-019-0072-8. g 5. Pammi M, Holland L, Butler G, Gacser A, Bliss JM. 2013. Candida parapsilosis is a signiﬁcant neonatal pathogen: a systematic review and meta-analysis. Pediatr Infect Dis J 32:E206–E216. https://doi.org/10.1097/INF.0b013e3182863a1c. 12. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng QD, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improve- ment. PLoS One 9:e112963. https://doi.org/10.1371/journal.pone.0112963. Downloaded from https://journals.asm.org/journal/mra on 22 March 2023 by 139.222.123.80. p g 6. Gardes M, Bruns TD. 1993. Steve A. James,a Andrea Telatin,a David Baker,a Rhiannon Evans,a Paul Clarke,b,c Lindsay J. Hall,a,d Simon R. Cardinga,c Short-read Illumina sequencing was performed using a modiﬁed 20-fold dilution of DNA Prep (Flex) reagent and run on a NextSeq 500 sequencer, producing 7,418,311 paired-end 150-bp reads. Nanopore sequencing was obtained from two methods. First, a novel modiﬁed Illumina DNA Prep (Flex) approach used symmetrical 24-base barcoded primers and a long-range polymerase (8). Libraries were pooled and size selected on a SageELF 0.75% cassette, fractions from 4 kb and above were pooled, and long-read sequencing was performed using a MinION sequencer (Oxford Nanopore Technologies [ONT]), ligation sequencing kit SQK-LSK109 (ONT), and ﬂow cell FLO-MIN106 R9.4.1 (ONT). This produced a total of 812,374 reads with an average read length of 4,591 bases. The second method followed the standard ligation protocol using the manufacturer’s recommendations and loading on a second ﬂow cell, producing 852,227 reads (average read length, 4,756 bases). Base calling was performed using Guppy (ONT; v.3.6.0) in high-accuracy mode (model dna_r9.4.1_450bps_hac). March 2023 Volume 12 Issue 3 Announcement Microbiology Resource Announcements Raw short- and long-read polishing, including the removal of adapters and low-quality bases, was performed using SeqFu 1.16 (9) and fastp 0.23 (10). In addition, long reads of ,1 kb in length were discarded. The genome was assembled using Flye 2.9.1 (11), polished with four rounds of Pilon 1.24 (12), and reﬁned using RagTag (13). The genome assembly comprised eight chromosome-sized contigs of .890 kb and a linear mitochondrial genome (29,583 bp). The total size of the genome was 13,082,726 bp, the N50 value was 2,085,264 bp, and the G1C content was 38.70%. The largest contig in the assembly was 3,026,395 bp. Genome completeness was estimated at 93.0% using BUSCO v5.4.4 (14). Dependencies and scripts are available at https://github.com/quadram-institute-bioscience/ont-candida. Data availability. This whole-genome shotgun project has been deposited at DDJB/ENA/GenBank (BioProject number PRJEB56866 and assembly accession number CAMXCU010000000.1). The version described in this paper is version 1. The raw reads were deposited at SRA (accession numbers ERX9916594 and ERX9917623). ACKNOWLEDGMENTS We gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC). This research was funded by BBSRC Core Capability Grant BB/ CCG1860/1 and BBSRC Institute Strategic Programme Grant Gut Microbes and Health (BB/ R012490/1) and constituent project BBS/E/F/000PR10353. REFERENCES ITS primers with enhanced speciﬁcity for basi- diomycetes–application to the identiﬁcation of mycorrhizae and rusts. Mol Ecol 2:113–118. https://doi.org/10.1111/j.1365-294x.1993.tb00005.x. 13. Alonge M, Lebeigle L, Kirsche M, Aganezov S, Wang X, Lippman ZB, Schatz MC, Soyk S. 2021. Automated assembly scaffolding elevates a new tomato system for high-throughput genome editing. bioRxiv. https://doi .org/10.1101/2021.11.18.469135. p g j 7. White TJ, Bruns TD, Lee SL, Taylor JW. 1990. Ampliﬁcation and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p 315–322. In Innis MA, Gelfand DH, Sninsky JJ (ed), PCR protocols: a guide to meth- ods and applications. Academic Press, San Diego, CA. 14. Manni M, Berkeley MR, Seppey M, Simao FA, Zdobnov EM. 2021. BUSCO update: novel and streamlined workﬂows along with broader and deeper phy- logenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol 38:4647–4654. https://doi.org/10.1093/molbev/msab199. 8. Baker DJ, Aydin A, Le-Viet T, Kay GL, Rudder S, Martins LD, Tedim AP, Kolyva A, Diaz M, Alikhan NF, Meadows L, Bell A, Gutierrez AV, Trotter AJ, March 2023 Volume 12 Issue 3 10.1128/mra.01273-22 2 10.1128/mra.01273-22 2
https://openalex.org/W2913296433	https://europepmc.org/articles/pmc6533219?pdf=render	English	null	Clinical implementation of pre-treatment DPYD genotyping in capecitabine-treated metastatic breast cancer patients	Breast cancer research and treatment	2,019	cc-by	4,828	Breast Cancer Research and Treatment (2019) 175:511–517 https://doi.org/10.1007/s10549-019-05144-9 Breast Cancer Research and Treatment (2019) 175:511–517 https://doi.org/10.1007/s10549-019-05144-9 BRIEF REPORT Clinical implementation of pre-treatment DPYD genotyping in capecitabine-treated metastatic breast cancer patients Received: 13 January 2019 / Accepted: 21 January 2019 / Published online: 12 February 2019 © The Author(s) 2019 Abstract Purpose Metastatic breast cancer (mBC) patients with DPYD genetic variants linked to loss of dihydropyrimidine dehydro- genase (DPD) activity are at risk of severe capecitabine-associated toxicities. However, prospective DPYD genotyping has not yet been implemented in routine clinical practice. Following a previous internal review in which two patients underwent lengthy hospitalisations whilst receiving capecitabine, and were subsequently found to be DPD deficient, we initiated routine DPYD genotyping prior to starting capecitabine. This study evaluates the clinical application of routine DPYD screening at a large cancer centre in London. Methods We reviewed medical records for consecutive patients with mBC who underwent DPYD genotyping before com- mencing capecitabine between December 2014 and December 2017. Patients were tested for four DPYD variants associated with reduced DPD activity. Results Sixty-six patients underwent DPYD testing. Five (8.4%) patients were found to carry DPYD genetic polymorphisms associated with reduced DPD activity; of these, two received dose-reduced capecitabine. Of the 61 patients with DPYD wild- type, 14 (23%) experienced grade 3 toxicities which involved palmar–plantar erythrodysesthesia (65%), and gastrointestinal toxicities (35%); no patient was hospitalised due to toxicity. Conclusions Prospective DPYD genotyping can be successfully implemented in routine clinical practice and can reduce the risk of severe fluoropyrimidine toxicities. Introduction Breast cancer is the most common malignancy among women accounting for 23% of new cancer cases and 15% of cancer-related deaths annually in the world [1]. Capecit- abine, an orally administered pro-drug of 5-fluoruracil has a pivotal role in the therapeutic armamentarium for metastatic breast cancer (mBC) usually as monotherapy but also in combination with lapatinib [2]. Fluoropyrimi- dines are considered to be safe and well tolerated with a side-effect profile that includes diarrhoea, mucositis, pal- mar–plantar erythrodysesthesia (PPE), bone marrow sup- pression and nausea. Although the majority of patients receiving fluoropyrimidines experience mild toxicities, usually managed with supportive measures, approximately 10–30% of patients will develop severe (grade ≥ 3) toxici- ties which can result in lengthy hospitalisations or death in 0.5–1% of cases [3–5]. Based on our initial experience with two mBC patients who were hospitalised and retrospectively found to be DPD deficient, we previously conducted an internal review including 48 patients and found that: (a) none of the other patients who were receiving capecitabine during that time (December 2012 to December 2013) underwent lengthy hos- pital admission for capecitabine toxicities and (b) the cost of the inpatient stay far outweighed the total cost of testing all those patients for DPD (£15 525 versus £1575 based on the cost of the test at that time) [33]. Fluoropyrimidine toxicities have been strongly associ- ated with reduced activity of the enzyme dihydropyrimi- dine dehydrogenase (DPD), which is the rate-limiting enzyme in the catabolism of fluoropyrimidines [6–9]. DPD catabolises 80% of 5-fluoruracil (5-FU), into the non-cyto- toxic metabolite 5-fluoro-5,6-dihydrouracil (dHFU) and patients with partial deficiency have 1.5-times increased 5-FU exposure when treated with standard doses [6, 10]. Approximately, 3–5% of the North American and Euro- pean population have partial DPD deficiency, whereas complete deficiency is much rarer with a prevalence of 0.01–0.1% [11–13]. Following this, routine DPYD screening prior to pre- scribing capecitabine was initiated at Guy’s and St Thomas’ NHS Trust (GSTT) in December 2014 using an in-house developed genotyping assay [34]. In this retrospective observational study, we describe our centre’s experience and clinically evaluate prospective DPYD genotyping for metastatic breast cancer patients treated with capecitabine. In particular, we evaluated the feasibility of implementing DPYD tests in routine clinical practice, treatment decisions and outcomes based on the pharmacogenetic test results, and its utility in preventing severe toxicities. Abstract Keywords DPYD genotyping · DPYD screening · Fluoropyrimidines · Toxicities · Metastatic breast cancer · Capecitabine Keywords DPYD genotyping · DPYD screening · Fluoropyrimidines · Toxicities · Metastatic breast cancer · Capecitabine Anthony Marinaki Tony.Marinaki@viapath.co.uk Eleni Karapanagiotou eleni.karapanagiotou@gstt.nhs.uk Dionysis Papadatos‑Pastos Dionysis.Papadatos‑Pastos@uclh.nhs.uk 1 Breast Unit, Guy’s and St Thomas’ NHS Foundation Trust and King’s Biomedical Centre, 4th Floor, Bermondsey Wing, Great Maze Pond, London SE1 9RT, UK 2 Purine Research Laboratory, Viapath, Guy’s and St Thomas’ NHS Foundation Trust, Westminster Bridge Road, London SE1 7EH, UK * Janine Mansi Janine.mansi@gstt.nhs.uk Chara Stavraka chara.stavraka@gstt.nhs.uk Athanasios Pouptsis th.pouptsis@gmail.com Leroy Okonta leroy.okonta@nhs.net Karen DeSouza Karen.DeSouza@bsuh.nhs.uk Philip Charlton Philip.charlton@nhs.net Matthaios Kapiris Matthaios.kapiris@gstt.nhs.uk 1 Breast Unit, Guy’s and St Thomas’ NHS Foundation Trust and King’s Biomedical Centre, 4th Floor, Bermondsey Wing, Great Maze Pond, London SE1 9RT, UK 2 Purine Research Laboratory, Viapath, Guy’s and St Thomas’ NHS Foundation Trust, Westminster Bridge Road, London SE1 7EH, UK (0121 3456789) 3 512 Breast Cancer Research and Treatment (2019) 175:511–517 guidance on appropriate dose reductions in patients har- bouring these polymorphisms to prevent severe toxicities [24] Despite growing evidence supporting the significant clinical and financial benefits of routine DPYD genotype screening prior to fluoropyrimidine administration, this is not yet the standard of care [27–32]. The ability to pre- dict and prevent severe capecitabine-associated toxicities is of paramount importance particularly in metastatic breast cancer patients, as a number of alternative treat- ment options are available. Such toxicities can result in death or pose significant delays to consequent treatment and may even compromise patients’ fitness to receive fur- ther treatment. Introduction The activity of DPD has been found to be regulated at the genetic, transcriptional (by transcription factors SP1 and AP3) and post-transcriptional level (microRNA 27-a and microRNA 27-b) [14–16]. The most prevalent cause of DPD deficiency though, is the presence of deleteri- ous polymorphisms in its encoding DPYD gene, which have received much interest as predictive biomarkers for fluoropyrimidine-induced toxicities [13, 17–21]. Four DPYD genetic variants have been established as clinically relevant and associated with severe toxicities: DPYD2A (IVS14 + 1G > A, c.1905 + 1G > A, or rs3918290), c.2846A > T (p.D949V or rs67376798), c.1679T > G (rs55886062, DPYD13, p.I560S), and c.1236G > A (rs56038477, p.E412E, Hap B3) [13, 22–25]. A fifth variant c.1601G > A (p.S534N, DPYD4, or rs1801158), has been associated with impaired DPD activity [26] but its clinical relevance remains inconclusive [13]. Current evidence suggests that heterozygous carriers of these variants have an average decrease in the DPD activity of approximately 25% (c.2846A > T, c.1236G > A) and 50% (DPYD2A, c.1679T > G) [24]. The Clinical Pharmaco- genetics Implementation Consortium (CPIC) has issued Statistical analysis modifications as per our institutional guidelines as follows: heterozygous c.1905 + 1G > A for treatment with 50% dose reduction, heterozygous c.2846A > T and heterozygous c.1601G > A for treatment with 25% dose reduction [34]. Three of these patients did not receive capecitabine, and an alternative regimen was prescribed by their treating physician. Treatment outcomes for the DPYD variant car- riers are summarised in Table 2. The other two patients received a 50% dose reduction of capecitabine during their first cycle of treatment with no complications. The c.2846A < T variant carrier had a subsequent dose increase to 75% on cycle 2 which was tolerated very well. Con- versely, on increasing the dose for the c.1905 + 1G > A carrier she developed grade 3 toxicities (PPE, diarrhoea, nausea, neutropaenia) requiring hospitalisation for 10 days and treatment cessation. Descriptive statistics were generated to characterise the study cohort in terms of clinicopathological parameters. Categorical outcomes were presented as a frequency and proportion. The SPSS statistical package version 25 (IBM SPSS Inc., USA).fi Official approval for the use of retrospective data was granted by Guy’s and St Thomas’ Clinical Audit Office. Data were handled in accordance with the Declaration of Hel- sinki and all patients had provided informed consent prior to DPYD testing. Prospective DPYD genotyping At GSTT routine, DPYD genotype screening was imple- mented in December 2014 for all mBC patients who were thought suitable to receive capecitabine. Patients were tested for the following four DPYD genetic variants which are prevalent in the British population and associated with severe capecitabine-associated toxicities: DPYD2A (IVS14 + 1G > A, c.1905 + 1G > A, or rs3918290), c.2846A > T (p.D949V or rs67376798), c.1679T > G (rs55886062, DPYD13, p.I560S) and c.1601G > A (S534N, DPYD4, or rs1801158) [34, 35]. The pharmacogenetic tests were performed in our institutional laboratory and their pro- spective nature was determined by comparing the genotyp- ing date with the date of treatment initiation. 1 3 3 Breast Cancer Research and Treatment (2019) 175:511–517 513 Table 1 Patient demographic and clinical characteristics ECOG Eastern Cooperative Oncology Group Performance Status Characteristic Number (%), N = 66 Sex Male 2 (3%) Female 64 (97%) Mean age, years 58 (28–85) ECOG performance status 0 22 (33%) 1 24 (36%) 2 9 (14%) 3 2 (3%) Not specified 9 (14%) DPYD status Wild type 61 (92%) Heterozygous c.1601G > A 2 Heterozygous c.2846A < T 2 Heterozygous c.1905 + 1G > A 1 Capecitabine received 63 (95%) Single agent 49 (22%) Combination with lapatinib 14 (78%) Table 1 Patient demographic and clinical characteristics Patient population We retrospectively identified metastatic breast cancer patients who were prescribed capecitabine (monotherapy or in combination with lapatinib) between December 2014 and December 2017 at GSTT. Electronic patient records were reviewed, and informa- tion collected on demographic and clinical characteristics including age, gender, ECOG performance status, prescribed treatment regimen, performance of pre-treatment DPYD genotyping test, DPYD genotype outcome, time of treat- ment initiation, nature and grade of capecitabine-associated toxicities, dose modifications, reasons for treatment discon- tinuation and hospitalisation. Toxicities were graded accord- ing to the NCI—CTCAE v4.0. The feasibility of the routine application of DPYD pre-treatment testing in clinical prac- tice was evaluated by assessing the percentage of patients screened for DYPD when a prescription of capecitabine was given. The turnaround time of the test was also evaluated as well as the presence of associated treatment delays. Patients who did not receive capecitabine due to non DPYD-related reasons were not included in the analysis. ECOG Eastern Cooperative Oncology Group Performance Status Results C2 dose increased to 75%: no toxicities F female, ECOG PS Eastern Cooperative Oncology Group Performance Status, C cycle, DR dose reduction Table 3 Capecitabine-related toxicities and dose modifications in DPYD wild-type patients Toxicity ≥ grade 3 Number (%) (N = 61) Overall toxicity 14 (23%) Gastrointestinal toxicities 5 (8%) Palmar-plantar erythrodysesthesia 9 (15%) Haematological 0 (0%) Capecitabine-related hospital admissions 0 (0%) Treatment discontinuation because of capecitabine induced adverse events 2 (3%) Capecitabine related deaths 0 (0%) Dose reduction on treatment outset 25% 6 (10%) 50% 3 (5%) Table 3 Capecitabine-related toxicities and dose modifications in DPYD wild-type patients Our patients were screened at our in-house facility for four DPYD variants with an assay that had a combined predictive value of > 99% and negative predictive value of 80% [34]. Only two of the five patients identified as carri- ers of DPYD polymorphisms received a reduced dose of capecitabine; this was 50% in both patients, but of note was the severe toxicity when the dose was increased by 25% for one of these patients. The consequence of giving full dose capecitabine, without the knowledge of the DPD deficiency, could have resulted in very severe morbidity or even death. The patients who did not receive capecitabine carried c.2846A > T and c.1601G > A polymorphisms, for which a recommendation of 25% dose reduction is advised. The variability seen in the treatment decisions made by the phy- sicians could reflect uncertainty due to the lack of adequate safety data in the literature at that time. Only recently have large studies emerged evaluating the clinical relevance and providing safety outcomes for commonly screened variants [13, 27, 28, 32]. Although the Clinical Pharmacogenetics Implementation Consortium (CPIC) has issued comprehen- sive guidance on dose adjustments, there is a need for more real-world safety data to identify the optimal dosing for each genotype [24, 28]. Results Between December 2014 and December 2017, a total of 72 consecutive mBC patients were considered for capecit- abine as a monotherapy or in combination with lapatinib and tested for DPD deficiency. All patients had a pre- treatment DPYD test (100%). Of these 72 patients, 5 did not receive capecitabine due to poor performance status or disease complications, and 1 patient died 6 days after starting cycle 1 due to variceal bleeding. None of these six patients had a DPYD variant and they were excluded from the analysis. The final analysis was on the remaining 66 patients. Patient characteristics are summarised in Table 1. Five of 66 (8%) were found to be heterozygous variant allele carriers (Table 1) with their pharmacogenomic test results being accompanied by recommendations for dose Of the 61 patients with a wild-type DPYD genotype, 14 (23%) experienced capecitabine-related adverse events (G > 3) such as PPE and gastrointestinal symptoms includ- ing diarrhoea and mucositis (Table 3). In two patients, treatment was stopped, however, none of these patients required hospitalisation. A total of 9 patients (15%) required a dose reduction at the outset of treatment due to comorbidities or poor performance status. There was no death associated with capecitabine treatment in our patient cohort. The test turnaround time for the DPYD genotyping results was 2–3 working days and did not cause delays in treatment initiation. The cost of the test was £57.56 per patient. 1 3 3 Breast Cancer Research and Treatment (2019) 175:511–517 514 Table 2 Treatment outcomes based on pharmacogenomic results for patients carrying DPYD polymorphisms F female, ECOG PS Eastern Cooperative Oncology Group Performance Status, C cycle, DR dose reduction No. Patient summary DPYD variant Treatment outcome 1 56F, ECOG PS1 Heterozygous c.2846A < T Capecitabine not given Alternative treatment 2 42F, ECOG PS 2 Heterozygous c.1601G > A Capecitabine not given Alternative treatment 3 62F, ECOG PS 1 Heterozygous c.1601G > A Capecitabine not given Alternative treatment 4 45F, ECOG PS 0 Heterozygous c.1905 + 1G > A C1 DR 50% with no complications, C2 dose increased to 75%: admit- ted with G3 toxicities (PPE, diarrhoea, nausea, neutropaenia), treat- ment stopped, and patient recovered 5 59F, ECOG PS 0 Heterozygous c.2846A < T C1 DR 50%: no complications. Discussion In this observational study, we evaluated the feasibility and usefulness of routine prospective DPYD genotyping for the prevention of severe toxicities in mBC patients treated with capecitabine. Our results show that the implementation of DPYD screening in clinical practice was feasible and well accepted by clinicians, as every patient who was considered for capecitabine in our institution was successfully screened. The rapid turnaround time and relatively low cost of the test contributed to this, although these factors may vary across different treatment centres globally. This is in keeping with outcomes from large prospective multicentre studies sup- porting the feasibility and cost-effectiveness of prospective DPYD genotyping for patients receiving fluoropyrimidine- based treatment across all tumour types [27, 28]. From the patients found to be wild-type DPYD, only 23% developed grade 3 toxicities which were managed with sup- portive measures or dose reductions but did not result in hos- pitalisation. It has been previously reported that patients who do not carry DPYD variants can still experience severe side effects. This can be due to the sensitivity of the assay, the presence of unrecognised and hence unscreened polymor- phisms, or post-transcriptional modifications and variation in other genes influencing fluoropyrimidine drug metabo- lism [14, 15, 24, 28, 30]. DPD phenotyping testing can pre- dict DPD activity more accurately than DPYD genotyping, however, its cost and lengthy turnaround time make it dif- ficult to implement in clinical practice [36]. Similarly, not all patients carrying deleterious DPYD variants will experi- ence severe toxicity at standard doses, which may result in This evaluation is limited by its relatively small sample size. The low number of DPYD variant carriers precludes a formal evaluation of the effect of DPYD screening on capecitabine-induced toxicities. 1 3 3 Breast Cancer Research and Treatment (2019) 175:511–517 515 their undertreatment. Careful dose titration upon an initial dose reduction has been suggested by CPIC to address this issue [24]. treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study. J Clin Oncol 19(8):2282–2292. https://doi.org/10.1200/jco.2001.19.8.2282 treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study. J Clin Oncol 19(8):2282–2292. https://doi.org/10.1200/jco.2001.19.8.2282 4. p g j 4. Discussion Van Cutsem E, Twelves C, Cassidy J, Allman D, Bajetta E, Boyer M, Bugat R, Findlay M, Frings S, Jahn M, McKendrick J, Oster- walder B, Perez-Manga G, Rosso R, Rougier P, Schmiegel WH, Seitz JF, Thompson P, Vieitez JM, Weitzel C, Harper P (2001) Oral capecitabine compared with intravenous fluorouracil plus leucovorin in patients with metastatic colorectal cancer: results of a large phase III study. J Clin Oncol 19(21):4097–4106. https ://doi.org/10.1200/jco.2001.19.21.4097 Despite the growing amount of evidence favouring the routine implementation of DPYD genotype screening in clinical practice [27, 28, 31], this remains a subject of debate and controversy. Utilising pharmacogenomics to pre- vent avoidable toxicities, and death, is of paramount impor- tance for patient care. This has even greater implications for patients with mBC for whom a plethora of therapeutic options are available. Our study demonstrates that routine implementation of DPYD genotyping in this patient popula- tion is feasible and can guide treatment decisions in a per- sonalised manner. g j 5. Mikhail SE, Sun JF, Marshall JL (2010) Safety of capecitabine: a review. Expert Opin Drug Saf 9(5):831–841. https://doi. org/10.1517/14740338.2010.511610l g 6. Diasio RB, Harris BE (1989) Clinical pharmacology of 5-fluo- rouracil. Clin Pharmacokinet 16(4):215–237. https://doi. org/10.2165/00003088-198916040-00002l 7. Longley DB, Harkin DP, Johnston PG (2003) 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 3(5):330–338. https://doi.org/10.1038/nrc1074 Data availability The datasets during and/or analysed during the cur- rent study are available from the corresponding author on reasonable request. 8. van Kuilenburg AB, Haasjes J, Richel DJ, Zoetekouw L, Van Lenthe H, De Abreu RA, Maring JG, Vreken P, van Gennip AH (2000) Clinical implications of dihydropyrimidine dehydrogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPD gene. Clin Cancer Res 6(12):4705–4712 Conflict of interest The authors declare no conflict of interest. Ethical approval All procedures performed in studies involving human participants were in accordance with the Ethical Standards of the Insti- tutional and/or National Research Committee and with the 1964 Hel- sinki Declaration and its later amendments or comparable ethical stand- ards. 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Approximate survey propagation for statistical inference Fabrizio Antenucci, Florent Krzakala, Pierfrancesco Urbani et al. - This content was downloaded from IP address 128.178.116.185 on 27/12/2022 at 09:27 Journal of Statistical Mechanics: Theory and Experiment Journal of Statistical Mechanics: Theory and Experiment You may also like Engineering non-equilibrium quantum phase transitions via causally gapped Hamiltonians Masoud Mohseni, Johan Strumpfer and Marek M Rams - Local entropy as a measure for sampling solutions in constraint satisfaction problems Carlo Baldassi, Alessandro Ingrosso, Carlo Lucibello et al. - Approximate survey propagation for statistical inference Fabrizio Antenucci, Florent Krzakala, Pierfrancesco Urbani et al. - PAPER • OPEN ACCESS You may also like Engineering non-equilibrium quantum phase transitions via causally gapped Hamiltonians Masoud Mohseni, Johan Strumpfer and Marek M Rams - Local entropy as a measure for sampling solutions in constraint satisfaction problems Carlo Baldassi, Alessandro Ingrosso, Carlo Lucibello et al. - Approximate survey propagation for statistical inference Fabrizio Antenucci, Florent Krzakala, Pierfrancesco Urbani et al. - You may also like Engineering non-equilibrium quantum phase transitions via causally gapped Hamiltonians Masoud Mohseni, Johan Strumpfer and Marek M Rams - Local entropy as a measure for sampling solutions in constraint satisfaction problems Carlo Baldassi, Alessandro Ingrosso, Carlo Lucibello et al. - Approximate survey propagation for statistical inference Fabrizio Antenucci, Florent Krzakala, Pierfrancesco Urbani et al. - You may also like ∗Author to whom any correspondence should be addressed. Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Disordered systems insights on computational hardness J. Stat. Mech. (2022) 11401 This content was downloaded from IP address 128.178.116.185 on 27/12/2022 at 09:27 This content was downloaded from IP address 128.178.116.185 on 27/12/2022 at 09:27 David Gamarnik1, Cristopher Moore2 and Lenka Zdeborov´a3,∗ 1 Operations Research Center and Sloan School of Management, MIT, Cambridge, MA 02139, United States of America 2 Santa Fe Institute, Santa Fe, NM 87501, United States of America 3 SPOC Laboratory, ´Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Route Cantonale, CH-1015 Lausanne, Switzerland E-mail: gamarnik@mit.edu, moore@santafe.edu and lenka.zdeborova@epﬂ.ch 1 Operations Research Center and Sloan School of Management, MIT, Cambridge, MA 02139, United States of America 2 Santa Fe Institute, Santa Fe, NM 87501, United States of America 3 SPOC Laboratory, ´Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Route Cantonale, CH-1015 Lausanne, Switzerland E-mail: gamarnik@mit.edu, moore@santafe.edu and lenka.zdeborova@epﬂ.ch 1 Operations Research Center and Sloan School of Management, MIT, Cambridge, MA 02139, United States of America 2 Santa Fe Institute, Santa Fe, NM 87501, United States of America 3 SPOC Laboratory, ´Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Route Cantonale, CH-1015 Lausanne, Switzerland E-mail: gamarnik@mit.edu, moore@santafe.edu and lenka.zdeborova@epﬂ.ch Received 18 October 2022 Accepted for publication 18 October 2022 Published 24 November 2022 Received 18 October 2022 Accepted for publication 18 October 2022 Published 24 November 2022 Online at stacks.iop.org/JSTAT/2022/114015 https://doi.org/10.1088/1742-5468/ac9cc8 Online at stacks.iop.org/JSTAT/2022/114015 https://doi.org/10.1088/1742-5468/ac9cc8 (2022) 114015 Abstract. In this review article we discuss connections between the physics of disordered systems, phase transitions in inference problems, and computational hardness. We introduce two models representing the behavior of glassy systems, the spiked tensor model and the generalized linear model. We discuss the random (non-planted) versions of these problems as prototypical optimization problems, as well as the planted versions (with a hidden solution) as prototypical problems in statistical inference and learning. Based on ideas from physics, many of these problems have transitions where they are believed to jump from easy (solvable in polynomial time) to hard (requiring exponential time). We discuss several emerging ideas in theoretical computer science and statistics that provide rigor- ous evidence for hardness by proving that large classes of algorithms fail in the conjectured hard regime. This includes the overlap gap property, a particular mathematization of clustering or dynamical symmetry-breaking, which can be used to show that many algorithms that are local or robust to changes in their input fail. We also discuss the sum-of-squares hierarchy, which places bounds 1742-5468/22/114015+41$33.00 © 2022 IOP Publishing Ltd and SISSA Medialab srl Disordered systems insights on computational hardness on proofs or algorithms that use low-degree polynomials such as standard spec- tral methods and semideﬁnite relaxations, including the Sherrington–Kirkpatrick model. Throughout the manuscript we present connections to the physics of disordered systems and associated replica symmetry breaking properties. on proofs or algorithms that use low-degree polynomials such as standard spec- tral methods and semideﬁnite relaxations, including the Sherrington–Kirkpatrick model. Throughout the manuscript we present connections to the physics of disordered systems and associated replica symmetry breaking properties. Keywords: cavity and replica method, message-passing algorithms, statistical inference, typical-case computational complexity https://doi.org/10.1088/1742-5468/ac9cc8 1. Introduction Computational complexity theory [1] aims to answer the question of what problems can be solved by computers. More speciﬁcally, it aims to classify computational problems according to the resources (usually time or memory) needed to solve them, and how these resources scale with the problem size. Computationally hard problems are those that can be solved in principle but require prohibitively large amounts of resources, such as a running time that grows exponentially with the problem size. J. Stat. Mech. (2022) 114 The most iconic result of computational complexity theory is the existence of so- called NP-complete problems [2]. These problems, of which hundreds have been iden- tiﬁed, are all hard unless P = NP, in which case they are all easy. But if P = NP, anything which is easy to check would be easy to ﬁnd. All modern cryptosystems would be breakable; it would be easy to ﬁnd short proofs of unsolved mathematics problems or elegant theories to explain empirical data, without any need for insight or intuition. Even evolution would gain shortcuts: it would be easy to design proteins with cer- tain structures, rather than having to search for them by exploring a vast space of possible amino acid sequences. This would violate many of our deepest beliefs about the nature of search, proof, and even creativity. For these and other reasons, resolv- ing the P ̸= NP conjecture is considered the most important problem of theoretical computer science, and one of the most important open problems in mathematics more generally. at. Mech. (2022) 114015 Since we believe some problems are computationally hard, the question becomes the nature of this hardness. What is it about a problem’s structure that defeats polynomial- time algorithms? Since the late 1980s and early 1990s (e.g. [3–5]), some researchers have looked to the physics of disordered systems as one source of hardness. This comes very naturally since, for many canonical models such as spin glasses, ﬁnding a ground state is easily shown to be NP-hard (i.e. at least as hard as any NP-complete problem). Physical dynamics is itself computationally limited by the locality of interactions, and physics-based algorithms such as Markov chain Monte Carlo and simulated anneal- ing are subject to the same limits. In glassy systems these algorithms often get stuck in metastable states, or take exponential time to cross free energy barriers. Contents J. Stat. Mech. (2022) 114015 Contents 1. Introduction ...................................................................... ...3 2. Two problems in optimization and inference: deﬁnitions...................4 2.1. The spiked tensor model and spin glasses..............................................4 2.2. The generalized linear model and perceptrons........................................6 3. Hardness of optimizing p-spin models: the overlap gap property and implications.....................................................................7 3.1. p-spin model, ground states and algorithms...........................................8 3.2. OGP and its variants ......................................................................8 3.3. e-OGP as an algorithmic barrier to stable algorithms...............................9 3.4. Connections with replica symmetry, symmetry breaking and the clustering (shattering) property........................................................12 4. Statistical and computational trade-oﬀs in inference and learning......14 4.1. The minimum mean-squared error ....................................................14 4.2. AMP and its state evolution............................................................16 4.3. The phase diagrams and the hard phase.............................................17 4.4. Is the hard phase really hard?..........................................................21 4.5. The hard phase is glassy, causing hurdles to gradient-based algorithms.......21 5. Polynomial proofs: the sum-of-squares hierarchy...........................22 5.1. Proofs and refutations ...................................................................23 5.2. From proofs to algorithms: semideﬁnite programming ............................24 5.3. Sum-of-squares lower bounds: enter the charlatan .................................25 5.4. What does sum-of-squares understand?..............................................27 5.5. Relaxation and the Sherrington–Kirkpatrick model ...............................29 5.6. Beyond degree 2...........................................................................31 5.7. Pseudocalibration and clever planted models .......................................33 5.8. Optimal algorithms and the curious case of tensor PCA .........................35 6. Conclusion ........................................................................ .38 Acknowledgments................................................................ 38 References......................................................................... 38 https://doi.org/10.1088/1742-5468/ac9cc8 2 Disordered systems insights on computational hardness Disordered systems insights on computational hardness https://doi.org/10.1088/1742-5468/ac9cc8 2.1. The spiked tensor model and spin glasses One of the models we will consider from the statistics and computational perspective is a natural variant of the spin glass model with a ‘planted signal’ to be learned or reconstructed—physically, a low-energy state built into the landscape. It is called the spiked tensor model or tensor PCA, and is deﬁned as follows. Given a hidden vector u ∈RN, we observe the following tensor: at. Mech. (2022) 114015 Y = λu⊗p + J. (1) Y = λu⊗p + J. (1) Here u⊗p is the p-fold tensor outer product of u, and J is a N × · · · × N tensor describing the noise. We will assume that the entries Ji1,...,ip with 1 ⩽i1 < i2 < . . . < ip ⩽N are drawn i.i.d. from some common distribution with mean zero and variance σ2, such as the normal distribution N (0, 1). The other entries of J are ﬁxed by a symmetry assumption, Ji (1) i ( ) = Ji1 i for all permutations σ of [p] = {1, 2, . . ., p}. Here u⊗p is the p-fold tensor outer product of u, and J is a N × · · · × N tensor describing the noise. We will assume that the entries Ji1,...,ip with 1 ⩽i1 < i2 < . . . < ip ⩽N are drawn i.i.d. from some common distribution with mean zero and variance σ2, such as the normal distribution N (0, 1). The other entries of J are ﬁxed by a symmetry assumption, ( , ) y y y Jiσ(1),...,iσ(p) = Ji1,...,ip for all permutations σ of [p] = {1, 2, . . ., p}. σ(1), , σ(p) , , p We can think of λ as a signal-to-noise ratio, parameterizing how strongly the signal u aﬀects the observation Y compared to the noise J. In order to look for phase transitions in the hardness of reconstructing the planted vector u, we will allow λ to scale in various ways with N. We can also let J’s variance σ2 vary with N, but in most of the paper we will take it to be 1. We can consider variants of this problem where diﬀerent types of restrictions are placed on u. One is to take u ∈SN where SN is the N-dimensional sphere {u : ∥u∥2 = N}. 1. Introduction Unless there is some miraculous algorithmic shortcut for exploring glassy landscapes—which seems unlikely, except for a few isolated cases—it seems likely that no polynomial-time algorithms for these problems exist. In this paper we review some current areas of research on the connections between theory of disordered systems and computational hardness, and attempts to make this physical intuition mathematically rigorous. We will discuss two types of compu- tational problems: optimization problems where one aims to minimize an objective function (such as the energy) over a set of variables, and signal recovery or infer- ence problems where a signal is observed but obscured by noise, and the task is to reconstruct it (at least approximately) from these observations. In section 2 we deﬁne canonical examples of both these problems, stressing their relationship to disordered 3 Disordered systems insights on computational hardness systems studied in physics as well as their broad applicability to modelling various com- putational tasks. In section 3 we discuss recent results on computational hardness of optimization problems based on the overlap gap property, which formalizes the idea that solutions are widely separated from each other by energy barriers. Section 4 switches to signal recovery/inference problems and presents a rather generic picture that emerges from the study of phase transition in those problems. Finally, section 5 discusses the sum-of-squares hierarchy, another approach to proving computational lower bounds. J. Stat. Mech. (2022) 1140 https://doi.org/10.1088/1742-5468/ac9cc8 2.1. The spiked tensor model and spin glasses Another choice is to take Boolean values on the N-dimensional hypercube or equivalently Ising spins, u ∈BN where BN = {±1}N. We can also impose sparsity by demanding that a fraction ρ of u’s entries are nonzero, writing u ∈BN,ρ where BN,ρ = {u ∈{±1, 0}N : ∥u∥1 = Nρ}. In terms of Bayesian inference, we take the uniform measure on each of these sets to be a prior on u. The variant p = 2, i.e. the spiked matrix model, is particularly widely studied. It is also known as the spiked covariance model, or as low-rank matrix estimation, since u ⊗u is a rank-1 approximation of Y [6, 7]. The general questions to be addressed in this model are (a) can we learn, or recon- struct, the planted vector u from the observation Y? and (b) can we do this with an eﬃcient algorithm, i.e. one whose running time is polynomial in N? (We assume p is a constant, so polynomial in N is equivalent to polynomial in the size Np of the observed data.) Since reconstructing u exactly is often impossible, we are interested in 4 4 Disordered systems insights on computational hardness approximate reconstruction, i.e. producing an algorithmic estimate ˆu = ˆu(Y ) which has a nontrivial correlation with the ground truth u: for instance, by having an overlap (1/N)⟨ˆu, u⟩bounded above zero with high probability. approximate reconstruction, i.e. producing an algorithmic estimate ˆu = ˆu(Y ) which has a nontrivial correlation with the ground truth u: for instance, by having an overlap (1/N)⟨ˆu, u⟩bounded above zero with high probability. Question (a) is an information-theoretic or statistical question, unconcerned with computational resources. Using the theory of Bayesian inference we can write the posterior distribution, P(z\|Y ) = 1 Z P(z) P(Y \|z) where (2) P(Y \|z) = 1⩽i1<i2<...<ip⩽N N (Yi1,...,ip −λzi1zi2 . . . zip, 1), (3) (2) J. Stat. Mech. (2022) 1140 (3) where for concreteness we considered the elements of the noise J to be Gaussian with variance 1. (Due to universality properties, e.g. [7], this is not very restrictive for what follows.) Note that the partition function or normalization factor Z depends both on the observed tensor Y, the prior P(z), and the parameters λ, σ of the likelihood P(Y\|z). In our notation we drop this explicit dependence. The posterior distribution P(z\|Y) is an exponentially complicated object. https://doi.org/10.1088/1742-5468/ac9cc8 2.1. The spiked tensor model and spin glasses However, for several natural loss functions including the overlap ⟨ˆu, u⟩and the ℓ2 error ∥ˆu −u∥2, the best possible estimator ˆu depends only on the marginals P(zi\|Y). Thus question (b) boils down to whether, given Y, we can approximate these marginals with a polynomial- time algorithm. Mech. (2022) 114015 Another common approach in statistics is the maximum likelihood estimator4 (MLE) where we set ˆu to the z that maximizes P(Y\|z). In the Gaussian case (3), we have P(Y \|z) ∝exp ⎡ ⎣−1 2 1⩽i1<i2<...<ip⩽N Yi1,...,ip −λzi1zi2 . . . zip 2 ⎤ ⎦ = exp −1 p! 1 2∥Y ∥2 + λ2 2 ∥z∥2p −2⟨Y , z⊗p⟩ , (4) (4) where in the limit of large N we ignore terms with repeated indices, and where ⟨Y , z⊗p⟩= 1⩽i1<i2<...<ip⩽N Yi1,...,ipzi1zi2 . . . zip. (5) (5) Since ∥Y∥2 is ﬁxed by the observed data, and since ∥z∥2 = N if z ∈SN or BN (or ρN if it is in BN,ρ) then the MLE is the z that maximizes (5). But this is exactly the ground state of a p-spin model with coupling tensor Y, with spherical or Ising spins if z is in SN or BN respectively. In particular, if λ = 0 so that Y = J, we have a p-spin model with Gaussian random couplings and Hamiltonian E(z) = − 1⩽i1<i2<...<ip⩽N Ji1,...,ipzi1zi2 . . . zip. (6) (6) 4 It should be noted that while the MLE and similar extremization-based approaches are very popular in statistics, they are typically suboptimal in high-dimensional settings: that is, they do not optimize the overlap or minimize the ℓ2 error. 4 It should be noted that while the MLE and similar extremization-based approaches are very popular in statistics, they are typically suboptimal in high-dimensional settings: that is, they do not optimize the overlap or minimize the ℓ2 error. https://doi.org/10.1088/1742-5468/ac9cc8 5 Disordered systems insights on computational hardness Studying the optimization landscape of this un-planted problem may seem irrelevant to the inference problem of reconstructing u from Y. But in addition to being physically natural, as a generalization of the Sherrington–Kirkpatrick model [8] which corresponds to the case p = 2 and z ∈BN, it serves both as a starting point for the inference problem and as a null model where there is no signal at all. 2.2. The generalized linear model and perceptrons Another class of problems we will consider in this paper is the generalized linear model (GLM). Again, a planted vector u ∈RN is observed through a set of noisy observations, but this time through approximate linear combinations Y1, . . ., YP: . (2022) 114015 Yi ∼Pout Yi \| N a=1 Jiaua . (7) Yi ∼Pout Yi \| N a=1 Jiaua . (7) Here J ∈RP×N is a known matrix whose entries are i.i.d. with zero mean and variance σ2, and Pout is some noisy channel. In other words, f(j) = ⟨j, u⟩is an unknown linear function from RN to R, and our goal is to learn this function—that is, to reconstruct u—from noisy observations of its values f(j1), . . ., f(jP) at P random vectors where ji is the ith row of J. In machine learning we would say that the set of tuples (ji, Yi) are the training data, and by learning u we can generalize to f(j) for new values of j. Here J ∈RP×N is a known matrix whose entries are i.i.d. with zero mean and variance σ2, and Pout is some noisy channel. In other words, f(j) = ⟨j, u⟩is an unknown linear function from RN to R, and our goal is to learn this function—that is, to reconstruct u—from noisy observations of its values f(j1), . . ., f(jP) at P random vectors where ji is the ith row of J. In machine learning we would say that the set of tuples (ji, Yi) are the training data, and by learning u we can generalize to f(j) for new values of j. The main questions for the GLM are the same as for the spiked tensor model: (a) whether it is information-theoretically possible to learn the signal u given J and Y, and (b) whether there are eﬃcient algorithms that do that. Again Bayesian inference aims at computing the marginals of a posterior P(z\|Y , J) = 1 Z P(z) P i=1 Pout Yi \| N a=1 Jiaua . (8) (8) Here the partition function Z depends implicitly on the matrices Y and J as well as on the parameters of the probability Pout and of the prior P(z). 2.1. The spiked tensor model and spin glasses Thus in addition to the reconstruction problem where we assume that Y is drawn from the planted model (1) and we want to learn u, we will also consider the detection problem. That is, given Y, we want to determine whether it is drawn from the planted model, or the un-planted model where Y = J. Like reconstruction, this hypothesis testing problem may or may not be information-theoretically possible. If it is, it may or may not have a polynomial-time algorithm that succeeds with high probability. J. Stat. Mech. (2022) 114 In the literature there are many variants of the spiked tensor model. The signal can be of higher rank, i.e. j u⊗p j for multiple planted vectors uj, or one can plant a subspace rather than a vector. In addition to being non-Gaussian, the noise can be nonadditive, binary or sparse. And the observation could consist of multiple tensors with diﬀerent p rather than a single Y. All these variants have their own interest and applications; see examples in e.g. [7, 9]. In what follows we will also sometimes refer to sparse versions of the spiked matrix model, such as the stochastic block model which is popular in network science as a model of community structure (see e.g. [10]). Mech. (2022) 114015 https://doi.org/10.1088/1742-5468/ac9cc8 2.2. The generalized linear model and perceptrons As in tensor PCA, u can Here the partition function Z depends implicitly on the matrices Y and J as well as on the parameters of the probability Pout and of the prior P(z). As in tensor PCA, u can https://doi.org/10.1088/1742-5468/ac9cc8 6 6 Disordered systems insights on computational hardness be restricted to SN, BN or some other set, and we will assume that its Bayesian prior is uniform over this set. be restricted to SN, BN or some other set, and we will assume that its Bayesian prior is uniform over this set. Another family of estimators minimize some loss function ℓ, perhaps with a regularization term with strength λ: L(z) = P i=1 ℓ Yi, N a=1 Jiaza + λ N a=1 r(za). (9) (9) In a linear regression context, J is the observed data and Y the observed dependent variable, and (9) seeks to minimize the empirical risk ℓ. A typical regularization term might be r(za) = \|za\|, giving the ‘lasso’ or L1 regularization λ∥z∥1 which pushes z towards sparse vectors. J. Stat. Mech. (2022) 11401 J. Stat. Mech. (2022) The GLM captures many versions of high-dimensional linear regression, and covers a broad range of applications and situations. In signal processing or imaging u would be the N-dimensional signal/image to be reconstructed from measurements Y, where J is the measurement matrix and the channel Pout typically consists of additive Gaussian noise. In compressed sensing we consider the under-determined case N > P, but with a sparse prior on the signal u. at. Mech. (2022) 114015 Just as for the spiked tensor model the signal u can be seen as a planted solution to recover from Y and J. The version of the model where the distribution of Y is independent of u is well known in the statistical physics literature as the perceptron. The variant with z ∈SN is the spherical perceptron [11], and z ∈BN gives the binary perceptron [11, 12]. The perceptron model is particularly important as its study started the line of work applying physics of disordered systems to understanding supervised learning in artiﬁcial neural networks. The recent major success of methods based on deep learning [13] only added importance and urgency to this endeavour. ch. (2022) 114015 https://doi.org/10.1088/1742-5468/ac9cc8 3. Hardness of optimizing p-spin models: the overlap gap property and implications In this section we discuss the algorithmic hardness of the problem (6) of ﬁnding near ground states of p-spin models using the overlap gap property (OGP). The OGP is a property of solution space geometry which roughly speaking says that near opti- mal solutions should be either close or far from each other. It is intimately related to the replica symmetry breaking (RSB) property and the clustering (also some- times called shattering) property exhibited by some constraint satisfaction problems. In fact it emerged directly as way to establish the presence of the shattering property in constraint satisfaction problems [14, 15]. There are important distinctions, how- ever, between RSB, clustering and OGP, which we will discuss as well. A survey of OGP-based methods is in [16]. Our main focus is to illustrate how OGP presents a barrier to a certain class of algorithms as potential contenders for ﬁnding near ground states. Loosely speaking, it is the class of algorithms exhibiting input stability (noise insensitivity), thus revealing deep and intriguing connections with a rich ﬁeld of Fourier analysis of Boolean functions [17]. Many important algorithms are special https://doi.org/10.1088/1742-5468/ac9cc8 7 Disordered systems insights on computational hardness cases of this class, including approximate message passing (AMP) [18], low-degree polynomials [19, 20], Langevin dynamics [19], and low-depth Boolean circuits [21]. OGP was also established to be a barrier for certain types of quantum algorithms, speciﬁcally quantum approximate optimization algorithms (QAOA) [22–24], using a slightly diﬀerent implementation of the stability argument. We will therefore conclude that the values produced by these algorithms are bounded away from optimality. We will discuss various extensions of the OGP, including the multi-overlap gap property (m-OGP), which will allow us to bring the algorithmic barriers to the known algo- rithmic thresholds. In the case of the p-spin models these thresholds are achieved by AMP. It is entirely possible that models in the OGP regime do not admit any poly- nomial time algorithms, which at this stage is evidenced by just the lack of those. Proving this say modulo P ̸= NP assumption does not yet appear to be within the reach of the known techniques. J. Stat. Mech. (2022) 1140 3.1. p-spin model, ground states and algorithms at. Mech. (2022) 114015 We recall that our focus is the optimization problem (6). The optimization is over choice of z in some space ΘN which for the purposes of this section is either SN or BN. The former is referred to as spherical p-spin model and the latter is called the Ising p-spin model. We assume that the variance σ2 N of the i.i.d. entries of the tensor J is N−(p+1). A series of groundbreaking works by Parisi [25, 26], followed by Guerra-Toninelli [27], Talagrand [28], and Panchenko [29–31] led to proof of the existence and a method for computing a deterministic limit of (6) in probability as N →∞. We denote this limit by ηp,OPT in either case, where the choice of ΘN will be clear from the context. The value of this limit arises as a solution of a certain variational problem over the space of one-dimensional probability measures. The measure which provides the solution to this variational problem is called the Parisi measure which we denote by μ. The algorithmic goal under consideration is the goal of constructing a solution z ∈ΘN which achieves near optimality, namely the value close to ηp,OPT when the tensor J is given as an input. Ideally, we want an algorithm A which for every constant ϵ > 0 produces a solution ˆz ≜A(J) satisfying ⟨J, ˆz⊗p⟩⩾(1 −ϵ)ηOPT in polynomial (in N) time. This was achieved in a series of important recent developments [32–34], when the associated Parisi measure μ is strictly increasing. This monotonicity property is related to the OGP as we will discuss below. 3.2. OGP and its variants The following result states the presence of the OGP for the p-spin models. Theorem 1. For every even p ⩾4, ΘN = BN or ΘN = SN, there exists ηp,OGP < ηp,OPT, 0 < ν1 < ν2 < 1 and c > 0 such that with probability at least 1 −exp(−cN) for large enough N the following holds. For every z1, z2 ∈ΘN satisfying ⟨J, z⊗p j ⟩⩾ηp,OGP, j = 1, 2 Theorem 1. For every even p ⩾4, ΘN = BN or ΘN = SN, there exists ηp,OGP < ηp,OPT, 0 < ν1 < ν2 < 1 and c > 0 such that with probability at least 1 −exp(−cN) for large enough N the following holds. For every z1, z2 ∈ΘN satisfying ⟨J, z⊗p j ⟩⩾ηp,OGP, j = 1, 2 1 N \|⟨z1, z2⟩\| /∈(ν1, ν2). https://doi.org/10.1088/1742-5468/ac9cc8 https://doi.org/10.1088/1742-5468/ac9cc8 8 8 Disordered systems insights on computational hardness Here ⟨x, y⟩denotes the inner product 1⩽i⩽N xiyi. Namely, modulo an exponentially in N unlikely event, the normalized angle (overlap) between any two solutions with value at least ηp,OGP cannot fall into the interval (ν1, ν2). The model exhibits an overlap gap. Here ⟨x, y⟩denotes the inner product 1⩽i⩽N xiyi. Namely, modulo an exponentially in N unlikely event, the normalized angle (overlap) between any two solutions with value at least ηp,OGP cannot fall into the interval (ν1, ν2). The model exhibits an overlap gap. p,OGP ( ) The values ηp,OGP and νj (and in fact the optimal values ηp,OPT themselves) are in general diﬀerent for Ising and spherical models and their precise values are of no algorithmic signiﬁcance. While the result is only known to hold for even p ⩾4, it is expected to hold for all p ⩾3. It is conjectured not to hold when p = 2 [26] for the Ising case and the AMP algorithm achieving the near ground state value in this case is eﬀective modulo this conjecture [33]. It does not hold when p = 2 for the spherical case for a trivial reason as in this case the problem corresponds to optimizing a quadratic form over sphere SN. The proof of this theorem 1 for the Ising case can be found in [35], and is obtained by a detailed analysis of the variational problem associated with pairs of solutions z1, z2 within a certain proximity to optimality. Furthermore, when t1 = 0, t2 = 1, it holds 1 N \|⟨z1, z2⟩\| ∈[0, ν1]. Furthermore, when t1 = 0, t2 = 1, it holds 1 N \|⟨z1, z2⟩\| ∈[0, ν1]. The probability event above is with respect to the joint randomness of J and ˜J. Theorem 2 says that the OGP holds for pairs of solutions with values above ηp,OGP across the entire interpolated sequence of instances J(t). Furthermore, at the extremes, that is for the pair of instances J and ˜J, these solutions must have overlap at most ν1. We note that the overlap value 1 is trivially achievable when t1 = t2 by taking two identical solutions z1 = z2 with value at least ηp,OGP. The proof for the Ising case can be found in [18], and for the spherical case in [19], and it is a rather straightforward extension of theorem 1 by appealing to the chaos property exhibited by many glassy models [37, 38]. https://doi.org/10.1088/1742-5468/ac9cc8 3.2. OGP and its variants The proof for the spherical case can be found in [36]. J. Stat. Mech. (2022) 1140 In order to use this result as an algorithmic barrier, we need to extend this theorem to the following ensemble variant of the OGP which we dub e-OGP. For this purpose it will be convenient to assume that the distribution of the entries of J is Gaussian. Consider an independent pair of tensors J, ˜J ∈RN⊗p with Gaussian entries. Introduce the following natural interpolation between the two: J(t) = √1 −tJ + √ t˜J, t ∈[0, 1]. The distribution of J(t) is then identical to one of J and ˜J for every t. Theorem 2. For every even p ⩾4, ΘN = BN or ΘN = SN, for the same choice of parameters ηp,OGP, ν1, ν2 as in theorem 1 the following holds with probability at least 1 −exp(−cN) for some c and large enough N. For every t1, t2 ∈[0, 1] and every z1, z2 ∈ ΘN satisfying ⟨J(tj), z⊗p j ⟩⩾ηp,OGP, j = 1, 2 we have . (2022) 114015 1 N \|⟨z1, z2⟩\| /∈(ν1, ν2). 3.3. e-OGP as an algorithmic barrier to stable algorithms We now discuss how the presence of the e-OGP presents an algorithmic barrier to a class of algorithms we loosely deﬁne as stable (noise-insensitive) algorithms. This part will be discussed rather informally, as each concrete instantiation of the arguments is model and algorithm dependent. We think of algorithms as mappings of the form A(J) →ΘN which 9 9 Disordered systems insights on computational hardness map instances (tensors) J into a solution z = A(J) in the solution space ΘN. In some cases the algorithms can take advantage of an additional randomization with functions now taking the form A(J, ω), where ω is a sample corresponding to the randomization seed. For simplicity, we stick with non-randomized versions A : RN⊗p →ΘN. Informally, we say that the algorithm A is stable (noise-insensitive), if a small change in J results in a small change in the output. Namely, ∥A(J1) −A(J2)∥is likely to be small with respect to the natural metric on ΘN when ∥J1 −J2∥2 is small. The choice of metric on ΘN is driven by the space itself and can be Hamming distance when ΘN = BN or L2 norm when it is SN. The ‘likely’ is in reference to the randomness of the tensor J. The following theorem stated informally shows why the presence of the e-OGP presents a barrier to stable algorithms. J. Stat. Mech. (2022) 1140 Theorem 3 (Informal). For every stable algorithm A and every ϵ > 0, ⟨J, (A(J))⊗p⟩⩽ ηp, OGP + ϵ w.h.p. as N →∞. Namely, this theorem states that stable algorithm cannot overcome the OGP barrier. t. Mech. (2022) 114015 Proof sketch: we provide an outline of a simple proof of this theorem. The stability of the algorithm can sometimes be used to establish the concentration of its value around expectation, namely that ⟨J, (A(J))⊗p⟩≈E⟨J, (A(J))⊗p⟩as N →∞. This is not the case universally, but for simplicity let us assume this for now. Then it suﬃces to establish the claim E⟨J, (A(J))⊗p⟩⩽ηp, OGP + ϵ. Suppose not. Then we have E⟨J, (A(J))⊗p⟩⩾ ηp, OGP + ϵ implying E⟨J(t), A(J(t))⟩⩾ηp, OGP + ϵ for every t in the interpolation path. We will obtain a contradiction. By the second part of theorem 2 we then must have w.h.p. and in expectation 1 N \|⟨A(J(0)), A(J(1))⟩\| ⩽ν1, namely namely 1 N ∥A(J(0)) −A(J(1))∥2 ⩾ √ 2 −2ν1. https://doi.org/10.1088/1742-5468/ac9cc8 3.3. e-OGP as an algorithmic barrier to stable algorithms Here we assume that we use L2 for ΘN and the norm of every solution produced by the algorithm is √ N (which is the case when say ΘN = BN). On the other hand trivially 1 N \|⟨A(J(0)), A(J(0))⟩\| = 1 > ν2, implying 1 N ∥A(J(0)) −A(J(1))∥2 = 0 ⩽ √ 2 −2ν2. Stability of the algorithm A implies then the existence of time τ such that Stability of the algorithm A implies then the existence of time τ such that 1 N \|⟨A(J(0)), A(J(τ))⟩\| ∈(ν1, ν2), □ which is a contradiction to the ﬁrst part of theorem 2 (ﬁgure 1). □ which is a contradiction to the ﬁrst part of theorem 2 (ﬁgure 1). which is a contradiction to the ﬁrst part of theorem 2 (ﬁgure 1). The proof above is just an outline of the main ideas that have diﬀerent speciﬁc implementations for speciﬁc problems. The earliest application of this idea was in [39], in a diﬀerent context of ﬁnding large independent sets in sparse random graphs. The method was used to show that local algorithms, appropriately deﬁned, are stable, where 10 Disordered systems insights on computational hardness Figure 1. The smaller circle represents ηp,OGP-optimal solutions at distance ⩽√2 −2ν2 from A(J(0)). The complement to the larger circle represents ηp,OGP- optimal solutions at distance ⩾√2 −2ν2 from A(J(0)). As distance between the cir- cle boundaries is √2 −2ν1 −√2 −2ν2 ≜κ, at some instance t the distance between ‘successive’ solutions A(J(t)) and A(J(t + δt)) has to be at least κ, contradicting stability. Disordered systems insights on computational hardness J. Stat. Mech. (2022) 1140 at. Mech. (2022) 114015 Figure 1. The smaller circle represents ηp,OGP-optimal solutions at distance ⩽√2 −2ν2 from A(J(0)). The complement to the larger circle represents ηp,OGP- optimal solutions at distance ⩾√2 −2ν2 from A(J(0)). As distance between the cir- cle boundaries is √2 −2ν1 −√2 −2ν2 ≜κ, at some instance t the distance between ‘successive’ solutions A(J(t)) and A(J(t + δt)) has to be at least κ, contradicting stability. J denotes random graph connectivities. In the context of spin glasses, it was shown in [18] that the AMP algorithm is stable and thus cannot overcome ηp,OGP barrier. This was generalized in [19] where algorithms based on low-degree polynomials were shown to be stable. https://doi.org/10.1088/1742-5468/ac9cc8 3.3. e-OGP as an algorithmic barrier to stable algorithms In the same paper Langevin dynamics was shown to be stable for spherical spin models when the running time is linear in N. Extending the limitation of the Langevin dynamics beyond linear bound is an interesting open problem. A natural conjecture is that the Langevin dynamics produces a value at most ηp,OGP when run for NO(1) time. p, By leveraging the multi-e-OGP method, which involves studying overlap patterns of more than two solutions, the barrier ηp,OGP and its analogues for other models can be pushed to the value achievable by the state of the art algorithms. These algorithms are AMP in the p-spin Ising case [34] and the spherical p-spin model case [32], simple greedy algorithms for the case of random K-SAT problem and the case of independent sets in sparse random graphs. The implementation of the multi-e-OGP for spin glass models was done by Huang and Sellke [40], who have implemented a very ingenious version of the multi-OGP, called branching-OGP. This version was motivated by the ultrametric structure of the solution space of p-spin models, widely conjectured to hold. The implementation for the random K-SAT was done by Bresler and Huang [41], and for independent sets in sparse random graphs by Wein in [20]. [ ] Arguably the strongest implication of the OGP as an algorithmic barrier is its usage for establishing the state of the art lower bounds on depth of polynomial size Boolean 11 Disordered systems insights on computational hardness circuits. There is a long history in the theoretical computer science literature on estab- lishing such lower bounds for various problems. In the context of constraint satisfaction problems, the prior state of the art result was achieved by Rossman [42, 43] (see also extensions in [44, 45]), who established a depth lower bound Θ(log n/(κn log log n)) for poly-size circuits deciding the presence of an independent set of size kn in graphs with n nodes. When the depth of the circuit is bounded by an n-independent constant, he showed that the size of the circuit has to be at least nΩ(logn). This was done in the regime of random graphs where the typical value of kn grows at most logarithmically in n. Using the OGP method this bound was improved to Θ(log n/log log n), though for the search as opposed to the decision problem [21]. 3.3. e-OGP as an algorithmic barrier to stable algorithms Similarly, when the depth of the circuit is at most a constant, a stretched exponential lower bound exp(nΩ(1)) on the size was established as well. It is in the context of this problem where the concentration around expectation adopted in the proof sketch does not hold, and furthermore, the stabil- ity property does not hold w.h.p. Instead the idea was to establish that circuits with small depth have stability property with at least sub-exponentially small probability. On the other hand, the stability can occur only for the event which is complementary to the OGP, and this complement event holds with exponentially small probability, thus leading to a contradiction. J. Stat. Mech. (2022) 1140 at. Mech. (2022) 114015 A similar application of the OGP based method shows that poly-size circuits producing solutions larger than ηp,OGP in p-spin models also have depth at least Θ(log n/log log n). Pushing this result towards the value algorithmically achievable by the AMP, say using the Huang and Sellke [40] is not immediate due to the overlap Lip- schitz concentration assumption required in [40]. This extension is an interesting open problem. p Broadly speaking a big outstanding challenge is the applicability of OGP or similar methods for models with a planted signal, which we discuss in the following sections. While a version of OGP takes place in many such models, its algorithmic implication is far narrower than in the settings discussed above, such as p-spin models and random constraint satisfaction problems. This presents an interesting and rather non-trivial challenge for future. https://doi.org/10.1088/1742-5468/ac9cc8 3.4. Connections with replica symmetry, symmetry breaking and the clustering (shattering) property For example, for the random K-SAT problem the onset of the clustering property is known to take place close to the threshold (2K/K)log K for the clauses to variables densities, when K is large, but only in the weak clustering sense discussed above: most but not necessarily all of the solutions can be split into clusters [46]. J. Stat. Mech. (2022) 114 Stat. Mech. (2022) 114015 [ ] As it turns out, these exceptions are not just a minor nuisance, and can have profound algorithmic implications. The so-called symmetric perceptron model is a good demonstration of this [47–51]. For this model, the weak clustering property is known to take place at all constraints to variables densities, yet polynomial time algo- rithms exist at some strictly positive density values [49]. The multi-OGP analysis conducted in [50] reveals that the gaps in the overlaps occur at densities higher than the known algorithmic thresholds and thus the thresholds for the weak clustering property and the OGP do not coincide and, furthermore, the weak clustering property is appar- ently not a signature of an algorithmic hardness. Whether the strong clustering property can be used as a ‘direct’ evidence of algorithmic hardness remains to be seen. For the further discussion of the connection between the OGP, the weak and strong clustering properties, and the algorithmic ramiﬁcations, we refer the reader to [16]. Mech. (2022) 114015 Next we discuss the connection between the OGP, replica symmetry, symmetry breaking and the Parisi measure μ. The Parisi measure μ arises in studying the Gibbs measure associated with Hamiltonian H. (Very) roughly speaking, it describes an over- lap structure of two nearly optimal solutions σ and τ chosen uniformly at random. This can be formalized by introducing a small positive temperature parameter in the Gibbs distribution, but we skip this formalism. The idea is that (1/N)\|⟨σ, τ⟩\| has the cumulative distribution function (CDF) described by μ in the large N limit. The support of μ is naturally some subset of [0, 1]. The source of randomness is dual here, one arising from the randomness of the Hamiltonians, and one arising from the sampling procedure. Whether μ is indeed the limit the CDF of the overlaps in the limit remains a conjecture, which has been conﬁrmed only for the spherical case. 3.4. Connections with replica symmetry, symmetry breaking and the clustering (shattering) property We discuss these connections rather informally now, leaving the technical aspects to other sources which we reference here. The OGP arose in connection with studying the replica symmetry, RSB and related properties of spin glasses and their variants. Speciﬁcally, it arose as a method of proving that the set of satisfying solutions of a random constraint satisfaction problem is clus- tered (sometimes called shattered), meaning that it can be partitioned into ‘connected’ components with order Θ(N) distance between them. How can one establish the exis- tence of such a clustering picture? If the model exhibits the OGP say with parame- ters ν1 < ν2, then clustering follows immediately, provided that solutions at distances √2 −2ν1 or larger exist, as in this case one deﬁnes clusters as the set of solutions which can be reached from each other by paths in the underlying Hamming cube. The fact that https://doi.org/10.1088/1742-5468/ac9cc8 12 Disordered systems insights on computational hardness distances between √2 −2ν2 and √2 −2ν1 do not exist between the pairs of solutions imply that at least two (but in fact many) clusters exist. distances between √2 −2ν2 and √2 −2ν1 do not exist between the pairs of solutions imply that at least two (but in fact many) clusters exist. There are several caveats associated with this connection between the OGP and the clustering property. First this connection is one directional, in the sense that the presence of clustering does not necessarily imply the OGP, for a very simple reason: the diameter of the cluster can in principle be larger than the distances between the clusters. In this case, while the clustering property takes place, the set of all normalized pairwise distances could potentially span the entire interval [0, 1] without any gaps. Therefore the path towards establishing algorithmic lower bounds is not entirely clear. Second, as it turns out in some models and in some regimes, the clustering pic- ture has been established for the ‘majority’ of the solution space, and not for the entire solution space. We will call it the weak clustering property, to contrast with the strong clustering property, which refers to a clustering property without exceptions. https://doi.org/10.1088/1742-5468/ac9cc8 3.4. Connections with replica symmetry, symmetry breaking and the clustering (shattering) property Loosely speaking the model is deﬁned to be in the replica symmetric regime (RS) if μ is just a δ mass at zero. Namely, the overlap (1/N)⟨σ, τ⟩is approximately zero with high probability, implying that typical pairs of solutions are nearly orthogonal to each other. Replica symmetry breaking (RSB) then refers to μ being distinct from this single- ton structure. Now if the model exhibits OGP, then a part of μ is ﬂat: the CDF of 13 Disordered systems insights on computational hardness the overlaps is constant on (ν1, ν2). Namely, the CDF is not strictly increasing. The absence of this ﬂat part of μ is exactly what was used in constructions of near opti- mal solutions in [32–34], (and the presence of the OGP is an algorithmic obstruction as we have discussed). So presumably, we could have used the ﬂatness of the Parisi measure as a ‘certiﬁcate’ of hardness. However, there are challenges associated with this alternative. First, as we have discussed, whether μ indeed describes the distri- bution of overlaps remains an open question, whereas the presence of the OGP has been conﬁrmed. More importantly though, even modulo the μ being the accurate descriptor of the overlaps, the connection between OGP and the ﬂatness of μ is one- directional. The ﬂatness of μ in some intervals (ν1, ν2) means only that the density of the overlaps falling into this interval is asymptotically zero after taking N to inﬁnity. It does not imply the absence of such overlaps. This is similar to the distinction between the weak and strong clustering property: most of the overlaps are outside of the ﬂat parts, but exceptions might exist. The presence of such exceptions is bad news for the eﬀorts of establishing algorithmic lower bounds. Not only the argument for proving the algorithmic lower bounds appears to break down, but also the presence of exceptions, namely a small number of overlaps falling into this interval, might be potentially a game changer, as we saw in the case of the symmetric perceptron model. J. Stat. Mech. (2022) 114 4. Statistical and computational trade-oﬀs in inference and learning In this section we move from optimization problems to statistical inference, in other words from the non-planted problems to the planted ones. We recall our working exam- ples deﬁned in section 2, that cover a large range of settings and applications, the spiked tensor model and the GLM. (2022) 114015 In order to describe the conjectured results on the algorithmic hardness of the planted problems we will ﬁrst discuss the Bayes-optimal inference of the planted conﬁguration from observations. We will then show how to analyze the performance of the Bayes- optimal inference in the large size limit N →∞and under the stated randomness of the generative model. We will then show that phase transitions in the capability of the Bayes-optimal estimator to reconstruct the signal have an intriguing algorithmic role as a suitable type of message passing algorithms are able to reach optimal perfor- mance for all parameters except in the metastable region of ﬁrst order phase transitions. This metastable region is then conjectured to be algorithmically hard—the hard phase. Section 5 will then present the currently strongest known method for showing evidence of such hardness in some cases. 4.1. The minimum mean-squared error In both the spiked tensor model and the GLM as deﬁned in section 2 the optimal inference of the planted signal u can be achieved by computing the marginals of the https://doi.org/10.1088/1742-5468/ac9cc8 14 Disordered systems insights on computational hardness posterior probability distribution posterior probability distribution posterior probability distribution posterior probability distribution P(z\|Y ) = 1 Z P(z)P(Y \|z). (10) P(z\|Y ) = 1 Z P(z)P(Y \|z). P(z\|Y ) = 1 Z P(z)P(Y \|z). (10) (10) Concretely, when aiming to ﬁnd an estimator ˆz that would minimize the mean-squared error to the signal u MSE(ˆz) = 1 N N i=1 (ui −ˆzi)2 (11) (11) J. Stat. Mech. (2022) 1140 we conclude that from all the possible estimators we should take ˆz to be the marginal of the posterior we conclude that from all the possible estimators we should take ˆz to be the marginal of the posterior ˆzi = EP(z\|Y )(zi). (12) ˆzi = EP(z\|Y )(zi). (12) We will call the MSE achieved by this estimator the minimum-MSE, abbreviated MMSE. In the large size limit N →∞computing marginals over P(z\|Y) with z ∈RN is in general exponentially costly in N, and thus potentially computationally hard even in the speciﬁc probabilistic generative models from section 2. Mech. (2022) 114015 However, for the spiked tensor model as well as for the GLM tools from the theory of spin glasses come to the rescue and allow us to analyze the value of the MMSE in the larger size limit as well as design message passing algorithms with properties closely related to the approach to obtain the MMSE. Let us start by describing the form in which we obtain the asymptotic value of the MMSE. Replica theory allows us to derive an explicit formula for a function ΦRS(m), m ∈R, called the replica symmetric free entropy such that (2022) 114015 lim N→∞EY ,u,J log Z = max m ΦRS(m). (13) (13) We note that in physics it is more common to deﬁne the free energy which is just the negative of the free entropy. The average over Y, u, J applies to the GLM. In the spiked matrix model the Y can be dropped as in the deﬁnition we gave it explicitly depends on u and J. The function ΦRS(m) explicitly depends on the parameters of the prior, the likelihood and the ratio α = N/P, but in our notation we omit this dependence. https://doi.org/10.1088/1742-5468/ac9cc8 4.1. The minimum mean-squared error We then call m∗= argmax ΦRS(m) (14) m∗= argmax ΦRS(m) (14) and state a generic result for the MMSE that is given by the global maximizer of the replica symmetric free entropy and state a generic result for the MMSE that is given by the global maximizer of the replica symmetric free entropy lim N→∞MMSE = ρ −m∗ (15) lim N→∞MMSE = ρ −m∗ (15) where the constant ρ = E(u2 i) is simply the second moment of the signal components. where the constant ρ = E(u2 i) is simply the second moment of the signal components. ρ ( i) p y g p The derivations of these result and the explicit formulas for ΦRS(m) were given in the spin glass literature for many special cases and mostly without a rigorous justiﬁcation. In the general form considered in this paper and including rigorous proofs they were given for the spiked tensor model in [52], and for the GLM in [53]. For the purpose of https://doi.org/10.1088/1742-5468/ac9cc8 15 15 Disordered systems insights on computational hardness this paper we will stay on the abstract level expressed above because on this level the discussion applies to a broad range of settings and we do not want to obfuscate it with setting-dependent details. An important comment needs to be made here about the very generic validity of the replica symmetric result for the free entropy in the Bayes-optimal setting, i.e. when the prior and likelihood match the corresponding distributions in the model that generated the data. By the very nature of the Bayes’ formula the signal u has prop- erties interchangeable with properties of a random sample from the posterior P(z\|Y). This is true even at ﬁnite size N and even for models where J is not random and where the likelihood and the prior are not separable. A consequence of the inter- changeability is that under the averages over the posterior measure and the signal u we can replace the signal u for a random sample from the posterior and vice versa. This is called the Nishimori condition in the statistical physics literature [54, 55]. 4.1. The minimum mean-squared error A direct consequence of the Nishimori condition is that the magnetization (correlation between the signal and a random sample) and the overlap (correlation of two random samples) have to be equal, which in return means that the overlap distribution needs to be concentrated on a delta function and thus no RSB is possible in the Bayes-optimal setting. The Nishimori conditions also play a key role in the proof techniques used to establish the above results rigorously in [52, 53]. J. Stat. Mech. (2022) 1140 at. Mech. (2022) 114015 It also important to note that what we discuss in this section is limited to the large size limit N →∞with parameters scaling in such a way with N for the MMSE to go from ρ to 0 as the signal-to-noise ratio α increases from 0 to large O(1) values. This imposes scaling on the λN for the spiked tensor model that is O(N (1−p)/2). This will be in particular important for our claims about the optimality of the AMP algorithm that will be restricted to this regime and will not necessarily apply to performance of AMP for much larger signal to noise ratios. ch. (2022) 114015 https://doi.org/10.1088/1742-5468/ac9cc8 4.2. AMP and its state evolution The proofs of state evolution have been extended to a broader setting [59–61]. for a function fSE that depends on the parameters of the models, but not any longer of any high-dimensional quantity. The state evolution of AMP is a crucial contribution that came from mathematical developments of the theory [57, 58] and was not known in its current form in the statistical physics literature before that. The proofs of state evolution have been extended to a broader setting [59–61]. What makes the state evolution particularly appealing in the statistical physics context is its connection to the computation of the MMSE. The ﬁxed points of the expression (18) can be expressed at the stationary points of the replica symmetric free entropy m = fSE (m) ⇔ ∂ΦRS(m) ∂m = 0 (19) (19) S(m) is indeed the same free entropy as in equation (13). where ΦRS(m) is indeed the same free entropy as in equation (13). (2022) 114015 Since the signal u is unknown the corresponding initialization is mt=0 = 0 (this is for prior distribution with zero mean) and thus the performance of AMP is given by the stationary point of the free entropy that is reached by iterating (18) initialized at mt=0 = 0. The performance of AMP at convergence thus corresponds to the local maximum mAMP of the free entropy ΦRS(m) that has the largest error. The corresponding MSE is then (20) MSEAMP = ρ −mAMP. 4.2. AMP and its state evolution In the previous section we analyzed the MMSE as it would be achieved by the exact computation of the posterior average. This is, however, in general computationally demanding and thus a next natural question is whether we can reach this MMSE com- putationally eﬃciently. Message passing algorithms provide an algorithmic counter-part of the replica method. In particular, the approximate message passing algorithm (AMP) that is an extension of the TAP equations [56] to the general setting of the spiked tensor model and the GLM is of interest to us in this paper. AMP is an iterative algorithm that aims to compute the Bayes-optimal estimator ˆz. Schematically the update of AMP at time step t for the AMP’s estimate zt AMP ∈RN can be written for both the considered models as zt+1 AMP = F(zt AMP) (16) zt+1 AMP = F(zt AMP) (16) for an update function F(.) that depends on Y, parameters of the prior and the likelihood, and for the GLM also on J. for an update function F(.) that depends on Y, parameters of the prior and the likelihood, and for the GLM also on J. The key property that makes AMP so theoretically attractive is that in the large size limit the accuracy of the AMP estimator can be tracked via low-dimensional set 16 Disordered systems insights on computational hardness of equations called state evolution. To state this we introduce the correlation between AMP estimate and the signal at iteration t of equations called state evolution. To state this we introduce the correlation between AMP estimate and the signal at iteration t mt N = 1 N N i=1 ui (zt AMP)i (17) mt N = 1 N N i=1 ui (zt AMP)i (17) The state evolution implies that this quantity in the large size limit mt = limN→∞mt N behaves as The state evolution implies that this quantity in the large size limit mt = limN→∞mt N behaves as mt+1 = fSE (mt), (18) mt+1 = fSE (mt), (18) J. Stat. Mech. (2022) 114 for a function fSE that depends on the parameters of the models, but not any longer of any high-dimensional quantity. The state evolution of AMP is a crucial contribution that came from mathematical developments of the theory [57, 58] and was not known in its current form in the statistical physics literature before that. https://doi.org/10.1088/1742-5468/ac9cc8 https://doi.org/10.1088/1742-5468/ac9cc8 4.3. The phase diagrams and the hard phase We have seen in the previous two subsections that the values of the MMSE as well as the MSE obtained by the AMP algorithm can both be deduced from the extremizers of the free entropy function ΦRS(m). While the MMSE is given by the global maximizer of ΦRS(m), the MSE reached by the AMP algorithm is given by the maximizer having the smallest m. In the following we will consider all the extremizers of ΦRS(m) as this will allow us to understand the resulting overall picture. We will discuss how the extremizers depend on some kind of signal to noise ratio α. This signal to noise ratio can be simply the value of α = λ in the spiked matrix model, or the sample complexity ratio α = P/N in the GLM. / Depending on the other parameters of the model we can observe a number of scenar- ios, we will discuss several of them below and refer to examples where they appear. In 17 Disordered systems insights on computational hardness the following sketches all the colored curves are extremizers of ΦRS(m). Those in blue are the global maximizers of the free entropy corresponding to the MMSE. No algorithmic procedure can achieve an error lower than the MMSE. When the AMP algorithm does not achieve the MMSE, the MSE it reaches at its ﬁxed point corresponds to a maximizer of the free entropy of a higher error MSEAMP depicted in green. In red we depict the other extremizers of the free entropy, in dashed red the minimizers, and in full red the other maximizers. The region of error between the green and the blue curve are values of the MSE that are information-theoretically reachable, but the AMP algorithm does not reach them. We call this region the hard phase, and its boundaries on the signal-to-noise ratio axes: αIT for the information theoretic threshold where the values of the two maximizers of ΦRS(m) switch order, and αalg above which AMP reaches the MMSE. The hard phase exists in between these two thresholds, αIT < α < αalg. A third threshold αs marks the spinodal point at which the lower-error maximizer of the free entropy ceases to exist, this point does not have signiﬁcant algorithmic consequences for ﬁnding the signal. In other cases there may be no phase transition at all or a second order (continuous) phase transition marked by αc. J. 4.3. The phase diagrams and the hard phase Colors explained in the text. (Left) A case without a phase transition. (Right) A case with a ﬁrst order phase transition. Figure 3. Extremizers of the replica symmetric free entropy when m = 0 is a stationary point for all α. Colors explained in the text. (Left) A case with a (contin- uous) 2nd order phase transition. (Right) A case with a (discontinuous) ﬁrst order phase transition. Disordered systems insights on computational hardness Figure 2. Extremizers of the replica symmetric free entropy when neither m = 0 nor m = ρ are stationary points. Colors explained in the text. (Left) A case without a phase transition. (Right) A case with a ﬁrst order phase transition. J. Stat. Mech. (2022) 114 Figure 2. Extremizers of the replica symmetric free entropy when neither m = 0 nor m = ρ are stationary points. Colors explained in the text. (Left) A case without a phase transition. (Right) A case with a ﬁrst order phase transition. at. Mech. (2022) 114015 Figure 3. Extremizers of the replica symmetric free entropy when m = 0 is a stationary point for all α. Colors explained in the text. (Left) A case with a (contin- uous) 2nd order phase transition. (Right) A case with a (discontinuous) ﬁrst order phase transition. Figure 3. Extremizers of the replica symmetric free entropy when m = 0 is a stationary point for all α. Colors explained in the text. (Left) A case with a (contin- uous) 2nd order phase transition. (Right) A case with a (discontinuous) ﬁrst order phase transition. ch. (2022) 114015 Figure 3. Extremizers of the replica symmetric free entropy when m = 0 is a stationary point for all α. Colors explained in the text. (Left) A case with a (contin- uous) 2nd order phase transition. (Right) A case with a (discontinuous) ﬁrst order phase transition. case for instance in the symmetric stochastic block model with more than 4 groups, see e.g. ﬁgure 3 in [62] for a speciﬁc example. In this case the threshold at which the ﬁxed point at m = 0 ceases to be a maximum and start to be a minimum is the well- known Kesten–Stigum threshold [63], marked αc on the lhs of the ﬁgure, and αalg on the rhs of the ﬁgure. https://doi.org/10.1088/1742-5468/ac9cc8 4.3. The phase diagrams and the hard phase Stat. Mech. (2022) 1140 at. Mech. (2022) 114015 The physical interpretation of the cases where the hard phase exists is the one of ﬁrst order phase transition in a high-dimensional (mean-ﬁeld) system. The αIT corresponds to the thermodynamic phase transition while αs and αalg are the spinodals, i.e. the boundaries of the metastable regions. In the hard phase the thermodynamic equilibrium corresponds to the higher free entropy branch depicted in blue, and the green ﬁxed point corresponds to the metastable state. In the region αs < α < αIT the AMP algorithm ﬁnds the thermodynamic equilibrium, but this state is split into exponentially many separated states, each corresponding to the metastable branch (full red). In the language of replica- symmetry breaking this phase corresponds to the dynamical-1RSB phase (d-1RSB). In the d-1RSB phase the AMP algorithm reached optimal performance in terms of ﬁnding the signal, however, sampling the posterior measure in the d-1RSB region is conjectured computationally hard. In ﬁgure 2 we depict one possible structure of extremizers of the free entropy ΦRS(m) for models where neither m = 0 nor m = ρ are ﬁxed points for α > 0. On the left-hand side of ﬁgure 2 we depict a case without a phase transition. This situation arises for instance in generalizes linear models with Gaussian prior and a sign activation function, corresponding to the spherical teacher-student perceptron, see e.g. center of ﬁgure 2 in [53] for a concrete example. On the right-hand side of ﬁgure 2 we depict a case with a ﬁrst order phase transitions. Such as situation arises for instance spiked matrix model where the prior is sparse with non-zero mean, see e.g. rhs of ﬁgure 4 in [7] for a concrete example. In ﬁgure 3 we depict another possible structure of extremizers of the free entropy ΦRS(m) for models where m = 0 is a ﬁxed point. On the left of ﬁgure 3 there is a situation with a second order phase transition as is the case for instance in the symmetric stochastic block model with two groups, see e.g. ﬁgure 1 in [62] for a speciﬁc example. On the right of ﬁgure 3 there is a situation with a ﬁrst order phase transition as is the 18 Disordered systems insights on computational hardness Figure 2. Extremizers of the replica symmetric free entropy when neither m = 0 nor m = ρ are stationary points. 4.3. The phase diagrams and the hard phase When m = 0 and MMSE = ρ is the thermodynamic equilibrium no correlation with the signal can be obtained and the phase α < αIT is in this case referred to as the undetectable region. In this phase the planted model is contiguous to the non-planted model in the sense that all high-probability properties in the planted model are the same in the non-planted one other [64]. This is the setting that is most often explored in the sum-of-squares approach of section 5. In ﬁgure 4 we depict yet another possible structure of extremizers of the free entropy ΦRS(m) for models where m = ρ is a ﬁxed point and thus where exact recovery of the signal with MMSE = 0 is possible for suﬃciently large signal-to-noise ratios. On the right of ﬁgure 4 we depict a case with a ﬁrst order phase transition. Such a situation 19 Disordered systems insights on computational hardness Figure 4. Extremizers of the replica symmetric free entropy when m = ρ is a sta- tionary point for all α. Colors explained in the text. (Left) A case with a 2nd order phase transition. (Right) A case with a ﬁrst order phase transition. Disordered systems insights on computational hardness J. Stat. Mech. (2022) 1140 Figure 4. Extremizers of the replica symmetric free entropy when m = ρ is a sta- tionary point for all α. Colors explained in the text. (Left) A case with a 2nd order phase transition. (Right) A case with a ﬁrst order phase transition. at. Mech. (2022) 114015 arises e.g. in the GLM with binary prior and sign activations, corresponding to the teacher-student binary perceptron, see left-hand side of ﬁgure 2 in [53]. On the left of ﬁgure 4 we depict a case with a second order phase transition, this arises e.g. in the GLM with Laplace prior and no noise, corresponding to the minimization of the ℓ1 regularization, see e.g. ﬁgure 3 in [65]. The examples we depict in this section do not exhaust all the possible scenarios one encounters in computational problems. Some of those we did not cover include the planted locked constraint satisfaction problems where both m = 0 and m = ρ ﬁxed points exist and an all-to-nothing ﬁrst order phase transition happens between these two ﬁxed points [66]. 4.4. Is the hard phase really hard? A fundamental question motivating the discussion of this paper is for what class of algorithms is the hard phase computationally inaccessible? An important evidence towards the hardness is summarized in [69] where it is shown that a very broad range of algorithms related structurally to the AMP cannot improve over the AMP that uses the Bayes-optimal parameters. Eﬀorts to prove lower bounds are considerable, as discussed in section 5. A number of authors put forward a conjecture that in settings where the large-size limit and randomness is taken in such a way that AMP and the Bayes-optimal solution are related in the way we describe above, then AMP is optimal among a large class of algorithms. But could this possibly be all polynomial algorithms? J. Stat. Mech. (2022) 114 It is important to note that there are problems with the phenomenology leading to the hard phase yet for which polynomial algorithms to ﬁnd the signal exist never-the- less. One of them is that planted XOR-SAT problem [66, 70] that is mathematically a linear problem in the Boolean algebra and can thus always be solved using Gaussian elimination. Gaussian elimination, however, runs with time larger than linear in the size of the system and is not robust to noise where we plant a solution that violates a small fraction of clauses. A more surprising and recent example is given by the noise- less phase retrieval problem for Gaussian matrix J where the so-called LLL algorithm also works in polynomial time down to the information-theoretic threshold [71, 72]. The phase retrieval problem is NP-hard, unlike the planted XOR-SAT. Again the LLL is based on linear algebra and thus in some sense related to Gaussian eliminations, it is not robust to noise, or runs in time that is polynomial with an exponent considerably larger than one. at. Mech. (2022) 114015 The existence of these examples makes it clear that in some cases other algo- rithms can perform better than AMP with the Bayes-optimal parameters in the high- dimensional limit. It is thus more reasonable to conjecture that the AMP algorithm may be optimal among those polynomial ones that are required to be robust to noise? Or among those that run with resources linear with the input size of the problem (i.e. quadratic in N)? 4.4. Is the hard phase really hard? We also want to note here another case that is often cited as an example where other algorithms beat AMP. This is the spiked tensor model for p ⩾3. However, in this case the algorithmic threshold happens at λN ∼N−p/4 while the information theoretic one at λN ∼N (1−p)/2. We do not expect AMP to be in general optimal for other scalings than the information-theoretic one, we thus do not consider this as a counter-example to the conjecture of optimality of AMP. Our conjectures about optimality of AMP restrict to the information-theoretic scaling. Disordered systems insights on computational hardness Disordered systems insights on computational hardness https://doi.org/10.1088/1742-5468/ac9cc8 4.3. The phase diagrams and the hard phase Both m = 0 and m = ρ ﬁxed point also exist for instance in the GLM with Gaussian prior and absolute value activation corresponding to the phase retrieval problem. In that case there is a second order phase transition from the undetectable phase to a detectable one and later on a ﬁrst order phase transition to exact recovery, see e.g. left-hand side of ﬁgure 5 in [53]. Another interesting and very generic case is depicted e.g. in ﬁgure 6 of [7] for the spiked matrix model with a symmetric Rademacher–Bernoulli prior. In this case the undetectable phase (m = 0 ﬁxed point) is followed by a phase where a correlation with the signal is detectable but small, and where AMP reaches a small but suboptimal correlation to the signal. The position of the ﬁrst order phase transition can be either before or after the detectability threshold (as in the left or right of the lower part of ﬁgure 6 in [7]). While this may seem a rare scenario, results in [67] (see ﬁgure 2) actually indicate that it is likely very generic and that often the size of the region where detection is possible but sub-optimal is very thin. Yet another interesting example of a phase transition in a planted problem is the planted matching problem where the phase transition is inﬁnite order, i.e. all the deriva- tives of the order parameter m exist at the transition from partial recovery phase to exact recovery phase [68]. https://doi.org/10.1088/1742-5468/ac9cc8 20 4.5. The hard phase is glassy, causing hurdles to gradient-based algorithms From the physics point of view the conjecture of optimality of AMP is very intriguing. It needs to be stressed that the state evolution that rigorously tracks the performance of the AMP algorithm corresponds to the replica symmetric branch of the free entropy while RSB is needed to describe the physical properties of the metastable state [73]. 21 Disordered systems insights on computational hardness Disordered systems insights on computational hardness Physically, and following the success of survey propagation [74] in solving the ran- dom K-SAT problem, one may have hoped that including the glassiness in the form of the algorithm, as done in [75], would improve the performance. This is, however, not happening and is rigorously precluded by the proof of [76]. So in a sense while AMP follows the non-physical solution for the metastable state, this solution has fundamental meaning in terms of being the best solution achievable by a computationally tractable algorithm. It is interesting to note that early work in statistical physics indeed dismissed the replica symmetric spinodal as non-physical, see [77], and presumed that algorithms will be stopped by the glassiness of the metastable phase. This is a nice example where the later state-evolution proof takes over the early physical intuition about what is the relevant algorithmic threshold. J. Stat. Mech. (2022) 1140 At the same time, the physics intuition of the glassiness stopping the dynamics for signal-to-noise ratios larger than where the replica symmetric appears was not wrong. It simply does not apply to the AMP algorithm that does not correspond to a physical dynamics as it does not perform a walk in the space of possible signals but rather iterates marginals over the signal components. If we consider now instead phys- ical dynamics such and Monte-Carlo Markov chains (MCMC) or algorithms updating the signal estimate based on possibly noisy gradient descent the early intuition of [77] turned out to be completely correct in the sense that these algorithms actually per- form considerably worse than AMP when the hard phase is present. Interestingly this was not expected in some works, e.g. [62] conjectured that MCMC performs as well as message passing in the stochastic block model, which turns out to be wrong [78]. Very clear-cut examples of gradient-based Langevin algorithms performing worse than AMP are given for the mixed spiked matrix-tensor model in [79] and for the phase retrieval in [80]. at. Mech. 4.5. The hard phase is glassy, causing hurdles to gradient-based algorithms (2022) 114015 [ ] The phase retrieval example is particularly relevant due to its interpretation as a neural network and given that gradient descent is the working horse of the current machine learning revolution. One may ask whether some key parts of the current machine learning tool-box such as over-parameterization and stochasticity in gradient descent are not a consequence of mitigation of the hurdles that gradient descent encounters due to glassiness of the landscape. Some resent works on the phase retrieval problem do point in that direction [81, 82]. https://doi.org/10.1088/1742-5468/ac9cc8 https://doi.org/10.1088/1742-5468/ac9cc8 5. Polynomial proofs: the sum-of-squares hierarchy In the absence of a proof that P ̸= NP, we have no hope of proving that problems in a certain parameter range truly require exponential time. There may in fact be no hard regimes. But we can try to gather the eﬃcient algorithms we know of into large families—each characterized by a particular strategy or kind of reasoning, or which can only ‘understand’ certain things about their input—and show that no algorithm in these families can succeed. In the previous section, we discussed how the OGP can be used to defeat algorithms that are stable to noise or small perturbations in their input. https://doi.org/10.1088/1742-5468/ac9cc8 22 Disordered systems insights on computational hardness Here we discuss classes of algorithms that have an algebraic ﬂavor. We will focus on the sum-of-squares hierarchy, and brieﬂy discuss its cousin the low-degree likelihood ratio. Many of the best algorithms we know of are captured by these classes, including powerful generalizations of spectral algorithms and classic approximation algorithms. Thus if we can show that they fail to solve certain problems, or more precisely that they require polynomial ‘proofs’ or ‘likelihood ratios’ of high degree, this constitutes additional evidence that these problems are hard. There are types of reasoning that these systems have diﬃculty with, as our ﬁrst example will illustrate. This leaves open the possibility that some very diﬀerent algorithm could eﬃciently solve problems in what we thought was a hard regime. However, these other types of reasoning seem ﬁne-tuned and fragile, and only work in noise-free settings. For a wide variety of noisy problems, algorithms associated with sum-of-squares are conjectured to be optimal [83]. J. Stat. Mech. (2022) 1140 5.1. Proofs and refutations , ht such that k i=1 gi(x)fi(x) = t j=1 hj(x)2 + 1, (23) (23) where 1 on the right-hand side can be replaced by any positive constant. In other words, we ﬁnd a linear combination of the fi that is strictly positive everywhere, so they can never be zero simultaneously. Any unsatisﬁable system of polynomial equations {fi(x) = 0} has a refutation of this form [84, 85]. A logician would say that the SoS proof system is complete. J. Stat. Mech. (2022) 1140 Now, we say a SoS proof is of degree d if the polynomials gifi and h2 j on the left and right sides of (23) have maximum degree d. Thus our example (22) is a proof of degree d = 4. (By convention d is always even: the hj have degree at most d/2 = 2.) As we increase d, we obtain a hierarchy of increasingly powerful proof systems. at. Mech. (2022) 114015 In some cases the lowest possible degree of an SoS proof is much larger than the degree of the original constraints fi, since we may need high-degree coeﬃcients gi to create the right cancellations so that the sum can be written as a sum of squares. As we will see below, if the necessary degree grows with the size of the problem, we can interpret this as evidence that the problem is computationally hard. 5.1. Proofs and refutations At its heart, the sum-of-squares (SoS) hierarchy is a way of constructing refutations of constraint satisfaction or optimization problems: proofs that a solution does not exist, or that no solution achieves a certain value of the objective function. It comes with a dual problem, of evading refutation by ﬁnding a pseudoexpectation: a ﬁctional distribution of solutions that looks reasonable as long as we only ask about polynomials up to a certain degree. If a pseudoexpectation can be constructed that ‘fools’ polynomials up to degree d, then any refutation must have degree greater than d. h. (2022) 114015 Let us look at an example. Consider three variables x, y, z ∈{±1}. Is it possible for them to sum to zero? This problem may seem trivial, but bear with us. Algebraically, we are asking whether the following system of polynomials has a solution, x2 −1= 0 y2 −1= 0 z2 −1= 0 x + y + z = 0. (21) x2 −1= 0 y2 −1= 0 z2 −1= 0 x + y + z = 0. (21) Here is a proof, that the motivated reader can verify, that no solution exists: Here is a proof, that the motivated reader can verify, that no solution exists: 1 8 x2 + 3(y2 + z2) + 4(xy + xz + 3yz) −3 (x2 −1) + y2 + 3(x2 + z2) +4(yz + xy + 3xz) −3) (y2 −1) + z2 + 3(x2 + y2) + 4(xz + yz + 3xy) −3 (z2 −1) + (x + y + z)2 = 1 8 (x + y + z)2 −1 2 + 1. (22) (22) If the constraints (21) hold, then the left-hand side of (22) is identically zero. On the other hand, the right-hand side is the square of a polynomial plus 1, giving the contradiction 0 ⩾1. We will reveal below how we constructed this proof. https://doi.org/10.1088/1742-5468/ac9cc8 23 23 Disordered systems insights on computational hardness More generally, suppose we have a set of polynomials f1(x), . . ., fk(x) over n variables x1, . . ., xn. We wish to prove that there is no x ∈Rn such that fi(x) = 0 for all i. A sum- of-squares proof consists of additional polynomials g1, . . . , gk and h1, . . . 5.2. From proofs to algorithms: semideﬁnite programming Of course, the existence of an SoS proof does not necessarily make it easy to ﬁnd. Algorithmically, how would we search for these polynomials? If we choose some order- ing for the monomials up to some degree, writing a symbolic vector m = (1, x, y, z, x 2, xy, xz, y 2, . . .), then we can represent a polynomial q as a vector q of its coeﬃcients and write q(x) as an inner product ⟨q \| m⟩. Multiplying two polynomials is a bilinear operation, and the sum on the right-hand side of (23) can be written t j=1 hj(x)2 = t j=1 ⟨m \| hj⟩⟨hj \| m⟩= ⟨m\|H\|m⟩ where H = t j=1 \|hj⟩⟨hj\|. (24) (24) This bilinear form H is positive semideﬁnite, which we denote H ⪰0. This bilinear form H is positive semideﬁnite, which we denote H ⪰0. With this abstraction, the problem of ﬁnding SoS proofs asks for a positive semideﬁ- nite matrix that matches the left-hand side of (23). To nail this down, for a polynomial q let qu denote the coeﬃcient of each monomial u. Then summing over all the cross-terms in the product of two polynomials p, q gives u = v,w : vw=u pvqw. (25) (pq)u = v,w : vw=u pvqw. (25) /doi.org/10.1088/1742-5468/ac9cc8 24 https://doi.org/10.1088/1742-5468/ac9cc8 24 Disordered systems insights on computational hardness Disordered systems insights on computational hardness Since for any two monomials s, t the entry Hs,t = ⟨s\|H\|t⟩must equal the coeﬃcient of u = st on the left-hand side of (23), for any s, t such that st ̸= 1 we have Since for any two monomials s, t the entry Hs,t = ⟨s\|H\|t⟩must equal the coeﬃcient of u = st on the left-hand side of (23), for any s, t such that st ̸= 1 we have i v,w : vw=st (gi)s(fi)t = Hs,t, (26) and for s = t = 1 we have i (gi)1(fi)1 = 1 + H1,1. and for s = t = 1 we have and for s = t = 1 we have i (gi)1(fi)1 = 1 + H1,1. (27) s = t = 1 we have i (gi)1(fi)1 = 1 + H1,1. i (gi)1(fi)1 = 1 + H1,1. (27) i (gi)1(fi)1 = 1 + H1,1. 5.2. From proofs to algorithms: semideﬁnite programming (27) For a given set {fi}, these constraints are linear in the coeﬃcients of the {gi}. Adding the semideﬁniteness constraint H ⪰0 to this linear system of equations makes this a case of semideﬁnite programming or SDP [86–89]. For a given set {fi}, these constraints are linear in the coeﬃcients of the {gi}. Adding the semideﬁniteness constraint H ⪰0 to this linear system of equations makes this a case of semideﬁnite programming or SDP [86–89]. J. Stat. Mech. (2022) 114 SDP can be solved up to arbitrarily small error in polynomial time whenever the number of constraints and the dimension of the matrices is polynomial. (There is an important caveat, namely that the coeﬃcients of the SoS proof need to be polynomi- ally bounded [90, 91].) Since the number of monomials over n variables of degree d is n+d−1 d = O(nd), this means that SoS proofs are easy to ﬁnd whenever the degree d is constant. t. Mech. (2022) 114015 On the other hand, if we can somehow prove that the lowest degree of any SoS proof grows with n, this rules out a large class of polynomial-time algorithms. When we can prove them, these SoS lower bounds are thus evidence of computational hardness. 5 Many concepts in theoretical computer science have become personiﬁed over the years: the Adversary, the Oracle, Arthur and Merlin, Alice, Bob, and Eve, and so on. We propose that the Charlatan be added to this cast of characters. Disordered systems insights on computational hardness Disordered systems insights on computational hardness Let us think of E as a bilinear form that takes two polynomials p, q of degree up to d/2 and returns E[pq] = ⟨p\|E\|q⟩. Then condition (3) corresponds to E being positive semideﬁnite, just as for H above. Since conditions (1) and (2) are linear, ﬁnding a pseudoexpectation is another case of semideﬁnite programming. In our example, since d = 2, the monomials that E needs to deal with are just 1, x, y, z. Without further ado, we present the Charlatan’s claim as a multiplication table of pseudoexpectations: (28) (28) J. Stat. Mech. (2022) 1140 (28) at. Mech. (2022) 114015 That is, they claim that x, y, z each have expectation E [x] = ⟨1\|E\|x⟩= 0; they each have variance E [x2] = ⟨x\|E\|x⟩= 1; and each distinct pair is negatively correlated, with E [xy] = ⟨x\|E\|y⟩= −1/2. As a result, E [x + y + z] = 0, and E [(x + y + z)p] = 0 for any linear function p, satisfying condition (2) above. That is, they claim that x, y, z each have expectation E [x] = ⟨1\|E\|x⟩= 0; they each have variance E [x2] = ⟨x\|E\|x⟩= 1; and each distinct pair is negatively correlated, with E [xy] = ⟨x\|E\|y⟩= −1/2. As a result, E [x + y + z] = 0, and E [(x + y + z)p] = 0 for any linear function p, satisfying condition (2) above. It is easy to check that this matrix of pseudomoments is positive semideﬁnite. Indeed its 3 × 3 part is the Gram matrix of three unit vectors that are 120◦apart. This is impossible for three real-valued variables in {±1}, but as far as quadratic polynomials of x, y, z are concerned, there is no contradiction. h. (2022) 114015 On the other hand, we already know that we can debunk the Charlatan’s claims if we ask about degree-4 polynomials. The left-hand side of (22) must have zero expectation since it is a linear combination of the fi. By linearity, this would imply that E 1 8 (x + y + z)2 −1 2 = −1 < 0. (29) (29) Thus there is no way to extend the pseudoexpectation in (28) from degree 2 to degree 4 without violating positive semideﬁniteness. https://doi.org/10.1088/1742-5468/ac9cc8 5.3. Sum-of-squares lower bounds: enter the charlatan To see how we might prove such a lower bound, let us return to our earlier problem. A Charlatan5 comes along and claims that the system (21) has not just one solution, but many. That is, they claim to know a joint probability distribution over reals x, y, z such that x 2 = y 2 = z 2 = 1 and x + y + z = 0. To convince you, they oﬀer to tell you the expectation E[q] of any polynomial q(x, y, z) you desire—but only for q of degree d or less, where in this case d = 2. Let us call the Charlatan’s claimed value for E [q] the pseudoexpectation, and denote it E [q]. How might you catch them in a lie? You are no fool; you know that the expec- tation of a sum is the sum of the expectations. Since the constraints fi(x) = 0 must hold identically, you also know that any q that has fi as a factor must have zero expectation. Finally, you are well aware that the square of any polynomial is everywhere nonnegative, and thus has nonnegative expectation. Putting this together, the pseudoexpectation must be a linear operator from the space of polynomials of degree d to R with the following properties: (a) E [1] = 1. (b) E [fiq] = 0 for any polynomial q(x) of degree d −deg(fi) or less. (c) E [q2] ⩾0 for any polynomial q(x) of degree d/2 or less. https://doi.org/10.1088/1742-5468/ac9cc8 25 https://doi.org/10.1088/1742-5468/ac9cc8 Disordered systems insights on computational hardness More generally, an SoS proof of the form (23) would imply Thus there is no way to extend the pseudoexpectation in (28) from degree 2 to degree 4 without violating positive semideﬁniteness. More generally, an SoS proof of the form (23) would imply E j h2 j = −1 < 0. (30) E j h2 j = −1 < 0. E j h2 j = −1 < 0. (30) (30) Thus for each degree d, there is an SoS proof if and only if there is no pseudoex- pectation. These two problems are dual SDPs; a solution to either is a certiﬁcate that the other has no solution. In particular, any degree at which the Charlatan can succeed is a lower bound on the degree a refuter needs to prove that no solution exists. In this example, we have shown that degree 4 is both necessary and suﬃcient to prove that no three variables in {±1} can sum to zero. 26 Disordered systems insights on computational hardness 5.4. What does sum-of-squares understand? The reader is probably wondering how the SoS framework performs on larger versions of our example. Suppose we have n variables x1, . . ., xn. If n is odd, clearly it is impossible to satisfy the system x2 i −1 = 0 for all i = 1, . . ., n n i=1 xi = 0. (31) x2 i −1 = 0 for all i = 1, . . ., n n i=1 xi = 0. (31) J. Stat. Mech. (2022) 114 To put it diﬀerently, if you take an odd number of steps in a random walk on the integers, moving one unit to the left or right on each step, there is no way to return to the origin. To put it diﬀerently, if you take an odd number of steps in a random walk on the integers, moving one unit to the left or right on each step, there is no way to return to the origin. It turns out [92–94] that any SoS proof of this fact requires degree n + 1. That is, the Charlatan can construct a pseudoexpectation for polynomials of degree d up to n −1. This includes the case n = 3 we studied above. How can the Charlatan do this? Since x2 i = 1 for all i, it suﬃces for them to construct pseudoexpectations for the multilinear monomials, i.e. those of the form xS = i∈S xi for some set S ⊂{1, . . ., n}. Furthermore, we can symmetrize over all permutations of the xi, and assume that E[xS] only depends on their degree \|S\|: semideﬁnite programming is a convex problem, so symmetric problems have symmetric solutions if any. Now let ak denote E[xS] for \|S\| = k. Equivalently, ak = E[x1x2 . . . xk]. We can compute ak as follows. Suppose I tell you that n/2 of the xi are +1, and n/2 are −1. (Do not ask whether n/2 is an integer.) If we choose a uniformly random set of k distinct vari- ables from among the xi, then ak is the average parity of their product. An enjoyable combinatorial exercise gives, for k even, (2022) 114015 ak = (−1)k/2 n/2 k/2 n k = (−1)k/2 (k −1)(k −3)(k −5) . . .1 (n −1)(n −3)(n −5) . . 5.4. What does sum-of-squares understand? The polynomials gi in (34) are guaranteed to exist because, in the ring of polynomials, the set {x2 i −1} spans the set of all polynomials that vanish on {±1}n. For the experts, {x2 i −1} is a Gr¨obner basis for this ideal. For instance, the reader can check that the three terms inside the square brackets in (22) sum to (w + 3)(w + 1)(w −1)(w −3) where w = x + y + z. The polynomials gi in (34) are guaranteed to exist because, in the ring of polynomials, the set {x2 i −1} spans the set of all polynomials that vanish on {±1}n. For the experts, {x2 i −1} is a Gr¨obner basis for this ideal. J. Stat. Mech. (2022) 114 Now we wish to show that some polynomial with w as a factor, say w2, is nonzero. To do this, we ﬁnd a polynomial q(w) that is everywhere positive and that coincides with w2 at the odd integers between −n and n. By polynomial interpolation, we can take q(w) to be even and of degree n + 1. For n = 3, for instance, we have q(w) = 1 8(w2 −1)2 + 1 ⩾1, (35) (35) which we have already written as a sum of squares. 2 which we have already written as a sum of squares. 2 have already written as a sum of squares. 2 Since the polynomial q(w) −w2 has these odd integers as roots, it is a multiple of the expression in (34). Putting this together for n = 3 gives Since the polynomial q(w) −w2 has these odd integers as roots, it is a multiple of the expression in (34). Putting this together for n = 3 gives 1 8(w + 3)(w + 1)(w −1)(w + 3) + w2 = q(w), (36) (36) (2022) 114015 which is exactly what we wrote in (22). Now recall that SoS refutations of degree d can be found in polynomial time only if d is a constant. This means that as far as SoS is concerned, proving that (31) is unsatisﬁable is hard. Clearly SoS does not understand parity arguments very well. Morally, this is because the matrix elements (32) are analytic functions of n: they cannot tell whether n is odd or even, or even whether n is an integer or not. https://doi.org/10.1088/1742-5468/ac9cc8 5.4. What does sum-of-squares understand? .(n −k + 1) (32) (32) and ak = 0 for k odd. and ak = 0 for k odd. k Again using the fact that x2 i = 1 for all i, for any two sets S, T we have xSxT = xS△T where △denotes the symmetric diﬀerence. Thus we deﬁne the pseudoexpectation as a bilinear operator that takes monomials xS, xT where \|S\|, \|T\| ⩽d/2, with matrix elements Again using the fact that x2 i = 1 for all i, for any two sets S, T we have xSxT = xS△T where △denotes the symmetric diﬀerence. Thus we deﬁne the pseudoexpectation as a bilinear operator that takes monomials xS, xT where \|S\|, \|T\| ⩽d/2, with matrix elements ⟨xS\|E\|xT⟩= E[xS xT] = E[xS△T] = a\|S△T\|, (33) (33) which generalizes (28) above. As long as d ⩽n −1, it turns out that this E is positive semideﬁnite [94]; its spectrum can be analyzed using representation theory [95]. Thus any SoS refutation of the system (31) must be of degree at least d = n + 1. which generalizes (28) above. As long as d ⩽n −1, it turns out that this E is positive semideﬁnite [94]; its spectrum can be analyzed using representation theory [95]. Thus any SoS refutation of the system (31) must be of degree at least d = n + 1. This lower bound is tight: any pseudoexpectation on Boolean variables x1, . . ., xn ∈ {±1} of degree n + 1 must be a true expectation, i.e. must correspond to an actual distribution over the hypercube [96]. Thus at degree n + 1, the Charlatan can no longer produce a convincing pseudoexpectaton unless solutions actually exist. If n is odd, there are no solutions, so by SDP duality there is a refutation of degree n + 1. 27 Disordered systems insights on computational hardness One way to construct a refutation is as follows. Let w denote i xi. First we ‘prove’ that w is an odd integer between −n and n by ﬁnding polynomials g1, . . ., gn such that n i=1 gi(x) (x2 i −1) = ...,n−2,n t=−n,−n+2,... (w −t). (34) (34) For instance, the reader can check that the three terms inside the square brackets in (22) sum to (w + 3)(w + 1)(w −1)(w −3) where w = x + y + z. 5.4. What does sum-of-squares understand? To put it diﬀerently, binomials like those in the numerator of ak in (32) will happily generalize to half-integer inputs with the help of the Gamma function. After all, there are 3 3/2 = 32/(3π) = 3.395 . . . ways to take three steps of a random walk and return to the origin. Th ‘h d ’ f hi l k S S l k lik k f The ‘hardness’ of this example may make SoS look like a very weak proof system. But parity is a very delicate thing. If n Boolean variables are represented as {0, 1}, then their parity is merely their sum mod 2; but if we represent them as spins ±1, the parity is their product, which is of degree n. When n is large, we would be amazed to ﬁnd such a term in the Hamiltonian of a physical system. No observable quantity depends on whether the number of atoms in a block of iron is odd or even. The situation seems similar to XORSAT, whose clauses are linear equations mod 2. Its energy landscape has many of the hallmarks of algorithmic hardness, with clusters, frozen variables, and large barriers between solutions [97]. See also the discussion in subsection 3.4 of section 3. In the noise-free case it can be solved in polynomial time using Gaussian elimination over Z2. But if we add any noise, for instance only requiring 28 Disordered systems insights on computational hardness that 99% of the XORSAT clauses be satisﬁed, its algebraic structure falls apart and this algorithmic shortcut disappears. So while parity and XORSAT are good cautionary tales, we should not think of them as representative of more generic problems. As we will see next, for many problems with noise, including those involving random matrices and tensors with planted structure, the SoS framework is associated with many algorithms that are conjectured to be optimal. 6 Thanks to Tselil Schramm for suggesting the name ‘relaxer’ for this rehabilitated version of the Charlatan. Perhaps ‘slacker’ would also work in contemporary English. 5.5. Relaxation and the Sherrington–Kirkpatrick model Above we referred to the pseudoexpectation as the work of a charlatan who falsely claims that an unsatisﬁable problem has many solutions. But there is another, less adversarial way to describe this character: rather than trying to fool us, they are a Relaxer who honestly solves a less-constrained problem, and thus proves bounds on the optimum of the original problem.6 J. Stat. Mech. (2022) 114 To celebrate the 40th anniversary that inspired this book, let us consider the Sher- rington–Kirkpatrick model. Given a coupling matrix J we can write the ground state energy of an Ising spin glass as E0 = −max x∈{±1}n i<j Jijxixj = −1 2max X∈C tr JX (37) (37) (where we take J to be symmetric and zero on the diagonal). In other words, the energy is quadratic in the spins, but linear in the products Xij = xixj. So we just have to maximize a linear function! This is exactly the maximization problem (6) when p = 2, ignoring the −1/2 factor. (2022) 114015 The tricky part is that we have to maximize tr JX over a complicated set. In (37), C is the set of matrices X = \|x⟩⟨x\| corresponding to actual spin conﬁgurations, namely symmetric rank-1 matrices with ±1 entries and +1s on the diagonal. We would get the same maximum if we deﬁned C to be the polytope of all convex linear combinations of such matrices. But this so-called cut polytope has exponentially many facets, making this maximization computationally infeasible [98]. In the worst case where J is designed by an adversary, it is NP-hard since, for instance, it includes Max Cut as a special case [99]. We can relax this problem by allowing X to range over some superset C′ of C. Then the maximum of tr JX will be greater than or equal to the true maximum over C, providing a lower bound on E0. A hopeful goal is to ﬁnd a set C′ whose structure is simple enough to perform this maximization eﬃciently, while giving a bound that is not too far from the truth. The ﬁrst attempt we might make is to allow X to range over all positive semideﬁnite matrices with trace n. Call this set C0: (38) C0 = {X : X ⪰0 and tr X = n}. 6 Thanks to Tselil Schramm for suggesting the name ‘relaxer’ for this rehabilitated version of the Charlatan. https://doi.org/10.1088/1742-5468/ac9cc8 C0 = {X : X ⪰0 and tr X = n}. (38) https://doi.org/10.1088/1742-5468/ac9cc8 5.5. Relaxation and the Sherrington–Kirkpatrick model Perhaps ‘slacker’ would also work in contemporary English. https://doi.org/10.1088/1742-5468/ac9cc8 29 Disordered systems insights on computational hardness Disordered systems insights on computational hardness Then (39) max x∈C0 tr JX = nλmax max x∈C0 tr JX = nλmax (39) where λmax is J’s most positive eigenvalue. For the SK model where the Jij are Gaussian with mean 0 and variance 1/n, the Wigner semicircle law tells us that, in the limit of large n, the spectrum of J is supported on [−2, 2]. Thus where λmax is J’s most positive eigenvalue. For the SK model where the Jij are Gaussian with mean 0 and variance 1/n, the Wigner semicircle law tells us that, in the limit of large n, the spectrum of J is supported on [−2, 2]. Thus lim n→∞E0/n ⩾−λmax 2 = −1. lim n→∞E0/n ⩾−λmax 2 = −1. (40) J. Stat. Mech. (2022) 1140 (40) This is fairly far from Parisi’s solution E0/n = −0.7632 [100, 101]. Can we get a better bound with some other choice of C′? This is fairly far from Parisi’s solution E0/n = −0.7632 [100, 101]. Can we get a better bound with some other choice of C′? We can tighten our relaxation by adding any constraint that holds for the true set of matrices C. Let us start with the constraint that X’s diagonal entries are 1. This gives a set of matrices sometimes called the elliptope [102], C2 = {X : X ⪰0 and Xii = 1 for all i}. (41) (41) We might hope that maximizing tr JX over C2 rather than C0 gives a better bound on the energy. Unfortunately, this is not the case: for any constant ε > 0, with high probability there is an X ∈C2 such that tr JX ⩾2 −ε. We will sketch the proof of [103]. We might hope that maximizing tr JX over C2 rather than C0 gives a better bound on the energy. Unfortunately, this is not the case: for any constant ε > 0, with high probability there is an X ∈C2 such that tr JX ⩾2 −ε. We will sketch the proof of [103]. First let vλ denote the eigenvector of J with eigenvalue λ, normalized so that \|vλ\|2 = 1. Let m denote the number of eigenvalues in the interval [2 −ε, 2]. These eigenvalues span a low-energy subspace where E0 ≈−1. 5.5. Relaxation and the Sherrington–Kirkpatrick model Now deﬁne Y as (2022) 114015 Y = n m λ∈[2−ε,2] \|vλ⟩⟨vλ\|. (42) Y = n m λ∈[2−ε,2] \|vλ⟩⟨vλ\|. (42) That is, Y is n/m times the projection operator onto this subspace. Thus Y ⪰0 and tr JY ⩾(2 −ε)n. That is, Y is n/m times the projection operator onto this subspace. Thus Y ⪰0 and tr JY ⩾(2 −ε)n. ( ) We can write Y’s diagonal entries as ( ) We can write Y’s diagonal entries as ) an write Y’s diagonal entries as Yii = n m λ (vλ)2 i. Yii = n m λ (vλ)2 i. (43) (43) Since the distribution of Gaussian random matrices is rotationally invariant, the vλ are distributed as a uniformly random set of m orthonormal vectors in n dimensions. Thus the (vλ)2 i are asymptotically independent, and are 1/n on average. As a result, each Yii is concentrated around 1. To turn Y into an X such that Xii = 1 holds exactly, deﬁne D as the diagonal matrix Dii = Yii and let X = D−1/2Y D−1/2. (44) X = D−1/2Y D−1/2. (44) Clearly X ⪰0. Moreover, since D itself is close to the identity, we have tr JX = tr JY up to a vanishing error term. Since X ∈C2, we have shown that C2 does not give a bound any better than the simple spectral bound provided by C0. The alert reader will note that C2 is exactly the set of pseudoexpectations E that a degree-2 charlatan can choose from. If Xij = E[xixj], then X ⪰0 and Xii = E[x2 i ] = 1. 30 Disordered systems insights on computational hardness So whether we regard X as the solution to a relaxed problem or a false claim about the covariances E[xixj], we have shown that degree-2 SoS proofs cannot establish a bound better than E0/n > −1 on the SK ground state energy. That is, they are incapable of refuting the claim that there are states with energy −1 + ε or below, for arbitrarily small ε. (There is a subtlety here. The refuter’s goal is not to understand the typical ground state energy of the SK model, but to provide ironclad proofs for individual realizations J that their ground state energy is above a certain point. 7 This is usually presented the other way around. If we round the relaxed solution to ±1 spins by cutting Rn with a random hyperplane, the Goemans–Williamson algorithm gives a cut that is at least 0.878 times the optimum, and the unique games conjecture implies that this cannot be improved. The same argument [105] implies an upper bound on the relaxed solution. (Thanks to Tim Kunisky for pointing this out). 5.5. Relaxation and the Sherrington–Kirkpatrick model On the other hand, for the SK model it was recently shown [106] that higher-degree SoS does not improve our bounds on the ground state energy, as we will see next. 5.5. Relaxation and the Sherrington–Kirkpatrick model What we have shown is that, for most realizations J, there is no degree-2 proof that its ground state energy is noticeably above −1.) J. Stat. Mech. (2022) 1140 ) We should also note that, just as C is the set of matrices X = \|x⟩⟨x\| where the xi = ±1 are Ising spins, C2 is the set of matrices X = \|x⟩⟨x\| where the xi are n-dimensional vectors with \|xi\|2 = 1. So while Ising spins cannot achieve the covariances Xij = E[xixj] that the Charlatan claims, these vector-valued spins can achieve them in the sense that Xij = ⟨xi \| xj⟩. This is the heart of the Goemans–Williamson approximation algorithm for Max Cut [104]—or, in physics terms, bounding the ground-state energy of an antiferromagnet. In Max Cut, our goal is to assign a spin xi = ±1 to each vertex, and maximize the number w of edges whose spins are opposite. For a graph with m edges and adjancency matrix A, this is Mech. (2022) 114015 w = 1 2(m −⟨x\|A\|x⟩). (45) w = 1 2(m −⟨x\|A\|x⟩). (45) If we relax this problem by letting the xi be unit-length vectors in Rn instead of just ±1, this becomes an SDP that we can solve in polynomial time. It can be shown that this relaxation increases w by a factor of at most 1/0.878 = 1.138 . . ., so the optimum of this relaxation is not too far from that of the original problem. If we relax this problem by letting the xi be unit-length vectors in Rn instead of just ±1, this becomes an SDP that we can solve in polynomial time. It can be shown that this relaxation increases w by a factor of at most 1/0.878 = 1.138 . . ., so the optimum of this relaxation is not too far from that of the original problem. We do not know whether going to higher-degree SoS improves this approximation ratio. If we assume the unique games conjecture (a plausible strengthening of P ̸= NP) then no polynomial-time algorithm can do better than Goemans–Williamson [105].7 This suggests that going to degree 4, 6, and so on does not give a better algorithm, but even for degree 4 this is an open question. https://doi.org/10.1088/1742-5468/ac9cc8 5.6. Beyond degree 2 Can SoS proofs of some constant degree d > 2 prove a tighter bound on the ground state energy E0 of the Sherrington–Kirkpatrick model? Do higher-degree polynomials help us go beyond the simple spectral bound E0 ⩾−1? The Charlatan’s job for d = 4 is already quite interesting. In addition to provid- ing X ∈C2, they now have to provide an n 2 -dimensional matrix X (4), with rows and https://doi.org/10.1088/1742-5468/ac9cc8 31 Disordered systems insights on computational hardness columns for each pair (i, j), such that columns for each pair (i, j), such that columns for each pair (i, j), such that X(4) (i,j),(k,ℓ) = E[xixjxkxℓ]. (46) X(4) (i,j),(k,ℓ) = E[xixjxkxℓ]. (46) Thus X(4) must have the symmetries of a symmetric four-index tensor, Thus X(4) must have the symmetries of a symmetric four-index tensor, X(4) (i,j),(k,ℓ) = X(4) (i,k),(j,ℓ) = X(4) (i,ℓ),(j,k). (47) (47) In addition, X(4) needs to be consistent with the degree-2 pseudexpectations and the constraint x2 i = 1. Thus In addition, X(4) needs to be consistent with the degree-2 pseudexpectations and the constraint x2 i = 1. Thus J. Stat. Mech. (2022) 1140 X(4) (i,j),(i,k) = E x2 ixjxk = E[xjxk] = Xjk (48) X(4) (i,j),(i,j) = E x2 ix2 j = 1. (49) (48) (49) (We saw these relations in section 5.4 where we wrote xSxT = xS△T.) Finally, as always X(4) must be positive semideﬁnite, (We saw these relations in section 5.4 where we wrote xSxT = xS△T.) Finally, as always X(4) must be positive semideﬁnite, X(4) ⪰0. (50) X(4) ⪰0. (50) X(4) ⪰0. (50) X(4) ⪰0. (50) Mech. (2022) 114015 The energy E = −(1/2)tr JX is still a function of the second-order pseudoexpectation X. But not all matrices X in C2 can be extended to fourth order in this way: the set C4 = {X ∈C2 : ∃X(4) such that (47)–(50) holds} (51) (51) is a proper subset of the elliptope C2. In other words, armed with degree-4 SoS proofs, a refuter can prove some new constraints on the covariances Xij = xixj that go beyond Xii = 1 and X ⪰0. is a proper subset of the elliptope C2. In other words, armed with degree-4 SoS proofs, a refuter can prove some new constraints on the covariances Xij = xixj that go beyond Xii = 1 and X ⪰0. https://doi.org/10.1088/1742-5468/ac9cc8 5.6. Beyond degree 2 For example, consider any three Ising spins, xi, xj, and xk. Their products (xixj, xjxk, xixk) can only take the values (1, 1, 1), (1, −1, −1), (−1, 1, −1), and (−1, −1, 1). Thus the expectation of their products (Xij, Xjk, Xik) must lie in the convex hull of these four vectors, namely the tetrahedron with these four vertices. The facets of this tetrahedron are the linear inequalities Xij + Xjk + Xik + 1 ⩾0 (52) Xij −Xjk −Xik + 1 ⩾0 (53) −Xij + Xjk −Xik + 1 ⩾0 (54) −Xij −Xjk + Xik + 1 ⩾0. (55) Xij + Xjk + Xik + 1 ⩾0 Xij −Xjk −Xik + 1 ⩾0 −Xij + Xjk −Xik + 1 ⩾0 −Xij −Xjk + Xik + 1 ⩾0. (52) (53) (54) (55) We have already seen a pseudoexpectation in C2 that violates the ﬁrst of these inequali- ties—namely (28) where Xij = Xjk = Xik = −1/2. Thus we cannot prove these inequali- ties with degree-2 sum-of-squares. But we can prove them with degree 4, and we already have! After all, we can rewrite (52) as We have already seen a pseudoexpectation in C2 that violates the ﬁrst of these inequali- ties—namely (28) where Xij = Xjk = Xik = −1/2. Thus we cannot prove these inequali- ties with degree-2 sum-of-squares. But we can prove them with degree 4, and we already have! After all, we can rewrite (52) as E[xixj + xjxk + xixk + 1] ⩾0. (56) E[xixj + xjxk + xixk + 1] ⩾0. (56) https://doi.org/10.1088/1742-5468/ac9cc8 https://doi.org/10.1088/1742-5468/ac9cc8 oi.org/10.1088/1742-5468/ac9cc8 32 32 Disordered systems insights on computational hardness Disordered systems insights on computational hardness But if x2 i = x2 j = x2 k = 1 this is equivalent to But if x2 i = x2 j = x2 k = 1 this is equivalent to E (xi + xj + xk)2 ⩾1. (57) Looking again at our proof (22) that no three spins can sum to zero, the reader will see that we in fact proved that (x + y + z)2 ⩾1 whenever x2 = y2 = z2 = 1. The symmetry operations x →−x, y →−y, and z →−z give similar proofs of (53)–(55). Thus any matrix X that violates these ‘triangle inequalities’ can be refuted by degree- 4 sum-of-squares. 5.6. Beyond degree 2 More generally, since any t + 1 pseudoexpectation on t spin variables is a true expectation [96], any linear inequality on the covariances of t spins—or equiv- alently any inequality that involves a t × t principal minor of X—can be proved with degree t + 1 sum-of-squares. J. Stat. Mech. (2022) 114 Perhaps these and other degree-4 constraints will ﬁnally give a better bound on E0? Sadly—or happily if you love computational hardness—they do not. In fact, no constant degree can refute the claim that some spin conﬁguration lies in the low-energy subspace, and thus prove a bound tighter than tr JX ⩽2 or E0 ⩾−1. at. Mech. (2022) 114015 One intuition for this is that for natural degree-2 pseudoexpectations, like the X we constructed above (44) by projecting onto the low-energy subspace, triangle inequalities and their generalizations already hold with room to spare. In the SK model we typically have E[xixj] = O(1/√n), so (52)–(55) all read 1 + O(1/√n) ⩾0. Thus, with perhaps a slight perturbation to make it positive deﬁnite and full rank, X is already deep inside the elliptope C2, and is not refuted by the additional inequalities we can prove with low-degree SoS proofs. ch. (2022) 114015 There are several ways to make this intuition rigorous. One is to explicitly construct higher-degree pseudoexpectations X (4), X (6), and so on that extend X in a natural way, somewhat like a cluster expansion in physics. For instance, we could deﬁne X(4) (i,j),(k,ℓ) = XijXkℓ+ XikXjℓ+ XiℓXjk −2 n m=1 XimXjmXkmXℓm. (58) (58) This expression has the permutation symmetry of (47). The ﬁrst three terms look like Wick’s theorem or Isserlis’ theorem for the moments of Gaussian variables [107]; the reader can check that by cancelling two of these terms when k = ℓ, the sum over m ensures the consistency relations (48) and (49) to leading order. A small perturbation then satisﬁes these conditions exactly [108] and it is relatively easy to show that the result is positive semideﬁnite; see also [109]. A similar approach works for degree 6 [110]. https://doi.org/10.1088/1742-5468/ac9cc8 https://doi.org/10.1088/1742-5468/ac9cc8 5.7. Pseudocalibration and clever planted models While constructions like (58) could probably be carried out for higher degree, the recent proof [106] that no constant degree of SoS can improve the bound on E0 comes from a diﬀerent direction called pseudocalibration [111, 112]. In pseudocalibration, the Charlatan claims that the data is generated by a planted model where the claimed solution is built in, rather than the (true) null model. In the Sherrington–Kirkpatrick model this means pretending that the couplings J have been chosen so that some Boolean vector x ∈{±1}n achieves the spectral bound E0 = −1. 33 Disordered systems insights on computational hardness If we can construct a pseudoexpectation around this idea, then low-degree SoS cannot tell the diﬀerence between the null model and the planted model. In particular, it cannot prove that the planted solution does not exist. Following [112], we can brieﬂy describe pseudocalibration as follows. We consider two joint distributions on a signal x and observed data Y. In both cases, we choose x from a prior P(x). In the null model, we choose Y independently of x with probability P0(Y); in the planted model, we choose Y with probability P1(Y\|x). Thus P0(x, Y ) = P0(Y ) P(x) P1(x, Y ) = P1(Y \| x) P(x) = P1(Y ) P1(x \| Y ), J. Stat. Mech. (2022) 114 where P1(Y ) = Ex∼P(x)P1(Y \| x) is Y’s likelihood in the planted model. (Y ) = Ex∼P(x)P1(Y \| x) is Y’s likelihood in the planted model. In the Charlatan’s ﬁrst attempt, they deﬁne the pseudoexpectation of a function q(x) as its true expectation given Y, but reweighted to change the null model into the planted one: E[q(x) \| Y ] = Ex∼P(x) P1(x, Y ) P0(x, Y ) q(x) = Ex∼P(x) P1(Y ) P1(x \| Y ) P0(Y ) P(x) q(x) = P1(Y ) P0(Y ) Ex∼P1(x \| Y )q(x). (59) = P1(Y ) P0(Y ) Ex∼P1(x \| Y )q(x). (59) (59) That is, the pseudoexpectation of q(x) is its true expectation in the posterior distribution P1(x\|Y), multiplied by the likelihood ratio P1(Y)/P0(Y). (2022) 114015 This pseudoexpectation is proportional to a true expectation, albeit over another distribution. Thus it is positive semideﬁnite, E[q2] ⩾0. Similarly, if P(x) and therefore P(x\|Y) are supported on x satisfying some constraint fi(x) = 0, then E[fiq] = 0 for any q. 5.7. Pseudocalibration and clever planted models To do this for q(x) of degree d, they typically need to preserve terms in Y up to some suﬃcient degree D > d. J. Stat. Mech. (2022) 114 Showing that E remains positive semideﬁnite after this projection, and that it contin- ues to satisfy the constraints E[fi] = 0, can involve summing over many combinatorial terms. This was ﬁrst done for the planted Clique problem [111]. While each applica- tion since then has involved special-purpose calculations, several conjectures [112] oﬀer general principles by which this program might be extended. at. Mech. (2022) 114015 The projection of E[1 \| Y ] = P1(Y )/P0(Y ) into low-degree polynomials in Y is of its own interest: it is the low-degree likelihood ratio. If it is usually close to 1 in the null model but is large in the planted model, then it provides a polynomial-time hypothesis test for distinguishing between these two. Thus showing that it has bounded variance in the null model is in itself evidence of computational hardness [113]. In particular, [114] showed that the degree-D likelihood ratio fails to improve the bound on the SK model for any D = o(n/log n). This does not in itself prove that SoS fails up to this degree, but the two approaches are closely related. We conclude this section by discussing the choice of planted model. Proving that refutation is hard might require a clever way to hide a solution, as opposed to the standard spiked matrices and tensors. For instance, to prove their SoS lower bounds on the Sherrington–Kirkpatrick model, [106] related a planted model proposed by [109] where a random subspace (i.e. the low-energy subspace) contains a Boolean vector to a model of Gaussian random vectors, where in the planted case these vectors belong to two parallel hyperplanes. More generally, there is a long history in physics and computer science of ‘quiet’ planting, in order to make the solution as diﬃcult as possible to detect [66, 115]. The quieter the planting, the harder it is to distinguish from the null model. In this case, we want the planting to be computationally quiet [114], and in particular to match the low- degree moments of the null distribution. 5.7. Pseudocalibration and clever planted models Moreover, (59) gives any function of x and Y the expectation over the null model that it would have in the planted model, This pseudoexpectation is proportional to a true expectation, albeit over another distribution. Thus it is positive semideﬁnite, E[q2] ⩾0. Similarly, if P(x) and therefore P(x\|Y) are supported on x satisfying some constraint fi(x) = 0, then E[fiq] = 0 for any q. q Moreover, (59) gives any function of x and Y the expectation over the null model that it would have in the planted model, EY ∼P0E[q(x, Y )] = E(x,Y )∼P0 P1(x, Y ) P0(x, Y ) q(x, Y ) = E(x,Y )∼P1q(x, Y ). (60) (60) where we took the average over Y as well as x. On the other hand, for individual Y we have some trouble. For instance, (59) gives E[1 \| Y ] = P1(Y )/P0(Y ), the likelihood ratio instead of 1. This would make it easy to catch the Charlatan whenever the null and planted models can be distinguished information-theoretically. Moreover, while the planted model guarantees that Y has a solution x, most Y drawn from the null model have no such solution. In that case we have P1(Y) = 0, and the posterior distribution P1(x\|Y) is undeﬁned. We can ﬁx both these problems by projecting E[q(x) \| Y ] into the space of low-degree polynomials, both in x and in Y. In other words, we take its Taylor series in x and Y up to some degree. For Boolean variables, this is equivalent to keeping just the low- frequency part of the Fourier spectrum; in some cases, we might project onto a suitable 34 Disordered systems insights on computational hardness set of orthogonal polynomials. This preserves the appearance (60) of the planted model for functions of low degree in x and Y. If all goes well, this projection smooths the likelihood ratio, keeping it concentrated around its expectation 1. It also smooths the posterior distribution P1(x\|Y) as a function of Y, extending it from the small set of Y produced by the planted model (for instance, the few instances of the SK model where E0 = −1) to the more generic Y produced by the null model. However, the Charlatan has to preserve enough of the dependence on Y to make E[q \| Y ] convincing. 5.7. Pseudocalibration and clever planted models For instance, rather than the usual spiked model where we add a rank-1 perturbation to a Gaussian random matrix J—which disturbs the entire spectrum—we can plant a large eigenvalue more quietly by increasing the eigenvalue of a speciﬁc eigenvector [116]. https://doi.org/10.1088/1742-5468/ac9cc8 5.8. Optimal algorithms and the curious case of tensor PCA We have talked a lot about what SoS algorithms cannot do. But for many problems they seem to be optimal, performing as well as any polynomial-time algorithm can. For Max Cut and the Sherrington–Kirkpatrick model, we have seen evidence that this is the case even at degree 2. 35 Disordered systems insights on computational hardness Thus in many cases, SoS algorithms seem to succeed or fail at the same place where physics suggests a hard/easy transition. Even when these thresholds do not coincide exactly, they often have the same scaling and thus diﬀer by a constant. For example, degree-2 SoS—also known as the Lov´asz ϑ function—can refute graph colorings in random regular graphs within a factor of 4 of the Kesten–Stigum transition [117], and it is possible that higher-degree SoS does better. While refuting the existence of a planted solution lets SoS solve the detection problem—distinguishing the null from a planted model—a reﬁnement of this idea often yields algorithms for reconstruction as well. Roughly speaking, if we can refute the existence of a solution when it does not exist, we can often ﬁnd it when it does [112]. J. Stat. Mech. (2022) 114 To see how this works, consider a planted model, and let x∗denote the ground truth. Let φ(x) be some polynomial for which φ(x∗) ⩽φ∗: for instance, in PCA, φ(x) could be the ℓ2 distance between the signal matrix \|x⟩⟨x\| and the observed matrix Y. Now suppose there is a degree-d refutation of the claim that there are any good solutions far from the ground truth: that is, a proof that if φ(x) ⩽φ∗then \|x −x∗\|2 ⩽ε. Then any degree-d pseudoexpectation must claim that \|E[x] −x∗\|2 ⩽ε, and E[x] is a good estimate of x∗. at. Mech. (2022) 114015 This approach yields eﬃcient algorithms for many problems [118, 119], including tensor PCA [120]. But for tensor PCA in particular, a curious gap appeared between algorithms and physics. Recall from section 2.1 that tensor PCA, a.k.a. the spiked tensor model, is a planted model of p-index tensors deﬁned by h. (2022) 114015 (61) Y = λu⊗p + J. Here λ is the signal-to-noise ratio, the planted vector u is normalized so that \|u\|2 = n, and the noise tensor J is permutation-symmetric with Gaussian entries N (0, 1). The information-theoretic transition occurs at λ = λcn−(p−1)/2 for a constant λc depending on p and u’s prior [52, 121]. 8 Our notation ≳and ≲suppresses logarithmic factors. These are consequences of matrix Chernoﬀbounds, and could probably be removed. https://doi.org/10.1088/1742-5468/ac9cc8 5.8. Optimal algorithms and the curious case of tensor PCA The best known polynomial-time algorithms, on the other hand, require a consider- ably larger signal-to-noise ratio, λ≳n−p/4. One such algorithm, called ‘tensor unfolding,’ reinterprets Y as a matrix and iteratively applies PCA to it. For p = 4, for instance, we treat Y as an n2 × n2 matrix Yij,kℓand ﬁnd its leading eigenvector v. Since v ≈u ⊗u, we then treat v as an n × n matrix and estimate u as its leading eigenvector. At each stage we unfold the tensor into a matrix which is as square as possible. /4 Other algorithms, that also succeed for λ≳n−p/4, can be derived directly from sum- of-squares [122]. Conversely, SoS lower bounds suggest that there is no polynomial-time algorithm if λ ≲n−p/4, so this appears to be the algorithmic threshold [123].8 On the other hand, physics-based algorithms such as belief propagation and its asymptotic cousin AMP, as well as Langevin dynamics, all fail unless λ≳n−1/2, mak- ing these algorithms suboptimal whenever p ⩾3 [121, 124]. This does not contradict conjectures of optimality from section 4.4 as those were restricted to the scaling of parameters corresponding to the information-theoretical regime which in this case is https://doi.org/10.1088/1742-5468/ac9cc8 36 Disordered systems insights on computational hardness λ ≈n−(p−1)/2. Never-the-less, focusing on the regime discussed here, does sum-of-squares know something that physics does not? ( 1)/2 λ ≈n−(p−1)/2. Never-the-less, focusing on the regime discussed here, does sum-of-squares know something that physics does not? ( )/ This conundrum has a satisfying answer [125]: in the scaling regime λ≳n−(p−1)/2 we were using the wrong physics. Belief propagation keeps track of pairwise correlations. When we compute the Bethe free energy, we pretend that the Gibbs distribution, i.e. the posterior distribution P(x\|Y), has the form P(x) = i μi(xi) × (i,j) μij(xi, xj) μi(xi) μj(xj) (62) (62) J. Stat. Mech. (2022) 1140 where μi and μij are one- and two-point marginals. Minimizing the resulting free energy is equivalent to ﬁnding ﬁxed points of belief propagation [126]. where μi and μij are one- and two-point marginals. Minimizing the resulting free energy is equivalent to ﬁnding ﬁxed points of belief propagation [126]. But when p ⩾3, it becomes vital to consider correlations between clusters of p vari- ables. This gives rise to a hierarchy of free energies due to [127]. For p = 3, for instance, we assume that the Gibbs distribution has the form at. Mech. https://doi.org/10.1088/1742-5468/ac9cc8 5.8. Optimal algorithms and the curious case of tensor PCA (2022) 114015 P(x) = i μi × (i,j) μij μi μj × (i,j,k) μijk μi μj μk μij μjk μik (63) (63) (where for readability we suppress (xi), (xi, xj), and so on). At each level of this approx- imation, we correct for overcounting smaller clusters. Taking the logarithm of this expression and averaging over x gives an inclusion-exclusion-like formula for the entropy. (where for readability we suppress (xi), (xi, xj), and so on). At each level of this approx- imation, we correct for overcounting smaller clusters. Taking the logarithm of this expression and averaging over x gives an inclusion-exclusion-like formula for the entropy. ch. (2022) 114015 There are several ways one might turn this into a spectral algorithm. One is to write an iterative algorithm to minimize the free energy. This gives rise to a generalization of belief propagation in which each variable sends messages to clusters of up to p −1 variables with which it interacts [128, 129]. One could then linearize this message-passing algorithm around a trivial ﬁxed point, producing a operator analogous to the non- backtracking operator for belief propagation [130, 131]. An alternate approach is to compute the Hessian of the free energy at a trivial ﬁxed point, generalizing the use of the Bethe Hessian for spectral clustering in graphs [132]. This gives rise to the following operator. For a set U = {s1, . . ., sp} with \|U\| = p, let YU denote Ys1,...,sp. Fix ℓ⩾p/2. Then deﬁne the following n ℓ -dimensional operator, whose rows and columns are indexed by sets S, T with \|S\| = \|T\| = ℓ: MS,T = YS△T if \|S △T\| = p 0 otherwise, (64) MS,T = YS△T if \|S △T\| = p 0 otherwise, MS,T = YS△T if \|S △T\| = p 0 otherwise, (64) where △again denotes the symmetric diﬀerence. where △again denotes the symmetric diﬀerence. where △again denotes the symmetric diﬀerence. The spectral norm of M can be used as a test statistic to distinguish the planted model from the null model where λ = 0. In addition, the leading eigenvector of M points approximately to the minimum of the free energy, and a voting procedure yields a good estimate of the signal u. This yields polynomial-time algorithms for detection and reconstruction whenever λ≳n−p/4, matching the SoS threshold. Thus the marriage of algorithms and statistical physics is redeemed [125]. 6. Conclusion What does the future hold? As our understanding of algorithms deepens, we hope to understand the universal characteristics that make problems easy or hard, unifying larger and larger classes of polynomial-time algorithms and connecting them rigorously with physical properties of the energy landscape. Very recently, [136] connected the low- degree likelihood ratio with the Franz–Parisi potential, adding to the evidence that free energy barriers imply computational hardness. We will know much more in a few years than we know now. J. Stat. Mech. (2022) 1140 5.8. Optimal algorithms and the curious case of tensor PCA 37 Disordered systems insights on computational hardness The same analysis matches a continuum of subexponential-time algorithms at smaller values of λ [133] and yields a simpler refutation of random constraint satisfaction prob- lems at high clause densities [134]. These ‘Kikuchi matrices’ have additional applications, e.g. [135]. Acknowledgments Mech. 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https://openalex.org/W4388446607	https://bmcnephrol.biomedcentral.com/counter/pdf/10.1186/s12882-023-03352-6	English	null	Correction: Atypical histological abnormalities in an adult patient with nephronophthisis harboring NPHP1 deletion: a case report	BMC nephrology	2,023	cc-by	407	Correction: Atypical histological abnormalities in an adult patient with nephronophthisis harboring NPHP1 deletion: a case report Maiko Akira1, Hitoshi Suzuki1*, Arisa Ikeda1, Masako Iwasaki1, Daisuke Honda1, Hisatsugu Takahara1, Hisaki Rinno1, Shigeki Tomita2 and Yusuke Suzuki3 BMC Nephrology BMC Nephrology BMC Nephrology Akira et al. BMC Nephrology (2023) 24:327 https://doi.org/10.1186/s12882-023-03352-6 Open Access Open Access Reference 1. Akira M, et al. Atypical histological abnormalities in an adult patient with nephronophthisis harboring NPHP1 deletion: a case report. BMC Nephrol. 2021;22:261. https://doi.org/10.1186/s12882-021-02466-z. The original article can be found online at https://doi.org/10.1186/s12882- 021-02466-z. shitoshi@juntendo.ac.jp 1 Department of Nephrology, Juntendo University Urayasu Hospital, 2‑1‑1 Tomioka, Urayasu‑Shi, Chiba 279‑0021, Japan 2 Department of Pathology, Juntendo University Urayasu Hospital, Chiba, Japan 3 Department of Nephrology, Juntendo University Faculty of Medicine, Tokyo, Japan © 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. Correction: Nephrology 22, 261 (2021) https://doi.org/10.1186/s12882-021-02466-z Following publication of the original article [1], we found that there were misprint in the Fig. 2a. The correct Fig. 2 is given below: g g The original article has been corrected. . Open Access T d d Page 2 of 2 Akira et al. BMC Nephrology (2023) 24:327 Akira et al. BMC Nephrology Fig. 2 Genetic analysis of the NPHP1 gene. a No exons from NPHP1 were amplified in this patient. b Multiplex ligation-dependent probe amplification analysis indicated complete deficiency of the NPHP1 gene Fig. 2 Genetic analysis of the NPHP1 gene. a No exons from NPHP1 were amplified in this patient. b Multiplex ligation-dependent probe amplification analysis indicated complete deficiency of the NPHP1 gene
https://openalex.org/W4225552022	https://zenodo.org/records/6407875/files/WJARR-2022-0156.pdf	English	null	Mental health well -being of Frontline Health Care Professionals (FHCP) managing Covid-19 cases in two selected tertiary hospitals in Bayelsa and Edo States, Nigeria	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	7,479	Abstract This study investigated the mental health well-being of frontline health care professionals managing COVID-19 cases in two selected tertiary hospitals in Bayelsa and Edo States. The descriptive analytical survey design was adopted. The population of this study comprises of all health care professionals managing COVID -19 cases in Bayelsa and Edo States. The purposive sampling technique was utilized to sample 181 respondents made up of 39 from Niger Delta University Teaching Hospital (NDUTH) Okolobiri, Bayelsa and 142 from Irrua Specialist Hospital, Edo State. The instrument for data collection is a questionnaire titled “Warwick-Edinburgh Mental Well-being Scale (WEMWBS) and New Well-being Measure Tools adapted by the researchers”. The responses were structured into Likert four-point scale of Every time (ET), Often (O), Rarely (R) and Never (N). The instrument was validated by the researchers and other experts. The reliability of the instrument was ascertained using the Cronbach alpha statistic to obtain a reliability coefficient of 0.87. One research question was raised and three hypotheses formulated to guide the direction of this study. The ethical clearance was obtained from relevant authorities. The data collected were analyzed using descriptive statistics of mean, standard deviation, bar chart and inferential statistics of t-test, and One-way analysis of variance. The study found among others that participants in Bayelsa and Edo States have a negative mental health well-being in managing COVID 19 cases. It was concluded amongst others the need for training and re-training of health care professionals, incentives and allowances be given to health care professionals, etc. Keywords: Mental Health Well-being; Frontline Health Care Professionals; Tertiary Hospitals; COVID-19 Mental health well -being of Frontline Health Care Professionals (FHCP) managing Covid-19 cases in two selected tertiary hospitals in Bayelsa and Edo States, Nigeria Esther Emarobebh Muojekwu (FRSPH) 1, , Deliverance Brotobor 2 and Omotayo Bunmi Omojola 3 1 Department of Mental Health and Psychiatric Nursing, Niger Delta University, Amassoma Bayelsa State, Nigeria. 2 Department of Nursing, Ambrose Ali University, Ekpoma, Edo State, Nigeria. 3 Department of Clinical and Administration, Wellstar Hospitals Ltd. Abuja, Nigeria. World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 Publication history: Received on 11 January 2022; revised on 27 February 2022; accepted on 01 March 2022 Article DOI: https://doi.org/10.30574/wjarr.2022.13.3.0156 Mental health well -being of Frontline Health Care Professionals (FHCP) managing Covid-19 cases in two selected tertiary hospitals in Bayelsa and Edo States, Nigeria Esther Emarobebh Muojekwu (FRSPH) 1, , Deliverance Brotobor 2 and Omotayo Bunmi Omojola 3 1 Department of Mental Health and Psychiatric Nursing, Niger Delta University, Amassoma Bayelsa State, Nigeria. 2 Department of Nursing, Ambrose Ali University, Ekpoma, Edo State, Nigeria. 3 Department of Clinical and Administration, Wellstar Hospitals Ltd. Abuja, Nigeria. World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 Publication history: Received on 11 January 2022; revised on 27 February 2022; accepted on 01 March 2022 Article DOI: https://doi.org/10.30574/wjarr.2022.13.3.0156 Mental health well -being of Frontline Health Care Professionals (FHCP) managing Covid-19 cases in two selected tertiary hospitals in Bayelsa and Edo States, Nigeria Esther Emarobebh Muojekwu (FRSPH) 1, , Deliverance Brotobor 2 and Omotayo Bunmi Omojola 3 World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 Publication history: Received on 11 January 2022; revised on 27 February 2022; accepted on 01 March 2022 Article DOI: https://doi.org/10.30574/wjarr.2022.13.3.0156 epartment of Mental Health and Psychiatric Nursing, Niger Delta University, Amassoma Bayelsa State, Nigeria. opyright © 2022 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attr Corresponding author: Muojekwu Esther Emarobebh Department of Mental Health and Psychiatric Nursing, Niger Delta University, Amassoma Bayelsa State, Nigeria. Corresponding author: Muojekwu Esther Emarobebh Copyright © 2022 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. or(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 no significant difference in the mental health coping strategies for FHCP managing COVID-19 cases based on marital status in two tertiary hospitals Bayelsa and Edo States. no significant difference in the mental health coping strategies for FHCP managing COVID-19 cases based on marital status in two tertiary hospitals Bayelsa and Edo States. At the time of this study over 218 countries of the world were affected including Nigeria; as of 23rd November 2020 the global prevalence of COVID 19 reported cases was over 54 million out of which 1.4 million deaths occurs worldwide, in Africa the reported cases were over 1.2 million, where as in Nigeria total cases of 66 thousand occurred with over 1 thousand deaths in the country [1, 2, 3]. Moreover, in the study areas; Bayelsa and Edo the prevalence as of 23rd November 2020 reported cases were 445 & 2,694 respectively. This has attributed to 21 deaths in Bayelsa and 111 deaths in Edo within this period [2, 1]. The upsurge in the incidence of COVID-19, the critical conditions, deaths, unpreparedness, emergencies etc., could possibly impact on the mental well-being of the healthcare professionals at the frontline managing COVID-19. These could be as result of associated stress of workload available at the isolation centers. Evidences revealed that the frontline healthcare professionals might be exposed to various mental disorders including; anxiety, depression, fear, panic attack, burnout, grief etc., and these could influence on their mental well-being, thereby affecting healthcare delivery to the patients [4, 4, 6]. The current global health crises have some features that exposes the healthcare professionals working at different isolation centers of the country, as they faced huge burden on their total well-being including; physical, emotional and Psychosocial health. The issue of occupational hazard associated with or experienced by healthcare workers has been a challenge among these working population in the time past. This is due to the type of enormous hazardous procedures involved in this environment that could jeopardize the workers’ health. The Frontline Health Care Professionals (FHCP) managing COVID-19 cases in Nigeria are not left out, especially this time of the outbreak that could require more efforts, skills and attention. World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 Several evidences from the literature revealed that FHCP are exposed to both physical, biological, chemical, ergonomics and mental hazards given to the huge number of patients, workload, shortage of staffs, demand of work etc. [4,5,6]. Although, little or no attention is given to this aspect of mental well-being this period, since optimum mental health well-being would ensure utmost productivity in terms of service delivery, quality care and positive care outcome of patients. Hence, this could pose greater health implications on the health care workers’ mental well-being, which would also affect the care rendered to their patients. Study carried out by United Nation Development Programme [UNDP] [7] shows that the pandemic is capable of causing devastating psycho-social, economic, political crisis etc. In addition, studies from World Health Organization and Loiwal [4,5] shows that the health care professionals at the frontline tackling the COVID-19 pandemic are exposed to mental disorders like anger, depression, grief, burnout. However, despite what outcome anticipated being the aftermaths, it’s necessary to note that COVID-19 could negatively impact on both mental and psychosocial well-being of the FHCP managing COVID-19 cases. Therefore, based on the above facts the need to investigate on the mental health well-being of FHCP so as to provide relevant answers and coping strategies to assist them cope with the associated burden of COVID-19 while caring for their patients. 1. Introduction The world is in the scourge of Corona virus (COVID-19) Pandemic. This COVID-19 outbreak is a public health emergency of international concern that involves both the current and anticipated consequences. These consequences could impact on the health workers’ mental health and well-being. The frontline health care professionals (FHCP) are providers of care to patients on admission for COVID-19; to ensure health is restored and the virus is contained. This study examined the Mental Health Well-being of FHCP managing COVID-19 Cases in the two Tertiary Hospitals of Bayelsa and Edo States, Southern Nigeria. One corresponding research question was raised and three hypotheses formulated to ascertain the differences that exist between the variables. These differences when identified will provide baseline information on the topic. The study addressed the following hypotheses: H1: There is no significant difference in mental health disorder that could affect the mental health of FHCP managing COVID-19 Cases based on age in the two Tertiary Hospitals of Bayelsa and Edo States; H2: There is no significant difference in the factors responsible for mental disorders associated with FHCP managing COVID-19 cases based on gender in two tertiary hospitals in Bayelsa and Edo States; H3: There is 1.1. Mental Health Well-being Furthermore, evidence has shown that mental health well-being of an individual is centered on three distinctive mechanisms of which include the following; first, emotional well-being; this has to do with happiness, interest in life and satisfaction. Secondly, psychological well-being which involves liking most part of one’s own personality, being good at managing the responsibilities of daily lives and being satisfied with one’s life. Finally, in this category is the social well-being; which has to do with positive functioning and it involves one’s contribution to the society; i.e. social integration, social actualization and social coherence [11, 12]. Therefore, it is clear to note that one’s mental health could be affected by what happens in their environment. For instance, events, disasters and crises due to an outbreak, work environment or condition (stressors) as in the case of health care workers facing tremendous stress at the frontline of this disease outbreak. mental disorders found among health care professionals managing the pandemic in various isolation centers [10]. Vinkers et al [11] posit that the health care workers are more susceptible and exposed to stress that could affect their mental health well-being. Furthermore, evidence has shown that mental health well-being of an individual is centered on three distinctive mechanisms of which include the following; first, emotional well-being; this has to do with happiness, interest in life and satisfaction. Secondly, psychological well-being which involves liking most part of one’s own personality, being good at managing the responsibilities of daily lives and being satisfied with one’s life. Finally, in this category is the social well-being; which has to do with positive functioning and it involves one’s contribution to the society; i.e. social integration, social actualization and social coherence [11, 12]. Therefore, it is clear to note that one’s mental health could be affected by what happens in their environment. For instance, events, disasters and crises due to an outbreak, work environment or condition (stressors) as in the case of health care workers facing tremendous stress at the frontline of this disease outbreak. Furthermore, studies have recognized that health care professionals are exposed to health challenges like anxiety, fears, and this is due to shortage of staffs, lack of PPE and family challenges, burnout, and excessive workload owing to the increasing number of patients in their care [13,14,15]. 1.1. Mental Health Well-being Also, studies conducted in China revealed that health care professionals managing COVID-19 cases are at a higher risk of stress that exposed their mental health to mental disorders like anxiety, depression, sleep disturbances etc., [15,16]. Hence, the health care professionals’ managing COVID-19 pandemic are not left out in this case, as enormous unprecedented events and the impacts on the general well-being of the population which has great health consequences that could impose detrimental effects on their mental health well-being. 1.2. Mental Health Disorders and COVID-19 Mental health disorders are ranges of conditions that influences on individual’s mood, thinking and behavior. According to Frich and Frich [17] there are behaviors or psychological syndrome or patterns associated with distress or disability or increased risks of suffering, death, pain or loss of freedom. The emotional, psychological and social aspect of an individual well-being could be affected which could result to changes in their behavior. Also, mental health disorders are characterized by addition of abnormal thoughts, emotions, behavior and relationship with others; which includes a range of conditions with various symptoms [18]. These symptoms may include the following; depression, anxiety, mood swing, burnout, fear etc., [18]. Generally, COVID-19 pandemic has a devastating effect on the FHCP’s mental health, though not just on their mental health. This is due to the current measures in place for containing the virus including social distancing, isolation and others. The FHCP are separated from their families and loved ones to face these crises; thereby experiencing the followings: boredom; loneliness and burnout; anxiety; fear; depression; panic attack; sleep disturbances and distress. A study by Pappa et al [19] revealed that huge proportion of frontline health care professional’s mental health well-being are jeopardized by COVID-19 pandemic. Similar evidence from virus outbreak revealed that frontline health care professionals reported challenges like; stigmatization, isolation, suffering, loneliness and sadness [20]. The W.H.O has also made efforts to contain the spread of the virus through measures like; identification, testing infected persons, and also on the verge of developing drugs and vaccines for treatment. Meanwhile, to achieve the W.H.O goal of containing the spread of the virus, the health care professionals are needed at the frontline to help manage, control and mitigate the effects of the disease, so as to contain the virus from spreading. Although, the main focus is on preventing the transmission of the virus, containing the infection and saving lives, little or no attention is placed on the mental health of these health care professionals managing infected persons at the various isolation centers or hospitals. The evidences so far have shown that almost every aspect of the FHCP well-being is jeopardized during this period and this is likely to impact on the workers’ health afterwards. However, it is clear that COVID-19 pandemic could cause a rapid widespread of fear, anxiety, depression, loneliness, panic attack, sadness etc. 1.1. Mental Health Well-being The concept of mental health well-being as defined by the world health organization [W.H.O] [8] “is a state of well-being in which the individual realizes his or her own abilities, can cope with the normal stressors of life, can work productively and fruitfully and its able to make a contribution to his or her community. Well-being is a state of being or feeling comfortable, happy and healthy or feeling well. There are some characteristics that can properly define well-being. These includes the following; having a quality mental health, optimum life and ability to manage or cope with stress that interferes with daily activities. Therefore, mental health wellbeing could be defined as a state of comfort where an individual identifies their potential on how to cope and work maximally under stressful condition of life, so as to positively impact their society. In addition the concept of mental well-being as addressed by Galderisi, et al [9] “ is a dynamic state of internal equilibrium which enables individuals to use their abilities in harmony with universal values of the society, basic cognitive and social skills , ability to recognize, express and modulate one’s own emotion, as well as empathize with others, flexibility and ability to cope with adverse life events and function in social roles; and harmonious relationship between body and mind represents important components of mental health which contributes to varying degrees to the state of internal equilibrium. This implies that a mentally healthy individual can equally experience normal human emotions like; fear anger sadness worry anxiety grief etc. Also, at the same time having enough strength to recover to the dynamic state of internal equilibrium. Thus, is likely that when an individual or a health care worker have no resilience to recover from the dynamic state of equilibrium, then problem can set in and expose the person to unhealthy mental health or mental disorder. It is quite indicative that with the alarming increase in the number of new cases and mortality daily also attributed to the cause of 46 World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 mental disorders found among health care professionals managing the pandemic in various isolation centers [10]. Vinkers et al [11] posit that the health care workers are more susceptible and exposed to stress that could affect their mental health well-being. 1.4. Mental Health Coping Strategies for FHCP The frontline health care professionals’ managing COVID-19 disease need some coping strategies to help them face the challenges of stress at the work place or build their resilience towards the effects of stress on their mental well-being. However, intervention should be focused on providing coping strategies; including preventive measures and training of health care professionals on other measures to help them cope with the crisis at the frontline. The world health organization [WHO] [4] recommendation on psychological and mental health measures to support the workers this period through promoting the mental health well-being is focused on; improving the worker’s skills through information, communication and social supports system. FHCP are encouraged to adopt positive behaviors or attitudes that would promote a positive mental well-being. These includes; maintaining healthy lifestyle, resting, virtually getting in touch with families and friends, ensuring team work and connecting with colleagues and other health care teams as form of social supports while at the workplace (hospitals). Moreover, studying the differences that exist between marital status of FHCP; study from Liu et al [28] revealed that the female married HCPs’ in general are known for lack of supports, this due to work-family conflict that exist among them. That is, the married HCPs’ lack coping strategies. In contrast, Badahdah et al [31] reported that marital status has no impact on the mental well-being of FHCP managing COVID-19 disease, since the married HCPs’ reported less stress. Lazarus and Folkman [32] highlighted two forms of coping strategies identified as; emotional focused coping strategies and problem focused coping strategies. The emotional coping strategies (accepting responsibilities, distancing and positive reappraisal), the individual worker manages his or her emotional response to threat or challenges at the work environment that lead to stress. Whereas, problem focused coping strategies which includes; confrontive coping strategies, self-control, plan problem solving and seeking social supports helps the worker to adjust or reduce the threats in the work environment in a suitable way. However, for the FHCP to cope in the face of COVID -19 Challenges or threats in their workplace, there is need the workers engage in cognitive-behavioral approaches which may likely help them restructure their emotions and behavior towards the threat. The FHCP need to be properly equipped with social supports needs and skills that would enhance their ability of utilizing resilience whilst managing COVID-19 cases at the different isolation Centre’s or hospitals. 1.4. Mental Health Coping Strategies for FHCP Thus, this would help the FHCP maintain a positive mental health that would equally improve their health and well-being. Moreover, supports from both Governmental and non- governmental organizations in terms of providing resources: these could be work equipment’s like PPEs to ensure the workers safety, provision of their basic needs and incentives etc. Also, supports from colleagues and families are required this period, for example emotional supports. Finally, to ameliorate the devastating effect of COVID-19 on the FHCP mental well-being, there is need their adhere to certain coping strategies including; the best coping strategies that suit each individual worker; improve their stress management skills and also adhere to the recommended WHO strategies, so as to maintain a positive mental well-being not just during the pandemic but in the long run. 1.3. Factors of Mental Health Disorders Nevertheless, there are some responsible factors of psychosocial stress among the FHCP which includes but not limited to; work condition and environment; workplace discrimination or stigmatization; management issues; lack of motivation and institutional/governmental policies. A study on COVID-19 parameters/stress at the workplace on different working population reveals that occupational stress across all sectors of the workforce are caused by factors such as: workload, role conflict, role ambiguity, lack of social support, lack of motivation, career, etc., [21]. The impact of occupational stress on the FHCP managing COVID-19 cases in the different hospitals could be detrimental to their mental health well-being, of which could have a long-term effect on the workers’ mental well-being. 47 World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 Several studies have shown that stress among the FHCP during the pandemic could occur as a result of managerial and organizational factors including: lack of PPE’s; lack of information or communication; shortage of equipment for managing patients e.g. (ventilators and bed space); shortage of staff and excessive workload; unpreparedness or fear of facing challenges of the pandemic. This could also compromise their mental well-being and adjustment in the workers’ daily social and family lifestyles [14,13,15]. Also, there are other biological factors of mental health disorders such as; age, gender etc. Although, few studies have shown the differences in age and mental health disorders; such as depression and anxiety. Neuber et al, [22] and Lowe et al [23] noted that older health care professionals (HCPs’) experiences lesser stressor, negative emotions and better mental health well-being, whereas the younger HCPs’ are at greater risk of mental health disorders. This is due to the optimistic nature of adults in managing their emotions. Whereas, Bruine de bruine [24] posit that the older adults are at lower risk of mental disorder. On the contrary, studies have also shown that there is no difference in the age that could be affected by mental disorders [25,26]. This means that all age groups are at risk irrespective of being old or young. Although, studies showed that difference exist between genders of FHCP; that is the female HCPs’ are likely to be exposed to the risk of infections, stress or other associated factors including mental disorders [27,28,29,30]. On the contrary, Bahahdah [31] noted that there is no difference between genders of FHCP managing COVID-19 disease. World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 Scale (WEMWBS); the new well-being measure tool (Tennant et al [33] was adapted by the researchers”. The responses were structured into Likert four-point scale of Every time (ET), Often (O), Rarely (R) and Never (N). The instrument was validated by the researchers and other experts in the field of mental health well-being and stress. The reliability of the instrument was ensured through the Cronbach alpha statistic and a reliability coefficient of 0.87 was obtained. A research question and three hypotheses were formulated for this study. A written consent was sought from respective participants before collection of data. Duration for the distribution and retrieval of questionnaire i.e. data collection lasted for four (4) weeks (2nd July – 28th July 2021). 2.1. Statistical analysis Descriptive statistics of mean and standard deviation were used to calculate for socio-demographic characteristics and the mental health well-being of FHCP. Whereas, inferential statistics of t-test, and One-way analysis of variance were used to test the hypotheses, and also drawn conclusions in this study with a significant level of P< 0.05. SPSS software version 20 was used for data analysis. Table 1 shows the mental health well-being of FHCP managing COVID-19 Cases in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria. The data reveals that items numbers 1,2,3,4,6,8,9,11 and 13 have mean scores less than the 2. Material and methods This study was a descriptive analytical survey conducted in two tertiary hospitals in southern Nigeria; Niger Delta University Teaching Hospital Bayelsa State (NDUTH) and Irrua Specialist Hospital Edo State, Nigeria. The total population of this study comprises of all FHCP managing COVID-19 cases in the two hospitals. A purposive sampling technique was used to draw a sample of 39 from NDUTH and 142 from Irrua Specialist hospitals making a total of 181 sample size. The instrument used for data collection was a questionnaire titled “Warwick-Edinburgh Mental Well-being 48 World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 criterion mean of 2.50 which means that these items were rejected by the participants. While items 5,7,10,12 and 14 have mean scores above 2.50 which means that the participants accepted these items. The participants in Bayelsa State had a grand mean of 2.38 which is less than the 2.50 criterion mean. This means that the participants in Bayelsa State rejected most of the items listed on the table. This means that the participants in Bayelsa State have a negative mental health well-being in managing COVID 19 cases. Whereas in Edo state the result reveals that items 1,6,7,8,10,12,13 and 14 have mean scores less that the required minimum mean criterion of 2.50, this means that the items were rejected by the respondents. While, items numbers 2,3,4,5,9 and 11 have mean scores above 2.50 which means that these items were accepted by the respondents. The participants in Edo State had a grand mean of 2.43 which is less than the 2.50 criterion mean. This means that participants in Edo State also have a negative mental health well-being in managing COVID 19 cases. Figure 1 Demographic Characteristics of Participants Figure 1 Demographic Characteristics of Participants Figure 1 Demographic Characteristics of Participants Figure 1 shows the demographic characteristics of participants in this study. From the above result, for Gender 131 representing (71.98%) of the respondents were female and 51 (28.02%) were male, 75 representing (41.21%) were between the ages of 25-30 years, 44 (24.18%) were within the ages of 31-35 years, between the ages of 36-40 represent 45(24.73%), between the ages of 41-45 represent 14 (7.69%) and 4 (2.20%) were within the age range of 46 and above. Also, 105 representing (57.69%) were married, 73 representing (40.11%) of the participants were single, while 4 (2.20%) were divorced. H01: There is no significant difference in mental health disorder that could affect the mental health of FHCP managing C0VID-19 Cases based on age in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria. 3. Results 3. Results Research Question One: What is the mental health well-being of FHCP managing COVID-19 Cases in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria? Table 1 Mental Health Well-being of FHCP managing COVID-19 Cases in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria S/No Item Description Bayelsa Edo Mean Decision Mean Decision 1 I am positive that my tomorrow will be better 2.44 Reject 2.22 Reject 2 I have been helpful in assisting the patients 2.21 Reject 2.51 Accept 3 I have been feeling relaxed at work 2.33 Reject 2.50 Accept 4 I have been feeling involved in other peoples affairs 2.03 Reject 2.58 Accept 5 I have had energy to spare during stress 2.79 Accept 2.50 Accept 6 I have been able to manage my worries myself without assistance 2.44 Reject 2.34 Reject 7 I have been able to express my ideas for better understanding 2.54 Accept 2.43 Reject 8 I have been feeling good about myself 2.08 Reject 2.35 Reject 9 I am confident of my skills 2.23 Reject 2.61 Accept 10 I have good interpersonal relationship with my colleagues at work 2.62 Accept 2.38 Reject 11 I have been able to take decision concerning my life 2.31 Reject 2.50 Accept 12 I have been feeling loved by my loved ones 2.64 Accept 2.35 Reject 13 I have been interested in learning new things 2.21 Reject 2.37 Reject 14 I have been feeling happy with people around me 2.50 Accept 2.42 Reject Grand Mean 2.38 2.43 (X) (X) Research Question One: What is the mental health well-being of FHCP managing COVID-19 Cases in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria? bl l l h ll b f h l f l d d Research Question One: What is the mental health well-being of FHCP managing COVID-19 Case Hospitals of Bayelsa and Edo States, Nigeria? 49 Table 2 shows ANOVA analysis of mental health disorder that could affect the mental health of FHCP managing COVID- 19 cases based on age in tertiary hospitals in Bayelsa and Edo States, Nigeria. The results indicate that F (4, 177) = 0.002, P=1.000 which is significant at 0.05 alpha level. The sig value of 1.000 is statistically higher than 0.05 alpha level. Hence, the null hypothesis is accepted. This implies that there is no significant difference in mental health disorder that could affect the mental health of FHCP managing COVID - 19 cases based on age in Bayelsa and Edo States in Southern Nigeria. H02: There is no significant difference in the factors responsible for mental disorders associated with FHCP managing COVID-19 cases based on gender in two tertiary hospitals in Bayelsa and Edo States. World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 Between Groups 38.152 2 19.076 0.003 0.997 Within Groups 1291587.041 179 7215.570 Total 1291625.192 181 able 4 One-Way Analysis of Variance (ANOVA) of Mental Health Coping Strategies for FHCP Managin ased on marital status in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria Analysis of Variance (ANOVA) of Mental Health Coping Strategies for FHCP Managing COVID-19 Case status in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria Table 4 shows ANOVA analysis of mental health coping strategies for FHCP managing COVID-19 cases based on marital status in two tertiary hospitals in Bayelsa and Edo States. The above results indicate that F (2, 179) = 0.003, P=0.997 which is significant at 0.05 alpha level. The sig value of 0.997 is statistically higher than 0.05 alpha level. Hence, null hypothesis is accepted. This means that there is no significant difference in the mental health coping strategies for FHCP managing COVID-19 cases based on marital status in Bayelsa and Edo States. World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 able 2 One-Way Analysis of Variance (ANOVA) of Mental Health Disorder That Could Affect the Ment anaging COVID-19 Cases Based on Age in the two Tertiary Hospitals of Bayelsa and Edo States, Nige Table 2 One-Way Analysis of Variance (ANOVA) of Mental Health Disorder That Could Affect the Mental Health of FHCP Managing COVID-19 Cases Based on Age in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria. alysis of Variance (ANOVA) of Mental Health Disorder That Could Affect the Mental Health of FHCP Cases Based on Age in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria. Table 2 One-Way Analysis of Variance (ANOVA) of Mental Health Disorder That Could Affect the Mental Health of FHCP Managing COVID-19 Cases Based on Age in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria. Sum of Squares Df Mean Square F Sig. Between Groups 59.302 4 14.825 0.002 1.000 Within Groups 1401905.468 177 7920.370 Total 1401964.769 181 α= 0.05 Table 2 shows ANOVA analysis of mental health disorder that could affect the mental health of FHCP managing COVID- Table 2 shows ANOVA analysis of mental health disorder that could affect the mental health of FHCP managing COVID- 19 cases based on age in tertiary hospitals in Bayelsa and Edo States, Nigeria. The results indicate that F (4, 177) = 0.002, P=1.000 which is significant at 0.05 alpha level. The sig value of 1.000 is statistically higher than 0.05 alpha level. Hence, the null hypothesis is accepted. This implies that there is no significant difference in mental health disorder that could affect the mental health of FHCP managing COVID - 19 cases based on age in Bayelsa and Edo States in Southern Nigeria. H02: There is no significant difference in the factors responsible for mental disorders associated with FHCP managing COVID-19 cases based on gender in two tertiary hospitals in Bayelsa and Edo States. affect the mental health of FHCP managing COVID - 19 cases based on age in Bayelsa and Edo States in Southern Nigeria. H02: There is no significant difference in the factors responsible for mental disorders associated with FHCP managing COVID-19 cases based on gender in two tertiary hospitals in Bayelsa and Edo States. H02: There is no significant difference in the factors responsible for mental disorders associated with FHCP managing COVID-19 cases based on gender in two tertiary hospitals in Bayelsa and Edo States. World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 50 World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 Table 3 t-test Analysis of Factors Responsible for Mental Disorders Associated with FHCP Managing COVID-19 Cases Based on Gender in two Tertiary Hospitals in Bayelsa and Edo States Gender N Mean Standard Deviation Df tcal Sig (2-tailed) Decision Male 51 31.90 61.94 180 0.244 0.808 Accept Null Hypothesis Female 131 28.42 94.32 (X) s of Factors Responsible for Mental Disorders Associated with FHCP Managing COVID-19 Cases wo Tertiary Hospitals in Bayelsa and Edo States Table 3 t-test Analysis of Factors Responsible for Mental Disorders Associated with FHCP Mana Based on Gender in two Tertiary Hospitals in Bayelsa and Edo States Table 3 shows the t-test analysis of factors responsible for mental disorders associated with FHCP managing COVID-19 cases based on gender in two tertiary hospitals in Bayelsa and Edo States. The results reveals that t-calculated is 0.244 and sig value is 0.808 at 0.05 level of significance. The sig value of 0.808 is statistically higher than critical sig value of 0.05. This means that the null hypothesis which states that there is no significant difference in the factors responsible for mental disorders associated with FHCP managing COVID-19 Cases based on gender in Bayelsa and Edo States is accepted. That is there is no statistically significant difference in the factors responsible for mental disorders associated with FHCP managing COVID-19 cases in Bayelsa and Edo States. H03: There is no significant difference in the mental health coping strategies for FHCP managing COVID-19 cases based on marital status in two tertiary hospitals Bayelsa and Edo States, Nigeria. Table 4 One-Way Analysis of Variance (ANOVA) of Mental Health Coping Strategies for FHCP Managing COVID-19 Cases Based on marital status in the two Tertiary Hospitals of Bayelsa and Edo States, Nigeria Sum of Squares Df Mean Square F Sig. 5. Conclusion The study conclude that a negative mental health well-being exists among FHCP Managing COVID-19 Cases in the selected tertiary hospitals in Bayelsa and Edo States. The FHCP are faced with some challenges at the frontline which has negatively impacted on their mental well-being. The challenges are demand of work, shortage of staff, burnout, stress etc. Also, the findings in the study indicated there is no difference in the marital status and coping strategies of FHCP managing COVID-19 cases, which has led to a gap in this study. Therefore, due to the enormous negative impacts of this virus on the mental well-being of FHCP, there is need to ensure a positive mental health well-being of these professionals at the frontline. Thus, it is recommended that FHCP should adhere to the best coping strategies that suit them the most; they should improve their stress management skills and there is need for relevant authorities or employers to provide them with PPEs, training and retraining packages, incentives and technologies to connect them to their families and loved ones so as to enhance a positive mental health well-being of all FHCP throughout the period of COVID -19 and afterwards. 4. Discussion of Findings coping strategies for FHCP managing COVID-19 Cases based on marital status in Bayelsa and Edo States, Nigeria. Other literatures in this regard has noted the differences that exist in the mental health coping strategies of FHCP managing COVID-19 [31,28]. Thus, no study exists on the differences in marital status and coping strategies that has been conducted in the two states in Nigeria. This study finding shows there is no significant difference in the marital status and coping strategies of FHCP managing COVID-19 cases; therefore, this has created a gap in this present study. Acknowledgments We would want to thank all the frontline health care professionals who took part in this study freely and selflessly. we commend their efforts and dedication during this period. Statement of informed consent Informed consent was obtained from all individual participants included in the study. Statement of ethical approval Ethical approval was sought from the College of health sciences (CHS) NDU, NDUTH and Irrua specialist hospital ethical committees (CHSEC, 15/6/2020). 4. Discussion of Findings The findings in Table 1 shows that FHCP managing COVID-19 Cases in tertiary hospitals in Bayelsa and Edo States have a negative mental health well-being. This implies that the FHCP are faced with some challenges such as; shortage of staff, demand of work, burnout and stress at the frontline which has negatively impacted on their mental health well- being. This finding is in agreement with previous studies by Lai et al and Zhang et al, [15,16] who concluded in their separate studies that health care professionals managing COVID-19 disease are at higher risk of stress that exposed them to mental health disorders like anxiety, depression, sleep disturbances, etc. The results in table 2 shows that there is no significant difference in mental health disorders that could affect the mental well-being of FHCP managing COVID- 19 Cases in Bayelsa and Edo States. This finding is in conformity with evidences from Qiu et al, and Wang et al [25,26] in their separate studies it was concluded that, there is no difference in the age at which health care professionals could be affected by mental disorders such as depression and anxiety etc. This means that all age groups are at risks irrespective of the age differences. Furthermore, result in table 3 shows there is no statistical significant difference in factors responsible for mental disorders associated with FHCP managing COVID-19 Cases based on gender in tertiary hospitals in Bayelsa and Edo States. This finding is in conformity with study by Badahdah et al [31] which noted that, there is no difference between genders of FHCP managing COVID-19 disease. However, COVID-19 impact on the mental well-being of FHCP affects all regardless of their gender differences. Table 4 shows there is no statistical significant difference in the mental health 51 World Journal of Advanced Research and Reviews, 2022, 13(03), 045–054 coping strategies for FHCP managing COVID-19 Cases based on marital status in Bayelsa and Edo States, Nigeria. Other literatures in this regard has noted the differences that exist in the mental health coping strategies of FHCP managing COVID-19 [31,28]. Thus, no study exists on the differences in marital status and coping strategies that has been conducted in the two states in Nigeria. This study finding shows there is no significant difference in the marital status and coping strategies of FHCP managing COVID-19 cases; therefore, this has created a gap in this present study. Funding This study received no particular grant from funding agencies. Disclosure of conflict of interest Disclosure of conflict of interest The authors declare no conflict of interest. The authors declare no conflict of interest. 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https://openalex.org/W4362426811	https://figshare.com/articles/journal_contribution/Supplementary_Table_3_from_Gene_Expression_Signatures_for_Tumor_Progression_Tumor_Subtype_and_Tumor_Thickness_in_Laser-Microdissected_Melanoma_Tissues/22441813/1/files/39892696.pdf	English	null	Supplementary Table 3 from Gene Expression Signatures for Tumor Progression, Tumor Subtype, and Tumor Thickness in Laser-Microdissected Melanoma Tissues	null	2,023	cc-by	5,891	1 1 1 No. Affymetrix ID Gene Symbol GeneDescription GOTermsBP Q value 1. 209351_at KRT14 keratin 14 structural constituent of cytoskeleton, intermediate filament, epidermis development <0.01 2. 204268_at S100A2 S100 calcium binding protein A2 biological process unknown, calcium ion binding, cellular component unknown <0.01 3. 33323_r_at SFN stratifin/14-3-3σ regulation of progression through cell cycle, extracellular space, cytoplasm, cell proliferation, protein kinase C inhibitor activity, protein domain specific binding <0.01 4. 33322_i_at SFN stratifin/14-3-3σ regulation of progression through cell cycle, extracellular space, cytoplasm, cell proliferation, protein kinase C inhibitor activity, protein domain specific binding <0.01 5. 201820_at KRT5 keratin 5 structural constituent of cytoskeleton, intermediate filament, epidermis development <0.01 6. 209125_at KRT6A keratin 6A structural constituent of cytoskeleton, intermediate filament, ectoderm development <0.01 7. 209260_at SFN stratifin/14-3-3σ regulation of progression through cell cycle, extracellular space, cytoplasm, cell proliferation, protein kinase C inhibitor activity, protein domain specific binding <0.01 8. 213680_at KRT6B keratin 6B structural constituent of cytoskeleton, intermediate filament, ectoderm development <0.01 9. 202286_s_at TACSTD2 tumor-associated calcium signal transducer 2 receptor activity, cytosol, integral to plasma membrane, cell surface receptor linked signal transduction, sensory perception, visual perception, cell proliferation, membrane <0.01 10. 200606_at DSP desmoplakin structural constituent of cytoskeleton, cytoskeleton, intermediate filament, cell-cell adherens junction, epidermis development <0.01 11. 206400_at LGALS7 lectin, galactoside- binding, soluble, 7 (galectin 7) sugar binding, extracellular space, nucleus, apoptosis, heterophilic cell adhesion <0.01 2 12. 205916_at S100A7 S100 calcium binding protein A7 (psoriasin 1) calcium ion binding, epidermis development <0.01 13. 202917_s_at S100A8 S100 calcium binding protein A8 (calgranulin A) calcium ion binding, extracellular space, inflammatory response <0.01 14. 205900_at KRT1 keratin 1 complement activation, lectin pathway, receptor activity, structural constituent of cytoskeleton, protein binding, protein binding, sugar binding, cytoskeleton, intermediate filament, <0.01 15. 204855_at SERPINB5 serpin peptidase inhibitor, clade B (ovalbumin), member 5 serine-type endopeptidase inhibitor activity, cell motility <0.01 16. 203074_at ANXA8 annexin A8 calcium ion binding, calcium- dependent phospholipid binding, blood coagulation, negative regulation of coagulation <0.01 17. 204734_at KRT15 keratin 15 structural constituent of cytoskeleton, intermediate filament, epidermis development <0.01 18. 39248_at AQP3 aquaporin 3 transporter activity, membrane fraction, integral to plasma membrane, transport, excretion, membrane <0.01 19. 217528_at CLCA2 chloride channel, calcium activated, family member 2 <0.01 20. 206032_at DSC3 desmocollin 3 calcium ion binding, protein binding, membrane fraction, cytoskeleton, plasma membrane, intercellular junction, cell adhesion, homophilic cell adhesion, integral to membrane <0.01 21. 1 212236_x_at KRT17 keratin 17 structural constituent of cytoskeleton, intermediate filament, epidermis development <0.01 22. 202504_at TRIM29 tripartite motif-containing 29 transcription factor activity, intracellular, transcription from RNA polymerase II promoter, zinc ion binding, metal ion binding <0.01 23. 203407_at PPL periplakin structural constituent of cytoskeleton, protein binding, cytoskeleton, keratinization <0.01 24. 204971_at CSTA cystatin A (stefin A) cysteine protease inhibitor activity, intracellular <0.01 25. 206276_at LY6D lymphocyte antigen 6 complex, locus D protein binding, membrane fraction, cell adhesion, membrane <0.01 3 3 26. 221854_at PKP1 plakophilin 1 (ectodermal dysplasia/skin fragility syndrome) signal transducer activity, nucleus, cytoskeleton, intermediate filament, plasma membrane, cell adhesion, signal transduction, cell-cell signaling, intermediate filament binding, desmosome, structural constituent of epidermis <0.01 27. 205064_at SPRR1B small proline-rich protein 1B (cornifin) structural molecule activity, cytoplasm, intermediate filament, epidermis development, keratinization <0.01 28. 206165_s_at CLCA2 chloride channel, calcium activated, family member 2 <0.01 29. 206033_s_at DSC3 desmocollin 3 calcium ion binding, protein binding, membrane fraction, cytoskeleton, plasma membrane, intercellular junction, cell adhesion, homophilic cell adhesion, integral to membrane <0.01 30. 210020_x_at CALML3 calmodulin-like 3 calcium ion binding <0.01 31. 217744_s_at PERP PERP, TP53 apoptosis effector unknown <0.01 32. 204952_at LYPD3 LY6/PLAUR domain containing 3 unknown <0.01 33. 209863_s_at TP73L tumor protein p73-like transcription factor activity, nucleus, transcription, apoptosis, induction of apoptosis, Notch signaling pathway <0.01 34. 206642_at DSG1 desmoglein 1 calcium ion binding, cytoskeleton, intercellular junction assembly, homophilic cell adhesion, calcium-dependent cell-cell adhesion, <0.01 35. 209800_at KRT16 keratin 16 structural constituent of cytoskeleton, intermediate filament, cytoskeleton organization and biogenesis, cell proliferation, epidermis development <0.01 36. 201983_s_at EGFR epidermal growth factor receptor MAP/ERK kinase kinase activity, epidermal growth factor receptor activity, negative regulation of progression through cell cycle, regulation of peptidyl-tyrosine phosphorylation, regulation of nitric-oxide synthase activity, actin filament binding <0.01 4 37. 204455_at DST dystonin actin binding, integrin binding, structural constituent of cytoskeleton, calcium ion binding, integrin-mediated signaling pathway, intermediate filament cytoskeleton organization and biogenesis <0.01 38. 209126_x_at KRT6B keratin 6B structural constituent of cytoskeleton, intermediate filament, ectoderm development <0.01 39. 213796_at SPRR1A small proline-rich protein 1A structural molecule activity, epidermis development, keratinization <0.01 40. 212242_at TUBA1 tubulin, alpha 1 (testis specific) nucleotide binding, GTPase activity, structural molecule activity, protein binding, GTP binding, microtubule, microtubule-based movement, <0.01 41. 201131_s_at CDH1 cadherin 1, type 1, E- cadherin calcium ion binding, protein binding, homophilic cell adhesion, integral to membrane <0.01 42. 1 219936_s_at GPR87 G protein-coupled receptor 87 rhodopsin-like receptor activity, receptor activity, signal transduction, G-protein coupled receptor protein signaling pathway, <0.01 43. 213287_s_at KRT10 keratin 10 structural molecule activity, intermediate filament, epidermis development <0.01 44. 214164_x_at CA12 carbonic anhydrase XII carbonate dehydratase activity, one-carbon compound metabolism, zinc ion binding, integral to membrane, lyase activity, metal ion binding <0.01 45. 206166_s_at CLCA2 chloride channel, calcium activated, family member 2 unknown <0.01 46. 210397_at DEFB1 defensin, beta 1 extracellular region, response to pest, pathogen or parasite, defense response to bacteria, innate immune response, <0.01 47. 205157_s_at KRT17 keratin 17 structural constituent of cytoskeleton, intermediate filament, epidermis development <0.01 48. 205595_at DSG3 desmoglein 3 (pemphigus vulgaris antigen) calcium ion binding, protein binding, cytoskeleton, intercellular junction, cell adhesion <0.01 5 49. 215867_x_at AP1G1 adaptor-related protein complex 1, gamma 1 subunit transporter activity, binding, Golgi stack, coated pit, protein complex assembly, intracellular protein transport <0.01 50. 203963_at CA12 carbonic anhydrase XII carbonate dehydratase activity, one-carbon compound metabolism, zinc ion binding, integral to membrane, <0.01 51. 214580_x_at KRT6A keratin 6A structural constituent of cytoskeleton, intermediate filament, ectoderm development <0.01 52. 207023_x_at KRT10 keratin 10 structural molecule activity, intermediate filament, epidermis development <0.01 53. 209212_s_at KLF5 Kruppel-like factor 5 (intestinal) DNA binding, RNA polymerase II transcription factor activity, nucleus, transcription, regulation of transcription, <0.01 54. 203917_at CXADR coxsackie virus and adenovirus receptor receptor activity, protein binding, plasma membrane, integral to plasma membrane, cell adhesion <0.01 55. 219597_s_at DUOX1 dual oxidase 1 unknown <0.01 56. 209771_x_at CD24 CD24 antigen (small cell lung carcinoma cluster 4 antigen) plasma membrane, humoral immune response <0.01 57. 217272_s_at SERPINB1 3 serpin peptidase inhibitor, clade B (ovalbumin), member 13 serine-type endopeptidase inhibitor activity, cellular component unknown, response to UV, regulation of proteolysis and peptidolysis <0.01 58. 205051_s_at KIT v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog nucleotide binding, receptor signaling protein tyrosine kinase activity, receptor activity, vascular endothelial growth factor receptor activity, ATP binding, signal transduction, <0.01 59. 218677_at S100A14 S100 calcium binding protein A14 calcium ion binding <0.01 60. 201667_at GJA1 gap junction protein, alpha 1, 43kDa (connexin 43) signal transducer activity, protein binding, integral to plasma membrane, connexon complex, cell-cell signaling, heart development, junction assembly, <0.01 61. 209604_s_at GATA3 GATA binding protein 3 transcription factor activity, nucleus, transcription, regulation of transcription, DNA-dependent, transcription from RNA polymerase II promoter, defense response, <0.01 6 62. 1 209719_x_at SERPINB3 serpin peptidase inhibitor, clade B (ovalbumin), member 3 serine-type endopeptidase inhibitor activity <0.01 63. 210715_s_at SPINT2 serine peptidase inhibitor, Kunitz type, 2 serine-type endopeptidase inhibitor activity, extracellular region, soluble fraction, cell motility <0.01 64. 202597_at IRF6 interferon regulatory factor 6 transcription factor activity, nucleus, transcription, regulation of transcription <0.01 65. 201286_at SDC1 syndecan 1 integral to plasma membrane, cytoskeletal protein binding, membrane <0.01 66. 202267_at LAMC2 laminin, gamma 2 structural molecule activity, protein binding, basement membrane, laminin-5, cell adhesion, heparin binding, epidermis development <0.01 67. 204870_s_at PCSK2 proprotein convertase subtilisin/kexin type 2 proprotein convertase 2 activity, subtilase activity, protein binding, proteolysis and peptidolysis, cell- cell signaling, <0.01 68. 216379_x_at CD24 CD24 antigen (small cell lung carcinoma cluster 4 antigen) plasma membrane, humoral immune response <0.01 69. 221841_s_at KLF4 Kruppel-like factor 4 (gut) transcription factor activity, nucleus, transcription, negative regulation of cell proliferation, transcriptional activator activity, transcriptional repressor activity <0.01 70. 209873_s_at PKP3 plakophilin 3 structural molecule activity, protein binding, nucleus, cytoskeleton, intercellular junction, cell adhesion <0.01 71. 203961_at NEBL nebulette actin binding, structural constituent of muscle, actin cytoskeleton <0.01 72. 205185_at SPINK5 serine peptidase inhibitor, Kazal type 5 serine-type endopeptidase inhibitor activity, antimicrobial humoral response, anti- inflammatory response <0.01 73. 218002_s_at CXCL14 chemokine (C-X-C motif) ligand 14 chemotaxis, inflammatory response, signal transduction, cell-cell signaling, chemokine activity <0.01 74. 213496_at LPPR4 unknown <0.01 75. 210633_x_at KRT10 keratin 10 structural molecule activity, intermediate filament, epidermis development <0.01 76. 209720_s_at SERPINB3 serpin peptidase inhibitor, clade B (ovalbumin), serine-type endopeptidase inhibitor activity <0.01 7 member 3 77. 203726_s_at LAMA3 laminin, alpha 3 receptor binding, structural molecule activity, basement membrane, epidermis development, regulation of cell adhesion, regulation of cell migration, <0.01 78. 207324_s_at DSC1 desmocollin 1 calcium ion binding, protein binding, cytoskeleton, gap junction, cell adhesion, <0.01 79. 205014_at FGFBP1 fibroblast growth factor binding protein 1 unknown <0.01 80. 201015_s_at JUP junction plakoglobin structural molecule activity, membrane fraction, soluble fraction, cytoplasm, cytoskeleton, cell adhesion <0.01 81. 204379_s_at FGFR3 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism) MAPKKK cascade, nucleotide binding, skeletal development, protein-tyrosine kinase activity, receptor activity, fibroblast growth factor receptor activity, JAK-STAT cascade, <0.01 82. 266_s_at CD24 CD24 antigen (small cell lung carcinoma cluster 4 antigen) plasma membrane, humoral immune response <0.01 83. 218186_at RAB25 RAB25, member RAS oncogene family nucleotide binding, GTP binding, intracellular protein transport, small GTPase mediated signal transduction <0.01 84. 1 220414_at CALML5 calmodulin-like 5 calcium ion binding, signal transduction, epidermis development <0.01 85. 204990_s_at ITGB4 integrin, beta 4 receptor activity, protein binding, cell-matrix adhesion, integrin- mediated signaling pathway, integrin complex <0.01 86. 206385_s_at ANK3 ankyrin 3, node of Ranvier (ankyrin G) protein binding, cytoskeleton, protein targeting, signal transduction <0.01 87. 203287_at LAD1 ladinin 1 structural molecule activity, basement membrane <0.01 88. 209211_at KLF5 Kruppel-like factor 5 (intestinal) DNA binding, RNA polymerase II transcription factor activity, nucleus, transcription, regulation of transcription <0.01 89. 202712_s_at CKMT1B creatine kinase, mitochondrial 1B creatine kinase activity, mitochondrion <0.01 90. 40016_g_at MAST4 microtubule associated serine/threonine kinase family member 4 unknown <0.01 8 91. 219476_at C1orf116 chromosome 1 open reading frame 116 unknown <0.01 92. 206164_at CLCA2 chloride channel, calcium activated, family member 2 unknown <0.01 93. 203638_s_at FGFR2 fibroblast growth factor receptor 2 nucleotide binding, protein- tyrosine kinase activity, receptor activity, fibroblast growth factor receptor activity, cell growth, <0.01 94. 209610_s_at SLC1A4 solute carrier family 1 (glutamate/neutral amino acid transporter), member 4 membrane fraction, integral to plasma membrane, dicarboxylic acid transport, neutral amino acid transport <0.01 95. 202350_s_at MATN2 matrilin 2 calcium ion binding, extracellular matrix <0.01 96. 203535_at S100A9 S100 calcium binding protein A9 (calgranulin B) signal transducer activity, calcium ion binding, inflammatory response, cell-cell signaling <0.01 97. 214549_x_at SPRR1A small proline-rich protein 1A structural molecule activity, epidermis development <0.01 98. 204718_at EPHB6 EPH receptor B6 nucleotide binding, protein- tyrosine kinase activity, receptor activity, ephrin receptor activity, transmembrane receptor protein tyrosine kinase signaling pathway <0.01 99. 206561_s_at AKR1B10 aldo-keto reductase family 1, member B10 (aldose reductase) aldehyde reductase activity, electron transporter activity, aldehyde metabolism, <0.01 100. 219410_at TMEM45 A transmembrane protein 45A integral to membrane <0.01 101. 220225_at IRX4 iroquois homeobox protein 4 transcription factor activity, nucleus, regulation of transcription <0.01 102. 212811_x_at SLC1A4 solute carrier family 1 (glutamate/neutral amino acid transporter), member 4 membrane fraction, integral to plasma membrane, dicarboxylic acid transport, neutral amino acid transport <0.01 103. 205109_s_at ARHGEF4 Rho guanine nucleotide exchange factor (GEF) 4 Rho guanyl-nucleotide exchange factor activity, intracellular signaling cascade <0.01 104. 210413_x_at SERPINB4 serpin peptidase inhibitor, clade B (ovalbumin), member 4 serine-type endopeptidase inhibitor activity, immune response, regulation of proteolysis and peptidolysis <0.01 105. 201287_s_at SDC1 syndecan 1 integral to plasma membrane, cytoskeletal protein binding, <0.01 106. 1 211906_s_at SERPINB4 serpin peptidase inhibitor, clade B (ovalbumin), member 4 serine-type endopeptidase inhibitor activity, immune response, regulation of proteolysis and peptidolysis <0.01 9 107. 218796_at C20orf42 chromosome 20 open reading frame 42 unknown <0.01 108. 218980_at FHOD3 formin homology 2 domain containing 3 unknown <0.01 109. 219497_s_at BCL11A B-cell CLL/lymphoma 11A (zinc finger protein) nucleic acid binding, transcription, <0.01 110. 214599_at IVL involucrin cornified envelope, structural molecule activity, cytosol, keratinocyte differentiation, <0.01 111. 60474_at C20orf42 chromosome 20 open reading frame 42 unknown <0.01 112. 211361_s_at SERPINB1 3 serpin peptidase inhibitor, clade B (ovalbumin), member 13 serine-type endopeptidase inhibitor activity, regulation of proteolysis and peptidolysis <0.01 113. 204400_at EFS embryonal Fyn-associated substrate protein binding, cell adhesion, intracellular signaling cascade <0.01 114. 205363_at BBOX1 butyrobetaine (gamma), 2-oxoglutarate dioxygenase (gamma- butyrobetaine hydroxylase) 1 iron ion binding, gamma- butyrobetaine dioxygenase activity, oxidoreductase activity, oxidoreductase activity, <0.01 115. 205220_at GPR109B G protein-coupled receptor 109B rhodopsin-like receptor activity, receptor activity, signal transduction, G-protein coupled receptor protein signaling pathway <0.01 116. 220289_s_at AIM1L absent in melanoma 1-like unknown <0.01 117. 219395_at RBM35B RNA binding motif protein 35A unknown <0.01 118. 219528_s_at BCL11B B-cell CLL/lymphoma 11B (zinc finger protein) nucleic acid binding, tregulation of transcription <0.01 119. 213385_at CHN2 chimerin (chimaerin) 2 GSH3/SH2 adaptor activity, GTPase activator activity, intracellular signaling cascade <0.01 120. 204869_at PCSK2 proprotein convertase subtilisin/kexin type 2 proprotein convertase 2 activity, subtilase activity, protein binding, proteolysis and peptidolysis, <0.01 121. 200965_s_at ABLIM1 actin binding LIM protein 1 actin binding, cytoskeleton organization and biogenesis, actin cytoskeleton <0.01 122. 219461_at PAK6 p21(CDKN1A)-activated kinase 6 nucleotide binding, protein serine/threonine kinase activity, protein amino acid phosphorylation <0.01 123. 211597_s_at HOP transcription factor activity, regulation of transcription, DNA- dependent <0.01 124. 214734_at SLAC2-B protein binding, intracellular protein transport, Rab GTPase binding <0.01 10 125. 209301_at CA2 carbonic anhydrase II carbonate dehydratase activity, cytoplasm, lyase activity, <0.01 126. 209270_at LAMB3 laminin, beta 3 structural molecule activity, protein binding, basement membrane, cell adhesion, <0.01 127. 204636_at COL17A1 collagen, type XVII, alpha 1 structural molecule activity, extracellular matrix, intercellular junction, phosphate transport, cell-matrix adhesion <0.01 128. 203453_at SCNN1A sodium channel, nonvoltage-gated 1 alpha ion channel activity, protein binding, membrane fraction, amiloride-sensitive sodium channel activity <0.01 129. 206677_at KRTHA1 keratin, hair, acidic, 1 structural constituent of cytoskeleton, intermediate filament, epidermis development <0.01 130. 39729_at PRDX2 peroxiredoxin 2 electron transporter activity, cytoplasm, response to oxidative stress, oxidoreductase activity, regulation of apoptosis <0.01 131. 1 204942_s_at ALDH3B2 aldehyde dehydrogenase 3 family, member B2 aldehyde dehydrogenase [NAD(P)+] activity, alcohol metabolism <0.01 132. 212810_s_at SLC1A4 solute carrier family 1 (glutamate/neutral amino acid transporter), member 4 membrane fraction, integral to plasma membrane, transport, dicarboxylic acid transport, neutral amino acid transporter activity, <0.01 133. 205807_s_at TUFT1 tuftelin 1 extracellular region, bone mineralization, <0.01 134. 208153_s_at FAT2 FAT tumor suppressor homolog 2 (Drosophila) protein binding, cell adhesion, homophilic cell adhesion, integral to membrane <0.01 135. 220723_s_at FLJ21511 unknown <0.01 136. 209699_x_at AKR1C2 aldo-keto reductase family 1, member C2 electron transporter activity, lipid metabolism, canalicular bile acid transport, oxidoreductase activity <0.01 137. 218552_at ECHDC2 enoyl Coenzyme A hydratase domain containing 2 unknown <0.01 138. 203797_at VSNL1 visinin-like 1 calcium ion binding <0.01 139. 219995_s_at FLJ13841 <0.01 140. 220016_at AHNAK AHNAK nucleoprotein (desmoyokin) neurogenesis <0.01 141. 204750_s_at DSC2 desmocollin 2 protein binding, cytoskeleton, intercellular junction, cell adhesion, homophilic cell adhesion, <0.01 142. 219825_at CYP26B1 cytochrome P450, family 26, subfamily B, monooxygenase activity, iron ion binding, electron transport, <0.01 11 polypeptide 1 membrane 143. 220403_s_at P53AIP1 unknown <0.01 144. 207109_at POU2F3 POU domain, class 2, transcription factor 3 transcription factor activity, nucleus, regulation of transcription from RNA polymerase II promoter, <0.01 145. 207463_x_at PRSS3 protease, serine, 3 (mesotrypsin) chymotrypsin activity, trypsin activity, calcium ion binding, proteolysis and peptidolysis, <0.01 146. 206122_at SOX15 SRY (sex determining region Y)-box 15 transcription factor activity, RNA polymerase II transcription factor activity, regulation of transcription from RNA polymerase II promoter, <0.01 147. 207720_at LOR loricrin structural constituent of cytoskeleton <0.01 148. 209602_s_at GATA3 GATA binding protein 3 transcription factor activity, nucleus, transcription from RNA polymerase II promoter, morphogenesis <0.01 149. 205490_x_at GJB3 gap junction protein, beta 3, 31kDa (connexin 31) connexon complex, cell communication, perception of sound, <0.01 150. 206884_s_at SCEL sciellin cytoplasm, zinc ion binding, epidermis development <0.01 151. 213050_at COBL cordon-bleu homolog (mouse) unknown <0.01 152. 203780_at EVA1 epithelial V-like antigen 1 protein binding, cytoskeleton, cell adhesion, homophilic cell adhesion <0.01 153. 203126_at IMPA2 inositol(myo)-1(or 4)- monophosphatase 2 magnesium ion binding, phosphate metabolism, signal transduction, inositol-1(or 4)- monophosphatase activity, <0.01 154. 211734_s_at FCER1A Fc fragment of IgE, high affinity I, receptor for, alpha polypeptide receptor activity, receptor signaling protein activity, immune response <0.01 155. 203021_at SLPI secretory leukocyte peptidase inhibitor serine-type endopeptidase inhibitor activity <0.01 156. 209792_s_at KLK10 kallikrein 10 chymotrypsin activity, trypsin activity, proteolysis and peptidolysis, negative regulation of progression through cell cycle <0.01 157. 1 221666_s_at PYCARD PYD and CARD domain containing protein binding, induction of apoptosis, cell cycle, signal transduction, caspase activator activity, negative regulation of progression through cell cycle 0.01 12 158. 215704_at FLG filaggrin structural molecule activity, intermediate filament 0.01 159. 204508_s_at CA12 carbonic anhydrase XII carbonate dehydratase activity, zinc ion binding, integral to membrane 0.01 160. 208228_s_at FGFR2 fibroblast growth factor receptor 2 nucleotide binding, protein- tyrosine kinase activity, receptor activity, fibroblast growth factor receptor activity, cell growth, 0.01 161. 217707_x_at SMARCA 2 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 DNA binding, transcription coactivator activity, regulation of transcription from RNA polymerase II promoter, 0.01 162. 219764_at FZD10 frizzled homolog 10 (Drosophila) receptor activity, G-protein coupled receptor activity, G- protein coupled receptor protein signaling pathway, Wnt receptor activity 0.01 163. 201425_at ALDH2 aldehyde dehydrogenase 2 family (mitochondrial) aldehyde dehydrogenase (NAD) activity, aldehyde dehydrogenase [NAD(P)+] activity, electron transporter activity, 0.01 164. 206125_s_at KLK8 kallikrein 8 (neuropsin/ovasin) chymotrypsin activity, trypsin activity, proteolysis and peptidolysis 0.01 165. 210347_s_at BCL11A B-cell CLL/lymphoma 11A (zinc finger protein) nucleic acid binding, regulation of transcription 0.01 166. 219197_s_at SCUBE2 signal peptide, CUB domain, EGF-like 2 unknown 0.01 167. 205403_at IL1R2 interleukin 1 receptor, type II receptor activity, interleukin-1, Type II, blocking receptor activity, immune response, 0.01 168. 218963_s_at KRT23 keratin 23 (histone deacetylase inducible) structural molecule activity, intermediate filament 0.01 169. 205990_s_at WNT5A wingless-type MMTV integration site family, member 5A receptor binding, signal transduction, frizzled-2 signaling pathway, cell-cell signaling 0.01 170. 215243_s_at GJB3 gap junction protein, beta 3, 31kDa (connexin 31) connexon complex, cell communication, connexon channel activity 0.01 171. 213992_at COL4A6 collagen, type IV, alpha 6 extracellular matrix structural constituent, collagen, cell adhesion, extracellular matrix organization and biogenesis 0.01 172. 206378_at SCGB2A2 secretoglobin, family 2A, member 2 steroid binding 0.01 173. 206023_at NMU neuromedin U receptor binding, r signal transduction 0.01 13 174. 205174_s_at QPCT glutaminyl-peptide cyclotransferase (glutaminyl cyclase) protein modification, proteolysis and peptidolysis, glutaminyl- peptide cyclotransferase activity, transferase activity 0.01 175. 211002_s_at TRIM29 tripartite motif-containing 29 transcription factor activity, transcription from RNA polymerase II promoter 0.01 176. 207789_s_at DPP6 dipeptidylpeptidase 6 catalytic activity, dipeptidyl- peptidase IV activity, proteolysis and peptidolysis 0.01 177. 205778_at KLK7 kallikrein 7 chymotrypsin activity, trypsin activity, proteolysis and peptidolysis 0.01 178. 214370_at S100A8 S100 calcium binding protein A8 (calgranulin A) calcium ion binding, inflammatory response 0.01 179. 202890_at MAP7 microtubule-associated protein 7 unknown 0.01 180. 1 204503_at EVPL envoplakin structural molecule activity, cytoskeleton, epidermis development 0.01 181. 203815_at GSTT1 glutathione S-transferase theta 1 glutathione transferase activity, response to stress 0.01 182. 212543_at AIM1 absent in melanoma 1 unknown 0.01 183. 219909_at MMP28 matrix metallopeptidase 28 peptidoglycan metabolism, metalloendopeptidase activity, calcium ion binding, extracellular matrix, proteolysis and peptidolysis, 0.01 184. 220013_at ABHD9 abhydrolase domain containing 9 unknown 0.01 185. 221796_at NTRK2 neurotrophic tyrosine kinase, receptor, type 2 nucleotide binding, transmembrane receptor protein tyrosine kinase activity, protein amino acid phosphorylation, transmembrane receptor protein tyrosine kinase signaling pathway, neurogenesis, 0.01 186. 219630_at PDZK1IP1 PDZK1 interacting protein 1 integral to membrane 0.01 187. 214451_at TFAP2B transcription factor AP-2 beta (activating enhancer binding protein 2 beta) transcription factor activity, transcription coactivator activity, regulation of transcription from RNA polymerase II promoter, neurogenesis 0.01 188. 203887_s_at THBD thrombomodulin transmembrane receptor activity, calcium ion binding, blood coagulation, membrane 0.01 189. 202005_at ST14 suppression of tumorigenicity 14 (colon chymotrypsin activity, trypsin activity, proteolysis and 0.01 14 14 carcinoma, matriptase, epithin) peptidolysis 190. 208650_s_at CD24 CD24 antigen (small cell lung carcinoma cluster 4 antigen) humoral immune response 0.01 191. 216905_s_at ST14 suppression of tumorigenicity 14 (colon carcinoma, matriptase, epithin) chymotrypsin activity, trypsin activity, proteolysis and peptidolysis 0.01 192. 205470_s_at KLK11 kallikrein 11 proteolysis and peptidolysis 0.01 193. 211653_x_at AKR1C2 aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase 2, bile acid binding protein, 3-alpha hydroxysteroid dehydrogenase, type III) binding, electron transporter activity, steroid metabolism, canalicular bile acid transport, oxidoreductase activity, 0.01 194. 210461_s_at ABLIM1 actin binding LIM protein 1 actin binding, cytoskeleton organization and biogenesis, actin cytoskeleton 0.01 195. 219010_at C1orf106 chromosome 1 open reading frame 106 unknown 0.01 196. 213421_x_at PRSS3 protease, serine, 3 (mesotrypsin) chymotrypsin activity, trypsin activity, calcium ion binding, extracellular region, proteolysis and peptidolysis 0.01 197. 204151_x_at AKR1C1 aldo-keto reductase family 1, member C1 aldo-keto reductase activity, binding, electron transporter activity, oxidoreductase activity, 20-alpha-hydroxysteroid dehydrogenase activity, 0.01 198. 204105_s_at NRCAM neuronal cell adhesion molecule neuron migration, protein binding, central nervous system development, cell-cell adhesion, positive regulation of neuron differentiation 0.01 199. 220724_at FLJ21511 unknown 0.01 200. 204777_s_at MAL mal, T-cell differentiation protein lipid raft polarization, induction of apoptosis, signal transduction, central nervous system development, structural constituent of myelin sheath, cell differentiation 0.01 201. 218657_at RAPGEFL 1 Rap guanine nucleotide exchange factor (GEF)- like 1 unknown 0.01 15 202. 1 210058_at MAPK13 mitogen-activated protein kinase 13 nucleotide binding, protein serine/threonine kinase activity, MAP kinase activity, protein- tyrosine kinase activity, protein amino acid phosphorylation, cell cycle, protein kinase cascade, 0.01 203. 39249_at AQP3 aquaporin 3 transporter activity, membrane fraction, excretion 0.01 204. 222303_at ETS2 v-ets erythroblastosis virus E26 oncogene homolog 2 (avian) transcription factor activity, regulation of transcription 0.01 205. 204753_s_at HLF hepatic leukemia factor double-stranded DNA binding, regulation of transcription, transcription from RNA polymerase II promoter, 0.01 206. 209442_x_at ANK3 ankyrin 3, node of Ranvier (ankyrin G) ü protein binding, üGO:0005856: cytoskeleton, protein targeting, signal transduction, 0.01 207. 201328_at ETS2 v-ets erythroblastosis virus E26 oncogene homolog 2 (avian) transcription factor activity, regulation of transcription 0.01 208. 206030_at ASPA aspartoacylase (Canavan disease) aminoacylase activity, aspartate catabolism, acting on ester bonds 0.01 209. 213217_at ADCY2 adenylate cyclase 2 (brain) adenylate cyclase activity, cAMP biosynthesis, intracellular signaling cascade 0.01 210. 219411_at ELMO3 engulfment and cell motility 3 (ced-12 homolog, C. elegans) cytoskeleton, GO:0006915: apoptosis, 0.01 211. 63825_at ABHD2 abhydrolase domain containing 2 catalytic activity, integral to membrane 0.01 212. 213618_at CENTD1 centaurin, delta 1 unknown 0.01 213. 204751_x_at DSC2 desmocollin 2 calcium ion binding, protein binding, cytoskeleton, intercellular junction, cell adhesion 0.01 214. 203700_s_at DIO2 deiodinase, iodothyronine, type II thyroxine 5'-deiodinase activity, thyroid hormone generation, selenium binding, hormone biosynthesis 0.02 215. 207076_s_at ASS argininosuccinate synthetase urea cycle, nucleotide binding, argininosuccinate synthase activity, protein binding, amino acid biosynthesis, ligase activity 0.02 216. 216918_s_at DST dystonin actin binding, integrin binding, structural constituent of cytoskeleton, calcium ion binding, integrin-mediated signaling pathway, hemidesmosome, intermediate filament cytoskeleton 0.02 16 organization and biogenesis 217. 208156_x_at EPPK1 epiplakin 1 cytoskeleton 0.02 218. 209603_at GATA3 GATA binding protein 3 transcription factor activity, transcription, regulation of transcription, transcription from RNA polymerase II promoter, 0.02 219. 201250_s_at SLC2A1 solute carrier family 2 (facilitated glucose transporter), member 1 transporter activity, glucose transporter activity, 0.02 220. 213929_at unknown 0.02 221. 203215_s_at MYO6 myosin VI microfilament motor activity, actin binding, calmodulin binding, structural constituent of muscle, actin filament-based movement, 0.02 222. 205709_s_at CDS1 CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 1 magnesium ion binding, diacylglycerol cholinephosphotransferase activity, phosphatidate cytidylyltransferase activity, phospholipid biosynthesis, 0.02 223. 201829_at NET1 neuroepithelial cell transforming gene 1 regulation of cell growth, guanyl- nucleotide exchange factor activity, signal transduction 0.02 224. 206363_at MAF v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian) chromatin, DNA binding, RNA polymerase II transcription factor activity, 0.02 225. 1 220646_s_at KLRF1 killer cell lectin-like receptor subfamily F, member 1 sugar binding, integral to plasma membrane, cell surface receptor linked signal transduction, MHC class I receptor activity 0.02 226. 208407_s_at CTNND1 catenin (cadherin- associated protein), delta 1 structural molecule activity, protein binding, cytoskeleton, cell-cell adhesion 0.02 227. 204675_at SRD5A1 steroid-5-alpha-reductase, alpha polypeptide 1 (3- oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1) 3-oxo-5-alpha-steroid 4- dehydrogenase activity, electron transporter activity, cell-cell signaling, integral to membrane, oxidoreductase activity 0.02 228. 219850_s_at EHF ets homologous factor unknown 0.02 229. 205455_at MST1R macrophage stimulating 1 receptor (c-met-related tyrosine kinase) protein-tyrosine kinase activity, receptor activity, macrophage colony stimulating factor receptor activity, cell motility, signal transduction, positive regulation 0.02 17 of cell proliferation 230. 210059_s_at MAPK13 mitogen-activated protein kinase 13 nucleotide binding, protein serine/threonine kinase activity, MAP kinase activity, protein- tyrosine kinase activity, protein amino acid phosphorylation, cell cycle, 0.02 231. 213425_at WNT5A wingless-type MMTV integration site family, member 5A receptor binding, signal transduction, frizzled-2 signaling pathway, cell-cell signaling 0.02 232. 213506_at F2RL1 coagulation factor II (thrombin) receptor-like 1 receptor activity, receptor binding, signal transduction, G- protein coupled receptor protein signaling pathway, blood coagulation, thrombin receptor activity 0.02 233. 202018_s_at LTF lactotransferrin serine-type endopeptidase activity, humoral immune response, metal ion binding 0.02 234. 204351_at S100P S100 calcium binding protein P calcium ion binding, nucleus, calcium-dependent protein binding 0.02 235. 210128_s_at LTB4R leukotriene B4 receptor nucleotide binding, receptor activity, leukotriene receptor activity, cell motility, signal transduction, G-protein signaling, coupled to IP3 second messenger (phospholipase C activating) 0.02 236. 209590_at BMP7 bone morphogenetic protein 7 (osteogenic protein 1) ossification, cytokine activity, growth factor activity, cell differentiation, growth 0.02 237. 203962_s_at NEBL nebulette actin binding, actin cytoskeleton, regulation of actin filament length 0.02 238. 213369_at PCDH21 protocadherin 21 unknown 0.02 239. 203786_s_at TPD52L1 tumor protein D52-like 1 unknown 0.02 240. 205653_at CTSG cathepsin G cathepsin G activity, chymotrypsin activity, trypsin activity, proteolysis and peptidolysis, 0.02 241. 211986_at AHNAK AHNAK nucleoprotein (desmoyokin) nucleus, neurogenesis 0.02 242. 218736_s_at PALMD palmdelphin unknown 0.02 243. 219529_at CLIC3 chloride intracellular channel 3 voltage-gated chloride channel activity, ion transport, chloride transport, signal transduction 0.02 244. 202489_s_at FXYD3 FXYD domain containing ion transport regulator 3 ion channel activity, chloride channel activity 0.02 of cell proliferation 18 245. 216594_x_at AKR1C1 aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1, 20- alpha (3-alpha)- hydroxysteroid dehydrogenase) aldo-keto reductase activity, electron transporter activity, oxidoreductase activity, trans-1,2- dihydrobenzene-1,2-diol dehydrogenase activity 0.02 246. 1 206470_at PLXNC1 plexin C1 unknown 0.02 247. 212848_s_at C9orf3 chromosome 9 open reading frame 3 aminopeptidase activity, membrane alanyl aminopeptidase activity, proteolysis and peptidolysis, metallopeptidase activity, 0.02 248. 221815_at ABHD2 abhydrolase domain containing 2 catalytic activity, integral to membrane 0.02 249. 204136_at COL7A1 collagen, type VII, alpha 1 serine-type endopeptidase inhibitor activity, protein binding, collagen type VII, basement membrane, cell adhesion 0.02 250. 201189_s_at ITPR3 inositol 1,4,5-triphosphate receptor, type 3 inositol 1,4,5-triphosphate- sensitive calcium-release channel activity, calcium ion transport, signal transduction 0.02 251. 205554_s_at DNASE1L 3 deoxyribonuclease I-like 3 DNA binding, endonuclease activity, DNA catabolism, apoptosis, hydrolase activity 0.02 252. 205694_at TYRP1 tyrosinase-related protein 1 monooxygenase activity, copper ion binding, melanin biosynthesis from tyrosine, metabolism 0.02 253. 207134_x_at TPSB2 tryptase beta 2 chymotrypsin activity, extracellular region, proteolysis and peptidolysis, tryptase activity 0.02 254. 204948_s_at FST follistatin protein binding, development, activin inhibitor activity 0.03 255. 201136_at PLP2 proteolipid protein 2 (colonic epithelium- enriched) protein binding, ion transport, chemotaxis,: cytokine and chemokine mediated signaling pathway, chemokine binding 0.03 256. 209386_at TM4SF1 transmembrane 4 L six family member 1 unknown 0.03 257. 203240_at FCGBP Fc fragment of IgG binding protein unknown 0.03 258. 205251_at PER2 period homolog 2 (Drosophila) signal transducer activity transcription, regulation of transcription, DNA-dependent, signal transduction 0.03 259. 203081_at CTNNBIP 1 catenin, beta interacting protein 1 regulation of transcription, DNA- dependent, signal transduction, development, 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205349_at GNA15 guanine nucleotide binding protein (G protein), alpha 15 (Gq class) nucleotide binding, GTPase activity, signal transducer activity, GTP binding, heterotrimeric G-protein complex, signal transduction, phospholipase C activation, 0.03 20 elevation of cytoplasmic calcium ion concentration 276. 201613_s_at RUVBL1 RuvB-like 1 (E. coli) nucleotide binding, regulation of cell growth, DNA helicase activity, protein binding, DNA recombination, regulation of transcription from RNA polymerase II promoter, chromatin modification 0.03 277. 201984_s_at EGFR epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) MAP/ERK kinase kinase activity, epidermal growth factor receptor activity, negative regulation of progression through cell cycle, regulation of peptidyl-tyrosine phosphorylation, regulation of nitric-oxide synthase activity, actin filament binding 0.03 278. 203691_at PI3 peptidase inhibitor 3, skin-derived (SKALP) serine-type 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metal ion binding 0.04 313. 205128_x_at PTGS1 prostaglandin- endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) prostaglandin biosynthesis, prostaglandin-endoperoxide synthase activity, lipid metabolism, oxidoreductase activity, metal ion binding 0.04 314. 219496_at C2orf26 chromosome 2 open reading frame 26 unknown 0.05 315. 219680_at NOD9 unknown 0.05 316. 204469_at PTPRZ1 protein tyrosine phosphatase, receptor- type, Z polypeptide 1 carbonate dehydratase activity, transmembrane receptor protein tyrosine phosphatase activity, integral to plasma membrane, protein amino acid dephosphorylation, central 0.05 23 nervous system development, 317. 205239_at AREG amphiregulin (schwannoma-derived growth factor) cytokine activity, cell-cell signaling, growth factor activity, cell proliferation 0.05 318. 220484_at MCOLN3 mucolipin 3 cation channel activity, cation transport 0.05 319. 206643_at HAL histidine ammonia-lyase histidine ammonia-lyase activity, histidine 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https://openalex.org/W2179107360	https://www.nature.com/articles/srep13967.pdf	English	null	Real-time Detection of Breast Cancer Cells Using Peptide-functionalized Microcantilever Arrays	Scientific reports	2,015	cc-by	10,562	Real-time Detection of Breast Cancer Cells Using Peptide- functionalized Microcantilever Arrays received: 29 January 2015 accepted: 12 August 2015 Published: 05 October 2015 Hashem Etayash1,2, Keren Jiang2, Sarfuddin Azmi1, Thomas Thundat2 & Kamaljit Kaur1,3 Hashem Etayash1,2, Keren Jiang2, Sarfuddin Azmi1, Thomas Thundat2 & Kamaljit Kaur1,3 Ligand-directed targeting and capturing of cancer cells is a new approach for detecting circulating tumor cells (CTCs). Ligands such as antibodies have been successfully used for capturing cancer cells and an antibody based system (CellSearch®) is currently used clinically to enumerate CTCs. Here we report the use of a peptide moiety in conjunction with a microcantilever array system to selectively detect CTCs resulting from cancer, specifically breast cancer. A sensing microcantilever, functionalized with a breast cancer specific peptide 18-4 (WxEAAYQrFL), showed significant deflection on cancer cell (MCF7 and MDA-MB-231) binding compared to when exposed to noncancerous (MCF10A and HUVEC) cells. The peptide-functionalized microcantilever allowed efficient capture and detection of cancer cells in MCF7 spiked human blood samples emulating CTCs in human blood. A detection limit of 50–100 cancer cells mL−1 from blood samples was achieved with a capture yield of 80% from spiked whole blood samples. The results emphasize the potential of peptide 18-4 as a novel peptide for capturing and detecting cancer cells in conjunction with nanomechanical cantilever platform. The reported peptide-based cantilever platform represents a new analytical approach that can lead to an alternative to the various detection platforms and can be leveraged to further study CTCs. In-vivo examinations of breast cancer is mainly implemented through techniques like mammography (an x-ray of the breast), ultrasound exams, magnetic resonance imaging (MRI) and/or [18F]fluorode- oxyglucose positron emission tomography, which are typically followed by ex vivo biopsy and further checkups1. A simple blood test to detect circulating tumor cells (CTCs) that flow in the bloodstream of cancer patients due to cell shedding from primary tumors could complement other detection methods for disease diagnosis. In recent years, molecular and clinical findings have revealed that cancer cells may invade into the blood circulation at early stages of tumor development, emphasizing the importance of sensitive and specific detection of CTCs in the blood1. Developing a sensitive and accurate tool for detec- tion of CTCs would provide valuable information on cancer prognosis, diagnosis, monitoring of tumor sensitivity to anticancer drugs, as well as, in personalization of anticancer therapy1,2. y g p py Numerous approaches have been developed for reliably identifying and quantifying CTCs in blood samples3–8. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 Real-time Detection of Breast Cancer Cells Using Peptide- functionalized Microcantilever Arrays The presence of CTCs or cancer cells in blood (∼ hundreds per mL) is masked by nor- mal blood cells that appear at a billion times higher concentration, making their detection challenging. The classical methods for isolation and enumeration of CTCs are time consuming and cannot be used for easy, routine screening to determine disease recurrence and response to treatments. Evolving tech- nologies in the past few years have allowed identification and quantification of CTCs with applicable 1Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. 2Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, T6G 2V4, Canada. 3Chapman University School of Pharmacy (CUSP), Harry and Diane Rinker Health Science Campus, Chapman University, Irvine, California, 92618-1908, USA. Correspondence and requests for materials should be addressed to K.K. (email: kkaur@chapman.edu) Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 1 www.nature.com/scientificreports/ Figure 1. Schematic showing principle of microcantilever sensor operation. (a) Microcantilever coated with non-specific reference peptide (Ref-1or Ref-2) shows no response to the presence of normal or cancer cells (no deflection). (b) Microcantilever functionalized with cancer targeting peptide (18-4 or cRGDfC) demonstrates a strong response (deflection) to cancerous cells due to peptide-cancer cell interactions. PSD, Position Sensitive Detector. Figure 1. Schematic showing principle of microcantilever sensor operation. (a) Microcantilever coated with non-specific reference peptide (Ref-1or Ref-2) shows no response to the presence of normal or cancer cells (no deflection). (b) Microcantilever functionalized with cancer targeting peptide (18-4 or cRGDfC) demonstrates a strong response (deflection) to cancerous cells due to peptide-cancer cell interactions. PSD, Position Sensitive Detector. specificity and sensitivity. Methods such as the immunohistochemistry (IHC)9, flow cytometry (FC)10 and the polymerase chain reactions (PCR)11 are very sensitive and compliant approaches for detections. However, with respect to their applicable use, they continue to suffer from numerous constrains such as the need for the trained cytologist to handle the sample assessments, time-consumption associated with the handling and pre-treatment procedures, as well as the cross-reactivity of the antibodies and nucleo- tides used during the detections6,12. Other alternative label-free biosensing technologies to the classical approaches of CTCs detection are under development, such as nanowire sensor13, the graphene oxide nano-sheets14, the electro-impedance cytometry15 and microcantilevers16–18. One platform based on the immunomagnetic beads conjugated with an antibody to EpCAM (CellSearch®, VeridexTM, Warren, PA), is now clinically used for enumeration of CTCs from human blood samples19. Real-time Detection of Breast Cancer Cells Using Peptide- functionalized Microcantilever Arrays Majority of these advanced detection platforms rely on antibody and/or oligonucleotide probes for recognition, identification, and quantification of the target cells.fi qi g In this study, we report the development of a peptide-based microcantilever array sensor for efficient capture of intact representative cancer cells at low concentrations without pre-requisite labeling or sam- ple processing (Fig. 1). The microcantilever array was functionalized separately with two cancer targeting peptides, namely, a decapeptide 18-4 (WxEAAYQrFL) with an additional C-terminal cysteine or a cyclic RGD peptide (cRGDfC)20 using the thiol group of cysteine residue. Peptide 18-4 is a proteolytically stable engineered breast cancer targeting peptide derived from a 12-mer peptide p160 that was identified using in vivo phage display for cancer targeting21–23. Peptide 18-4 exhibits high affinity for breast cancer cell lines (MCF7, MDA-MB-231, and MDA-MB-435), most likely through a receptor-mediated mechanism, with almost no binding to the noncancerous cells (MCF10A and HUVECs). RGD is a well-studied tumor homing peptide that interacts with specific integrin receptors (α vβ 3) overexpressed on several tumor epithelial cells24,25. However RGD also targets non-tumorigenic tissues as it is recognized by several integrins (8 out of 24 heterodimers) and is therefore deemed less specific. To explore whether cancer cells can be selectively captured with these peptides, breast cancer cells (MCF7 or MDA-MB-231) alone or in combination with non-cancerous MCF10A (derived from the same breast tissue as MCF7) were spiked into a buffer or blood solution to obtain mimics of CTCs in human blood. The cancer cells were detected by recording the nanomechanical bending of the cantilevers in real-time based on the surface stress induced by adhesion of the cancer cells to the immobilized peptides. Results and Discussion First, we aimed to assess and compare the binding efficiency of the designed peptide-based microcantilever sensor (peptide 18-4 sensor) to other peptide-sensors including cRGDfC sensor against the human adenocarcinoma breast cell line MCF7, which is a good mimic for circulating breast tumor cells in human blood. The thiolated peptides (Table S1) were chemically synthesized and independently immobilized on cantilever beams in arrays using the tip-dipping method as described in the material and methods section. Cancer cells were spiked into PBS (25 cells mL−1, pH ∼ 7.4) and were allowed to flow through the microcantilever array. Reference cantilevers functionalized with control peptides were treated with the same concentra- tion of cancer cells and subjected to nanomechanical readings for comparisons. Results of the analysis revealed significant beam deflection for peptide 18-4 functionalized sensor with approximate deflection of 120 ± 7 nm achieved after sample introduction (Fig. 2a). The deflection, however, showed to be slightly less in case of cRGDfC functionalized array with a deflection distance of 102 ± 3 nm. Compared to the peptide 18-4 and RGD sensors, the reference cantilevers (ref. 1 and ref. 2) exhibited insignificant bending when subjected to the cancer cells, indicating weak binding properties of the control peptides.fi j g g p p p p Further insight on the peptide binding efficiency to cancer cells was gained by estimating the capture yield of cancer cells of each peptide sensor. Figure 2b displays the capture yield (%) of the peptide can- tilever sensors in contrast to the reference cantilevers. The calculated capture efficiency was found to be around 80 ± 4% in the case of peptide 18-4 sensor and almost 60 ± 6% for cRGDfC sensor. In contrast to the sensing peptides, the reference cantilevers showed only 14–19% for both ref. 1 and ref. 2. The cantilever results demonstrated that peptide 18–4 sensor has better binding affinity to cancer cell lines than the cRGDfC sensor. These results match well with the previous studies of peptide array whole cell binding assay for screening of cancer targeting peptides using fluorescence microscopy21,22. Specificity of Peptide-functionalized Microcantilevers. In order to determine the specificity of the designed sensor, we applied the peptide 18-4 functionalized cantilever array to distinguish between cancerous and non-cancerous cell lines in real-time (Fig. 3). Results and Discussion Functionalization of Microcantilevers. Microcantilevers in an array were functionalized with self-assembled monolayers (SAMs) of cancer cell binding peptides, which act as specific ligands for can- cer cells. As illustrated in Fig. 1, the detection principle is based on static mode of cantilever operation, Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 2 www.nature.com/scientificreports/ which means that cantilever beam bends as a result of changes in the surface stress generated by ana- lyte-ligand interactions26. Specifically in this study, selective adsorption of the cancer cells to the immo- bilized peptide on the surface of the cantilevers results in a decrease in the surface free energy which in turn leads to generate a differential surface stress between the functionalized and non-functionalized sides of the lever. This differential surface stress causes cantilever to deflect or bend by a certain extent that can be expressed according to Stoney’s formula27. An in-house built microcantilever array sensor was used for the cantilever experiments (Supplementary Information Figure S1).fi y g One of the essential parameters that determine the efficiency of the cell capture on the microcanti- lever system is the flow velocity of the sample throughout the system28. Therefore, in order to optimize the flow rate, a number of experiments were conducted to determine the sensor capture efficiency at different flow velocities. We spiked cancer cell lines (MCF7) into phosphate buffered saline (PBS) at ∼ 100 cells mL−1 and dispensed on a peptide (18-4) coated microcantilever array as a function of flow rates ranging from 1 to 5 mL h−1. The capture yield was calculated for each flow rate and results were charted as shown in Figure S2. We found that the estimated capture efficiency increased by decreasing the flow velocity of the samples, indicating an inverse proportion of the capture yield to the sample flow velocity. The capture yield was significantly enhanced at 1 mL h−1 flow rate (81%) compared to that at faster flow rate of 5 mL h−1 (54%). Based on these results that suggest enhanced binding of the cancer cells to the immobilized peptide with increased incubation time, the subsequent studies were performed using a flow rate of 1–2 mL h−1. Cancer Cell Binding to the Peptide-functionalized Microcantilevers. Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 Results and Discussion Each cantilever differential deflection represents an average calculation of eight replicates and error bars indicate standard deviations, P < 0.05. Figure 2. Label-free real time detection of cancer cells using peptide-functionalized cantilever array. (a) Real time detection of MCF7 human breast cancer cells mimicking the circulating tumor cells at 25 ± 5 cells mL–1 using peptide-functionalized cantilever array. Cantilevers were functionalized with four different peptides, two cancer-targeting peptides (18-4 and RGD) and two non-specific reference peptides (ref. 1 and ref. 2). (b) Capture yield of cancer cells corresponding to each peptide sensor. Each cantilever differential deflection represents an average calculation of eight replicates and error bars indicate standard deviations, P < 0.05. spectroscopy33. Furthermore, Mannoor et al. showed bioselective recognition of pathogenic bacteria at a single-cell level using peptide assembled onto a wireless graphene nanosensor34. Here we have employed a cancer-targeting peptide, engineered from a phage display library and synthetic peptide array library for breast cancer cell binding, as a sensing molecule to detect cancer cells in a cantilever array for the first time.hfl spectroscopy33. Furthermore, Mannoor et al. showed bioselective recognition of pathogenic bacteria at a single-cell level using peptide assembled onto a wireless graphene nanosensor34. Here we have employed a cancer-targeting peptide, engineered from a phage display library and synthetic peptide array library for breast cancer cell binding, as a sensing molecule to detect cancer cells in a cantilever array for the first time.hfl i The differential deflection of the peptide microcantilever sensor to cancer cells was also explored by injecting samples with different ratios of cancer cells (MCF7) to noncancerous (MCF10A) cells. Figure 3b demonstrates cantilevers deflection after injection of cancer cells only (100 cells mL−1) as well as after dilution with MCF10A (MCF7:MCF10A; 1:0, 3:1, 2:2, 1:3, 0:1). The cantilevers selectively responded to MCF7 cells and showed amplitude of deflection proportionally scaled with concentration of the MCF7 cells in the sample (Fig. 3c). In a co-culture of cancerous and noncancerous cells, the cantilever was able to detect cancer cells in the presence of ∼ 75% normal cells (MCF10A). Similarly, as the concentration of normal cells was increased, the deflection signal decreased (Fig. 3d) indicating the ability of peptide probes to discriminate between cell types. Results and Discussion Here the binding affinity of the peptide sensor was explored against two types of breast cancer cell lines, namely, MCF7 and MDA-MB-231 and two non-cancerous cell lines, MCF10A and HUVEC. MCF10A are non-cancerous cells derived from the same human mammary tissue as MCF7, whereas HUVEC are endothelial cells isolated from normal human umbilical vein. When cells were injected at a concentration of 100 ± 10 cells mL−1 sep- arately to each peptide cantilever, the cantilever showed significant deflection for cancerous cell binding (280 ± 25 nm) compared to non-cancerous cell binding (90 ± 15 nm, Fig. 3a). The variation in cantilever deflection upon binding cancerous or noncancerous cells is most likely due to the differential expression levels of specific peptide-binding receptors present in cancerous and noncancerous cells. We and others have shown that peptide 18-4 and the original lead peptide p160 enter cells by a receptor-mediated endocytosis21,23. The receptor is not known yet, however, it is clear that the receptor is overexpressed in breast cancer cells compared to normal cells. The results confirm our conjecture that peptide 18-4 binds breast cancer cells with high specificity. Previously we showed that a similar peptide, peptide 18 (WXEAAYQRFL), binds MDA-MB-435 breast cancer cells with an apparent Kd of 41.9 μM22.hi This finding highlights that such tumor binding peptides are not only useful for tumor imaging or targeted drug delivery, but can also be useful as recognition elements to develop peptide-based biosensor platforms for cancer cell detection in real-time. In recent years, several studies have explored the fea- sibility of using short-ligand peptides as molecular recognition elements in biosensing techniques and have validated the ability of natural and synthetic peptides to serve as robust biorecognition probes in biosensors29–32. We have recently shown that an antimicrobial peptide from class IIa bacteriocins can be used for the detection of Gram-positive Listeria monocytogenes at 1 bacterium μL−1 using impedance Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 3 www.nature.com/scientificreports/ Figure 2. Label-free real time detection of cancer cells using peptide-functionalized cantilever array. (a) Real time detection of MCF7 human breast cancer cells mimicking the circulating tumor cells at 25 ± 5 cells mL–1 using peptide-functionalized cantilever array. Cantilevers were functionalized with four different peptides, two cancer-targeting peptides (18-4 and RGD) and two non-specific reference peptides (ref. 1 and ref. 2). (b) Capture yield of cancer cells corresponding to each peptide sensor. Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 Results and Discussion (c) and (d) demonstrate the concentration dependence of cantilever response to the number of cancerous or non-cancerous cells, respectively, present in the co-culture sample (100 cells mL−1). The representative graphs show an increase in cantilever deflection with an increase in the number of MCF7 cells (c), and a decrease in cantilever deflection with an increase in the number of MCF10A (d) in the media. Each cantilever differential deflection represents an average calculation of eight replicates and error bars indicate standard deviations. Figure 3. Specific binding of peptide-cantilevers to breast cancer cells. (a) Label-free real time recognition of cancer cell lines (MCF7 or MDA-MB-231, 100 ± 10 cells mL−1) from non-cancerous cell lines (MCF10A or HUVEC, 100 ± 10 cells mL−1) with microcantilever array functionalized with 18-4 cancer targeting peptide. (b) Cantilever deflection in response to a function of different concentration ratios of cancerous to non-cancerous cells (MCF7 to MCF10A) in PBS solution as indicated. (c) and (d) demonstrate the concentration dependence of cantilever response to the number of cancerous or non-cancerous cells, respectively, present in the co-culture sample (100 cells mL−1). The representative graphs show an increase in cantilever deflection with an increase in the number of MCF7 cells (c), and a decrease in cantilever deflection with an increase in the number of MCF10A (d) in the media. Each cantilever differential deflection represents an average calculation of eight replicates and error bars indicate standard deviations. ones36,37, and such receptors are being targeted for diagnosis and drug delivery using different types of ligands such as antibodies, aptamers, affibodies and peptides.h fi The sensitivity of detection is one of the key features for practical application of the sensor in med- ical and biological applications. To this end, the sensitivity of the peptide based cantilever array was determined by exposing the sensor to various concentrations of cancer cells (MCF7) spiked in PBS (5 ± 3–103 ± 10 cells mL−1) (Figure S3). The results showed the ability of sensor to detect as low as 25 ± 5 MCF7 cells per mL in pure buffer solution from the background deflection (baseline). The signal, however, was not distinguishable from the background at lower concentration, suggesting a minimum detection limit of 25 cells per mL. Several studies have shown that biosensor performances are often affected by the analyte transport in the vicinity of the sensing area28,38 as well as dispensing of the cells in the microfluidic system35,38. Results and Discussion The results suggest that the presence of normal cells (MCF10A used here) does not prevent cancer cells from binding to the immobilized probes; it might however, impede their transportation to the sensor probes at lower concentrations dropping the limit of detec- tion. Non-specific binding, sample delivery and improper cell dispersion (mixing) may also contribute to reduced capture sensitivity35. p y Peptide 18-4 binds to breast cancer cells most likely via a receptor-mediated mechanism22. We attrib- ute the variation in cantilever responses between cancerous and non-cancerous cells to the presence of different receptors or different expression levels of a specific receptor on surface of cancer cells. It is well known that certain receptors or/markers are over expressed on cancer cells and deficient in the normal Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 4 www.nature.com/scientificreports/ Figure 3. Specific binding of peptide-cantilevers to breast cancer cells. (a) Label-free real time recognition of cancer cell lines (MCF7 or MDA-MB-231, 100 ± 10 cells mL−1) from non-cancerous cell lines (MCF10A or HUVEC, 100 ± 10 cells mL−1) with microcantilever array functionalized with 18-4 cancer targeting peptide. (b) Cantilever deflection in response to a function of different concentration ratios of cancerous to non-cancerous cells (MCF7 to MCF10A) in PBS solution as indicated. (c) and (d) demonstrate the concentration dependence of cantilever response to the number of cancerous or non-cancerous cells, respectively, present in the co-culture sample (100 cells mL−1). The representative graphs show an increase in cantilever deflection with an increase in the number of MCF7 cells (c), and a decrease in cantilever deflection with an increase in the number of MCF10A (d) in the media. Each cantilever differential deflection represents an average calculation of eight replicates and error bars indicate standard deviations. Figure 3. Specific binding of peptide-cantilevers to breast cancer cells. (a) Label-free real time Figure 3. Specific binding of peptide-cantilevers to breast cancer cells. (a) Label-free real time recognition of cancer cell lines (MCF7 or MDA-MB-231, 100 ± 10 cells mL−1) from non-cancerous cell lines (MCF10A or HUVEC, 100 ± 10 cells mL−1) with microcantilever array functionalized with 18-4 cancer targeting peptide. (b) Cantilever deflection in response to a function of different concentration ratios of cancerous to non-cancerous cells (MCF7 to MCF10A) in PBS solution as indicated. Results and Discussion 1 to MCF7 or MCF10A cells. A sharp SPR signal was generated for specific interaction between the peptide 18-4 sensor and MCF7 cancer cells compared to the other signals. A low response was observed for peptide 18-4 binding to MCF10A cells followed by similar response signals by the reference sensor to both MCF7 and MCF10A cells. The responses, however, are likely related to non-specific interactions with the sensor surface since no clear differentiation exists between the two cell lines. In agreement with the cantilever results, peptide 18-4 SPR sensor exhibited highest signal to MCF7 cells indicating a specific interaction to the corresponding cells and confirming the applicability of the assay to distinguish between cancerous and noncancerous cells in real-time.h microcantilever system39. SPR is a highly sensitive method, however, piezoresistivity, the change in elec- trical resistivity under stress or deflection is a simple method that eliminates the complexity inherent to optical instruments such as SPR without the loss of sensitivity. The ligand peptide 18-4 was covalently immobilized on SPR gold slide using the thiol chemistry as described above. The peptide functionalized slide was inserted into the instrument and PBS solution was allowed to flow at a constant flow rate of 10 μL min−1. SPR slide functionalized with a reference peptide (ref. 1) was used at the same time on another SPR channel for comparison. The sensor selectivity to the target cells was measured by SPR reading after injecting samples of cancerous (MCF7) and noncancerous cells (MCF10A) simultaneously at a concentration of 100 cells mL−1. Figure 4a displays a typical SPR spectrum illustrating responses of the SPR sensor functionalized with peptide 18-4 or ref. 1 to MCF7 or MCF10A cells. A sharp SPR signal was generated for specific interaction between the peptide 18-4 sensor and MCF7 cancer cells compared to the other signals. A low response was observed for peptide 18-4 binding to MCF10A cells followed by similar response signals by the reference sensor to both MCF7 and MCF10A cells. The responses, however, are likely related to non-specific interactions with the sensor surface since no clear differentiation exists between the two cell lines. Results and Discussion Therefore in flow through systems like microcantilevers, it is possible to achieve a low detection limit by controlling the fluid delivery with a proper mixing regime. In addition, cantilever with a continuous fluidic flow can allow analysis of relatively large sample volumes with a few CTCs, thereby improving the detection limit, as opposed to other detection methods with fixed sample volumes. Cancer Cell Binding using Surface Plasmon Resonance. In order to validate microcantilever results, surface plasmon resonance (SPR) was utilized to study the specific recognition of cancer cells by surface immobilized cancer targeting peptides. SPR is routinely used as a standard characterization tool for bimolecular interactions and serves as a complementary transduction method to the piezoresistive Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 5 www.nature.com/scientificreports/ Figure 4. Direct discernment of cancer cells from non-cancerous cells by peptide-based SPR sensor. (a) SPR intensity signal resulting from interaction of 18-4 functionalized SPR chip (or functionalized with reference peptide) with MCF7 or MCF10A cells. (b) SPR sensitivity spectra for peptide 18-4 against various concentrations of cancer cells (MCF7) at a constant flow rate of 10 μL min−1. Figure 4. Direct discernment of cancer cells from non-cancerous cells by peptide-based SPR sensor. (a) SPR intensity signal resulting from interaction of 18-4 functionalized SPR chip (or functionalized with reference peptide) with MCF7 or MCF10A cells. (b) SPR sensitivity spectra for peptide 18-4 against various concentrations of cancer cells (MCF7) at a constant flow rate of 10 μL min−1. microcantilever system39. SPR is a highly sensitive method, however, piezoresistivity, the change in elec- trical resistivity under stress or deflection is a simple method that eliminates the complexity inherent to optical instruments such as SPR without the loss of sensitivity. The ligand peptide 18-4 was covalently immobilized on SPR gold slide using the thiol chemistry as described above. The peptide functionalized slide was inserted into the instrument and PBS solution was allowed to flow at a constant flow rate of 10 μL min−1. SPR slide functionalized with a reference peptide (ref. 1) was used at the same time on another SPR channel for comparison. The sensor selectivity to the target cells was measured by SPR reading after injecting samples of cancerous (MCF7) and noncancerous cells (MCF10A) simultaneously at a concentration of 100 cells mL−1. Figure 4a displays a typical SPR spectrum illustrating responses of the SPR sensor functionalized with peptide 18-4 or ref. Results and Discussion Figures (b) and (d) show the average deflection of peptide 18-4 coated cantilevers in both, whole blood and blood without plasma, respectively, based on three individual studies performed under the same conditions. The error bars indicate corresponding standard deviations. igure 5. Differential deflection of microcantilever arrays with MCF7 spiked into human blood samples Figure 5. Differential deflection of microcantilever arrays with MCF7 spiked into human blood samples. (a) Top figure shows results from injection of whole blood spiked with MCF7 cells, whereas (c) lower figure shows data from injection of blood without plasma where blood was first spiked with MCF7 or MCF10A cells followed by plasma removal. The system was first equilibrated by injection of blood samples free from cell lines, followed by injection of samples containing 25, 50 or 100 cells/mL. The control represents the response of a negative analogue of peptide 18-4 to ∼ 100 cells mL−1 spiked samples. Figures (b) and (d) show the average deflection of peptide 18-4 coated cantilevers in both, whole blood and blood without plasma, respectively, based on three individual studies performed under the same conditions. The error bars indicate corresponding standard deviations. Cancer Cell Detection in Whole blood Samples. To mimic the detection of CTCs from patient blood samples, the designed peptide-based microcantilever were exposed to MCF7 cells spiked in human blood samples. First, the blood was made less viscous by diluting it with buffer solution (90%) in order to facilitate the injection and diminish the viscosity effects. In addition, to enhance the sensitivity, the plasma was removed from the blood by centrifugation. Plasma is routinely removed from the blood for CTC enrichment from whole blood40,41. Blood samples spiked with different concentrations of MCF7 (25, 50 or 100 cells/mL) were allowed to flow over the peptide 18-4-cantilever (Fig. 5a,b). Likewise for blood without plasma samples, freshly obtained blood samples were first spiked with MCF7 or MCF10A (25, 50 or 100 cells/mL) followed by plasma removal. The resulting sample was allowed to flow over the peptide 18-4-cantilever for nanomechanical readings (Fig. 5c,d). The cantilever system was initially equilibrated by injecting the blood sample (with or without plasma) free from the cell lines, followed by injection of spiked blood samples. This was done to clearly observe the deflection after introduction of the spiked blood. Results and Discussion In agreement with the cantilever results, peptide 18-4 SPR sensor exhibited highest signal to MCF7 cells indicating a specific interaction to the corresponding cells and confirming the applicability of the assay to distinguish between cancerous and noncancerous cells in real-time.h The sensitivity of the peptide functionalized SPR sensor was evaluated by injecting serial concentra- tions of MCF7 cancer cells (5 ± 3 to 100 ± 10 cells mL−1) to the peptide 18-4 sensor at a fixed flow rate of 10 μL min−1. Figure 4b shows a representative SPR spectrum where an increase in SPR intensity was observed with an increase in concentration of the injected MCF7 cells. Similar to the microcantilever studies (Figure S3), the number of cells bound to the immobilized ligand is directly proportional to the number of cancer cells in the sample, up to a maximum of ∼ 500 cells mL−1, where the saturation takes place. 6 Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 www.nature.com/scientificreports/ Figure 5. Differential deflection of microcantilever arrays with MCF7 spiked into human blood samples. (a) Top figure shows results from injection of whole blood spiked with MCF7 cells, whereas (c) lower figure shows data from injection of blood without plasma where blood was first spiked with MCF7 or MCF10A cells followed by plasma removal. The system was first equilibrated by injection of blood samples free from cell lines, followed by injection of samples containing 25, 50 or 100 cells/mL. The control represents the response of a negative analogue of peptide 18-4 to ∼ 100 cells mL−1 spiked samples. Figures (b) and (d) show the average deflection of peptide 18-4 coated cantilevers in both, whole blood and blood without plasma, respectively, based on three individual studies performed under the same conditions. The error bars indicate corresponding standard deviations. Figure 5. Differential deflection of microcantilever arrays with MCF7 spiked into human blood samples. (a) Top figure shows results from injection of whole blood spiked with MCF7 cells, whereas (c) lower figure shows data from injection of blood without plasma where blood was first spiked with MCF7 or MCF10A cells followed by plasma removal. The system was first equilibrated by injection of blood samples free from cell lines, followed by injection of samples containing 25, 50 or 100 cells/mL. The control represents the response of a negative analogue of peptide 18-4 to ∼ 100 cells mL−1 spiked samples. Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 Results and Discussion Instead when the system was equilibrated by injecting PBS, followed by blood and then spiked blood, the deflection due to spiked blood was less apparent (Figure S4). We envisage the patient samples can be run in the clinics by equilibrating the system with normal human blood followed by injection of the patient blood in the cantilever flow through system to obtain a clear read out. Figure 5 shows the differential deflections of microcantilever arrays after injecting the blood (Fig. a,b) or blood Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 7 www.nature.com/scientificreports/ without plasma (Fig. c,d). An increase in cantilever deflection was observed with an increase in number of MCF7 cells in the sample. The MCF7 spiked blood samples, whole blood or blood without plasma, showed a substantial deflection compared to the MCF7 free specimens at a concentration of 100 ± 10 cancer cells mL−1. Blood without plasma showed higher response (85 ± 8 nm) compared to whole blood sample (62 ± 10 nm). In addition, for the blood without plasma specimen (Fig. 5c) the nanomechanical bending was significant (40 ± 10 nm) even at 50 ± 10 MCF7 cancer cells mL−1, suggesting interference from plasma components such as proteins and other interfering biomolecules.l p p p g Further, we examined the cancer cell capture from whole blood samples using fluorescence micros- copy with comprehensive image analysis. MCF7 (or MCF10A) and white blood cells (WBCs) in blood samples were fluorescently labeled green and red, respectively, followed by injection into the cantile- ver. The eight microcantilevers were exposed to two spike cell concentrations, 50 or 25 cells mL−1 of blood at 1 mL hr−1 (Fig. 6a). The captured cells were imaged using fluorescence microscopy. Figure 6b shows images of two of the eight cantilevers with captured MCF7 (or MCF10A) and hematological cells (WBCs). While the control peptide cantilevers captured almost no cancer cells, the peptide 18-4 cantilevers captured 5 ± 2 MCF7 cells/cantilever. Overall, the average number of captured MCF7 cells per 8 cantilevers, when seeded at a concentration of ∼ 50 cells/mL, was found to be 40 ± 7 cells/mL for the peptide 18-4 coated sensor which is significantly higher than the control sensor (7 ± 5 cells/mL). These results are statistically significant as determined using the unpaired student t-test (P = 0.008). Results and Discussion In contrast, at seeded concentration of 25 cancer cells/mL, poor significant difference was observed for the peptide 18-4 sensor compared to the control sensor (P = 0.056; n = 5). In addition, peptide 18-4 coated sensor captured only 9 ± 3 cells/mL MCF10A cells when exposed to ∼ 50 MCF10A cells/mL in blood sample. From the optical images of single cells (MCF7) bound to microcantilevers, we can clearly see the results correlate reasonably well with the cantilever deflection measurements (Fig. 2b). An increase in bound cancer cells was observed with an increase in its seeded concentration. At 50 cells/mL, high capture efficiency (80 ± 5%) was achieved using peptide 18-4 functionalized microcantilevers. It is inter- esting to note that a similar capture yield (80%) was obtained for MCF7 cells present in PBS (Fig. 2b) or blood samples (Fig. 6c), whereas the cantilever deflection was decreased when MCF7 cells were present in whole blood (Fig. 5a) compared to when present in PBS (Fig. 2a). This is likely due to the higher baseline deflection for the spiked whole blood sample, where other biomolecules or cells from the blood bind to the peptide cantilever before cancer cell binding. For instance, we observe that white blood cells contribute to the non-specific binding on the peptide 18-4 cantilevers with 21 ± 6 WBCs bound per 8 cantilevers.i Owing to the high specificity of peptide 18-4 to breast cancer cells, detection of cancer cells was achieved in buffer and blood samples at reasonable concentration levels. Although it has been very challenging to detect cancer cells in pure blood samples, we achieved the detection limit of approxi- mately 50 cell mL−1, which compares well with other reported data13. Typically, antibodies or nucle- otides are used as molecular recognition elements in cancer detection42,43. The only test that has been approved by the US Food and Drug Administration to measure CTCs in patients is the CellSearch® system (VeridexTM, Warren, PA). This system is based on epithelial cell adhesion molecule (EpCAM) recognition by anti-EpCAM antibody41. The system is very sensitive, achieves robust capture efficiency, and is used for clinical prediction of CTCs with enumeration count of 5 or more cancer cells per 7.5 mL human blood. Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 Results and Discussion It is reported, however, that even with CellSearch a number of cancer cells escape from the detection due to the lack of the EpCAM molecule or due to the multiple steps that are required for the enrichment process12. There are several other platforms that use EpCAM antibody and are under development, such as nanowire based platform and platinum microelelctrodes coupled with electro- chemical impedance44,45. Lee and co-workers reported an integrated nanowire based platform where the EpCAM antibody immobilized in the quartz nanowire arrays captures CTCs from blood samples and laser scanning cytometry is used to enumerate the CTCs44. Similarly, in another study EpCAM antibody is used to capture cancer cells and the binding event is monitored using highly sensitive electrochemical impedance sensor45. In this case, however, the detection sensitivity is dependent on the ionic strength of the sample and the frequency at which the electrical impedance is measured. p q y p More recently, a genetic-based approach is reported where nanoconstructs called “NanoFlares” are used to detect live circulating tumor cells from blood46. NanoFlares consist of gold nanoparticles func- tionalized with single-stranded DNA (antisense recognition motif) that binds to short DNA complement containing a fluorescent reporter, whose fluorescence is quenched when it is present near the gold par- ticle. In the presence of cancer cells the NanoFlares bind to target mRNA, and the fluorescent reporter is away from the gold nanoparticles displaying enhanced fluorescence which is quantified using flow cytometry. Other methods for CTC detection include capturing CTC based on the cell size difference47,48. CTCs are typically larger than peripheral blood cells and different filtration approaches are being devel- oped to isolate and detect CTCs47. p Our study explores an alternative selective biomolecule (peptide) to detect cancer cells in combination with highly sensitive microcantilevers. The technique not only detects cancer cells by peptide capture, it also sorts cells in a single step. Unlike other techniques, peptide-based cantilever arrays are very simple to prepare, can be readily fabricated on silicon wafers and/or other materials using conventional micro- fabrication techniques, are inexpensive and can be used in an array format to detect simultaneously several cancer phenotypes. The ultra-small size of cantilevers, which resembles a miniature diving panel, Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 8 www.nature.com/scientificreports/ Figure 6. Cancer cell capture on the peptide coated microcantilevers (eight) using fluorescence microscopy. Results and Discussion (a) Schematic of 8 cantilevers used to capture cancer cells when exposed to 50 or 25 cells/mL sample. (b) Fluorescence microscope images of the captured cells (MCF-7 or MCF10A) on the peptide- coated (18-4 or control) microcantilevers (two of the eight cantilevers are shown here). The eight cantilevers were exposed to MCF7 or MCF10A spiked whole blood samples (50 or 25 cells/mL). MCF7 or MCF10A cells and the white blood cells (WBCs) were stained separately, green and red, respectively, before mixing. Scale bar is 100 μm. (c) The average number of captured cells per 8 microcantilevers as computed from the set of fluorescence microscopy images. Assay was repeated five times, and mean ± SD is presented. The P values were computed using the unpaired t-test to signify the statistical difference between the comparable groups. Figure 6. Cancer cell capture on the peptide coated microcantilevers (eight) using fluorescence Figure 6. Cancer cell capture on the peptide coated microcantilevers (eight) using fluorescence microscopy. (a) Schematic of 8 cantilevers used to capture cancer cells when exposed to 50 or 25 cells/mL sample. (b) Fluorescence microscope images of the captured cells (MCF-7 or MCF10A) on the peptide- coated (18-4 or control) microcantilevers (two of the eight cantilevers are shown here). The eight cantilevers were exposed to MCF7 or MCF10A spiked whole blood samples (50 or 25 cells/mL). MCF7 or MCF10A cells and the white blood cells (WBCs) were stained separately, green and red, respectively, before mixing. Scale bar is 100 μm. (c) The average number of captured cells per 8 microcantilevers as computed from the set of fluorescence microscopy images. Assay was repeated five times, and mean ± SD is presented. The P values were computed using the unpaired t-test to signify the statistical difference between the comparable groups. Figure 6. Cancer cell capture on the peptide coated microcantilevers (eight) using fluorescence microscopy. (a) Schematic of 8 cantilevers used to capture cancer cells when exposed to 50 or 25 cells/mL sample. (b) Fluorescence microscope images of the captured cells (MCF-7 or MCF10A) on the peptide- coated (18-4 or control) microcantilevers (two of the eight cantilevers are shown here). The eight cantilevers were exposed to MCF7 or MCF10A spiked whole blood samples (50 or 25 cells/mL). MCF7 or MCF10A cells and the white blood cells (WBCs) were stained separately, green and red, respectively, before mixing. Scale bar is 100 μm. Methods id Peptide Design and Synthesis. Two cancer-targeting peptides, peptide 18-4 (WxEAAYQrFLC) and cRGD (cyclicRGDfC) and the corresponding negative control peptides, ref. 1 (XEPAYQRFTC) and ref. 2 (cyclicRADfC) were used in this study. In each peptide an additional cysteine residue has been added at the terminus in order to enable adequate anchoring to the cantilever gold interfaces and the SPR gold chips through the well-known gold-thiol chemistry immobilization method20,53 Peptides were synthe- sized chemically using standard N-Fmoc solid phase peptide synthesis as described previously21. Briefly, the first amino acid was coupled to a 2-chlorotrityl resin (NovaBiochem, San Diego, CA) at 5-fold excess using the N,N diisopropyl ethylamine (DIPEA) at room temperature. Further amino acids were added automatically using an automated peptide synthesizer (Tribute, Protein Technology, Inc., USA). The com- pleted peptide was ultimately released from the resin with a mixture of 90% trifluoroacetic acid (TFA), 9% dichloromethane (DCM), and 1% triisopropylsilane (∼ 10 mL) for 90 min at room temperature. The cleaved peptide combined with TFA was then concentrated, washed with diethyl either, dissolved in water and purified using reversed-phase HPLC (Table S1). Microcantilever Sensor Preparation. Microcantilever arrays (Concentris GmbH – Switzerland) of eight gold-coated cantilevers (500 μm long, 100 μm wide and 1 μm thick) were used in the experi- ments. The apex – top gold surfaces of the cantilevers (20 nm gold thickness) were functionalized with our designed thiolated peptides following the procedure described for gold-thiol chemistry immobili- zation20,53. Briefly, cantilevers were cleaned with Piranha solution (30% H2O2:96% H2SO4, vol/vol) for 15 minutes, rinsed three times with MilliQ-water (18 MW) followed by ethanol, and dried in air. The arrays were incubated in 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (10 mM, Gelest Inc. Frankfurt, Germany) for 20 minutes, rinsed with ethanol and dried in air in order to make the backside of the levers inert and reduce nonspecific binding to the silicon side. Subsequently the microcantilevers were coated with the peptides of interest. In order to make sure that only cantilevers tips were func- tionalized with the peptide, only the tips were dipped-in the peptide solution (1 mg/mL) and kept for 6 h; the process was also repeated once to ensure an adequate peptide coupling to the cantilever surface. Prior to use, the arrays were rinsed with 70% ethanol and copious amount of PBS solution to remove any physically adsorbed materials. Cantilever Setup and Deflection Detection. Results and Discussion (c) The average number of captured cells per 8 microcantilevers as computed from the set of fluorescence microscopy images. Assay was repeated five times, and mean ± SD is presented. The P values were computed using the unpaired t-test to signify the statistical difference between the comparable groups. allows the sensor to exhibit quick responses to the biological and chemical deviations for real-time, in-situ monitoring49. Peptide functionalized cantilever arrays can be developed to capture multiple recep- tors expressed on cancer cells increasing sensor sensitivity. Our future work will focus on obtaining a well-defined peptide array with different binding affinities for cancer cells and the normal haematological cells in blood samples. The approach will encompass exploring strategies such as employing different techniques for peptide immobilization, investigating multi-ligands for targeting and using other sensor Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 9 www.nature.com/scientificreports/ platforms in parallel to achieve better detection limits with high selectivity. Peptide 18-4 works well to capture the immortalized cells spiked into human blood. Future work warrants the evaluation of peptide 18-4 binding to patient derived CTCs to validate the peptide-based microcantilever approach. Currently peptides are being used clinically to detect cancer50–52. For instance, RGD that binds α vβ 3 integrins on cancer cell surface is used in cancer patients as a radiotracer to detect breast cancer lesions by positron emission tomography (PET)50. In conclusion, functionalization of microcantilevers with breast cancer-targeting peptide 18-4 has enabled label-free sensing platform for real-time detection of cancer cells in human blood samples. The peptide 18-4 functionalized cantilever sensor can detect cancer cells in whole blood which contains sig- nificantly large number of hematological cells. The achieved detection limits with the cantilever sensor are 25, 50, and 100 cancer cells/mL in buffer, blood without plasma and blood, respectively. The higher sensitivity toward the blood without plasma sample suggests that the microcantilever sensing can be further improved by removing the non-cellular components from the blood. Further a capture yield of 80% from spiked whole blood samples was achieved with the peptide 18-4 functionalized cantilevers, which is comparable to the antibodies based systems14. These results suggest that the peptide-based microcantilever sensor can be developed into a diagnostic platform for detection of circulating tumor cells as well as to monitor the therapeutic outcomes in cancer patients. Methods id All cantilever experiments were carried out using an in-house built microcantilever array sensor (Figure S1). Briefly, the cantilever setup consists of a fluidic cell within which the functionalized cantilever array was mounted. The cell is attached to an inlet port connected to a syringe pump for introduction of the sample and an outlet port which is attached to a fluid reservoir. To detect cantilever deflections, a low-power (∼ 1 mW) laser beam was reflected off the free-end of the cantilever and was focused onto a position sensitive detector (PSD Thorlabs. Inc. New Jersey, USA). Data from nano-mechanical cantilever deflections were recorded in real-time using a multifunctional data-acquisition board driven by LabView-based software. The functionalized cantilever array was initially placed in the fluidic cell and equilibrated in running phosphate buffered saline (PBS) at a constant flow rate of 5 mL h−1 until a stable baseline was achieved. It was then exposed to running PBS solution for approximately 50 scans followed by flow of sample solutions containing cancer cells. An optimum flow rate for detection was determined by exposing the peptide 18-4-functionalized cantilever to a solution of cancer cells at various flow velocities. The results led us to select a flow rate of 1–2 mL h−1 for all our subsequent experiments. The experiments were performed for four different peptides as indicated in the text and four different cell lines including the non-cancerous control cells. Cell Culture. The human breast cancer cell lines MCF7 and MDA-MB-231 (American Type Culture Collection, Manassas, VA) were cultured in DMEM medium containing 10% fetal bovine serum (FBS), Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 10 www.nature.com/scientificreports/ 100 IU mL−1 penicillin, and 100 IU mL−1 streptomycin. The human mammary epithelial cell line MCF- 10A was cultured in minimal essential growth medium (MEGM, Lonza, Cedarlane) supplemented with the same additives as mentioned before. Human umbilical vein endothelial cells (HUVEC), kind gift from the laboratory of Sandra Davidge, University of Alberta, were cultivated using endothelial cell growth medium (EGM, Lonza, Cedarlane) containing 20% FBS, 2 mM L−1 glutamine, 100 IU mL−1 penicillin, 100 IU mL−1 streptomycin, and 2 ng/mL basic fibroblast growth factor (Roche Diagnostics, Mannheim, Germany). All cell lines were cultivated at 37 °C in a 5% CO2–95% O2 incubator, and growth media were replaced every 48h. Cantilever and Cell Capture Assay. Methods id For running the cantilever assay and performing the capture efficiency experiments, cells were diluted in serum-free medium, starting at an initial concentration of 103 mL−1 determined by a hemocytometer. Subsequently the cells were centrifuged at 500 rpm, and re-suspended in phosphate buffered saline (PBS, 1 mL). Cells were aliquoted into a low-attachment 96-well plate to obtain a serial dilution of cells ranging from 100–5 cells mL−1 in each well (optical microscopy was used for cell counting). Mixed cells samples were obtained by following the same proto- col, where aliquots of MCF7 and MCF10A were mixed to give final ratios of 25:75, 50:50, and 75:25 for MCF7:MCF10A with 100 cells mL−1 in each well. Before the cell capture assay, MCF7 cells were stained with fluorescent dye (CyQUANT, green – Life Technologies Inc., Burlington, ON, Canada) following the manufacture’s protocol, washed with PBS, centrifuged and re-suspended at 100 cells/mL in PBS solution. The sample with seeded cells was then introduced into the cantilever sensor after calibrating the baseline with PBS solution for ∼ 20 min. After taking the deflection reading the cantilevers were scanned by a fluorescence microscope (Olympus America, Melville, NY, USA) and sets of images corresponding to the captured cells were taken at different positions. The images were imported and cell numbers were computed using ImageJ software package. The capture efficiency was defined as the ratio of the number of target cells captured to the number of target cells initially seeded. g g y Human blood samples were collected from healthy donors with informed consent and in accordance with the approved guidelines by the University of Alberta. All experiments were performed following protocols as approved by the ethics committee. All samples were collected in EDTA tubes and were pro- cessed within 3 h. Before each injection into the cantilever system, the samples (whole blood or the blood without plasma) were spiked with various concentrations of cells (25, 50, and 100 cell mL−1), diluted to 10% concentration in 1× PBS solution, and subjected to nanomechanical reading. Briefly, blood without plasma sample was prepared by first spiking the blood (1 mL) with cell lines (MCF7 or MCF10A), mixed with buffer (1:9, v/v), centrifuged at 800 × g for 10 minutes until the blood cells fall to the bottom of the tube54 followed by aspiration of the plasma and buffer, and finally re-suspending in 1× PBS solution (1 mL)40,41. Methods id Samples were injected into the cantilever system at a flow rate of 1 ml hr−1. The capture yield was determined as discussed above.h The MCF7 or MCF10A and centrifuged white blood cells (from 1 mL blood) were stained green and red, respectively, suspended in PBS solution (1 mL) and injected to the cantilever device. Note that in order to get the red probe (propidium iodide) inside the WBCs, the hematological cells were incu- bated with the stain for ∼ 1 h at room temperature. Captured cells were then examined using fluores- cence microscopy at 20× magnification and excitation wavelength of 488 nm (CyQUANT) and 543 nm (propidium iodide). The emitted fluorescence was detected through spectral detection channels between 500–530 nm and 555–655 nm for green and red fluorescence, respectively. Capture efficiency was defined as mentioned above. SPR Measurements. Surface Plasmon Resonance (SPR) measurements were carried out using a SRP Navi 200 instrument (BioNavis Ltd., Tampere, Finland) that uses the Kretscheman prism configura- tion having a goniometer with dual flow channels and cohesive peristaltic pump with 100 μL sampler loops. Briefly, the experiments were performed in angular scan mode in order to determine the SPR angular position changes in a real-time. The critical angle of total internal reflection was measured as the reflection index changes due to the surface absorption on the chip. A flow rate of 10 μL min−1 was used throughout the experiments with a sensor temperature fixed at 25 °C. A laser with a wavelength of 670 nm was used as a light source to excite the surface plasmon at the dielectric gold interface. A freshly cleaned gold-coated SPR chip (50 nm gold, 5 nm Titanium adhesion layer) was functionalized with peptide 18-4 (or peptide ref. 1) by immersing in peptide/PBS solution (1 mg mL−1) for 12 h at room temperature. The measurements started by introducing the peptide chip into the sample holder and run- ning 1× PBS solution at a 7.4 pH to stabilize a baseline. Two samples of PBS (1× ) solution, containing cancer cell line MCF-7 (100 cells mL−1) or the corresponding normal cell line MCF-10A were injected separately through the flow cell. A continuous scan was performed on a liquid range of 50–77° and the recorded data were processed using the BioNavis software package. Statistical Analysis. Methods id For all the experiments, signals of identically functionalized cantilevers were averaged and each experiment was performed at least three times. All data are presented as mean ± SD of the calculations throughout the manuscript. The statistical difference was tested either using the unpaired t-test or the one way ANOVA test as specified55. In all statistical analysis the significance level (P value) was set at 0.05. Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 11 www.nature.com/scientificreports/ References Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition BMC Cancer 12, 178 (2012). 13. Lee, S. K. et al. Nanowire substrate-based laser scanning cytometry for quantitation of circulating tumor cells. Nano Lett 12, 2697–2704 (2012). 14. Yoon, H. J. et al. 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M. et al. www.nature.com/scientificreports/ Introduction to health research methods: a practical guide. (Jones & Bartlett Learning, 2012). Acknowledgements g We acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for supporting this research. We also thank Canada Excellence Research Chair Program. Hashem Etayash is the recipient of a Ph.D. scholarship under the Libyan–North American Scholarship Program. We acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for supporting this research. We also thank Canada Excellence Research Chair Program. Hashem Etayash is the recipient of a Ph.D. scholarship under the Libyan–North American Scholarship Program. Author Contributions H.E., K.K. and T.T. designed the experiments and analyzed the results. H.E. and S.A. prepared samples and experimental setups. H.E. and K.J. performed the cantilever and SPR experiments. 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Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 12 www.nature.com/scientificreports/ Additional Information upplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/sre Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Etayash, H. et al. Real-time Detection of Breast Cancer Cells Using Peptide- unctionalized Microcantilever Arrays. Sci. Rep. 5, 13967; doi: 10.1038/srep13967 (2015). How to cite this article: Etayash, H. et al. Real-time Detection of Breast Cancer Cells Using Peptide functionalized Microcantilever Arrays. Sci. Rep. 5, 13967; doi: 10.1038/srep13967 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 5:13967 \| DOI: 10.1038/srep13967 13
https://openalex.org/W4313491718	http://musicologica.upol.cz/doi/10.5507/mo.2022.009.pdf	Czech	null	Letters of Václav Jan Tomášek from the Morawetz Collection	Musicologica Olomucensia	2,022	cc-by-sa	20,048	1 Předložená práce vznikla za finanční podpory Ministerstva kultury v rámci institucionálního fi- nancování dlouhodobého koncepčního rozvoje výzkumné organizace Národní muzeum (DKRVO 2019–2023/20.III.d, 00023272). Za přepisy, revizi přepisů, překlad dopisů a četné konzultace a rešerše děkuji Vlastě a Hubertu Reittererovým.h 3 Srov. Jana Vojtěšková, „Letters from the Morawetz Collection (Musicians of Czech Origin in European Centres at the Turn of the 18th and 19th Centuries) / Dopisy Morawetzovy sbírky (Hudebníci českého původu v evropských centrech na přelomu 18. a 19. století),“ Musicalia 13, no. 1–2 (2021): 6–39 (anglická verze), 40–46 (česká verze). Musicologica Olomucensia 34 – 2022/2 DOI: Musicologica Olomucensia 34 – 2022/2 DOI: 10.5507/mo.2022.009 Musicologica Olomucensia 34 – 2022/2 DOI: Musicologica Olomucensia 34 – 2022/2 DOI: 10.5507/mo.2022.009 6 Jana Vojtěšková, „Několik pramenů z doby působení Carla Marii von Webera ve Stavovském divadle / Several Sources pertaining to Carl Maria von Weber’s Work for the Estates Theatre in Prague,“ Musicalia 5, no. 1–2 (2013): 57–64 (česká verze), 65–77 (anglická verze). 5 Srov. Jana Vojtěšková, „The works of Jan Dismas Zelenka in 18th and 19th centuries in Prague,“ Clavibus unitis 4, no. 1 (2015): 85–90. j ý 2 Jana Vojtěšková, ed., Album Jana Ludevíta Procházky z let 1860–1888 / The Procházka Album (1860–1888) (Praha: KLP & NM, 2013). g 4 Jana Vojtěšková, „Dopisy Vojtěcha Matyáše Jírovce z Morawetzovy sbírky,“ Opus musicum 53, no. 4 (2021): 52–65.h p 12 Sammlung Fritz Donebauer-Prag. Briefe, Musik-Manuskripte * Portraits zur Geschichte der Musik und des Theaters. Versteigerung vom 6. bis 8. April 1908 durch J. A. Stargardt Verlagsbuchhandlung und Antiquariat, Berlin W. 35. Lützowstr. 47. 7 O Morawetzově sbírce srov. Jana Vojtěšková, „Sbírka Richarda Morawetze,“ Muzikologické fórum 1, no. 1 (2012): 47–54. Zde je uvedena také přehledná tabulka zastoupených pisatelů a adresátů. 8 Katalog vyšel pod názvem: Sammlung Fritz Donebauer-Prag. Briefe, Musik-Manuskripte * Portraits zur Geschichte der Musik und des Theaters. Versteigerung vom 6. bis 8. April 1908 durch J. A. Stargardt Verlagsbuchhandlung und Antiquariat, Berlin W. 35. Lützowstr. 47. Richard Batka, Aus der Musik- und Theaterwelt. Beschreibendes Verzeichnis der Autographen-Samm- lung Fritz Donebauer in Prag (Praha: Fritz Donebauer – Löwit & Lamberg, 1894); Beschreibendes Verzeichnis der Autographensammlung Fritz Donebauer in Prag (Praha: Fritz Donebauer – Löwit & Lamberg, 1900). 9 Jana Vojtěšková Jana Vojtěšková V letech 2003 a 2008 získalo Národní muzeum – České muzeum hudby velmi cennou hudební sbírku,1 jejímž majitelem byl po roce 1908 někdejší továrník v Úpici Richard Morawetz (1881–1965). Kolekce obsahuje unikátní notové i nenotové rukopisy a ikonografický materiál z doby od 18. do počátku 20. sto- letí. V oblasti korespondence se jedná o skutečně jedinečné prameny k dějinám české hudby, proto se věnuji jejich výzkumu a publikaci formou kritické edice. Vydáno bylo již album korespondence Ludevíta Procházky,2 dopisy Jana Václava Huga Voříška, Jiřího Antonína Bendy, Leopolda Koželuha, Antonína Františka Bečvařovského3 a Vojtěcha Matyáše Jírovce.4 Pojednán byl i krátký rukopisný záznam Jana Dismase Zelenky5 a dopisy Carla Marii von Webera.6 Tento článek se soustředí na dopisy Václava Jana Tomáška. g 4 Jana Vojtěšková, „Dopisy Vojtěcha Matyáše Jírovce z Morawetzovy sbírky,“ Opus musicum 53, no. 4 (2021): 52–65.h 5 Srov. Jana Vojtěšková, „The works of Jan Dismas Zelenka in 18th and 19th centuries in Prague,“ Clavibus unitis 4, no. 1 (2015): 85–90. 6 Jana Vojtěšková, „Několik pramenů z doby působení Carla Marii von Webera ve Stavovském divadle / Several Sources pertaining to Carl Maria von Weber’s Work for the Estates Theatre in Prague,“ Musicalia 5, no. 1–2 (2013): 57–64 (česká verze), 65–77 (anglická verze). 120 Dopisy Václava Jana Tomáška z Morawetzovy sbírky p 10 Album nazvané Correspondenz Stiepanek připravuje k vydání Kabinet pro studium českého divadla jako třetí svazek ediční řady Nota bene. 11 Srov pozn 8 9 Srov. pozn. 2. 11 Srov. pozn. 8. Morawetzově sbírce srov. Jana Vojtěšková, „Sbírka Richarda Morawetze,“ Muzikologické fórum o. 1 (2012): 47–54. Zde je uvedena také přehledná tabulka zastoupených pisatelů a adresátů. 13 O Tomáškovi srov. A. Simpson a K. DeLong, „Tomášek, Václav Jan Křtitel,“ in Grove Music Online, 24. února 2022, https://www.oxfordmusiconline.com/grovemusic/view/10.1093/gmo/ 9781561592630.001.0001/omo-9781561592630-e-0000028077;Markéta Kabelková, „Václav Jan Tomášek,“ in Die Musik in Geschichte und Gegenwart: Allgemeine Enzyklopädie der Musik: 21 Bände in zwei Teilen, eds. Friedrich Blume et al. (2. neubearb. Ausg. Kassel: Bärenreiter, 1994–); Kateřina Alexandra Šťastná, „Tomášek, Václav Jan Křtitel,“ in Český hudební slovník, dostupné z www.cesky- hudebnislovnik.cz/slovnik/; Marie Tarantová, „Václav Jan Tomášek ve staropražských hudebních salónech,“ Hudební věda 13, no. 1 (1976): 59–74. Morawetzova sbírka Základ Morawetzovy sbírky7 tvořila kolekce Friedricha Donebauera (1849–1916), který ji rozprodal v roce 1908 v berlínské aukci. Tehdy Museum Království čes- kého nemělo dostatečné prostředky na nákup, a proto památky přešly do soukro- mých rukou. Díky tištěným katalogům8 zjistíme, že se do Morawetzovy sbírky nedostaly zdaleka všechny Donebauerovy bohemikální prameny, nicméně docho- valo se podstatné jádro. Kromě dopisů z konce 18. století známe svědectví skla- datelů z následujícího století, a to i světových autorů, kteří měli vztah k českému prostředí – například Carla Marii von Webera, Hectora Berlioze nebo Richarda Wagnera. Morawetz zachoval také dvě významná alba korespondence a jiných písemností – album dr. Ludevíta Procházky9 a album Jana Nepomuka Štěpánka.10 K nejmladší vrstvě Morawetzovy sbírky patří např. tři rukopisy Leoše Janáčka. j y y p p p y J V současnosti je Morawetzova sbírka uložena v Českém muzeu hudby (dále jen ČMH) odděleně v pěti fondech – v Muzeu B. Smetany, Muzeu A. Dvořáka a ve fondu hudebnin, nenotových rukopisů a ikonografie. Jako kurátorka fondu nenotových rukopisů se věnuji převážně výzkumu korespondence, případně ji- ných nenotových rukopisných sdělení. Formou kritické edice postupně uveřejňuji dopisy z různých období, včetně překladu a komentářů k důležitému kontextu. Sám Donebauer dal zveřejnit dva katalogy, k nimž předmluvu napsal Richard Batka. První z nich vyšel v roce 1894 s rozsáhlým úvodem, druhý v roce 1900.11 Nejcennější pro nás je ovšem katalog, který vydal Stargardt v roce 1908 u pří- ležitosti již zmíněné berlínské aukce.12 Na základě katalogů můžeme porovnat, které předměty přešly do Morawetzovy sbírky a které získal Morawetz vlastní sběratelskou činností. Jeho kolekce měla za války i po ní neklidný osud a nyní ji známe již jen ve značně zredukované podobě. 121 Jana Vojtěšková Jana Vojtěšková Korespondence Václava Jana Tomáška tvoří významný konvolut v Mora- wetzově sbírce i v celém fondu nenotových rukopisů Českého muzea hudby. Tento článek předkládá kritickou edici jednotlivých dopisů, jejich překlad do češ- tiny a podrobné komentáře k jednotlivým dílům, jejich edicím, vydavatelům, knihkupcům, ale také žákům, hraběti Buquoyovi nebo básníkovi Johannu Wolf- gangu Goethovi. 14 Zdeněk Němec, ed., Vlastní životopis V. J. Tomáška (Praha: Topičova edice, 1941), 9. Václav Jan Křtitel Tomášek13 „Hudební skladatel hraběte Jiřího Buquoye, magister svobodných umění, za- sloužilý člen velkého nizozemského Hudebního spolku pro zvelebení umění hudebního, dopisující čestný člen u sv. Anny ve Vídni, čestný člen německého National-Verein für Musik und ihre Wissenschaft, čestný člen Hudebních spol- ků ve Vídni, Insbrucku, Pešti a Budíně, a čestný člen spolku ve Lvově.“ Takto charakterizoval sám sebe Tomášek v úvodu autobiografie.14 i Tomášek přišel do Prahy v roce 1790, studoval nejprve gymnázium a později (1794–1799) matematiku, historii, estetiku, filozofii a práva na Karlově univerzitě. Hned v prvním roce svého pražského pobytu, jak píše v autobiografii, navštívil představení Mozartova Dona Giovanniho. Mozart ho okouzlil a této orientaci zůstal věrný až do konce svého života. ý Po dokončení právnických studií nenastoupil Tomášek právnickou praxi, ale zahájil dráhu klavíristy, učitele klavíru a skladatele. Velký význam pro jeho klavírní techniku měl Jan Ladislav Dusík, s nímž se sešel v Praze a který talentovanému Tomáškovi předával své zkušenosti. Podněty mohl získat i od tehdy v Praze proslulého klavíristy a pedagoga Františka Xavera Duška. Roku 1801 Tomášek poslouchal přednášky teoretika a skladatele abbé Georga Josepha Voglera. U var- haníka a hudebního historika Johanna Nikolause Forkela si prohloubil vztah k dílu Johanna Sebastiana Bacha. Zaujali ho také saský dvorní kapelník Johann Gottlieb Naumann a Muzio Clementi, kteří navštívili Prahu. V roce 1806 se stal Tomáškovým zaměstnavatelem hrabě Jiří František August Buquoy / Georg Franz August von Buquoy (1781–1851), vědec, spi- sovatel, podnikatel, národohospodář a ekonom, v jehož paláci na Malé Straně 122 Dopisy Václava Jana Tomáška z Morawetzovy sbírky Tomášek žil a tvořil až do roku 1822, kdy se přestěhoval do domu v dnešní Tomášské ulici. Mimo jiné se výrazně podílel na produkcích hudebního salónu, který hrabě jednu sezónu provozoval. Jeho povinnosti nebyly velké, a tak se mohl po celý svůj život věnovat výhradně hudbě. V Buquoyových službách zůstal To- mášek až do smrti, svým způsobem ovšem omezen v možnosti odejít do většího hudebního centra a výrazněji absorbovat nové vlivy. Od roku 1824 po sňatku s Wilhelminou Ebertovou (sestrou básníka Karla Egona Eberta) se Tomášek intenzívněji věnoval vyučování. Tomáškovými žáky byli postupně mj. klavíristé Julius Schulhoff a Alexander Dreyschock, skladatelé Jan Václav Hugo Voříšek, Leopold Eugen Měchura, Jan Bedřich Kittl, Ludvík Ritter z Rittersberku, Hans Hampel či hudební teoretikové August Wilhelm Ambros a Eduard Hanslick. Václav Jan Křtitel Tomášek13 Wilhelmina byla dobrou zpěvačkou i klavíristkou a manželé zpočátku vedli bohatý společenský život, jejich salón se stal vyhledávaným místem představitelů pražské vědy a kultury. Manželské soužití s mnohem mladší partnerkou se ovšem neobešlo bez problémů a Tomášek se poté stáhl z pražského hudebního života. Nicméně jako proslulou osobnost ho v té době se zájmem vyhledávali hudebníci, kteří přijeli do Prahy (Clara Schumann, Richard Wagner, Hector Berlioz, Niccolò Paganini a Ole Bull). g Tomášek spolupracoval s nakladatelem Aloisem Klarem, vydavatelem pražské ročenky Libussa, se švagrem Rudolfem Glaserem, redaktorem časopisu Ost und West, kde obsáhle referoval o svých cestách po Čechách a Německu, scházel se s historikem Františkem Palackým a básníky Karlem Viktorem Hansgirgem i Václavem Hankou. V letech 1845–1850 publikoval v šesti dílech autobiografii, která se stala důležitým dokumentem o kulturním životě v Evropě na počátku 19. století. Zde se podrobně vyjadřuje o významných osobnostech ze světa hudby, s nimiž se setkal, o různých hudebních a společenských událostech (Vídeňský kongres 1814) a projevuje názory na umělecká díla nebo interpretaci. Autobio- grafii dovedl bohužel pouze do roku 1824, kdy vstoupil do manželství. i Z Tomáškova díla žijí do dnešních dnů především jeho klavírní skladby (eklo- gy, rapsodie a dithyramby) a písně, které v poslední době opět zažívají renesanci. V německých písních zhudebnil texty Klopstocka, Gellerta, Höltyho, Bürgera, Schillera a zejména Goetha. Posledně jmenované byly komponovány převážně v roce 1815 (stejně jako písně Schubertovy) a vydány vlastním nákladem v devíti svazcích v Praze. Tomášek je zaslal Goethovi, jenž si jich velmi cenil, jak vyplývá z dopisu zaslaného Tomáškovi samotným básníkem. Při osobním setkání v Che- bu a Mariánských Lázních pak měl možnost je Goethovi sám předvést. Kompozice na Goethovy texty tvoří vrchol Tomáškovy písňové tvorby. Au- tor vyšel z písně Mozartovy (Heidenröslein, An Linna) a přes široké hymnické 123 Jana Vojtěšková melodie podobné Beethovenovi (Nähe des Geliebten, Mignons Lied) se postupně propracoval k prvkům počínajícího romantismu (Schäfers Klagelied, Der Fischer). Celkově však hodnotí někteří muzikologové jeho písně jako méně invenční a po- stupně stagnující v užívaných melodických a harmonických idiomech.15 Ve tři- cátých letech se už v písňovém stylu neposunul dále a jeho tvorbu považovali současníci za konzervativní. České písně komponoval Tomášek na texty přítele Václava Hanky. Jsou spíše okrajovou tvorbou. Tomášek je složil, „aby úplně ne- zapomněl svůj mateřský jazyk,“ jak napsal v autobiografii. K nejzdařilejším patří Starožitné písně, op. p g p 16 Srov. Jana Vojtěšková, „Letters from the Morawetz Collection,“ 32–39. Václav Jan Křtitel Tomášek13 82, z roku 1835, na texty Královédvorského rukopisu, ani ty však nedosahují úrovně německých písní. Tomášek byl biedermeierskou osobností. Jako nejvýraznější představitel hud- by předsmetanovské se zvláště v klavírních skladbách a písních přiblížil raně romantické orientaci, ale nikdy ji ve svých dílech plně nerozvinul. Napsal pouze jedinou operu, Seraphine oder Grossmut und Liebe, která byla při prvním uvedení v roce 1811 dobře přijata diváky, dočkala se však jen čtyř prove- dení. Neuvedl ji ani nový kapelník Carl Maria von Weber, který se o ní údajně, jak píše Tomášek ve svých pamětech, před premiérou vyjadřoval s uznáním. Velkým problémem bylo patrně libreto Johanna Heinricha Dambecka. Tomášek je také autorem několika velkých chrámových děl, Mše Es dur (1813) a zejména Requiem c moll (1820) a Korunovační mše k intronizaci rakouského císaře Ferdinanda I. Dobrotivého, který se nechal korunovat na českého krále jako Ferdinand V. (1836). 15 Srov. A. Simpson a K. DeLong, viz pozn. 13. 16 Srov. Jana Vojtěšková, „Letters from the Morawetz Collection,“ 32–39. 15 Srov. A. Simpson a K. DeLong, viz pozn. 13. Tomáškovy dopisy V Morawetzově sbírce se dochovalo celkem devět dopisů psaných Tomáškem a dva adresované Tomáškovi. Psaní Jana Václava Huga Voříška Tomáškovi z roku 1818 bylo publikováno již dříve.16 Ostatní korespondence je zde editována v chro- nologickém pořadí, počínaje rokem 1811 a konče rokem 1846. g p p j První tři dopisy této edice byly adresovány nakladatelům Kühnelovi a Peter- sovi do Lipska. Tehdy již renomovaný skladatel nabízel k vydání klavírní eklogy, rapsodie a klavírní sonátu, dále písně a orchestrální ouvertury. Rád by vydal i svou mši, zmíněna je zde i Tomáškova opera Seraphine. Dopis Kühnelovi z prosince 1811 potvrzuje Tomáškovy vztahy s Carlem Marií von Weberem, který navštívil Prahu v době premiéry Tomáškovy opery Seraphine. Weber měl psaní nakladateli osobně předat a Tomášek doufal, že bude Kühnelovi o opeře referovat. Urguje tři autorské exempláře Eklog, op. 35, které Kühnel vydal v prosinci předchozího roku (1810), a žádá, aby je předal 124 Dopisy Václava Jana Tomáška z Morawetzovy sbírky zprostředkovateli pražského Widtmannova knihkupectví. Vyzývá Kühnela, aby neotálel s vydáním písní. Pravděpodobně šlo o Selma von Voß und zwey Gesänge mit Begleitung des Pianoforte, op. 34, jež vyšly u Kühnela v prosinci 1811 a v té době už mohly být vydány, ale Tomášek o tom nevěděl. y ý y y Následující dopis Kühnelovi nabízí několik zajímavých informací a podnětů k dalšímu bádání. Především píše o dílech, která podle současného tematického katalogu17 neumíme identifikovat nebo je neznáme, dále pak dosti podrobně popisuje vydavatelské praktiky. V červenci 1813 Tomášek sděluje obsah dopisu, na který nedostal odpověď. Udivuje ho, že Kühnel nevydal rapsodie. Z dopisu není zcela jednoznačné, zda šlo o op. 40, který vyšel v roce 1813 u Haaseho, nebo jiné nám neznámé skladby. Také ho překvapuje, že Kühnel nevydal Sonátu A dur,18 kterou věnoval abbé Voglerovi. Sděluje cenu za šest vokálních tercetů (dílo není obsaženo v současném tematickém katalogu VJT). S podivem zjistil, že v knihkupectví Calve se objevily exempláře rapsodií, které vydal Kühnel (zřejmě op. 41), protože dosud nedostal ani honorář, ani autorské exempláře. Poslal též opravu k několika ryteckým chybám, které se vyskytly ve vydaném exempláři, a žádal opravu v dotisku. Nabídl k tisku rovněž několik předeher. I zde stojí za zamyšlení, o jaká díla šlo. V úvahu přichází Ouverture in Es, op. 38, VJT 41 z r. [1811?] a předehra k opeře Seraphine, VTJ 39. 17 Markéta Kabelková, „Václav Jan Tomášek,“ (PhDiss., FF UK Praha, 2012). Dále citováno jako Kabelková, 2012 a díla se zkratkou VJT. 18 Pravděpodobně Sonate in A, op. 26, VJT 27, vydaná později [1816?] u Hoffmeistera v Lipsku chybně jako op. 48, s věnováním hraběti Leopoldu Kaunitzovi. 19 IV. sešitu, op. 51, VJT 54. 20 Op. 63, VJT 66, tyto eklogy už u Peterse nevyšly. 21 Mše Es dur, op. 46, VJT 49. 27 Gedichte von Goethe, op. 53–58, Heft 1–6, z roku 1815, vydané pravděpodobně v roce 1820. K pro- blematice Tomáškových písní na Goethovy texty srov. např. Theodora Straková, „Tomáškovy písně na Goethovy texty. Příspěvek k poznání vokální tvorby raného romantismu v našich zemích,“ Časopis Moravského muzea 40 (1955): 214–252; http://new.musicologicaolomucensia.upol.cz/ artkey/mus-201402-0002_vaclav-jan-tomasek-1774-1850-vsestranny-pisnovy-skladatel- -komparativni-analyza-vybranych-hudebnich-zpracova.php. Tomáškovy dopisy p p p Další dopis nakladateli z července 1818 je již adresován Carlu Friedrichu Petersovi, který převzal lipské nakladatelství po Kühnelovi. Týká se vydání šesti eklog19 a Tomášek opět urgoval autorské výtisky a honorář. Vypůjčil si tři exem- pláře od knihkupce pana Enderse, aby je mohl zaslat do Liski hraběti Pavlov- skému, jemuž je tisk věnován. Žádá o opravu několika tiskových chyb, které specifikuje. Zároveň se táže, zda má Peters zájem o V. sešit eklog.20 Tomášek také informuje o provedení své mše ve Vídni 17. a 30. května 1818.21 Mši kdysi nabídl Petersovi, ale ten mu vůbec neodpověděl. Vydal ji proto na své náklady v Praze a v předplatném udal 154 exemplářů. Lituje jen, že vydal pouze hlasy, ale ne partituru. Z července 1820 pochází dopis Johanna Wolfganga Goetha Tomáškovi. Jeho obsah byl odborné literatuře znám, avšak originál dochovaný v Morawetzově sbírce je dodnes považován za nezvěstný. Text dopisu byl poprvé otištěn v Ost p y gy 21 Mše Es dur, op. 46, VJT 49. 125 Jana Vojtěšková und West jako Zwei noch ungedruckte Briefe von Göthe an W. J. Tomaschek 22 a podle této verze pak v edici Goethes Werke 23 a Repertorium Goethe-Briefe.24 Obě souborné edice Goethových dopisů vycházely z upraveného přepisu v Ost und West. Podle Goethova originálu z Morawetzovy sbírky zde tedy poprvé předkládám dopis se zachováním všech detailů pravopisu a opravou chyb dřívější edice.i p p p y j Dopis uveřejnil sám Tomášek v autobiografii,25 faksimile publikovaly Lidové noviny.26 K obsahu postačí na tomto místě obecné shrnutí, že Goethe děkoval Tomáškovi za šest sešitů písní na básníkovy texty.27 Goethe si Tomáškova zhu- debnění svých jedenačtyřiceti básní opravdu cenil, což vyjádřil i při osobním setkání se skladatelem v Chebu a Mariánských Lázních.28 V Mariánských Lázních v srpnu 1822 se Tomášek setkal s několika osobnost- mi a dámami, o nichž píše ve svých pamětech.29 Dopis adresovaný nejmenované slečně v září 1822 mohl být určen nejspíše Amelii Illaire z Berlína. Tomášek jí poslal dvě písně na Schillerovy texty (Schillers Lyrische Gedichte I., Die Erwar- tung, Das Lied, op. 37, VJT 40), které vyšly v Lipsku u Friedricha Hoffmeistera po r. 1815. Adresátka měla zřejmě kontakt na Carla Friedricha Zeltera, který působil od r. 1800 jako dirigent a ředitel berlínské Sing-Akademie. Tomášek doufal, že nastuduje jeho Mši Es dur. Provedení se nepodařilo doložit a pravdě- podobně se neuskutečnilo. 22 „Zwei noch ungedruckte Briefe von Göthe an W. J. Tomaschek,“, Ost und West, Nr. 10 (3. 2. 1838): 42. 23 Goethes Briefe, IV. Abteilung, 33. Band. 25. April – 31. Oktober 1820, (Weimar: Hermann Göhlaus Nachfolger, 1905): 121–122. 26 Lidové noviny, 21. března 1932, strana 3. Ani toto faksimile neznala souborná edice Goethovy korespondence. p y y y p p p 28 K formální stránce dopisu je ještě třeba dodat, že je psán rukou Goethova písaře Johna, Goethe kromě vlastnoručního podpisu připojil ještě závěrečné ergebenst (nejoddaněji). 25 W. J. Tomaschek, „Selbstbiographie“, Libussa, Prag, roč. 4–9 (1845–1850), viz též srov. pozn. 14, strana 216. p p p p j j 29 Srov. Němec, Vlastní životopis, 244 ad. (viz pozn. 14). g 24 Srov. https://ores.klassik-stiftung.de/ords/f?p=402:2:::::P2_ID:10034 a viz též pozn. 64. 22 „Zwei noch ungedruckte Briefe von Göthe an W. J. Tomaschek,“, Ost und West, Nr. 10 (3. 2. 1838): 42. 23 Goethes Briefe, IV. Abteilung, 33. Band. 25. April – 31. Oktober 1820, (Weimar: Hermann Göhlaus Nachfolger, 1905): 121–122. 24 Srov. https://ores.klassik-stiftung.de/ords/f?p=402:2:::::P2_ID:10034 a viz též pozn. 64. 25 W. J. Tomaschek, „Selbstbiographie“, Libussa, Prag, roč. 4–9 (1845–1850), viz též srov. pozn. 14, strana 216. 26 Lidové noviny, 21. března 1932, strana 3. Ani toto faksimile neznala souborná edice Goethovy korespondence. 27 Gedichte von Goethe, op. 53–58, Heft 1–6, z roku 1815, vydané pravděpodobně v roce 1820. K pro- blematice Tomáškových písní na Goethovy texty srov. např. Theodora Straková, „Tomáškovy písně na Goethovy texty. Příspěvek k poznání vokální tvorby raného romantismu v našich zemích,“ Časopis Moravského muzea 40 (1955): 214–252; http://new.musicologicaolomucensia.upol.cz/ artkey/mus-201402-0002_vaclav-jan-tomasek-1774-1850-vsestranny-pisnovy-skladatel- -komparativni-analyza-vybranych-hudebnich-zpracova.php. 28 K formální stránce dopisu je ještě třeba dodat, že je psán rukou Goethova písaře Johna, Goethe kromě vlastnoručního podpisu připojil ještě závěrečné ergebenst (nejoddaněji). 29 Srov. Němec, Vlastní životopis, 244 ad. (viz pozn. 14). p p p p j j g j j v. Němec, Vlastní životopis, 244 ad. (viz pozn. 14). Tomáškovy dopisy Z dubna 1829 pochází Tomáškova žádost o zvýšení roční renty, adresovaná skladatelovu zaměstnavateli hraběti Jiřímu Františku Augustu Buquoyovi, do je- hož služeb vstoupil v roce 1806. Renta byla Tomáškovi vyplácena 23 let ve stále stejné výši, tj. 500 zlatých. Podle žádosti mu hrabě slíbil už v roce 1818 zvýšení na 600 zlatých, k tomu ale pravděpodobně nedošlo. Na základě žádosti z roku 1829 byla Tomáškovi pravděpodobně zvýšena renta na 550 zlatých konvenční 126 Dopisy Václava Jana Tomáška z Morawetzovy sbírky měny. Nemáme o tom sice přímý doklad, ale vyplývá to ze zápisu do královských zemských desek, kdy Buquoy zvyšuje Tomáškovi od 1. 11. 1844 doživotní rentu z 550 na 700 zlatých konvenční měny.30 Další Tomáškova korespondence pochází až ze čtyřicátých let. Dopis bás- níkovi a redaktorovi lipského listu Komet Rudolphu Hirschovi z dubna 1842 se týká vydání Tomáškovy písně Lied eines Alpenmädchens (č. 3 z Drei Gesänge mit Begleitung des Pianoforte, op. 96, VJT 99), kterou Hirsch vydal v rámci jím založeného Album für Gesang mit Original-Beiträgen v roce 1843 v nakladatelství C. H. Bösenberg. Zároveň Tomášek poslal příspěvek o Wenzelu Neukirchnerovi, fagotistovi a skladateli, který vystoupil v dubnu 1842 v Praze a chystal se na kon- certy v Drážďanech a v Lipsku.31 Tomášek také upozornil na mladou talentova- nou klavíristku Sophii Bohrer, kterou slyšel v Praze, a požádal Hirsche o výtisk Komety z roku 1840, v němž byla na článek s citátem „dvě zvířata z Betléma“ otištěna odpověď zaslaná z Prahy.32 Zajímavým dokladem je i dopis mladému Josefu Zvonařovi z listopadu 1844 s posudkem Zvonařových skladeb. Dvacetiletý Zvonař, tehdy student na varha- nické škole, zaslal Tomáškovi své rané kompozice, čtyřhlasé Salve regina, offerto- rium Exaudi Deus deprecationem meam a Te Deum laudamus. Tomášek je hodnotil velmi pozitivně. Napsal: „Rozpoznal jsem v nich racionálního skladatele, skuteč- nou vzácnost za našich dnů.“ Vzhledem k tomu, že šlo o juvenilia, pravděpodobně se nedochovala do dnešních dnů. Z ledna 1846 pochází Tomáškův dopis jeho žáku Alexandru Dreyschockovi. Svědčí o velmi blízkém vztahu učitele a žáka. Ostrým sarkasmem reaguje na ví- deňské kritiky Heinricha Josepha Adamiho a Carla Waltera, kteří se vyjadřovali s despektem o Dreyschockově umění. Kromě toho zmiňuje kritiku předehry k opeře Seraphina (Wiener allgemeine Musik-Zeitung, 6. 1. 1846), kde je uvedeno, že byla zahrána poněkud mdle. Celou situaci ale komentuje s nadhledem. 30 Srov. zápis do Zemských desek o zvýšení renty z 15. 7. 1848, zde s. 48-49. 31 Tomáškův článek byl publikován v Allgemeine musikalische Zeitung 29. 7. 1842. 32 Tento článek se vzhledem k nedostupnosti výtisku nepodařilo ověřit. Závěr Předložený článek spolu s edicí dává nahlédnout do Tomáškovy oficiální i sou- kromé korespondence. Poskytuje řadu detailních informací k jeho skladbám, k jejich vydávání, provozování a posuzování kritikou. Objasňuje skladatelovy kontakty s umělci, vydavateli, žáky a podrobně líčí Tomáškův vztah k mecenáši Buquoyovi. Vzhledem k tomu, že zvláště po roce 1824 jsme o Tomáškových aktivitách informováni velmi sporadicky, každý z těchto nových faktů je pro po- znání skladatelovy biografie vítaným obohacením. Dozvídáme se výši honorářů a vydavatelské lhůty, jmenovitě se seznamujeme s díly, která Tomášek nabízel k vydání, odkrývají se priority nakladatele a kontakty na knihkupce – zprostřed- kovatele při prodeji hudebních tisků. (A najevo vycházejí praktiky vydavatelů, kteří často zadržovali autorské honoráře i výtisky.) V korespondenci se poukazuje na Tomáškovy skladby dnes neznámé. Kromě těchto velmi cenných informací získáváme jasnější představu o skladateli jako o člověku velmi vzdělaném, s ži- votním nadhledem, schopném vtipných a sarkastických glos. To dokresluje také zajímavá reflexe básníka Johanna Wolfganga Goetha nebo Tomáškův posudek raných skladeb Josefa Zvonaře. Tomáškovy dopisy z Morawetzovy sbírky jsou jedinými korespondenčními sděleními tohoto autora, která v současné době uchovává Národní muzeum – České muzeum hudby. Doplněna o doklady z pozůstalosti Vlastimila Blažka, kde se mimo jiné dochoval zápis do zemských desek dokreslující Tomáškovu žádost o zvýšení doživotní renty, bezesporu patří k pramenům, které nelze při bádání o Václavu Janu Tomáškovi přehlédnout. Tomáškovy dopisy y p j Poslední dopis edice z prosince 1846 byl zřejmě adresován nakladateli Imma- nuelu Guttentagovi a týká se vydání Tomáškova zhudebnění Hymnu de Spiritu Sancto (Veni creator Spiritus), op. 80, VJT 83. Z konce Tomáškova života nemáme mnoho zpráv, a tak si každá zmínka o jeho činech zaslouží pozornost. Tomášek před vydáním skladby krátce navštívil Berlín. Nakladatele žádal, aby titulní list tisku obsahoval správně názvy všech spolků, které mu vzdávaly pocty. Zároveň upozorňuje na připravované berlínské vystoupení svého žáka Alexandra Drey- schocka, který má na repertoáru předehru k opeře Seraphine. Autor ji nabízí k případnému vydání v Guttentagově nakladatelství. 127 Jana Vojtěšková Jana Vojtěšková p y p p 39 Tomášek ve svém životopise napsal: „Při první hlavní zkoušce byl také přítomen K. M. Weber, jenž přijel do Prahy, aby zde uspořádal koncert a mimo to, aby dojednal s Liebichem podmínky ohledně navrhovaného mu kapelnického místa ve Stavovském divadle v Praze. Stál vedle mne u piana, četl se mnou partituru a nemohl se dosti vynadiviti svéráznému vedení basů a zcela zvláštní instrumentaci. Byl jsem však příliš zaměstnán, než abych mohl náležitě oceniti poklony, které mně skládal.“ Viz Němec 1941, 108, v pozn. 14. 37 Pravděpodobně Selma von Voß / und / zwey Gesänge / mit Begleitung des Pianoforte / komponirt und gewidmet / dem Herrn J. H. Dambek / von / W. I. Tomaschek, / Tonsetzer bei Herrn Georg Grafen v. Buquoy. / 34s Werk. Bei A Kühnel, / Bureau de Musique in Leipzig. / Pr. 16 Gr., VJT 37, komp. [1807–1808], vyd. [XII. 1811], ČMH III E 31. 36 Six Eclogues / pour le / Pianoforte, composées et dediés / a son Ami / J. T. Held / Docteur en Médicine / Par W. J. Tomaschek / Compositeur chez Mr. Georg le Conte de Buquoy. / Oeuvr. 35 / a Leipsic, chez A. Kühnel (Preis 16 gr.), VJT 38, komp. [1807–1808], vyšlo [XII 1810], srov. Kabelková 2012, 172; viz též recenze Allgemeine musikalische Zeitung 1811 (May), sl. 383–384. 33 Ambrosius Kühnel (1770–1813), lipský varhaník, který založil spolu s Franzem Antonem Hoff- meisterem v Lipsku v roce 1800 nakladatelství Bureau de musique. V roce 1814 nakladatelství převzal Carl Friedrich Peters. [ ], y [ ], 38 Premiéra opery Seraphine se uskutečnila se 15. prosince 1811 ve Stavovském divadle. 35 Carl Maria von Weber navštívil Prahu před premiérou Tomáškovy opery Seraphine oder Grossmut und Liebe, op. 36, VTJ 39. 34 Tužkou bylo později chybně připsáno: [an Peters]. 40 Knihkupectví Caspara Widtmanna (1745–1815) bylo v Praze na Malé Straně v Mostecké ulici. Firma existovala už od konce 18. století, v roce 1817 vydala Musicalien-Catalog (Intelligenz-Blatt Prager Zeitung 1817, no. 298 z 24. 10.). Firmu převzal v roce 1831 jeho syn Moritz, který se rovněž orientoval na hudebniny. 41 Údaje nakladatelství o přijetí dopisu. y 41 Údaje nakladatelství o přijetí dopisu. Ediční zásady Edice jednotlivých dopisů vychází z diplomatického přepisu, kde se editorské zpracování textu co nejvíce blíží originálu. Ponechány jsou pravopisné chyby, špatně použitá velká a malá písmena. Jen v případě, že by čtení bylo nesrozumi- telné nebo mohlo být chybně interpretováno, je upozorněno [!] nebo je správná verze uvedena v hranaté závorce. Omylem vynechaná slova, která jsou důležitá pro kontext, jsou doplněna v hranaté závorce. Škrtnutý text je ponechán. Neči- telná písmena nebo slova jsou označena […], škrtnutá nečitelná pak […]. Slova vložená shora nebo zdola jsou začleněna do textu s vyznačením \ / a / \. Zkratky nejsou doplňovány, pouze ve výjimečných případech jsou doplněny nebo vysvětle- ny v poznámkách pod čarou. Adresy na obálce nebo v úvodu dopisu jsou přepsány před vlastním textem dopisu. Jednotlivé řádky se vyznačují svislými čarami \|. V případě delších dopisů jsou označeny předěly mezi jednotlivými stranami \|\|. Názvy děl jsou ponechány ve tvaru, v němž je uvádí pisatel, správná verze se pak 128 Dopisy Václava Jana Tomáška z Morawetzovy sbírky nachází v poznámce pod čarou. Vodorovné čárky vyznačující zdvojení souhlásek se rozepisují typograficky menším typem druhého písmene: m = mm n = nn. * * * Tomášek, Václav Jan: [Kühnel, Ambrosius]33 Praha, 22. 12. 1811 (NM-ČMH G 13 662) Tomášek, Václav Jan: [Kühnel, Ambrosius]33 Praha, 22. 12. 1811 (NM-ČMH G 13 662) Prag am 22 Decembrio 811 Wohlgebohrner Geehrtester Herr!34 H. Karl Maria v. Weber, über dessen Verdienste als Komponist, und Klavierspieler Deutschland schon längst ein ehrenvolles Wort ausgesprochen hat, war so gefällig, dieses Schreiben an Sie zu bestellen,35 welches Sie an den Nachtrag der 3 noch mir schuldi- gen Exemplaire meiner Egloguen erinnert, die ich fast 9 Monate hindurch umsonst erwarte.36 Zugleich ersuche ich Sie, mit der Herausgabe der 3 von mir übernommenen Gesängen nicht länger zu säumen;37 – denn meiner Ansicht nach glaube ich bestimmt, daß gegenwärtiger Augenblick, welcher durch die bereits geschehene Aufführung mei- ner Oper38 mir sehr viele Musikfreunde zugeführt hat, selbst Ihrer merkantilischen Unternehmung nicht ohne günstigen Erfolg seyn kann. Herr v. Weber hat der ersten Production beigewohnt, und kann daher über mein Werk richtiger als Hundert andere sprechen.39 \|\| 33 Ambrosius Kühnel (1770–1813), lipský varhaník, který založil spolu s Franzem Antonem Hoff- meisterem v Lipsku v roce 1800 nakladatelství Bureau de musique. V roce 1814 nakladatelství převzal Carl Friedrich Peters. 129 Jana Vojtěšková Die 3 Exemplaires von Egloguen beliebe scher Buchhandlung40 zu übergeben, wel obbenannte Handlung beipacken wird. Ediční zásady Ich bin mit aller Achtung Ihr ergebenster 1811 Tomaschek41 d. 22. Dec. Prag. – 28. – Die 3 Exemplaires von Egloguen belieben Sie nur dem Comissionär von Widtmann- scher Buchhandlung40 zu übergeben, welcher Sie gewiß bei nächster Versendung an die obbenannte Handlung beipacken wird. Ich bin mit aller Achtung Ihr ergebenster Tomaschek 1811 Tomaschek41 d. 22. Dec. Prag. – 28. – Die 3 Exemplaires von Egloguen belieben Sie nur dem Comissionär von Widtmann- scher Buchhandlung40 zu übergeben, welcher Sie gewiß bei nächster Versendung an die obbenannte Handlung beipacken wird. Ich bin mit aller Achtung Ihr ergebenster Tomaschek 1811 Tomaschek41 d. 22. Dec. Prag. – 28. – Die 3 Exemplaires von Egloguen belieben Sie nur dem Comissionär von Widtmann- scher Buchhandlung40 zu übergeben, welcher Sie gewiß bei nächster Versendung an die obbenannte Handlung beipacken wird. Ich bin mit aller Achtung Die 3 Exemplaires von Egloguen belieben Sie nur dem Comissionär von Widtmann- scher Buchhandlung40 zu übergeben, welcher Sie gewiß bei nächster Versendung an die obbenannte Handlung beipacken wird. Ich bin mit aller Achtung Ih Tomaschek Tomaschek 1811 Tomaschek41 d. 22. Dec. Prag. – 28. – * * * Praha, 22. prosince 1811 Velevážený nejváženější pane!34 Pan Karl Maria v. Weber, o jehož zásluhách jako skladatele a klavíristy Německo už dávno vyslovilo uctivé slovo, byl tak laskav, že se postará o tento dopis Vám,35 jenž Vás upomene na dodání mně dosud dlužných tří exemplářů mých Eklog, které už téměř devět měsíců marně očekávám.36 Zároveň Vás žádám, abyste s vydáním tří písní, které jste ode mě převzal, déle neotálel;37 – neboť dle mého mínění s určitostí věřím, že současná chvíle, která mi právě uskutečněným uvedením mé opery38 získala velmi mnoho hudebních přátel, nemusí být bez příznivého úspěchu ani Vašemu obchodnímu podnikání. Pan v. Weber se účastnil prvního představení, a může se tedy o mém díle vyslovit správněji než stovky jiných.39 \|\| y p y ý Ty tři exempláře Eklog předejte laskavě zprostředkovateli Widtmannova knihkupec- tví,40 který je jistě přibalí k další zásilce shora jmenovanému obchodu. J Váš nejoddanější Váš Tomaschek 1811 Tomaschek41 22. prosince. Praha. – 28. – 1811 Tomaschek41 22. prosince. Praha. – 28. – 130 Dopisy Václava Jana Tomáška z Morawetzovy sbírky Autograf dopisu Václava Jana Tomáška Ambrosiu Kühnelovi z 22. 12. 1811, 1. strana (NM-ČMH G 13 662) Autograf dopisu Václava Jana Tomáška Ambrosiu Kühnelovi z 22. 12. 1811, 1. 44 Six Rapsodies pour le Pianoforté / composées et dediées / a son Ami / Francois Charles Fritsch / par / W. J. Tomaschek. / Compositeur chez Mr George le Comte de Bouquoy / Oeuvre 40. – JCH[?] Prague chez Haas, Rue de Jesuites Nro 186. VJT 43, [1810], vyšlo [1813]. Recenze Allgemeine musikalische Zeitung 1813, no. 7, July. Ediční zásady strana (NM-ČMH G 13 662) 131 Jana Vojtěšková Autograf dopisu Václava Jana Tomáška Ambrosiu Kühnelovi z 22. 12. 1811, 2. strana (NM-ČMH G 13 662) Autograf dopisu Václava Jana Tomáška Ambrosiu Kühnelovi z 22. 12. 1811, 2. strana (NM-ČMH G 13 662) 132 Dopisy Václava Jana Tomáška z Morawetzovy sbírky Tomášek, Václav Jan: [Kühnel, Ambrosius] Praha, 24. 7. 1813 (NM-ČMH G 13 663) 47 Není zřejmé, o jaké dílo šlo. Tematický katalog děl V. J. Tomáška, viz Kabelková 2012 uvádí jen Singterzetten, sine op., VJT 163, které obsahují pouze dva tercety. 46 Abbé Georg Joseph Vogler zemřel v roce 1814. Sonáta VJT 27 vyšla s věnováním A Mr. Le Comte Leopoldo de Kaunitz. p 43 Pravděpodobně šlo o rapsodie, které vydal Haas, viz dále. g , , J y 45 Sonate in A, op. 26, VJT 27, vydaná později [1816?] u Hoffmeistera v Lipsku chybně jako op. 48, srov. Kabelková 2012, 148. 42 Tento dopis není znám. 43 Pravděpodobně šlo o rapsodie, které vydal Haas, viz dále. 44 Six Rapsodies pour le Pianoforté / composées et dediées / a son Ami / Francois Charles Fritsch / par / W. J. Tomaschek. / Compositeur chez Mr George le Comte de Bouquoy / Oeuvre 40. – JCH[?] Prague chez Haas, Rue de Jesuites Nro 186. VJT 43, [1810], vyšlo [1813]. Recenze Allgemeine musikalische Zeitung 1813, no. 7, July. 45 Sonate in A, op. 26, VJT 27, vydaná později [1816?] u Hoffmeistera v Lipsku chybně jako op. 48, srov. Kabelková 2012, 148. 46 Abbé Georg Joseph Vogler zemřel v roce 1814. Sonáta VJT 27 vyšla s věnováním A Mr. Le Comte Leopoldo de Kaunitz. 47 Není zřejmé, o jaké dílo šlo. Tematický katalog děl V. J. Tomáška, viz Kabelková 2012 uvádí jen Singterzetten, sine op., VJT 163, které obsahují pouze dva tercety. 48 Viz pozn. 40. 42 Tento dopis není znám. 42 Tento dopis není znám. g 48 Viz pozn. 40. 42 Tento dopis není znám. 50 Pravděpodobně Six / Rapsodies / pour le / Pianoforte / composées et dediées / a son Ami / Joseph Reeger / par / W. I. Tomaschek. / Compositeur chez Mr. George le Comte de Bouquoy / Oeuvre 41. / Liv. II des Rapsodies. / A Leipzig. Chez A. Kühnel, Bureau de Musique. VJT 44 z roku [1810], které Kühnel vydal v [červnu 1813], viz Intelligenz_Blatt zur Allgemeinen Musikalischen Zeitung 1813, No VI (9. 6.), s. 9, též Kabelková 2012, 197–200. Tomášek, Václav Jan: [Kühnel, Ambrosius] Praha, 24. 7. 1813 (NM-ČMH G 13 663) Prag am 24tn July 1813 Wohlgebohrner Herr! Unbegreiflich ist es mir, daß Sie mein leztes Schreiben42 bisher nicht beantwortet haben. Bei Ihrer Pünktlichkeit, die sich in allen Ihren Geschäften von jeher bewährte, läßt mich auch dießmal nichts anderes vermuthen, als; daß Sie meinen Brief durch Verschulden des unordentlichen Postlaufs gar nicht erhalten haben, in welcher Voraussetzung ich manches nun in diesem Schreiben wiederholen muß. Vor allem mußte es mich befremden, daß Sie die ersten Rapsodien gleichsam die ersten Ideen dieser Musikgattung für Ihren Verlag nicht übernommen haben,43 welche nun bei H. Haas herausgekommen sind,44 und [hin]sichtlich ihrer äusern Schönheit und Korrektheit alles was bisher in Prag erschienen ist übertreffen.i f Nicht minder sonderbar mußte ich finden, daß Sie die Sonate in A Dur mir zurück geschickt haben.45 Die Dedication sie[?] dieser Sonate spricht eines Theils selbst aus, was ich von dieser Composition halte, und es konnte Sie nur das Drängen Ihrer Geschäfte daran hindern, das Werk zu prüfen, welches ich dem Abbe Vogler, dem größten izt lebenden Harmonisten zugedacht habe.46 Sie wünschen zu wissen, was ich für die 6 Singterzetten fo[r]dre.47 Wenn ich meinem sonst treuen Gedächtniße trauen darf, so habe ich in meinem vorlezten Schreiben für alle 6 das Honorar auf 6 Dukaten festgesezt, wo ich mich auch mit 27fr in Zwanzigern begnüge, welcher Preiß mit xx dem Fleyße, mit dem dieselben gearbeitet sind, in gar keinem Verhältniße stehet. \|\| Ich rathe Ihnen, diese Singterzetten sobald als möglich herauszugeben, indem ich un- zählig mal gefragt wurde, ob sie schon zu haben sind. Auch habe ich Ihnen in meinem Schreiben berichtet, daß H. Widtmann48 sich selbst angetragen hat, mir den Betrag 133 Jana Vojtěšková von 27f in Zwanzigern für die Egloguen49 aus zuzahlen, welchen Betrag ich auch von ihm richtig erhalten habe. Einer meiner Freunde zeigte mir gestern ein Exemplair von Rapsodien aus Ihrem Verlage,50 welches er bei Calve51 gekauft hat. – So wie mich diese Herausgabe erfreute, so mußte ich mich darüber nicht wenig verwundern, da ich bisher b weder Honorar, noch meine 6 Exemplaire erhalten habe, welches meines Erachtens wieder den ge[wöhnl]ichen Gang der Geschäfte ist. – Ich berichte es Ihnen, weil ich im Voraus überzeugt bin, daß Sie an dieser verkehrten Sendung gewiß keinen Antheil haben, sondern die Saumseligkeit des Fuhrmanns an dieser Unordnung allein schuld ist. 51 Calveho knihkupectví založil Johann Gottfried Calve roku 1786. Po jeho smrti převzal podnik Johann Koch, pak K. Barth a roku 1810 Friedrich Tempsky. Od svého založení knihkupectví sídlilo na Malém náměstí v Praze I. 49 Mohlo jít o honorář za Eklogy, op. 35, viz pozn. 36. Tyto opravy se spolu s dopisem nedochovaly. 53 V úvahu přichází Ouverture in Es, op. 38, VJT 41, z r. [1810, podle Tomáškovy autobiografie (Němec 1941: 101)] a předehra k opeře Seraphine, op. 36, VJT 39, která vyšla v Praze u Carla Wilhelma Enderse. (Ouverture in D op. 23, VJT 24, je datována až 12. 8. 1814). 54 Údaje nakladatelství o přijetí dopisu. W. J. Tomaschek m.p. 52 Tyto opravy se spolu s dopisem nedochovaly. Tomášek, Václav Jan: [Kühnel, Ambrosius] Praha, 24. 7. 1813 (NM-ČMH G 13 663) Im bei liegenden Zettel sind ein paar Stichfehler an gezeigt, die ich in den Rapsodien gefunden und welche ich Ihnen für die künftige Abdrucke mittheile.52 Ein paar Ouvertüren fürs ganze Orchester53 sind für Sie bereit, ich erwarte daher Ihren Entschluß, ob Sie dieselben zur Durchsicht verlangen. Auch dürfte es Ihnen leicht seyn, in Leipzig, wo soviel Gelegenheit ist Orchester Kamersinfonien zu hören, sich von Ihn ihrem Effekt zu überzeugen. f Ich verbleibe mit aller Achtung Ihr ergebenster Diener f Ich verbleibe mit aller Achtung Ihr ergebenster Diener W. J. Tomaschek m.p. W. J. Tomaschek m.p. d. 24. July Prag54 – 26. – d. 24. July Prag54 – 26. – * * * 134 Dopisy Václava Jana Tomáška z Morawetzovy sbírky Dopisy Václava Jana Tomáška z Morawetzovy sbírky Praha, 24. července 1813 Praha, 24. července 1813 Velevážený pane! Je mi nepochopitelné, že jste na můj poslední dopis42 dosud neodpověděl. Při Vaší pre- ciznosti, která se odjakživa osvědčuje ve všech Vašich obchodech, se i tentokrát mohu jen domnívat, že jste můj dopis vinou nepořádného chodu pošty vůbec nedostal, a s tímto předpokladem teď musím leccos v tomto psaní zopakovat. p p p p Především mě udivilo, že jste první Rapsodie, takřka první ideje tohoto hudebního druhu, pro své vydavatelství nepřevzal,43 ty, které teď vyšly u pana Haase44 a vzhle- dem k jejich vnější kráse a bezvadnosti překonávají vše, co dosud bylo v Praze vydáno. Neméně podivným jsem shledal, že jste Sonátu A dur45 poslal zpět. Věnování této sonáty dílem samo vypovídá, co si o této skladbě myslím, a jen naléhavost Vašich podniků Vám mohla zabránit si prozkoumat dílo, které jsem připsal abbé Voglerovi, největšímu nyní žijícímu harmonikovi.46 j Přejete si vědět, co žádám za šest vokálních tercetů.47 Pokud smím věřit své jinak věrné paměti, stanovil jsem v posledním dopise za všech šest honorář 6 dukátů, avšak spokojím se i s 27 fl. ve dvacetnících, což je cena, která není v žádném poměru k píli, s níž byly vypracovány. \|\| y y yp y \|\| Radím Vám, abyste tyto vokální tercety co nejdříve vydal, neboť jsem byl mnohokrát dotazován, zda už jsou k mání. Také jsem Vám ve svém psaní sděloval, že pan Widt- mann48 se sám nabídl vyplatit mi částku 27 f ve dvacetnících za Eklogy,49 a tento obnos jsem od něj také v pořádku obdržel. 24. července, Praha54 – 26. – 56 Carl Wilhelm Enders, narozen v Lipsku roku 1778, vedl jako Buch-, Kunst- und Musikalien- Händler v Praze knihkupectví od roku 1809. Enders vydával také litografické portréty českých skladatelů, včetně portrétu Tomáškova. Současníkem C. W. Enderse byl H. J. Enders, o jeho knihkupectví se nedochovalo mnoho zpráv. Vydal Tomáškův op. 71. Srov. Irena Janáčková, „Pražští vydavatelé Václava Jana Tomáška,“ Hudební věda 18, no. 2 (1981): 174. y 57 IV. sešit vydaný v roce 1818, VJT 54. Six Eglogues / pour le Pianoforte / composées et dediées / A Mon- sieur le Comte / Antoine Pawlowski / Monseigneur de Liski en Gallice / par / W. I. Tomaschek, / Com- positeur chez Mr. le Comte de Buquoy. / Oeuv. 51 / 4me Livr. des Eglogues / Leipzig, / au Bureau de Musique de C. F. Peters. / Pr. 1 Rthl. 59 U Peterse další eklogy nevyšly. Pátý sešit op. 63, VJT 66, r. [1819] a šestý sešit op. 66, VJT 69, [1819] vydal v Lipsku Frederic Hoffmeister. q 58 Rod Pawlowski sídlil v dominiu Liski v Haliči. 55 Carl Friedrich Peters (1779–1827), převzal v roce 1814 nakladatelství po Ambrosiu Kühnelovi, viz též pozn. 33. Tomášek, Václav Jan: [Kühnel, Ambrosius] Praha, 24. 7. 1813 (NM-ČMH G 13 663) Jeden z mých přátel mi včera ukázal exem- plář Rapsodií z Vašeho vydavatelství,50 který zakoupil u Calveho.51 – Ačkoli mě toto vydání potěšilo, musel jsem se nemálo podivovat, že jsem dosud nedostal ani honorář, ani své exempláře, což je podle mého názoru opět obvyklý obchodní postup. – Sděluji Vám to, neboť jsem předem přesvědčen, že na tomto převráceném zasílání jistě nemáte podíl Vy, nýbrž nepořádek zavinila jedině liknavost povozníka. Na přiloženém lístku je zaznamenáno několik ryteckých chyb, které jsem v Rapsodiích nalezl a sděluji Vám je pro příští dotisky.52 Je pro Vás připraveno několik předeher pro orchestr,53 očekávám tedy Vaše rozhodnutí, zda je žádáte k prohlédnutí. Také pro Vás může být snadné v Lipsku, kde je tolik pří- ležitosti slyšet orchestrální komorní sinfonie, se o jejich efektu přesvědčit. y Zůstávám s veškerou úctou Váš nejoddanější služebník W. J. Tomaschek 1813 Tomaschek 135 Jana Vojtěšková Tomášek, Václav Jan: [Peters, Carl Friedrich]55 Praha, 9. 7. 1818 (NM-ČMH G 13 664) 60 Missa con Graduale et Offertorio in Es, op. 46, z roku 1813, VJT 49, byla komponována na paměť porážky francouzského sboru u Chlumce v roce 1813. Dílo vyšlo nedlouho po dokončení Tomáškovým vlastním nákladem v hlasech, později je přijal do nákladu C. W. Enders v Praze. Mše byla věnována králi Friedrichu Augustovi I. Saskému. Srov. též dopis J. V. H. Voříška Tomáškovi, viz Vojtěšková, „Letters from the Morawetz Collection,“ 36–39. Č j p p j 62 „Kirchenmusik. Am 17. d. M. wurde in der Augustiner Hofpfarrkirche eine, für uns wenigstens neue Messe in Es, von der Composition des der musikalischen Welt ehrenvoll bekannten und von ihr nach Verdienst geschätzten Herrn Tomaschek aus Prag, aufgeführt, welche, wenn allenfalls noch Beweise erforderlich wären, die unläugbarste Burgschaft für des Tonsetzters erhabenen Künstlerberuf gewähren würde. Hohe Originalität der Ideen und Ausarbeitung, eine musterhafte Stimmenführung, die effectvollste Instrumentenbegleitung, und vor allem die beynahe mit einer asiatischen Verschwendung angebrachten Kunstschätze, welche unsere classische Altvordern ihren Lieblingen als Erbtheil hinterliessen, machen die charakteristischen Bestandtheile dieses treffli- chen Werkes aus, welches, nach einer Anhörung detaillieren zu wollen, an Verwegenheit gränzen würde. […].“ Allgemeine musikalische Zeitung, no. 22 (30. 5. 1818): sl. 191–192. Repríza je tam ale oznámena na 31. května. Tomášek, Václav Jan: [Peters, Carl Friedrich]55 Praha, 9. 7. 1818 (NM-ČMH G 13 664) Wohlgebohrner Herr! Durch Zufall sahe ich gestern bei H. Enders56 die neuen Eklogen von mir,57 welches mich sehr befremden mußte, als Sie von jeher der alten Sitte treu mir als Verfasser nebst dem Honorar die ersten 6 Exemplare überschickten. Von Ihrer Pünktlichkeit ganz überzeugt, kann ich diese inconvenienz auf Ihre Rechnung nicht schreiben, ein dritter, der die Versendungen zu besorgen hat, wird sie begangen haben. Herr Enders hatte die Gütte mir 3 Exemplare indessen zu borgen, welche ich am 13ten dieses durch Gefällig- keit eines Klaviermeisters, welcher nach Lisky reiset an den H. Grafen Pawlowski58 übersende. Um den Absatz dieses Werkes nicht aufzuhalten, und nicht zu lang ein Schuldner des H. Enders zu bleiben, bitte ich Sie nach Empfang dieser Zeilen mir sobald als möglich die 6 Exempl. wie auch das Honorar zu schicken, und mir zugleich zu berichten, ob es Ihnen recht ist, wenn ich Ihnen wieder 6 neue Eklogen übersende, welche in einigen Tagen fertig werden, welche \|\| wenn Sie meinen Worten trauen, den ersten 4 Heften gewiß nicht nachstehen, wenn sie dieselben nicht sogar übertreffen.59 Bei flüchtigem Durchblick sind in den letzten Eklogen folgende zwo Kleinigkeiten zu verbessern, als: auf der 8ten Seite im Trio der 2ten Ekloge muß es auf der 3ten Zeile in 2ten Takte der rechten Hand so stehen ˙bœ Œ dann auf der 20ten Seite muss es gleich oben apassionato und nicht apassconato heyßen. Üb d d Ekl hl S d S h l h d D k h Übrigens sind diese Eklogen sowohl von Seite des Stichs, als auch des Drucks sehr elegant aufgelegt, und auser den oben bemerkten paar Fehlern sehr korrekt, folglich für jeden Klavierspieler sehr einladend. 136 Dopisy Václava Jana Tomáška z Morawetzovy sbírky In der Kürze muß ich Ihnen berichten, daß meine Messe,60 welche ich Ihnen einmal zum Verlag angetragen, und vorüber Sie mir gar keine Antwort gegeben haben, den 17ten61May zu Wien in der Hofkirche aufgeführt wurde, und solchen Furore machte, daß sie gleich darauf den 30ten May auf allgemeines Verlangen wiederholt werden mußte. Ein paar Worte im Allgemeinen darüber finden Sie in der Wiener musikalischen Zeitung No. 22 dieses Jahrganges.62 \|\| g g g Ich habe dieses Werk auf meine Unkosten zu Prag aufgelegt, und es Sr. Majestät Ihrem gnädigsten König gewidmet, wofür mir als Zeichen des Allerhöchsten Wohlgefallens eine goldene emaillirte Dose zu Theil wurde. j 61 Číslice je přepsaná a není jednoznačně čitelná. 63 Partitura Tomáškovy Mše Es dur nevyšla za jeho života tiskem. Tomášek, Václav Jan: [Peters, Carl Friedrich]55 Praha, 9. 7. 1818 (NM-ČMH G 13 664) Uibrigens habe ich bei diesem Unter- nehmen schönes Geschäft gemacht, indem ich um den Pränumerazionspreis von 20 f. W. W. nur in Böhmen allein 154 Exemplare abgesetzt habe, wobei ich nichts anderes zu bedauern habe, als daß ich einigen meiner Freunde gefolgt, und dieses Werk nicht in Partitur sondern in Auflegstimmen herausgegeben habe, in welcher Form es nur allein von dem Chor, nicht aber von jenen benützt werden kann, die sich der Composition widmen, und kontrapunktisch gearbeitete Werke gerne studieren. Vielleicht werde ich dieses Werk noch in Partitur herausgeben.63 In Gewärtigung einer Antwort von Ihnen geharre ich mit aller Achtung geharre ich mit aller Achtung Ihr ergebenster Tomaschek. Tomaschek. Prag am 9ten July 818. * * * 137 Jana Vojtěšková Jana Vojtěšková Velevážený pane! Náhodou jsem včera uviděl u pana Enderse56 své nové Eklogy,57 což mě muselo velmi udivit, neboť jste mi odjakživa, věren starému zvyku jako autorovi, kromě honoráře posílal prvních 6 exemplářů. Zcela přesvědčen o Vaší preciznosti, nemohu tuto nepří- stojnost připisovat na Váš účet a dopustil se jí kdosi třetí, kdo má obstarávat zasílání. Pan Enders byl té dobroty, že mi prozatím 3 exempláře zapůjčil, které 13. tohoto měsíce laskavostí jednoho mistra klavíru, jenž cestuje do Liski, zašlu panu hraběti Pavlovskému.58 Abych odbyt tohoto díla nezadržel a nezůstal příliš dlouho dlužní- kem pana Enderse, prosím Vás, byste mi po obdržení těchto řádek co nejdříve poslal 6 exemplářů a honorář a zároveň mi sdělil, zda je Vám vhod, abych Vám opět poslal 6 nových Eklog, které budou hotovy v několika dnech, a které, pokud věříte mým slo- vům, jistě za prvními čtyřmi sešity nezaostávají, pokud je dokonce nepřekonávají.59 Při letmém pohledu je třeba v posledních Eklogách opravit následující dvě drobnosti, jako: na 8. stránce v Triu 2. Eklogy musí na 3. řádku v 2. taktu v pravé ruce stát ˙bœ Œ , pak na 20. stránce musí hned nahoře stát apassionato a nikoli apassconato. Ostatně je vydání těchto Eklog jak po stránce rytí, tak i tisku velmi elegantní, a kro- mě shora poznamenaných několika chyb velmi korektní, takže pro každého klavíristu velmi lákavé. V krátkosti Vám musím sdělit, že má Mše,60 kterou jsem Vám kdysi nabídl k vydání, na což jste mi vůbec neodpověděl, byla 17. května61 premiérována v dvorním kostele a vyvolala takové nadšení, že musela být hned 30. května na všeobecnou žádost opa- kována. Několik všeobecných slov k tomu naleznete ve Wiener musikalische Zeitung č. © Praha, 9. července 1818. 64 Dopis byl poprvé v upravené verzi publikován: „Zwei noch ungedruckte Briefe von Göthe an W. J. Tomaschek,“ Ost und West, Nr. 10 (3. 2. 1838): 42; český překlad dopisu uveřejnil Tomášek ve svých pamětech, srov. Němec 1941, 216; faksimile Lidové noviny, 21. března 1932, strana 3. Podle edice v Ost und West vyšlo in Goethes Werke, IV. Abteilung, 33. Band. Goethes Briefe, 25. April – 31. Oktober 1820, Weimar: Hermann Göhlaus Nachfolger 1905, 121–122. V Reper- torium der Goethe-Briefe je poznámka: Verbleib unbekannt, srov. https://ores.klassik-stiftung. de/ords/f?p=402:2:::::P2_ID:10034. Dopis zmíněn v knize Arnošt Vilém Kraus, Goethe a Čechy (Praha: Bursík a Kohout, 1893). Tomášek, Václav Jan: [Peters, Carl Friedrich]55 Praha, 9. 7. 1818 (NM-ČMH G 13 664) 22 tohoto ročníku.62 \|\| Vydal jsem toto dílo v Praze na vlastní náklady a věnoval Jeho Veličenstvu Jeho nej- milostivějšímu králi, za což mi byla na znamení Nejvyššího potěšení darována zlatá emailovaná dóza. Mimochodem jsem při této příležitosti udělal dobrý obchod, když jsem za předplatní cenu 20 zl. vídeňské měny jen v Čechách udal 154 exemplářů, přičemž nelituju ničeho víc, než že jsem poslechl některé své přátele a nevydal toto dílo v partituře, nýbrž jako hlasy, a v této podobě může být použitelné pouze pro sbor, nikoli však pro ty, kdo se věnují kompozici a rádi studují kontrapunkticky vypraco- vaná díla. Možná toto dílo v partituře ještě vydám.63 V očekávání odpovědi od Vás setrvávám se vší úctou Váš nejoddanější Tomášek. Tomášek. © Praha, 9. července 1818. 138 Dopisy Václava Jana Tomáška z Morawetzovy sbírky 65 Tomášek zhudebnil a vydal devět sešitů písní na Goetheho verše v roce 1815 jako Gedichte von Goethe, op. 53–61, Heft 1–9, z roku 1815, VJT 56–64, vydané asi v roce 1820. V tomto roce je Tomášek Goethemu zaslal. 66 Dopis psal Goethův písař John, podpis včetně „ergebenst“ rukou Goethovou. 67 Dopis mohl být určen Amelii Illaire z Berlína. Tomášek se s ní sešel v Mariánských Lázních v srpnu 1822, srov. Němec 1941, 244.i u Friedricha Hoffmeistera po r. 1815. 70 Carl Friedrich Zelter (1758–1832), německý skladatel a dirigent, od r. 1800 ředitel berlínské Sing-Akademie. p 68 Nilsen. Neidentifikovaná osoba. 67 Dopis mohl být určen Amelii Illaire z Berlína. Tomášek se s ní sešel v Mariánských Lázních v srpnu 1822, srov. Němec 1941, 244. 68 Nilsen. Neidentifikovaná osoba. 69 Schillers Lyrische Gedichte I. (1. Die Erwartung, 2. Das Lied), op. 37, VJT 40, které vyšly v Lipsku u Friedricha Hoffmeistera po r. 1815. 70 Carl Friedrich Zelter (1758–1832), německý skladatel a dirigent, od r. 1800 ředitel berlínské Sing-Akademie. Goethe, Johann Wolfgang von: [Tomášek, Václav Jan]64 Jena, 18. 7. 1820 (NM-ČMH G 13 684) Wie sehr ich Ihnen, mein Theuerster, für den Antheil an meinen Liedern65 danke und für die unermüdet fortgesetzte Behandlung derselben, möcht ich Ihnen mündlich aus- drucken und zwar aus doppelten Grunde. Denn ob ich gleich schon viel angenehme Stunden bey dem Vortrag Ihrer Lieder genoßen, so bin ich doch seit vielen Jahren überzeugt daß wohl nur der Tondichter selbst und allenfalls einige von seinem Sinne völlig durchdrungene Schüler uns wahrhaft und eindringlich mittheilen, was er in einen Gedicht gefunden, wie er es aufgenommen und was er hineingelegt. Sodann wünschte mit einfachen und treuen Worten aussprechen zu können, daß ich meinen so mannigfaltigen, unter den verschiedensten Anlässen entstandenen Liedern nur dann eine innere Uebereinstimmung und ideelle Ganzheit zuschreiben darf \|\| als der Tonkünstler sie auch in die E[i]nheit seines Gefühls hin nochmals aufnehmen und als wären sie ein Ganzes nach seiner Weise durchführen wollen. Hierüber ließe sich in Gegenwart gar freundlich handlen, da man in der Ferne immer nur im Allgemeinen verharren darf. Ich füge die besten Wünsche hinzu und bitte mich Herrn Grafen Bouqoy, deßen wahre Freundschaft ich mir schmeicheln darf gelegentlich zum Besten zu empfehlen und mich künftighin von Ihren neuesten Productionen, wenn sie sich auch nicht gerade auf mich bezögen, einiges erfahren zu laßen. Mit nochmaligem gefühlten Dank schließend und mich hochachtungsvoll unterzeichnend 66 g ergebenst J. W. v. Goethe. g ergebenst J. W. v. Goethe. Jena den 18n July 1820. 139 Jana Vojtěšková i 69 Schillers Lyrische Gedichte I. (1. Die Erwartung, 2. Das Lied), op. 37, VJT 40, které vyšly v Lipsku u Friedricha Hoffmeistera po r. 1815. 70 Carl Friedrich Zelter (1758–1832), německý skladatel a dirigent, od r. 1800 ředitel berlínské Sing-Akademie. 67 Dopis mohl být určen Amelii Illaire z Berlína. Tomášek se s ní sešel v Mariánských Lázních v srpnu 1822, srov. Němec 1941, 244. 68 Nilsen. Neidentifikovaná osoba. 69 Schillers Lyrische Gedichte I (1 Die Erwartung, 2 Das Lied), op 37, VJT 40, které vyšly v Lipsku 74 Po pobytu v Karlových Varech a v Chebu, kde se setkal s Goethem, léčil se Tomášek v srpnu 1822 v Mariánských Lázních. Zde se setkal s několika osobnostmi a dámami, o nichž píše dále. Srov. Němec 1941, 244. 73 Nepodařilo se zjistit, zda byla mše v Berlíně v té době provedena. O rok dříve však byla provede- na v Lipsku, srov. „Musikalischer Bericht aus Leipzig,“ Allgemeine musikalische Zeitung, Nro. 57 (18. 7. 1821): 454. j p 72 Míněna je berlínská Sing-Akademie, kde Zelter působil jako ředitel. Zelterova sbírka, která byla v r. 2001 vrácena do berlínské Staatsbibliothek z Kyjeva, v soupisu dochovaných hudebnin Tomáš- kovu mši neuvádí, srov. https://www.sing-akademie.de/99-1-Zelter-Collection.html. , 75 Emile von Schlabrendorf pocházela z německého rodu sídlícího ve Slezsku. Jana Vojtěšková Jana Vojtěšková Jak velmi Vám, můj nejdražší, děkuji za Váš zájem o mé písně 65 a za jejich neúnavné a vytrvalé zpracovávání bych se Vám rád vyslovil ústně, a to z dvojího důvodu. Neboť třebaže jsem již zažil při přednesu Vašich písní hodně příjemných chvil, jsem přece už mnoho let přesvědčen, že snad jen skladatel sám a po případě některý z jeho žáků, naprosto prodchnutých jeho duchem, může nám pravdivě a důrazně sdělit, co v té nebo oné básni nalezl, jak ji přijal a co do ní vložil. p Pak bych si přál moci prostými a věrnými slovy vyjádřit, že svým tak rozmanitým a z nejodlišnějších popudů vzniklým písním smím jen tehdy připisovat vnitřní souvis- lost a ideální celistvost, pokud je také skladatel znovu pojme do jednoty svého citu a jako by byly celek, provede je po svém. O tom by se dalo přátelsky jednat za přítomnosti, neboť na dálku musíme vždy setrvat jen na tom obecném. – Připojuji nejlepší přání a prosím, abyste mne příležitostně co nejvlídněji poručil panu hraběti Bouquoyovi, jehož opravdové přátelství mi lichotí, a i nadále mi poskytujte zprávy o Vašich nej- novějších výtvorech, třebaže by se nevztahovaly přímo ke mně. Končím opětováním procítěného díku a znamenám se v hluboké úctě 66 procítěného díku a znamenám se v hluboké úctě 66 procítěného díku a znamenám se v hluboké úctě 66 nejoddaněji J. W. Goethe. procítěného díku a znamenám se v hlub nejoddaněji J. W. Goethe. V Jeně 18. července 1820 nejoddaněji J. W. Goethe. Tomášek, Václav Jan: [neznámá]67 Praha, 2. 9. 1822 (NM-ČMH G 13 666) Wohlgebohrnes Fräulein! Eher als ich es glauben konnte, bin ich durch Vermittlung meines Freundes, Herrn Nilsen,68 im Stande, die bewußten Musikalien Ihnen zu zusenden. Sie finden dabei als Zugabe noch eine Piece, die Erwartung,69 und das Lied von Schiller enthaltend, zwo Compositionen, welche Ihnen nicht mißfallen dürften. Ich hoffe daß H. Zelter70 meine 140 Dopisy Václava Jana Tomáška z Morawetzovy sbírky Dopis Johanna Wolfganga Goetha Václavu Janu Tomáškovi z 18. 7. 1820, 1. strana (NM-ČMH G 13 684) Dopis Johanna Wolfganga Goetha Václavu Janu Tomáškovi z 18. 7. 1820, 1. strana (NM-ČMH G 13 684) 141 Jana Vojtěšková Dopis Johanna Wolfganga Goetha Václavu Janu Tomáškovi z 18. 7. 1820, 2. strana (NM-ČMH G 13 684) Dopis Johanna Wolfganga Goetha Václavu Janu Tomáškovi z 18. 7. 1820, 2. 76 Henriette Schadow (1784–1832), rozená Rosenstiel z Berlína, druhá manželka sochaře Johanna Gottfrieda Schadowa (1764–1850). 77 Henriette von Schuckmann pocházela ze starého pruského rodu. 71 Missa con Graduale ed Offertorio in Es, op. 46, VJT 49, z roku [1813]. Mše byla s velkým úspěchem uvedena v roce 1818 ve Vídni. Srov. Vojtěšková 2021, 36–39, zvl. pozn. 80. v r. 2001 vrácena do berlínské Staatsbibliothek z Kyjeva, v soupisu dochovaných hudebni kovu mši neuvádí, srov. https://www.sing-akademie.de/99-1-Zelter-Collection.html. yj , p ý mši neuvádí, srov. https://www.sing-akademie.de/99-1-Zelter-Collection.html. Jana Vojtěšková Rozdělení, kdy zpívají pouze čtyři sólové hlasy a kdy nastupuje celý sbor, je v sopránovém hlase průběžně označeno slovy sólo a tutti, opisovače je tedy třeba při vypisování pěveckých partů na to pouze upozornit, aby věděl, co má vzít a co vynechat. A tak zazní toto dílo také v Berlíně k poctě Boží, k níž bylo napsáno.73 Prosím Vás, abyste mě o řádném obdržení, přilo- ženého \|\| co nejdřív zpravila několika řádky, abych kvůli řádnému odevzdání nebyl zbytečně zneklidněn – že na onen oblažující okamžik v Mariánských Lázních74 ni- kdy nezapomenu, za to ručí můj cit a má paměť. Závěrem si odvažuji prosbu, abyste vyřídila mé poručení paní hraběnce Schlabrendorfové,75 paní von Schadow76 a slečně Schu[c]kmannové.77 Urozená slečno! Dříve než bych myslel, jsem prostřednictvím svého přítele pana Nilsena68 schopen Vám poslat dotyčné muzikálie. Najdete jako přídavek také jednu hudebninu obsahu- jící Očekávání 69 a Píseň na Schillera, dvě kompozice, které se Vám snad mohou líbit. Doufám, že pan Zelter70 nebude považovat mou Mši71 nehodnou toho, aby ji uvedl v život v prostorách, kde vládnou duchové Händela a Grauna.72 p Jelikož život hudebních děl v sobě zahrnuje rytmus a jeho pohyb, udal jsem ve varhan- ním partu přesná tempa podle Mälzelova metronomu. Rozdělení, kdy zpívají pouze čtyři sólové hlasy a kdy nastupuje celý sbor, je v sopránovém hlase průběžně označeno slovy sólo a tutti, opisovače je tedy třeba při vypisování pěveckých partů na to pouze upozornit, aby věděl, co má vzít a co vynechat. A tak zazní toto dílo také v Berlíně k poctě Boží, k níž bylo napsáno.73 Prosím Vás, abyste mě o řádném obdržení, přilo- ženého \|\| co nejdřív zpravila několika řádky, abych kvůli řádnému odevzdání nebyl zbytečně zneklidněn – že na onen oblažující okamžik v Mariánských Lázních74 ni- kdy nezapomenu, za to ručí můj cit a má paměť. Závěrem si odvažuji prosbu, abyste vyřídila mé poručení paní hraběnce Schlabrendorfové,75 paní von Schadow76 a slečně Schu[c]kmannové.77 A tak se loučím s uklidňující myšlenkou, že si při prozpívání těchto hudebních básní vzpomenete na jejich autora. S naprostou úctou Váš nejoddanější služebník Tomášek Praha, 2. září 1822 Praha, 2. září 1822 Moje adresa zní: Václav J. Tomášek skladatel pana hraběte George Buquoye v Praze Moje adresa zní: Václav J. Tomášek skladatel pana hraběte George Buquoye v Praze Tomášek, Václav Jan: [Buquoy, Georg August von]78 Praha, 14. 4. 1829 (NM-ČMH G 13 658) Hochgeborener Herr, Gnädigster Graf! 78 Jiří František August Buquoy (Georg Franz August von Buquoy), srov. úvod k edici. Jana Vojtěšková strana (NM-ČMH G 13 684) 142 Dopisy Václava Jana Tomáška z Morawetzovy sbírky Messe71 nicht für unwürdig halten wird, sie in den Räumen, wo Händels, Grauns Geister walten, ins Leben zu bringen.72 Da Rhythmus und seine Bewegung das Leben der Tonwerke in sich einschließt, so habe ich deßhalb in der Orgelparte die Tempos nach Mälzlischem Metronom genau angegeben. Die Austheilung, wann blos die vier Solostimmen [zu und] zu singen, und wann der volle Singchor einzutreten hat, ist in der Sopranstimme durchgehends mit den Worten Solo, und tutti angezeigt, der Kopist hat daher beim Auszug der Singparten für den Chor blos darauf zu sehen, um zu wissen, was er aufzunehmen, und auszulaßen hat. Und so erschalle dieß Werk auch in Berlin zu Ehre Gottes, zu welcher es geschrie- ben ist.73 – Ich ersuche Sie, mir den richtigen Empfang des Beiliegenden, sobald als \|\| möglich mit einigen Zeilen zu benachrichtigen, damit ich mich der richtigen Abgabe wegen nicht umsonst beunruhiget werde – daß ich der beseligenden Augenblicke zu Marienbad74 nie vergeßen werde, ist mein Gefühl und mein Gedächtniß Bürge da- für. Schließlich wage ich die Bitte, mich der Frau Gräfin Schlabrendorf,75Frau von Schadow,76 und dem Fräulein Schukmann77 zu empfehlen. Und so scheide ich mich dem trostreichen Gedanken, daß Sie beim Durchsingen dieser Tondichtungen, sich auch ihres Verfassers erinnern werden. Mit vollkommenster Achtung Ihr ergebenster Diener Tomaschek Prag am 2ten September 1822 Die Adresse an mich lautet: a Wenceslas J. Tomaschek Compositeur chez Mons: le Comte Die Adresse an mich lautet: a Wenceslas J. Tomaschek Compositeur chez Mons: le Comte George Buquoy a Prague * * * 71 Missa con Graduale ed Offertorio in Es, op. 46, VJT 49, z roku [1813]. Mše byla s velkým úspěchem uvedena v roce 1818 ve Vídni. Srov. Vojtěšková 2021, 36–39, zvl. pozn. 80. 143 Jana Vojtěšková Jana Vojtěšková Urozená slečno! Dříve než bych myslel, jsem prostřednictvím svého přítele pana Nilsena68 schopen Vám poslat dotyčné muzikálie. Najdete jako přídavek také jednu hudebninu obsahu- jící Očekávání 69 a Píseň na Schillera, dvě kompozice, které se Vám snad mohou líbit. Doufám, že pan Zelter70 nebude považovat mou Mši71 nehodnou toho, aby ji uvedl v život v prostorách, kde vládnou duchové Händela a Grauna.72 Jelikož život hudebních děl v sobě zahrnuje rytmus a jeho pohyb, udal jsem ve varhan- ním partu přesná tempa podle Mälzelova metronomu. 79 Od státního bankrotu roku 1811 platila v Rakousku do roku 1858 tzv. vídeňská měna. Roku 1816 vešly do oběhu stříbrné mince v hodnotě 20, 10, 5 a 3 krejcary a měděné mince v hodnotě 1, ½ a ¼ krejcaru. Jeden zlatý konvenční měny se dělil na tři dvacetníky (desítková soustava byla zavedena až 1857). Od roku 1816 byly také v oběhu nové bankovky. p 81 Angelica Catalani (1780–1849), italská koloraturní sopranistka, uspořádala 5. a 7. září 1818 ve Sta- vovském divadle dvě akademie, srov. Dalibor 6, no. 35 (10. 12. 1863): 273.; K. K. priv. Prager Zeitung 31 (10. 9. 1818, Nichtpolitischer Anhang): 1 otiskl anonymní báseň opěvující její umění. Jana Vojtěšková Als ich vor drey und zwanzig Jahren die Ehre hatte, von Euer Hochgeboren als Com- positeur im Hochdero Hause vorgestellt zu werden, erhielt ich von Euer Hochgeboren eine vom 30ten Jänner 1806 datirte Urkunde, vermög welcher mir eine jährliche Pen- 144 Dopisy Václava Jana Tomáška z Morawetzovy sbírky sion von 500f. zugesichert wird. – Die Zeit hat seitdem an dem Verhältniße des gang- baren Geldes gegen die Convenzionsmünze so viel geändert, daß ich vermög des damals in 147f bestehenden Kurses nach dem heutigen Verhältniße der Wiener Währung gegen die Conventionsmünze mich mit einer Pension von 136f in Zwanzigern zu begnügen hätte.79 Euer Hochgeboren beglückten mich im Verlaufe dieser geraumen Zeit so oft mit Hochdero Huld, und Anerkennung meines Wirkens in dem Gebiethe der Kunst, daß ich einen Frevel begehen würde, den Gedanken zu hegen, Hochdienselben könnten es verwägen, \dahin gelangen laßen daß/ mein künftiges Schicksal [an die] \von der buchstablichen Auslegung des Gesetzes abhängig gemacht werden könnte/ und des [?] Landrechtes zu [?], wo ich nach der Aussage meines Schwiegervaters nichts mehr als 136f in Zwanzigern als eine jährliche Pension zu erwarten hätte, ich daher schon itzt bei dem Unverhältniß des Geldes mit der Lebenssubsistenz vor der mich erwartenden Noth und Kummer schaudern muß. Wie könnte ich aber auch glauben, daß Euer Hoch- geboren bei Hochdero begründeten Ansichten über Recht und Billigkeit ein Unrecht der Zeit an mir auf eine so schmerzvolle Art strafen würden, da sich Hochdieselben gegen mich oft gnädig, bei meinen Leiden immer theilnehmend zeigten. Auch bedarf der Rechtliche meines Wissens, in solchen Fällen bei seinem Thun und Lassen keiner fremden Rücksichten, die Moral leitet seine Gedanken, und nicht seine Thaten. – Ich habe auf Hochdero ehrenvolle Aufforderung damals das jus, und mit ihm auch einen sichern glänzenden Erwerb aufgegeben, mich nach Hochdero Wunsche, ausschließlich der Kunst gewidmet; ich habe den Unterricht der hohen Familie im Clavierspiel nach und nach begonnen, und setze ihn nach meinem Gewißen treu und redlich fort.80 Daß ich während der Zeit mehrere sehr vortheilhafte Anträge aus Pohlen, Rußland, und Wien ausgeschlagen \abgelehnt/ habe, darf ich wohl hier berühren, um zu zeigen, wie genau ich es mit einem bereits eingegangenen Verhältniße nehme. Und dieß alles that, und thue ich aus einer Anhänglichkeit an Hochdieselben, und im Vetrauen, daß Eure Hochgeboren meine lebenslängliche Subsistenz auf eine ehrende Art sichern werden. y y y 80 Tomášek vyučoval děti hraběte a podle smlouvy směl od listopadu do března vyučovat v Praze soukromě, viz Kabelková 2012, 34. O rentě viz s. 34, pozn. 40. 82 Tomášek se oženil v roce 1824 s Wilhelminou Ebertovou. 83 Tomášek pracoval na Nauce o harmonii, srov. Němec 1941, 280. Jana Vojtěšková Daß ich dessen, was Euer Hochgeboren für mich thaten, und ferner noch thun wollen, nicht unwürdig bin, daß ich über mein Fach reiflich nachgedacht, darin auch Resultate ge- kommen, von denen meinen Vorgängern \und meinen Mitgenoßen/ nichts geträumet hat; dies darf ich mir mit gutem Gewissen gestehen, und habe nichts als den Wunsch noch zu hegen, daß sich ein Mezän fände, der mich unterstützte, um ungestöhrt meine Theorie vollenden, der Welt damit ein neues aus dem Naturgesetzen gefolgertes System der \Harmonie und des Kontrapunktes/ übergeben zu können.83 In der Composizion habe ich die Zeit her Vieles, nach dem Urtheile der Kritik bedeutendes geliefert, ich hätte \aber/ selbst bei der Beschränkheit meiner Zeit noch mehr schreiben können, wenn mich aber \nicht eben/ die peinvolle Ungenauigkeit nicht in eine Stimmung gebannt hätte, in der man wohl ein Requiem oder ein Miserere mit gutem Erfolg \außerdem aber nichts Anderes/ schaffen kann. Wie gern möchte ich zu dem Jubiläum auch ein Te Pension, denn der Künstler soll nicht rechnen, ihn gehet der Curs nichts an. Ich bin es ja in meinem Gewißen verbunden, sie lebenslänglich zu versorgen, denn ich habe sie aus ihrer carrière herausgerißen. Damit ich es nicht vergeße, mache ich mir hier den Knopf ins Sacktuch. Die Landrechte würden für sie nichts thun, wenn ich früher sterben möchte, diese sehen nur auf den Vortheil der Waisen, um recht viel für sie zu sparen. Seyn sie unbesorgt, dieser Tage sollen sie es haben.“ – p y g g Das war die Antwort von Euer Hochgeboren auf meine Bitte, sie war die Antwort des höchsten Seelenadels, die mich aller Zweifeln, aller Unruhe entheben, mich in meiner Liebe, und Anhänglichkeit für Hochdieselben noch mehr bestärken mußte. – Nun sind aber wieder eilft Jahre dahin, und ich bin in derselben Angst und Unruhe, die mit dem vorrückenden Alter immer mehr und mehr zunimmt. Freilich habe ich mir die bittersten Vorwürfe zu machen, seitdem meine Bitte nicht wieder erneuert zu haben, indem ich wohl einsehen sollte, daß Euer Hochgeboren bei dem Schwall von Geschäften, und andern geistigen Arbeiten ein mündlich angebrachtes Anliegen leicht vergeßen konnten. Jana Vojtěšková \|\| Daß es Euer Hochgeboren zu thun gesonnen sind, haben sich Hochdieselben im Jah- re 1818, als Catalani81 in Prag war, und ich nach einem Mittagmahle Hochderselben um die Bestimmung der mir zugesagten Pension bat, gegen mich deutlich in den huld- vollsten Ausdrücken folgends geäußert: „Ich werde nächsten Tage ihnen die landtäfliche Versicherung geben, und zwar: Ich gebe ihnen 600f in Zwanzigern als eine jährliche 145 Jana Vojtěšková Pension, denn der Künstler soll nicht rechnen, ihn gehet der Curs nichts an. Ich bin es ja in meinem Gewißen verbunden, sie lebenslänglich zu versorgen, denn ich habe sie aus ihrer carrière herausgerißen. Damit ich es nicht vergeße, mache ich mir hier den Knopf ins Sacktuch. Die Landrechte würden für sie nichts thun, wenn ich früher sterben möchte, diese sehen nur auf den Vortheil der Waisen, um recht viel für sie zu sparen. Seyn sie unbesorgt, dieser Tage sollen sie es haben.“ – Das war die Antwort von Euer Hochgeboren auf meine Bitte, sie war die Antwort des höchsten Seelenadels, die mich aller Zweifeln, aller Unruhe entheben, mich in meiner Liebe, und Anhänglichkeit für Hochdieselben noch mehr bestärken mußte. – Nun sind aber wieder eilft Jahre dahin, und ich bin in derselben Angst und Unruhe, die mit dem vorrückenden Alter immer mehr und mehr zunimmt. Freilich habe ich mir die bittersten Vorwürfe zu machen, seitdem meine Bitte nicht wieder erneuert zu haben, indem ich wohl einsehen sollte, daß Euer Hochgeboren bei dem Schwall von Geschäften, und andern geistigen Arbeiten ein mündlich angebrachtes Anliegen leicht vergeßen konnten. Ich bin es daher Euer Hochgeboren, mir, und meinem Weibe schuldig, mich an Hochdieselben schriftlich nun zu wenden, und zugleich zu bitten, Euer Hochgeboren möchten vor Allem gnädigst berücksichtigen, daß ich bereits fünf und fünfzig Jahre zähle, durch die Gicht sehr oft in meinem weitern Erwerb gehemmt bin, und bei der Hartnäckigkeit des sehr zu tief wurzelnden Uibels gewiß auf kein hohes Alter rechnen darf, – Hochdieselben wollen erwägen, daß ich für mein Weib zu sorgen habe,82 die ich mit Hochdero Vorwissen ehligte, und, um als gräfl. buquoischer Ton-\|\| dichter auf eine zwar frugale, jedoch aber anständige Art zu leben, bei meinem mit der Theuerung zu sehr divergirenden Einkommen, alles was ich nebstbei durch den Unterricht erwerbe \erringe/, zusetzen muß, folglich nichts ersparen kann. Jana Vojtěšková Ich bin es daher Euer Hochgeboren, mir, und meinem Weibe schuldig, mich an Hochdieselben schriftlich nun zu wenden, und zugleich zu bitten, Euer Hochgeboren möchten vor Allem gnädigst berücksichtigen, daß ich bereits fünf und fünfzig Jahre zähle, durch die Gicht sehr oft in meinem weitern Erwerb gehemmt bin, und bei der Hartnäckigkeit des sehr zu tief wurzelnden Uibels gewiß auf kein hohes Alter rechnen darf, – Hochdieselben wollen erwägen, daß ich für mein Weib zu sorgen habe,82 die ich mit Hochdero Vorwissen ehligte, und, um als gräfl. buquoischer Ton-\|\| mit Hochdero Vorwissen ehligte, und, um als gräfl. buquoischer Ton-\|\| dichter auf eine zwar frugale, jedoch aber anständige Art zu leben, bei meinem mit der Theuerung zu sehr divergirenden Einkommen, alles was ich nebstbei durch den Unterricht erwerbe \erringe/, zusetzen muß, folglich nichts ersparen kann. Daß ich dessen, was Euer Hochgeboren für mich thaten, und ferner noch thun wollen, nicht unwürdig bin, daß ich über mein Fach reiflich nachgedacht, darin auch Resultate ge- kommen, von denen meinen Vorgängern \und meinen Mitgenoßen/ nichts geträumet hat; dies darf ich mir mit gutem Gewissen gestehen, und habe nichts als den Wunsch noch zu hegen, daß sich ein Mezän fände, der mich unterstützte, um ungestöhrt meine Theorie vollenden, der Welt damit ein neues aus dem Naturgesetzen gefolgertes System der \Harmonie und des Kontrapunktes/ übergeben zu können.83 In der Composizion habe ich die Zeit her Vieles, nach dem Urtheile der Kritik bedeutendes geliefert, ich hätte \aber/ selbst bei der Beschränkheit meiner Zeit noch mehr schreiben können, wenn mich aber \nicht eben/ die peinvolle Ungenauigkeit nicht in eine Stimmung gebannt hätte, in der man wohl ein Requiem oder ein Miserere mit gutem Erfolg \außerdem aber nichts Anderes/ schaffen kann. Jana Vojtěšková Wie gern möchte ich zu dem Jubiläum auch ein Te 146 Dopisy Václava Jana Tomáška z Morawetzovy sbírky Deum schreiben,84 um es vielleicht Sr Majestät während seines Hierseyns überreichen zu können, aber es ist mir \aber/ bei der Unruhe, und der Angst vor der \trüben/ Zu- kunft nicht möglich, mich zu dem Jubel der Himmelsschaaren zu schwingen; Nur Euer Hochgeboren können meinen Geist aus den \seiner/ drey und zwanzig jähriger Haft befreyen wenn Hochdieselben mir die längst \mündlich und schriftlich/ versprochene Pension \urkundlich/ versichern, der Fesseln entledigt würde ich dem Ewigen nicht allein ein Te Deum sondern noch mehrere Hymnen \noch/ singen, und nie \dabei/ vergeßen, daß Euer Hochgeboren von jedem seiner \meiner/] Werke der Pflegevater sind, und sich den durch \Hochdenselben/ die Kunstwelt \dafür/ verpflichtet hat \ist/. Uibrigens ist der Unterricht bei der hohen Familie noch lange nicht beendet,85 daß schon itzt die Rede von der Beziehung einer Pension seyn könnte. Bei meiner öftern Kränklichkeit ist es wohl möglich, daß ich den Zeitpunkt des Pensiongenußes gar nicht erlebe, ich daher für den Augenblick nichts als ein beruhigtes Gemüth gewinne, wofür ich Euer Hochgeboren ewig dankbar \|\| bleiben muß \werde/. Indem ich mein unterthä- nigstes Anliegen noch einmal an Hochdero gefühlvolles Herz lege, und um eine baldige, gnadige Resoluzion bitte,86 verharre ich in tiefster Ehrfurcht g g Euer Hochgeboren unterthänigster Diener Wenzel Johann Tomaschek Prag am [14] April 829 Tužkou připsáno: Dem Grafen eigenhändig [über]lassen den 14ten April * * * Vysoce urozený, nejmilostivější hrabě! Když jsem měl před třiadvaceti lety tu čest být Vaší urozenosti v Jejím domě jako skladatel představen, obdržel jsem od Vaší urozenosti dokument, datovaný 30. lednem 1806, jímž se mi zaručovala roční renta 500 zl. – Čas od té doby změnil poměr běžných financí vůči konvenční minci tolik, že ze 147 zl. podle tehdejšího kurzu bych se měl spokojit podle dnešního poměru vídeňské měny vůči konvenční minci s rentou 136 zl. ve dvacetnících.79 Vaše urozenost mě v průběhu této dlouhé doby tak často obšťastnila svou přízní a uznáním mého konání v oblasti umění, že bych spáchal přečin, kdy- bych choval myšlenku, že by Vaše urozenost mohla uvažovat \dospět k tomu, že/ můj j j Když jsem měl před třiadvaceti lety tu čest být Vaší urozenosti v Jejím domě jako skladatel představen, obdržel jsem od Vaší urozenosti dokument, datovaný 30. lednem 1806, jímž se mi zaručovala roční renta 500 zl. 86 Tomáškovi byla renta pravděpodobně zvýšena na 550 zlatých, protože v r. 1844 uvedl Buquoy, že zvyšuje Tomáškovi rentu z 550 na 700 zlatých (viz dále záznam z r. 1844). O zvýšení v roce 1829 však neznáme žádný doklad. 84 Není známo, že by Tomášek v té době složil rekviem. Rekviem, op. 72, VJT 75 vydané u B. Schotta v Mohuči získalo imprimatur 1. 4. 1828. Poté žádnou kompozici tohoto druhu VJT neuvádí. 85 Srov. pozn. 80. 86 Tomáškovi byla renta pravděpodobně zvýšena na 550 zlatých, protože v r. 1844 uvedl Buquoy, že zvyšuje Tomáškovi rentu z 550 na 700 zlatých (viz dále záznam z r. 1844). O zvýšení v roce 1829 však neznáme žádný doklad. 84 Není známo, že by Tomášek v té době složil rekviem. Rekviem, op. 72, VJT 75 vydané u B. Schotta v Mohuči získalo imprimatur 1. 4. 1828. Poté žádnou kompozici tohoto druhu VJT neuvádí. 85 Srov pozn 80 85 Srov. pozn. 80. Jana Vojtěšková Buďte bez starostí, v těchto dnech to dostanete.“ – příští osud na učinit odvislým od doslovného výkladu zákona a [?] zemského práva, kdy bych podle výroku svého tchána nemohl očekávat roční rentu větší než 136 zl. ve dvacetnících, takže se už nyní při nepoměru peněz k životním potřebám musím hrozit očekávané bídy a starostí. Jak bych si ale také mohl myslet, že by mě Vaše uroze- nost při svých opodstatněných názorech na právo a spravedlnost tak bolestně trestala za bezpráví doby, když se ke mně často projevovala milostivě a vždy soucítila s mým utrpením. Spravedlivý také, podle mého vědomí, nepotřebuje v takových případech žádných cizích ohledů na to, co činí a poskytuje, morálka vede jeho myšlenky, a ne jeho činy. – Já se onehdy na čestnou výzvu Vaší urozenosti vzdal právnictví a tím také jistého skvělého výdělku, abych se na přání Vaší urozenosti věnoval výhradně umění; postupně jsem začal ve vznešené rodině vyučovat klavíru a podle svého svědomí v tom věrně a poctivě pokračuji.80 Smím zde jistě zmínit, že jsem během doby odvrhl \odmítl/ výhodné nabídky z Polska, Ruska a Vídně, abych ukázal, jak vážně vztah, do nějž jsem už vstoupil, chápu. A to vše jsem činil a činím z oddanosti k Vaší urozenosti a ve víře, že mi Vaše urozenost čestným způsobem zajistí doživotní existenci. \|\| ý p \|\| Že tak Vaše urozenost hodlá učinit, vyjádřila roku 1818, kdy byla v Praze Catalani81 a po jednom obědě jsem Vaši urozenost prosil o určení přislíbené renty, a Vaše urozenost mi jasně a nejvznešenějšími slovy sdělila následující: „V příštích dnech to dám stvrdit zemskými deskami, a sice: Poskytnu vám 600 zl., ve dvacetnících jako roční rentu, ne- boť umělec nemá počítat, jemu do kurzu nic není. Jsem přece svým svědomím zavázán vás doživotně zajistit, neboť jsem vás vytrhl z vaší kariéry. Abych na to nezapomněl, dělám si uzel na kapesníku. Zemské právo by pro vás nic neudělalo, kdybych zemřel dřív, to hledí jen na výhody sirotků, aby se pro ně hodně našetřilo. Buďte bez starostí, v těchto dnech to dostanete.“ – To byla odpověď Vaší urozenosti na mnou prosbu, byla to odpověď nejvyšší šlechetnosti duše, která mě zbavila všech pochybností, všeho neklidu a jen mě v mé oddanosti k Vaší urozenosti ještě posílila. – Nyní však uplynulo jedenáct let, a nacházím se ve těchže obavách a neklidu, které s postupujícím věkem stále víc narůstají. Jana Vojtěšková – Čas od té doby změnil poměr běžných financí vůči konvenční minci tolik, že ze 147 zl. podle tehdejšího kurzu bych se měl spokojit podle dnešního poměru vídeňské měny vůči konvenční minci s rentou 136 zl. ve dvacetnících.79 Vaše urozenost mě v průběhu této dlouhé doby tak často obšťastnila svou přízní a uznáním mého konání v oblasti umění, že bych spáchal přečin, kdy- bych choval myšlenku, že by Vaše urozenost mohla uvažovat \dospět k tomu, že/ můj 147 Jana Vojtěšková příští osud na učinit odvislým od doslovného výkladu zákona a [?] zemského práva, kdy bych podle výroku svého tchána nemohl očekávat roční rentu větší než 136 zl. ve dvacetnících, takže se už nyní při nepoměru peněz k životním potřebám musím hrozit očekávané bídy a starostí. Jak bych si ale také mohl myslet, že by mě Vaše uroze- nost při svých opodstatněných názorech na právo a spravedlnost tak bolestně trestala za bezpráví doby, když se ke mně často projevovala milostivě a vždy soucítila s mým utrpením. Spravedlivý také, podle mého vědomí, nepotřebuje v takových případech žádných cizích ohledů na to, co činí a poskytuje, morálka vede jeho myšlenky, a ne jeho činy. – Já se onehdy na čestnou výzvu Vaší urozenosti vzdal právnictví a tím také jistého skvělého výdělku, abych se na přání Vaší urozenosti věnoval výhradně umění; postupně jsem začal ve vznešené rodině vyučovat klavíru a podle svého svědomí v tom věrně a poctivě pokračuji.80 Smím zde jistě zmínit, že jsem během doby odvrhl \odmítl/ výhodné nabídky z Polska, Ruska a Vídně, abych ukázal, jak vážně vztah, do nějž jsem už vstoupil, chápu. A to vše jsem činil a činím z oddanosti k Vaší urozenosti a ve víře, že mi Vaše urozenost čestným způsobem zajistí doživotní existenci. \|\| Že tak Vaše urozenost hodlá učinit, vyjádřila roku 1818, kdy byla v Praze Catalani81 a po jednom obědě jsem Vaši urozenost prosil o určení přislíbené renty, a Vaše urozenost mi jasně a nejvznešenějšími slovy sdělila následující: „V příštích dnech to dám stvrdit zemskými deskami, a sice: Poskytnu vám 600 zl., ve dvacetnících jako roční rentu, ne- boť umělec nemá počítat, jemu do kurzu nic není. Jsem přece svým svědomím zavázán vás doživotně zajistit, neboť jsem vás vytrhl z vaší kariéry. Abych na to nezapomněl, dělám si uzel na kapesníku. Zemské právo by pro vás nic neudělalo, kdybych zemřel dřív, to hledí jen na výhody sirotků, aby se pro ně hodně našetřilo. Jana Vojtěšková Ovšemže si činím nejtrpčí výčitky, že jsem od té doby svou žádost nepřipomněl, vždyť jsem přece musel chápat, že Vaše urozenost při návalu povinností a další duševní činnosti mohla ústně přednesenou prosbu snadno zapomenout. Pročež jsem Vaší urozenosti, sobě i své ženě dlužen, abych se na Vaši urozenost nyní obrátil písemně, a zároveň poprosil, aby Vaše urozenost především milostivě vzala ohled na to, že už je mi padesát pět let, dna mě velmi často omezuje v dalším výdělku a při tvrdošíjnosti velmi hluboko vězícího ne- duhu jistě nemohu počítat s žádným vysokým věkem – Vaše urozenost kéž uváží, že se musím postarat o svou ženu,82 se kterou jsem se oženil s vědomím Vaší urozenosti, a že k tomu, abych jako hraběcí buquoyovský skladatel \|\| žil se svými, při zdražování příliš rozdílnými příjmy, sice skromně, avšak přesto slušně, musím to, co vydělám \čeho se domohu/ mimo, vyučováním, dosadit, a proto nemohu nic ušetřit. Že nejsem toho, co pro 148 Dopisy Václava Jana Tomáška z Morawetzovy sbírky mě Vaše urozenost učinila, a dále ještě činit bude, nehoden, že jsem o svém oboru mnoho přemýšlel a vzešly z toho také výsledky, o jakých se mým předchůdcům a souputníkům nesnilo, to si mohu s dobrým svědomím přiznat a nezbývá nic než vznést ještě přání, aby se nalezl mecenáš, který by mě podporoval, abych nerušen dokončil svou teorii a mohl předat světu nový systém \harmonie a kontrapunktu/, odvozený z přírodních zákonů.83 V kompozici jsem dosud dodal leccos, podle úsudku kritiky významného, mohl bych \však/ dokonce i při omezenosti času napsat ještě víc, kdyby mě však \právě/ trýz- nivá nejistota nepoutala v náladě, v níž se jistě s úspěchem dá napsat Requiem nebo Miserere, \krom toho však nic jiného/. Jak rád bych k jubileu také napsal Te Deum,84 a možná bych je Jeho Veličenstvu při jeho pobytu zde mohl předat, ale při neklidu a obavách z \pochmurné/ budoucnosti mi není možné povznést se k jásotu nebeských zástupů. Jen Vaše urozenost může mého ducha osvobodit z \jeho/ dvacetiletého vězení, pokud mi zajistí ústně a písemně dávno přislíbenou rentu dokumentem, a zbaven pout bych zpíval Věčnému nejen Te Deum, nýbrž ještě vícero hymnů, a nikdy nezapomněl, že Vaše urozenost je každému jeho \mému/ dílu pěstounem, a tím se \Vaší urozenosti/ je umělecký svět \za to/ zavázán. ý Ostatně výuka u vznešené rodiny ještě dávno není ukončena,85 aby už nyní mohla být řeč o výslužném. 87 Rudolf Hirsch (1816–1872), novinář, básník, skladatel. Studoval v Brně a ve Vídni práva. V roce 1840 odešel do Lipska, kde byl oblíben jako autor písňových textů, které nezřídka sám zhudebňo- val. V letech 1841–1843 byl redaktorem listu Komet (Kometa). V Lipsku založil Album für Gesang, kde publikoval mj. také Tomáškovy písně. První dva ročníky vyšly 1841–1843. 89 Václav Jan Tomášek: Lied eines Alpenmädchens, no. 3 z Drei Gesänge mit Begleitung des Pianoforte, op. 96, VJT 99. (1. Lied von einem Standbilde der Madonna, 2. Milde Stunde, 3. Lied eines Alpen- mädchens). Hirsch ji vydal v rámci Album für Gesang mit Original-Beiträgen von A. H. Chelard, Ferdinand David, … W. I. Tomaschek, … herausgegeben von Rudolph Hirsch. Zweiter Jahrgang, 1843. Leipzig, Verlag von C. H. Bösenberg. O písních srov. Wolfgang Antesberger, Die deutschspra- chigen Lieder für Pianoforte von Johann Wenzel Tomaschek, obsah dostupný z https://searchworks. stanford.edu/view/12686326. 90 Wenzel Neukirchner (1805–1889), fagotista a skladatel, pocházel z Nového Strašecí. V Praze vystoupil 8. 4. 1842, srov. Ant. Müller, „Kunst und Leben in Böhmen. Theaterbericht vom 8. April,“ Bohemia 15, no. 43 (10. 4. 1842): 3; o jeho posledním koncertu v Praze viz B: „Concert des Herrn Neukirchner,“, Bohemia 15, no. 47 (19. 4. 1842): 4. Tomášek o něm v souvislosti s pražským koncertem napsal: „Neukirchner gehört zu den in unseren Tagen seltenen Künstlern, die auf dem einzig wahren Wege ihre Kunstbildung verfolgen, die, fern von aller Anmassung, die Verherrli- chung der Kunst nie aus dem Auge verlieren, und die mühsam erworbene Technik nie als Zweck, sondern blos als Mittel betrachten; auch konnte er nur auf solchem Wege zu einer vollendeten Durchbildung des Tones gelangen. Der Umfang seines Instrumentes, vom Contra B bis zum e’, beträgt beinahe drei und eine halbe Oktave, eine gewiss bedeutende Tonmasse, die der Künstler mit einer unglaublichen Leichtigkeit und Ruhe beherrscht. Alle Töne sind von gleicher Färbung und gleicher Dichtigkeit, daher bei den tiefen Tönen durchaus kein Schnarren bemerkt wird. Die stets richtige Betonung und Sonderung der Perioden, die von der richtigen Vertheilung des Athems abhängt, dann die unendliche Nüancirung des Tones durch alle Gradationen des Piano und Forte, so wie es der Ausdruck erfordert, sind Vorzüge, die unsern Landsmann zu einem der grössten jetzt lebenden Virtuosen erheben, und die königlich würtembergische Musikkapelle kann mit Recht stolz darauf sein, Neukirchner zu ihren Mitgliedern zu zählen.“ Srov. Prag, Allgemeine musikalische Zeitung, no. 26 (29. 7. 1842): 532. 91 V Li k dě d b ě il 88 Podtrženo evidentně dodatečně, tužkou. g 91 V Lipsku pravděpodobně nevystoupil. Jana Vojtěšková Při mém častém churavění je docela dobře možné, že se doby, kdy budu moci výslužného užívat, ani nedožiji, proto pro tuto chvíli nezískám nic víc než klidnou mysl, za což zůstanu Vaší urozenosti navždy \|\| povděčen. Pročež svou nejpo- níženější prosbu ještě jednou Vaší urozenosti kladu na citlivé srdce, a prosím o brzké, milostivé rozhodnutí,86 zůstávám v nejhlubší úctě j Vaší urozenosti nejponíženější služebník Václav Jan Tomášek Praha, [14] dubna 1829 Tužkou připsáno: Hraběti vlastnoručně předáno 14. dubna j Vaší urozenosti nejponíženější služebník Václav Jan Tomášek Praha, [14] dubna 1829 Tužkou připsáno: Hraběti vlastnoručně předáno 14. dubna Vaší urozenosti nejponíženější služebník Václav Jan Tomášek Praha, [14] dubna 1829 T k Tužkou připsáno: Hraběti vlastnoručně předáno 14. dubna Tomášek, Václav Jan: Hirsch, Rudolph87 Praha, 15. 4. 1842 (NM-ČMH G 13 661) Von Prag An des Herrn Herrn Rudolph Hirsch Redakteurs Von Prag An des Herrn Herrn Rudolph Hirsch Redakteurs 87 Rudolf Hirsch (1816–1872), novinář, básník, skladatel. Studoval v Brně a ve Vídni práva. V roce 1840 odešel do Lipska, kde byl oblíben jako autor písňových textů, které nezřídka sám zhudebňo- val. V letech 1841–1843 byl redaktorem listu Komet (Kometa). V Lipsku založil Album für Gesang, kde publikoval mj. také Tomáškovy písně. První dva ročníky vyšly 1841–1843. 149 Jana Vojtěšková des Kometen Wohlgeboren in Leipzig Geehrtester Herr und Freund! Diesmal bin ich doch pünktlich in der Beantwortung Ihrer lieben Zeilen. Ich zweifle nicht, daß das Alpen-Mädchen88 schon bei Ihnen ist, wobei ich hoffe daß Ihnen der gesunde Gliedebau daran gefallen wird.89 Was den väterlichen Schutz anlangt, um den Sie mich für Ihre Lieder ansprechen, so dürfen Sie ganz beruhigt seyn, daß ich daran nichts versäume. Das alte Sprichwort eine Hand wäscht die andere, will ich durch das beiliegende Blättchen in Anwendung bringen, indem ich Sie ersuche es ungesäumt in Ihrem Blatte abdrucken zu laßen, damit die Dresdner und Leipziger auf die ausgezeichneten Vor- züge der Virtuosität vorbereitet würden, die Herr Neukirchner90 in seinen Conzerten nächster Tage darlegen wird.91 – Versäumen Sie es nicht, diesen wahrhaft seltenen Künstler zu hören. Sie klagen in Ihrem Briefe über Mangel an Zeit, ich aber verliere 90 Wenzel Neukirchner (1805–1889), fagotista a skladatel, pocházel z Nového Strašecí. V Praze vystoupil 8. 4. 1842, srov. Ant. Müller, „Kunst und Leben in Böhmen. Theaterbericht vom 8. April,“ Bohemia 15, no. 43 (10. 4. 92 Článek se nepodařilo dohledat. Periodikum je těžko dostupné. 93 Podtrženo tužkou později. 94 Sophia Bohrer (1828–1899), klavíristka, vystupovala jako zázračné dítě údajně od 7 let. Srov. Objekt-Metadaten (hfmt-hamburg.de). O jejím pražském vystoupení viz Prag, Allgemeine musi- kalische Zeitung, no. 26 (29. 7. 1842): 532. 95 S 89 g 95 Srov. pozn. 89. 93 Podtrženo tužkou později. 92 Článek se nepodařilo dohledat. Periodikum je těžko dostupné. Jana Vojtěšková 1842): 3; o jeho posledním koncertu v Praze viz B: „Concert des Herrn Neukirchner,“, Bohemia 15, no. 47 (19. 4. 1842): 4. Tomášek o něm v souvislosti s pražským koncertem napsal: „Neukirchner gehört zu den in unseren Tagen seltenen Künstlern, die auf dem einzig wahren Wege ihre Kunstbildung verfolgen, die, fern von aller Anmassung, die Verherrli- chung der Kunst nie aus dem Auge verlieren, und die mühsam erworbene Technik nie als Zweck, sondern blos als Mittel betrachten; auch konnte er nur auf solchem Wege zu einer vollendeten Durchbildung des Tones gelangen. Der Umfang seines Instrumentes, vom Contra B bis zum e’, beträgt beinahe drei und eine halbe Oktave, eine gewiss bedeutende Tonmasse, die der Künstler mit einer unglaublichen Leichtigkeit und Ruhe beherrscht. Alle Töne sind von gleicher Färbung und gleicher Dichtigkeit, daher bei den tiefen Tönen durchaus kein Schnarren bemerkt wird. Die stets richtige Betonung und Sonderung der Perioden, die von der richtigen Vertheilung des Athems abhängt, dann die unendliche Nüancirung des Tones durch alle Gradationen des Piano und Forte, so wie es der Ausdruck erfordert, sind Vorzüge, die unsern Landsmann zu einem der grössten jetzt lebenden Virtuosen erheben, und die königlich würtembergische Musikkapelle kann mit Recht stolz darauf sein, Neukirchner zu ihren Mitgliedern zu zählen.“ Srov. Prag, Allgemeine musikalische Zeitung, no. 26 (29. 7. 1842): 532. 91 V Li k dě d b ě il 150 Dopisy Václava Jana Tomáška z Morawetzovy sbírky bald den Begriff von der Zeit; – soviel ist wenigstens gewiß, daß ich für Andere, aber nicht für mich lebe. – Sie würden mich sehr verbinden, wenn Sie mir das Blatt des Kometen von 1840 verschaffen und schicken möchten, worin auf einen sehr unver- schämten Artikel unter dem Zitat „die beiden Thiere zu Bethlehem“ eine Erwiderung von Prag eingesandt, \abgedruckt/ wurde. \|\| Ich kann Ihnen das No des Blattes und auch nicht einmal den Monat anzeigen, worin die Erwiderung abgedruckt ist, doch dürfte sie in den Monaten Juni, Juli, August, höchstens noch im September92 zu finden seyn. Vor einer Stunde war die junge Sophie Bohrer93 bei mir, und spielte mehre Piecen aus verschiedenen Zeitaltern, wodurch sie sich als eine vorzügliche Pianistin erwies.94 Das Mädchen besitzt ein glänzendes Ta- lent, dabei ein ungehäures Gedächtniß, und für ihr Alter eine bewunderungswürdige Technik, mit richtiger Auffaßung und kräftigen Ton geschmückt. Leben Sie wohl, und sind Sie versichert, daß sobald mir der Abdruck von Hr. 96 Josef Leopold Zvonař (1824–1865), teoretik, pedagog a skladatel. Od roku 1844 Pietschův asistent na varhanní škole, kterou po jeho smrti dočasně vedl. 97 Autograf Salve Regina pro soprán, alt a varhany z r. 1864 se dochoval v ČMH (XXX C 53, opis O. Horníka XVIII D 185). Vzhledem k dataci jde zřejmě o jinou skladbu. Jana Vojtěšková Hoffmann zugeschickt wird, ich ihn gleich corrigiren werde.95 bald den Begriff von der Zeit; – soviel ist wenigstens gewiß, daß ich für Andere, aber nicht für mich lebe. – Sie würden mich sehr verbinden, wenn Sie mir das Blatt des Kometen von 1840 verschaffen und schicken möchten, worin auf einen sehr unver- schämten Artikel unter dem Zitat „die beiden Thiere zu Bethlehem“ eine Erwiderung von Prag eingesandt, \abgedruckt/ wurde. \|\| g g g \|\| Ich kann Ihnen das No des Blattes und auch nicht einmal den Monat anzeigen, worin die Erwiderung abgedruckt ist, doch dürfte sie in den Monaten Juni, Juli, August, höchstens noch im September92 zu finden seyn. Vor einer Stunde war die junge Sophie Bohrer93 bei mir, und spielte mehre Piecen aus verschiedenen Zeitaltern, wodurch sie sich als eine vorzügliche Pianistin erwies.94 Das Mädchen besitzt ein glänzendes Ta- lent, dabei ein ungehäures Gedächtniß, und für ihr Alter eine bewunderungswürdige Technik, mit richtiger Auffaßung und kräftigen Ton geschmückt. Leben Sie wohl, und sind Sie versichert, daß sobald mir der Abdruck von Hr. Hoffmann zugeschickt wird, ich ihn gleich corrigiren werde.95 g g Mit wahrem Freundschafts Gefühl Mit wahrem Freundschafts Gefühl Ihr ergebenster Diener Tomaschek Prag den 15ten April 1842 h ergebenster Diener Tomaschek Prag den 15ten April 1842 ergebenster Diener Tomaschek Prag den 15ten April 1842 * * * Z Prahy pánu panu Rudolfu Hirschovi redaktorovi Komety veleváženému Z Prahy pánu panu Rudolfu Hirschovi redaktorovi Komety veleváženému v Lipsku Nejváženější pane a příteli! Tentokrát přece jen odpovídám na Vaše milé řádky včas. Nepochybuji, že Alpská dívka88 už je u vás, přičemž doufám, že se Vám zdravá stavba jejích údů bude líbit.89 Pokud jde o otcovskou ochranu, kterou ode mě pro své písně vyžadujete, můžete být zcela klidný, že v tom nic nezanedbám. 151 Jana Vojtěšková 96 Josef Leopold Zvonař (1824–1865), teoretik, pedagog a skladatel. Od roku 1844 Pietschův asistent na varhanní škole, kterou po jeho smrti dočasně vedl. 97 A f l h d h l Č ( C 5 99 Autograf Te Deum laudamus pro sóla a sbor hlasů sopránových a altových s průvodem varhan, polnic a kotlů z roku 1865 se dochoval v NM-ČMH (XXX C 47, Horníkův opis XVIII D 188). Vzhledem k dataci jde zřejmě o jinou skladbu. Jana Vojtěšková Staré přísloví ruka ruku myje chci uplatnit přiloženým lístkem, v němž Vás žádám, abyste ho neprodleně ve svém listu dal otisknout, aby lidé v Drážďanech a Lipsku byli připraveni na přednosti virtuozity, kterou pan Neukirchner90 v příštích dnech při svých koncertech dosvědčí.91 – Nenechte si ujít, abyste tohoto vskutku vzácného umělce slyšel. Stěžujete si ve svém dopise na nedostatek času, já však brzy o čase ztratím po- jem; – jisté je přinejmenším jedno, že žiji pro druhé, avšak ne pro sebe. – Velmi si mě zavážete, pokud byste mi mohl sehnat a poslat výtisk Komety z roku 1840, v němž byla na velmi neomalený článek s citátem „dvě zvířata z Betléma“ otištěna odpověď, zaslaná z Prahy. \|\| Nemohu Vám sdělit číslo listu ani měsíc, kde je tato odpověď otištěna, ale musí se to nacházet v měsících červnu, červenci, srpnu, nanejvýš v září.92 Před hodinou u mě byla mladá Sophie Bohrerová,93 hrála mi několik skladeb z různých období a proká- zala se jako vynikající klavíristka.94 Ta dívka má skvělý talent, přitom ohromnou paměť a na svůj věk obdivuhodnou techniku, zdobenou správným pojetím a mocným tónem. Žijte blaze a buďte ujištěn, že jakmile mi pan Hoffmann pošle otisk, okamžitě provedu korekturu.95 p S opravdovým pocitem přátelství Váš nejoddanější služebník Tomášek Praha, 15. dubna 1842 nejoddanější služebník Tomášek Praha, 15. dubna 1842 Tomášek, Václav Jan: [Zvonař, Josef]96 Praha, 22. 11. 1844 (NM-ČMH G 13 665) Prag, am 22ten November 1844 Prag, am 22ten November 1844 g, Wohlgeborner Herr! Hier erhalten Sie Ihre mir mitgetheilten Compositionen wieder zurück. I h k t d i i ti l T t h li h i S lt h it i Wohlgeborner Herr! Hier erhalten Sie Ihre mir mitgetheilten Compositionen wieder zurück. g Hier erhalten Sie Ihre mir mitgetheilten Compositionen wieder zurück. g p Ich erkannte darin einen rationelen Tonsetzer, wahrlich eine Seltenheit in unsern Tagen. g Das vierstimmig behandelte Salve regina97 ist bei seiner Einfachheit von religiösem Gefühl duchdrungen, und kann daher seinen Zweck nicht verfehlen. 152 Dopisy Václava Jana Tomáška z Morawetzovy sbírky Das Offertorium „Exaudi Deus deprecationem meam“98 ist in rechtem Kirchenstyle abgefaßt, die Auffaßung des Textes und seine richtige Declamation, stehen mit der Orchesterpartie und dem Gesange im Einklange. Das Ganze krönt eine wacker durch- geführte Fuge. g f g Und was soll ich erst von dem großen Werke „Te Deum laudamus“ sagen! 98 Toto offertorium není uvedeno v soupisu díla, viz „Jos. Leopold Zvonař. K stému jubileu jeho narozeni a k šedesátému výročí jeho úmrtí,“ Podbrdský (Berounský) kraj 2, sešit 3 (1924–1925): 133–139. 106 Dreyschock vystoupil ve Vídni v lednu 1846 opakovaně. Čtvrtý koncert se konal 3. ledna a pátý koncert 17. ledna. Koncert na rozloučenou se uskutečnil 8. února v sále Gesellschaft der Musik- freunde. U příležitosti jeho vídeňských koncertů vydal J. P. Lyser delší životopisnou stať „Drey- schock: Ein Künstler-Portrait,“ Wiener-Zeitschrift für Kunst, Literatur, Theater und Mode XXXI, no. 28 (7. 2. 1846): 110–111. i 104 Tomáškův žák Hans Hampel (1822–1884), klavírista a skladatel. 105 Blanka byla dcerou Tomáškova bratra Jakuba. 100 Alexander Dreyschock (1818–1869), klavírista a skladatel, Tomáškův žák. 101 D i í á Jana Vojtěšková 99 – Das vom Anfange bis zum Ende mit einer großen Sorgfalt gearbeitet ist. Besonders ist in der Doppelfuge die Zusammenstellung des Textes sehr sinnreich, wo der Baß mit „In te domine speravi“ sein Hauptthema beginnt, worauf bald der Alt mit „non confundar in aeternum[“] sein zweites Thema hören läßt, wornach dann die beiden Themen in einer sehr glücklichen Wechselwirkung bis zu Ende des Werkes behandelt sind. g g Nehmen Sie also meinen herzlichen Dank für die Mittheilung Ihrer interessanten Werke, dem ich noch den Wunsch anreihe; der Himmel möge Ihnen bald Gelegenheit geben, um Ihr Talent geltend machen, und durch mehre [!] religiöse Tondichtungen zur Verherrlichung Gottes beitragen zu können. g g Mit vollkommenster Achtung Ihr ergebenster Diener Wenzel Johann Tomaschek m. p. ergebenster Diener Wenzel Johann Tomaschek m. p. * * * Praha, 22. listopadu 1844 Velevážený pane! Zde obdržíte zpět Vaše skladby, o nichž jste mi podal zprávu. Rozpoznal jsem v nich racionálního skladatele, skutečnou vzácnost za našich dnů. Čtyřhlase pojednané Salve regina97 je při jeho prostotě proniknuto náboženským du- chem, a nemůže se proto minout účinkem.f Rozpoznal jsem v nich racionálního skladatele, skutečnou vzácnost za našich dnů. Čtyřhlase pojednané Salve regina97 je při jeho prostotě proniknuto náboženským du- chem, a nemůže se proto minout účinkem. Offertorium „Exaudi Deus deprecationem meam“ 98 je vypracováno v pravém cír- kevním slohu, pochopení textu a jeho správná deklamace jsou v souladu s orchestrální složkou a zpěvem. Všechno korunuje řádně procítěná fuga. p p f g A co mám teprve říci o velkém díle „Te Deum laudamus“! 99 – Které je od začátku do konce pracováno s velkou pečlivostí. Zvláště ve dvojité fuze je sestavení textu velmi důmyslné, kde bas začíná se „In te domine speravi“ hlavní téma, po něm v altu zazní s „non confundar in aeternum“ druhé téma, načež se s oběma tématy ve velmi šťastném vzájemném působení pracuje až do konce. 153 Jana Vojtěšková Jana Vojtěšková Přijměte tedy můj srdečný dík za zprávu o Vašich zajímavých dílech, k čemuž ještě připojuji přání; kéž Vám nebe brzy poskytne příležitost, abyste svůj talent uplatnil, a vícerými duchovními díly mohl přispět k oslavě Boha. S dokonalou úctou Váš nejoddanější služebník Václav Jan Tomášek v. r. Přijměte tedy můj srdečný dík za zprávu o Vašich zajímavých dílech, k čemuž ještě připojuji přání; kéž Vám nebe brzy poskytne příležitost, abyste svůj talent uplatnil, a vícerými duchovními díly mohl přispět k oslavě Boha. 100 Alexander Dreyschock (1818–1869), klavírista a skladatel, Tomáškův žák. 101 Dopis není znám. 102 N id ifik á b i 103 Neidentifikovaná osoba. 100 Alexander Dreyschock (1818–1869), klavírista a skladatel, Tomáškův žák. 101 Dopis není znám. 102 Neidentifikovaná osoba. 103 Neidentifikovaná osoba. 104 Tomáškův žák Hans Hampel (1822–1884), klavírista a skladatel. 105 Blanka byla dcerou Tomáškova bratra Jakuba. 106 Dreyschock vystoupil ve Vídni v lednu 1846 opakovaně. Čtvrtý koncert se konal 3. ledna a pátý koncert 17. ledna. Koncert na rozloučenou se uskutečnil 8. února v sále Gesellschaft der Musik- freunde. U příležitosti jeho vídeňských koncertů vydal J. P. Lyser delší životopisnou stať „Drey- schock: Ein Künstler-Portrait,“ Wiener-Zeitschrift für Kunst, Literatur, Theater und Mode XXXI, no. 28 (7. 2. 1846): 110–111. Jana Vojtěšková Přijměte tedy můj srdečný dík za zprávu o Vašich zajímavých dílech, k čemuž ještě připojuji přání; kéž Vám nebe brzy poskytne příležitost, abyste svůj talent uplatnil, a vícerými duchovními díly mohl přispět k oslavě Boha. S dokonalou úctou Váš nejoddanější služebník Václav Jan Tomášek v. r. Tomášek, Václav Jan: Dreyschock, Alexander100 Praha, 14. 1. 1846 (NM-ČMH G 13 659) Prag am 14ten Jänner 1846 g J Mein lieber Alexander! Ihr letztes humorisches Schreiben machte mir sehr viel Freude.101 Ich las es Ihrer lie- ben Anna102 vor, als sie mich gestern mit ihrer Schwester Fanni103 besuchte. Anna ist wohl und ihr Aussehen sehr stattlich. So lang Sie auf Krebse stoßen, ist es noch gut, der Himmel bewahre Sie vor Scorpionen, wo man nicht so gut wegkommt. Ihr Brief ist keine Antwort auf mein Schreiben, denn er enthält nichts über Ham- pels Angelegenheit,104 und keine Erwähnung von meiner Nichte Blanka,105 die zu besuchen, ich Sie in meinem Briefe angelegentlichst ersuchte. Daß sie die Gemahlin des Amtskontroleur Herrn Herrmann im k. k. allgemeinen Krankenhause sei, und dort wohne, werden Sie doch nicht vergeßen haben. Schön und ersprießlich wäre es von Ihnen, wenn Sie bei der Gelegenheit ihr zwei Freikarten in das letzte Conzert antragen würden.106 Blanka schrieb mir im \|\| letzten Brief, daß sie alle Hofnung [!] Sie zu sehen und zu sprechen aufgeben muß. Mein lieber Alexander! Ihr letztes humorisches Schreiben machte mir sehr viel Freude.101 Ich las es Ihrer lie- ben Anna102 vor, als sie mich gestern mit ihrer Schwester Fanni103 besuchte. Anna ist wohl und ihr Aussehen sehr stattlich. So lang Sie auf Krebse stoßen, ist es noch gut, der Himmel bewahre Sie vor Scorpionen, wo man nicht so gut wegkommt. p g g Ihr Brief ist keine Antwort auf mein Schreiben, denn er enthält nichts über Ham- pels Angelegenheit,104 und keine Erwähnung von meiner Nichte Blanka,105 die zu besuchen, ich Sie in meinem Briefe angelegentlichst ersuchte. Daß sie die Gemahlin des Amtskontroleur Herrn Herrmann im k. k. allgemeinen Krankenhause sei, und dort wohne, werden Sie doch nicht vergeßen haben. Schön und ersprießlich wäre es von Ihnen, wenn Sie bei der Gelegenheit ihr zwei Freikarten in das letzte Conzert antragen würden.106 Blanka schrieb mir im \|\| letzten Brief, daß sie alle Hofnung [!] Sie zu sehen und zu sprechen aufgeben muß. 113 Neidentifikovaná osoba. Jana Vojtěšková 154 Dopisy Václava Jana Tomáška z Morawetzovy sbírky Die Bemerkung von Adami, daß er Ihnen die Bekanntschaft mit Dushek gern geschenkt hätte, ist der schlagendste Beweis von Adamis gewaltiger Ignoranz,107 Viele würden sich schämen, so eine Ungereimtheit zu denken, viel weniger sie drucken zu laßen.108 Und, wer ist denn der liebe künstlerisch gebildete Karl Walter? – Haben Sie ihn vielleicht beim Vorübergehen etwa auf sein musikalisches Hühner-Auge getreten?109 – Lieber Alexander! solche Geschöpfe muß es wegen der nöthigen Kontraste in der Welt auch geben. Wir hätten keine Madonnen, wenn es nicht auch Gefrieser gäbe. Trösten Sie sich darüber, wenn Sie von ein Paar dergleichen Schmeißfliegen geneckt werden. In einer Recension über Sie wird behauptet, daß meine Ouvertüre110 vom Orchester matt ausgeführt wurde; Ob ein verfehltes tempo, oder der \|\|Vortrag daran Schuld war, werden Sie mir bei Ihrer Heimkehr am besten sagen können.111 Ich habe nichts dagegen, wenn Sie die Ouverture nach Pest mitnehmen. Der ungarische Wein hat mehr Feuer als der öesterrechische, und Feuer bedarf dieses Werk bei seiner Production wenn es gehörig wirken soll. ß Nun noch einen Meisterkuß von Ihrem Meister Tomaschek * * * g miho článek vyšel ve Wiener allgemeine Theaterzeitung no. 5–6 (6. 1. 1846): 15. h g 108 Adamiho článek vyšel ve Wiener allgemeine Theaterzeitung no. 5–6 (6. 1. 1846): 15. h g 108 Adamiho článek vyšel ve Wiener allgemeine Theaterzeitung no. 5–6 (6. 1. 1846) y gh g 109 Carl Walter napsal: „Nicht ohne Grund zögerten wir gleich nach Dreyschock’s erstem Concert einen Bericht daruber zu bringen, es war uns um so mehr und wiederhohlte und gesicherte Ueberzeugung bey dem hier abzugebenden Ausspruche zu thun, da es einen Künstler zu beurthei- len galt, von dessen Ausgezeichnetheit das Gerücht schon so viel Wunderbares verkündiget, hatte, indeß wir gleich Anfangs die ekstatischen Erhebungen Dreyschock’s von Seite seiner Schüler und Anhänger nicht gänzlich gerechtfertigt fanden.“ Carl Walter, „Kunst Nachrichten. A. Dreyschock’s Concert,“ Wiener Zeitung, no. 358 (28. 12. 1845): 2841(3). 110 Zřejmě předehra k opeře Serafina, viz dopis Guttentagovi. i 111 „Den Beginn machte eine gediegene Ouverture Tomaschek’s, die jedoch etwas matt und zu wenig präcis vorgetragen wurde.“ (Dr. K–), Wiener allgemeine Musik-Zeitung, 6. 1. (1846): 11. Jednalo se o Dreyschockův 4. koncert ve Vídni, konaný 3. ledna v poledne v sále Musikvereinu. Tomáškova Ouvertura zazněla na začátku (s orchestrem Tonkünstlerverein), další program: J. L. Dusík, Kon- zertsatz; Dreyschock, Fugiertes Impromptu; Tomášek, Rapsodie č. fi 111 „Den Beginn machte eine gediegene Ouverture Tomaschek’s, die jedoch etwas matt und zu wenig präcis vorgetragen wurde.“ (Dr. K–), Wiener allgemeine Musik-Zeitung, 6. 1. (1846): 11. Jednalo se o Dreyschockův 4. koncert ve Vídni, konaný 3. ledna v poledne v sále Musikvereinu. Tomáškova Ouvertura zazněla na začátku (s orchestrem Tonkünstlerverein), další program: J. L. Dusík, Kon- zertsatz; Dreyschock, Fugiertes Impromptu; Tomášek, Rapsodie č. 2 h moll; Dreyschock, „In allen guten Stunden“ (mužský sbor); variace na God save the Queen; variace pro levou ruku Gruß aus Wien. – Wiener Zeitschrift, 5. 1. 1846, s. 17 (-sky) k Ouvertuře píše: „Sie war von Tomascheck, aber eigentlich von Mozart, und Tomascheck hatte nur die Mühe übernommen, sie zu kopiren. Wie würde Mozart sich freuen, könnte er erfahren, daß ein so großer Meister, wie Herr W. J. To- mascheck, es nicht unter seiner Würde hielt, der Zauberflöte-Ouvertüre seinen Namen vorzuset- zen.“ – Dreyschock hostoval ve Vídni od 11. 12. 1845, poslední koncert se uskutečnil 8. 2. 1846. 112 Viz pozn. 102.i g g Die herzlichsten Grüße von Anna112 und Marie,113 die sich freuen, Sie wieder zu sehen. Nun noch einen Meisterkuß von Ihrem Meister Tomaschek 107 Heinrich Joseph Adami (1807–1895), novinář, od 1832 redaktor Wiener Theaterzeitung (Bäuerles Theaterzeitung).h Jana Vojtěšková 2 h moll; Dreyschock, „In allen guten Stunden“ (mužský sbor); variace na God save the Queen; variace pro levou ruku Gruß aus Wien. – Wiener Zeitschrift, 5. 1. 1846, s. 17 (-sky) k Ouvertuře píše: „Sie war von Tomascheck, aber eigentlich von Mozart, und Tomascheck hatte nur die Mühe übernommen, sie zu kopiren. Wie würde Mozart sich freuen, könnte er erfahren, daß ein so großer Meister, wie Herr W. J. To- mascheck, es nicht unter seiner Würde hielt, der Zauberflöte-Ouvertüre seinen Namen vorzuset- zen.“ – Dreyschock hostoval ve Vídni od 11. 12. 1845, poslední koncert se uskutečnil 8. 2. 1846. 112 Viz pozn. 102. 113 N d fik b 155 Jana Vojtěšková Praha, 14. ledna 1846 j ý Vaše poslední humoristické psaní mi udělalo velkou radost.101 Předčítal jsem ho Vaší milé Anně,102 když mě včera navštívila se svou sestrou Fanni.103 Anna se má dobře a vypadá velmi znamenitě. Dokud budete narážet na raky, je to ještě dobré, ale střezte se škorpionů, těm tak snadno neutečete. p Váš dopis není odpovědí na můj dopis, protože o Hampelově záležitosti nic neobsa- huje,104 ani zmínku o mé neteři Blance,105 a ve svém dopise jsem Vás co nejnaléhavěji žádal, abyste ji navštívil. Přece jste nezapomněl, že je manželkou úředního kontro- lora pana Herrmanna v c. k. Všeobecné nemocnici a bydlí tam. Bylo by od Vás krásné a prospěšné, kdybyste jí při té příležitosti nabídl dvě volné vstupenky na poslední koncert.106 Blanka mi v posledním dopise \|\| psala, že se musí vzdát vší naděje, že Vás uvidí a promluví s Vámi. Adamiho poznámka, že by Vám seznámení s Dusíkem rád odpustil, je ten nejpádnější důkaz Adamiho ohromné ignorance,107 mnozí by se styděli si takový nesmysl jen myslet, a ještě spíš dát ho tisknout.108 A kdo je ten milý, umělecky vzdělaný Karl Walter? – Šlápl jste mu snad, když jste šel kolem, na jeho hudební kuří oko? 109 p j y j j – Milý Alexandře! Taková stvoření musejí kvůli nutnému kontrastu na světě také existovat. Neměli bychom žádné Madony, kdyby nebyly i ošklivé huby. Utěšte se tím, že vás poškádlilo jen pár takových masařek. V jedné recenzi o Vás se tvrdí, že má Předehra110 byla zahrána mdle. Jestli bylo na vině špatné tempo nebo přednes, mi po návratu budete nejlépe moci říct sám.111 Nemám nic proti tomu, když s sebou vezmete Předehru do Pešti. Jana Vojtěšková Uherské víno je ohnivější než rakouské a toto dílo při uvedení oheň potřebuje, pokud má náležitě zapůsobit. \|\| Nejsrdečnější pozdravy od Anny112 a Marie,113 které se těší, až Vás zase uvidí. Ještě jeden mistrovský polibek 156 156 Dopisy Václava Jana Tomáška z Morawetzovy sbírky 114 Zřejmě Immanuel Guttentag (1815/1817–1862), který získal v roce 1842 berlínské knižní na- kladatelství firmy Trautwein zaměřené na právnickou literaturu. Podnik vydával také hudebniny a byl posléze nazván po Immanuelu Guttentagovi. 115 Spolu s dopisem se nedochovalo. Vztahuje se zřejmě k Hymnu De Spiritu Sancto, viz dále pozn. 119. 116 August Eduard Grell (1800–1886), skladatel, varhaník, od roku 1853 ředitel berlínské Singaka- demie. 117 O Dreyschockově vystoupení v Drážďanech srov. např. Signale für die musikalische Welt, no. 10 (Februar 1847): 77 a o koncertě v Berlíně 27. 2. 1847 ibid., no. 13 (März 1847): 100–101. 120 Pravděpodobně skladatel Franz Wilhelm Ressel (1811–?). Pocházel Řasnice (Rückersdorf) na Li- berecku. Roku 1852 byl jmenován královským komorním hudebníkem v Berlíně. Kromě vlastních skladeb upravoval díla Mozarta či C. M. von Webera. 118 Předehra k opeře Seraphine vyšla v Praze u C. W. Enderse. Ouverture de l’Opera Seraphine par Mr. J. W. Tomaschek arrangée pour le Piano-Forte par Eduard Hofmann. NM-ČMH XVII F 78. g p p f 119 Hymnus de Spiritu Sancto (Veni creator Spiritus), op. 80, tisk: Berlin, T. Trautwein (J. Guttentag), VJT 83. Autograf a tisk dochován v NM-ČMH III F 101. 121 Plný titul vydané skladby zní: Hymnus / de spiritu sancto / festis Pentecostes diebus pro Graduali cantari solitus, / concentu musico redditus / a / Wenceslao J. Tomaschek / Sodali ad honores accito a societate mu- sicorum majore et ad literarum conmercium invitato a societate musicorum ad sanctam Annam Viennae, tum sodali honorario societatis Oenipotensis, sodali meritorum titulis nominato societatis ad musicen promovendam Batavorum majoris, nec non consociationis Germanorum gentis musicae ejusque disciplinae promovendae membro honorario, ac. Pestiensis et Budensis, et Leopolitanae in Galicia societatis musicae sodali. / Op. 80 / Proprietas editoris. / Inscriptus archivo societatis. / Berolini / Sumtibus T. Trautwein (J. Guttentag.) /Breite – Strasse No 8. / Pr. Partibus 1 Thlr 5 Sgr. / jeder einzelnen Stimme 2 ½ Sgr. Tomášek, Václav Jan: [Guttentag, Immanuel]114 Praha, 9. 12. 1846 (NM-ČMH G 13 660) Tomaschek. Prag am 9ten Dezember 1846. Alle lange Namens-Zusätze, die der Welt berichten sollen, was man ist, waren mir von jeher widrig, doch hier halte ich es für meine Pflicht, allen den Vereinen die mich ehren,121 meine Aufmerksammkeit öffentlich zu bezeugen. Mit unbegrenzter Hochachtung Ihr ergebenster Wenzel Joh. Tomaschek. Prag am 9ten Dezember 1846. Alle lange Namens-Zusätze, die der Welt berichten sollen, was man ist, waren mir von jeher widrig, doch hier halte ich es für meine Pflicht, allen den Vereinen die mich ehren,121 meine Aufmerksammkeit öffentlich zu bezeugen. Alle lange Namens-Zusätze, die der Welt berichten sollen, was man ist, waren mir von jeher widrig, doch hier halte ich es für meine Pflicht, allen den Vereinen die mich ehren,121 meine Aufmerksammkeit öffentlich zu bezeugen. Mit unbegrenzter Hochachtung Ihr ergebenster Wenzel Joh. Tomaschek. Mit unbegrenzter Hochachtung Ihr ergebenster Wenzel Joh. Tomaschek. Prag am 9ten Dezember 1846. * * * Velevážený pane! Naše společné zájmy jsou nyní v souladu, a tak ode mě obdržíte na důkaz přiložené a mnou vlastnoručně podepsané prohlášení, jímž se Vám vyhrazuje neomezené vlast- nictví díla.115 Že se poroučím poctivému knězi posvátného umění, myslím panu Grellovi,116 kterého jsem si za svého velmi letmého pobytu v Berlíně velmi oblíbil. p y Pokud [Grell] odsouhlasí klavírní výtah, můžete ho s plnou důvěrou a spolu s parti- turou předat k rytí. V naší době, kdy bude čtení partitury brzy patřit k vzácným hu- debním jevům, jsou klavírní výtahy nutné zlo, pročež proti tomu nemohu nic namítat. Pan Dreyschock, který už koncertuje v Drážďanech, navštíví také co nejdřív Berlín.117 Žádal mě o nějakou předehru, aby ji mohl na koncertě uvést. Dal jsem mu předehru ze své opery Serafina.118 Možná si ji poslechnete a budete se moci rozhodnout, zda byste mohl tuto skladbu potřebovat. Protože je partitura Hymnu119 napsána zřetelně, a jak věřím, také správně, mohl by se tam nalézt jistě odborník, jenž by se postaral o korekturu. Pan Ressel,120 jemuž jsem odpověděl na jeho dopis, se za mé přítomnosti, či dokonce písemně nabídl, že bude mé skladby pro Vaše vydavatelství korigovat. – Mimochodem Vás žádám, abyste titul vysázel k Hymnu tak, jak je na další straně. Všechny dlouhé přídavky ke jménu, které mají světu sdělit, čím kdo je, mi byly odjakživa protivné, avšak zde považuji za svou povinnost projevit veřejně svou pozornost všem spolkům, které mě ctí.121 Váš nejoddanější Václav Jan Tomášek. Václav Jan Tomášek. Praha, 9. prosince 1846. Praha, 9. Tomášek, Václav Jan: [Guttentag, Immanuel]114 Praha, 9. 12. 1846 (NM-ČMH G 13 660) Wohlgeborner Herr! Unsere wechselseitigen Interessen sind nun im Einklang gestimmt, und so empfangen Sie von mir zum Beweis den hier beiliegenden von mir eigenhändig ausgefüllten Re- vers, wodurch Ihnen das unbeschränkte Eigenthum über das Werk eingeräumt wird.115 Empfehlen Sie mich dem biedern Priester der heiligen Kunst, ich meine den Herrn Grell,116 der mir bei meinem zu flüchtigen Aufenthalt in Berlin sehr lieb geworden ist. Genehmigt er den Klavier-Auszug, so können Sie mit vollem Vertrauen ihn sammt der Partitur dem Stich übergeben. In unserer Zeit, wo das Lesen der Partitur bald zu den seltenen musikalischen Erscheinungen gehören wird, sind Klavierauszüge nothwen- diges Uibel, desshalb ich dagegen nichts einzuwenden habe. Herr Dreyschock, welcher bereits in Dresden Conzertirt wird auch ehestens Berlin besuchen.117 Er ersuchte mich um eine Ouverture um sie in einem Konzerte aufführen zu können. Ich gab ihm die Ouverture aus meiner Oper Seraphine.118 Vielleicht werden Sie dieselbe anhören, und sich entscheiden können, ob Sie diese Composition brauchen könnten. Da die Partitur der Hymne119 deutlich, und wie ich glaube auch correct geschrieben ist, so dürfte sich wohl ein Sachkündiger dort finden, der die Correctur besorgen würde. Herr Ressel,120 dem ich auf sein Schreiben geantwortet, hat sich bei meiner Anwesenheit, oder gar brieflich angetragen, meine Compositionen für Ihren Verlag zu corrigiren. – Uibrigens ersuche ich Sie den Titel, wie er auf nächster Seite geschrieben ist, auf die Hymne zu setzen. 114 Zřejmě Immanuel Guttentag (1815/1817–1862), který získal v roce 1842 berlínské knižní na- kladatelství firmy Trautwein zaměřené na právnickou literaturu. Podnik vydával také hudebniny a byl posléze nazván po Immanuelu Guttentagovi. 118 Předehra k opeře Seraphine vyšla v Praze u C. W. Enderse. Ouverture de l’Opera Seraphine par Mr. J. W. Tomaschek arrangée pour le Piano-Forte par Eduard Hofmann. NM-ČMH XVII F 78. g p p f 119 Hymnus de Spiritu Sancto (Veni creator Spiritus), op. 80, tisk: Berlin, T. Trautwein (J. Guttentag), VJT 83. Autograf a tisk dochován v NM-ČMH III F 101. 120 Pravděpodobně skladatel Franz Wilhelm Ressel (1811–?). Pocházel Řasnice (Rückersdorf) na Li- berecku. Roku 1852 byl jmenován královským komorním hudebníkem v Berlíně. Kromě vlastních skladeb upravoval díla Mozarta či C. M. von Webera. 157 Jana Vojtěšková Alle lange Namens-Zusätze, die der Welt berichten sollen, was man ist, waren mir von jeher widrig, doch hier halte ich es für meine Pflicht, allen den Vereinen die mich ehren,121 meine Aufmerksammkeit öffentlich zu bezeugen. Mit unbegrenzter Hochachtung Ihr ergebenster Wenzel Joh. 122 V pozůstalosti Vlastimila Blažka se mimo tento zápis dochovaly Tomáškovy žádosti o výplatu renty a jedna jízdenka. 123 Joseph Stossek, nar. 1798, právník a vedoucí obchodu. 124 Emanuel Danjczek viz např. https://anno.onb.ac.at/cgi-content/anno?aid=bru&datum= 18430816&query=%22Danjczek%22&ref=anno-search&seite=13. 125 August Seifert, nar. 1800, sazeč. Tomášek, Václav Jan: [Guttentag, Immanuel]114 Praha, 9. 12. 1846 (NM-ČMH G 13 660) prosince 1846. 158 Dopisy Václava Jana Tomáška z Morawetzovy sbírky 125 August Seifert, nar. 1800, sazeč. Zápis do Zemských desek o zvýšení Tomáškovy renty z roku 1844 (originál: NM-ČMH č. př. Blažek Vlastimil 58/1977)122 [Sechzehn Gulden] Da bei dem vorgerückten Alter und der sich öfters wiederhohlenden Kränklichkeit mein Compositeur Herr Wenzl Johann Tomaschek in der Zukunft daran verhindert sein dürfte durch seine Compositionen so viel zu erwerben, als es bisher der Fall war, und ich gerne demselben, falls mich der Tod früher übereilen sollte, ein Andenken hinterlas- sen möchte, so erhöhe ich hiemit seinen bisherigen jährlichen Gehalt von fünf hundert fünfzig Gulden Conventions Münze auf den von sieben hundert Gulden Conventions Münze auf seine ganze Lebenszeit, welchen erhöhten jährl. Gehalt p[e]r 700fr C. M. derselbe in den bisher üblichen Raten aus der gräflich Buquoyschen Hauptkassa vom 1ten November l[aufenden] J[ahres] angefangen zu beziehen hat. Damit aber Herr Wenzl Johann Tomaschek im Falle meines Absterbens auch gegen meine Erben gesi- chert, und diese Letztere gegen denselben rechtlich verbunden sind, so ordne ich hier letztwillig an, daß nach meinem Tode meine Erben verbunden sind, diesen erhöhten Gehalt von jährlichen 700fr C. M. dem besagten Herrn Wenzl Johann Tomaschek bis zu seinem Ableben in den üblichen Raten anstandslos aus der gräflich Buquoyischen Hauptkassa zu verabreichen. Urkund dessen meine eigenhändige Unterschrift und die Namensfertigung dreier ei- gends hiezu ersuchten Herren Zeugen. Georg August von Buquoy Georg August von Buquoy Prag am 8ten November 1844 Joseph Stossek123 als ersuchter Zeuge Joseph Stossek123 als ersuchter Zeuge August Seifert125 als ersuchter Zeuge Joseph Stossek123 als ersuchter Zeuge Emmanuel Danjczek JuDr.124 als ersuchter Zeuge August Seifert125 als ersuchter Zeuge 159 Jana Vojtěšková Jana Vojtěšková Ist der königl. böhmischen Landtafel am 15. Juli \|\|1848 Tom. 1337 Instr. sub lit. P 23 ämtlich eingetragen worden Joseph Mitteis126 Vizeregistrator. Joseph Mitteis126 Vizeregistrator. * * * [Šestnáct zlatých] Jelikož mému skladateli Janu Václavu Tomáškovi pro pokročilý věk a častou churavost bude pravděpodobně v budoucnu znemožněno, aby svými skladbami vydělával tolik jako dosud, a já bych mu rád pro případ, že by mě smrt zastihla dříve, zanechal vzpo- mínku, zvyšuji tímto jeho dosavadní roční plat z pěti set padesáti zlatých konvenční měny na sedm set zlatých konvenční měny doživotně, kterýžto zvýšený roční plat 700 zl. Cm. bude dostávat, počínaje 1. listopadem tohoto roku, v dosud obvyklých dáv- kách z hraběcí buquoyské hlavní pokladny. Aby však pan Václav Jan Tomášek v případě mého úmrtí byl zajištěn také vůči mým dědicům a tito vůči němu byli právně zavá- záni, nařizuji zde jako svou poslední vůli, že po mé smrti budou mí dědicové povinni tento zvýšený plat 700 zl. Cm. Zápis do Zemských desek o zvýšení Tomáškovy renty z roku 1844 (originál: NM-ČMH č. př. Blažek Vlastimil 58/1977)122 řečenému panu Václavu Janu Tomáškovi bez námitek v obvyklých splátkách z buquoyské hlavní pokladny poskytovat až do jeho smrti. Na doklad toho můj vlastnoruční podpis a jmenovité uvedení tří zvláště k tomu vy- žádaných pánů svědků. Praha, 8. listopadu 1844 Praha, 8. listopadu 1844 Joseph Stossek123 jako vyžádaný svědek August Seifert125 jako vyžádaný svědek Emmanuel Danjczek JuDr.124 jako vyžádaný svědek Joseph Stossek123 jako vyžádaný svědek Joseph Stossek123 jako vyžádaný svědek August Seifert125 jako vyžádaný svědek Úředně zaneseno do královských českých zemských desek 15. července 1848, sv. 1337 instr. pod písmenem P 23. Joseph Mitteis126 zástupce registrátora 126 Joseph Mitteis (1794–1868), na ředitele Zemských desek byl povýšen roku 1850. Roku 1846 dostal jako „Böhmischer Landtafel-Vice-Registrator“ velkou zlatou Čestnou medaili. Roku 1862, kdy byl penzionován, je uváděn jako Director des Prager Landtafel- und Grundbuchsamtes (ředitel pražských Zemských desek a pozemkového úřadu) a za zásluhy získal titul císařský rada. 160 Dopisy Václava Jana Tomáška z Morawetzovy sbírky Literatura Batka, Richard. Aus der Musik- und Theaterwelt. Beschreibendes Verzeichnis der Autographen- Sammlung Fritz Donebauer in Prag. Praha: Fritz Donebauer – Löwit & Lamberg, 1894; Beschreibendes Verzeichnis der Autographensammlung Fritz Donebauer in Prag. Praha: Fritz Do- nebauer – Löwit & Lamberg, 1900. Janáčková, Irena. „Pražští vydavatelé Václava Jana Tomáška,“ Hudební věda 18, no. 2 (1981): 174. Kabelková, Markéta. „Václav Jan Tomášek. In Die Musik in Geschichte und Gegenwart: A Kabelková, Markéta. „Václav Jan Tomášek. In Die Musik in Geschichte und Gegenwart: Allgemeine Enzyklopädie der Musik: 21 Bände in zwei Teilen, edited by Friedrich Blume et al., 2. neubearb. Ausg. Kassel: Bärenreiter, 1994. Kabelková, Markéta. „Václav Jan Tomášek.“ PhDiss., FF UK Praha, 2012. Kraus, Arnošt Vilém. Goethe a Čechy. Praha: Bursík a Kohout, 1893. Němec, Zdeněk, ed. Vlastní životopis V. J. Tomáška. Praha: Topičova edice, 1941. Simpson, A. a K. DeLong. „Tomášek, Václav Jan Křtitel.“ In Grove Music Online. https:// www.oxfordmusiconline.com/grovemusic/view/10.1093/gmo/9781561592630.001.0001/ omo-9781561592630-e-0000028077 (24. 2. 2022). Šťastná, Kateřina Alexandra. „Tomášek, Václav Jan Křtitel.“ In Český hudební slovník. www. ceskyhudebnislovnik.cz/slovnik/. Tarantová, Marie. „Václav Jan Tomášek ve staropražských hudebních salónech.“ Hudební věda 13, no. 1 (1976): 59–74. Vojtěšková, Jana, ed. Album Jana Ludevíta Procházky z let 1860–1888 / The Procházka Album (1860–1888). Praha: KLP & NM, 2013. Vojtěšková, Jana. „Letters from the Morawetz Collection (Musicians of Czech Origin in Eu- ropean Centres at the Turn of the 18th and 19th Centuries) / Dopisy Morawetzovy sbírky (Hudebníci českého původu v evropských centrech na přelomu 18. a 19. století),“ Musicalia 13 no. 1–2 (2021): 6–39 (anglická verze), 40–46 (česká verze). Vojtěšková, Jana. „Dopisy Vojtěcha Matyáše Jírovce z Morawetzovy sbírky,“ Opus musicum 53, no. 4 (2021): 52–65. Vojtěšková, Jana. „The works of Jan Dismas Zelenka in 18th and 19th centuries in Prague,“ Clavibus unitis 4, no. 1 (2015): 85–90. Vojtěšková, Jana. „Několik pramenů z doby působení Carla Marii von Webera ve Stavovském divadle / Several Sources pertaining to Carl Maria von Weber’s Work for the Estates Theatre in Prague,“ Musicalia 5, no. 1–2 (2013): 57–64 (česká verze), 65–77 (anglická verze). Vojtěšková, Jana. „Sbírka Richarda Morawetze,“ Muzikologické fórum 1, no. 1 (2012): 47–54. Abstract The article deals with the letters of Václav Jan Křtitel Tomášek from the Morawetz Collection, housed in the National Museum – Czech Museum of Music. In a critical edition, it publishes and comments on the letters Tomášek 161 Jana Vojtěšková addressed to the publishers Ambrosius Kühnel, Carl Friedrich Peters, and Im- manuel Guttentag, as well as to the poet and editor Rudolph Hirsch, Tomášek’s pupil Alexander Dreyschock and an unnamed (probably Amelia Illaire to Berlin). The edition also includes an appraisal of the compositions of Josef Zvonař and Tomášek’s request for an increase in his annual rent, addressed to Count George Franz August Buquoy in 1829. For the first time, a letter by Johann Wolfgang Goethe, the original of which is considered lost by the current edition of Goethe’s correspondence, is published here according to the original. Abstrakt Č Článek se zabývá dopisy Václava Jana Křtitele Tomáška z Morawetzovy sbírky, uložené v Národním muzeu – Českém muzeu hudby. V kritické edici zveřejňuje a komentuje dopisy, které Tomášek adresoval nakladatelům Ambrosiu Kühne- lovi, Carlu Friedrichu Petersovi a Immanuelu Guttentagovi, dále básníkovi a re- daktorovi Rudolphu Hirschovi, Tomáškovu žákovi Alexandru Dreyschockovi a nejmenované (nejspíše Amelii Illaire do Berlína). Edice obsahuje také posudek skladeb Josefa Zvonaře a Tomáškovu žádost o zvýšení roční renty, určenou hraběti Jiřímu Františku Augustu Buquoyovi v roce 1829. Poprvé je zde podle originálu publikován dopis Johanna Wolfganga Goetha, jehož originál je současnou edicí Goethovy korespondence považován za nezvěstný. Keywords Jiří František August Buquoy; Alexander Dreyschock; Johann Wolfgang Goe- the; Immanuel Guttentag; Rudolph Hirsch; Amelie Illaire; Ambrosius Kühnel; Morawetz Collection; National Museum – Czech Museum of Music; Carl Frie- drich Peters; Václav Jan Křtitel Tomášek; Josef Zvonař Klíčová slova Jiří František August Buquoy; Alexander Dreyschock; Johann Wolfgang Goe- the; Immanuel Guttentag; Rudolph Hirsch; Amelie Illaire; Ambrosius Kühnel; Morawetzova sbírka; Národní muzeum – České muzeum hudby; Carl Friedrich Peters; Václav Jan Křtitel Tomášek; Josef Zvonař Jana Vojtěšková Národní muzeum jana.vojteskova@nm.cz 162
https://openalex.org/W3135422985	https://www.mdpi.com/1660-4601/18/5/2515/pdf?version=1614931878	English	null	Feasibility, Acceptability, and Outcomes of a Yoga-Based Meditation Intervention for Hospice Professionals to Combat Burnout	null	2,020	cc-by	17,979	Article Feasibility, Acceptability, and Outcomes of a Yoga-Based Meditation Intervention for Hospice Professionals to Combat Burnout Marcel Allbritton 2, Rebecca Lehto 3 , Patrick Miller 4, Patricia McDaniel 4 and Michael Palett Carrie Heeter 1,, Marcel Allbritton 2, Rebecca Lehto 3 , Patrick Miller 4, Patricia McDanie 1 Department of Media and Information, Michigan State University, East Lansing, MI 48823, USA 2 Core Resonance Works, New Orleans, LA 70119, USA; marcel.allbritton@me.com 3 School of Nursing, Michigan State University, East Lansing, MI 48823, USA; lehtor@msu.edu 4 Northstar Care Community, Ann Arbor, MI 48130, USA; pmiller@hom.org (P.M.); pmcdaniel@hom.org (P.M.); mpaletta@hom.org (M.P.) Correspondence: carrie.heeter@gmail.com 1 Department of Media and Information, Michigan State University, East Lansing, MI 48823, USA 2 Core Resonance Works, New Orleans, LA 70119, USA; marcel.allbritton@me.com 3 School of Nursing, Michigan State University, East Lansing, MI 48823, USA; lehtor@msu.edu 4 Northstar Care Community, Ann Arbor, MI 48130, USA; pmiller@hom.org (P.M.); pmcdaniel@hom.org (P.M.); mpaletta@hom.org (M.P.) * Correspondence: carrie heeter@gmail com 1 Department of Media and Information, Michigan State University, East Lansing, MI 48823, USA 2 Core Resonance Works, New Orleans, LA 70119, USA; marcel.allbritton@me.com 3 School of Nursing, Michigan State University, East Lansing, MI 48823, USA; lehtor@msu.edu 4 Northstar Care Community, Ann Arbor, MI 48130, USA; pmiller@hom.org (P.M.); pmcdaniel@hom.org (P.M.); mpaletta@hom.org (M.P.) * Correspondence: carrie heeter@gmail com Abstract: (1) Background. This research examined the feasibility, acceptability and outcomes of delivering a 6-week yoga-based meditation intervention to clinical teams of hospice professionals (HPs) at a large non-proﬁt hospice organization. The intervention was designed to increase mind- body integration and combat burnout. This article was written for different audiences, including research scientists who study interoception, burnout, meditation, or yoga, designers of meditation interventions, and hospice organizations looking for ways to mitigate HP burnout. (2) Methods. The intervention was launched within clinical teams, beginning with a half-hour online introduction to the program and exposure to the week 1 meditation at each team’s monthly all-staff meeting. Throughout the program, HPs could access the meditations on their own via their workplace computers, tablets, and smartphones. Online pre- and post-intervention surveys were submitted by 151 HPs, 76 of whom were exposed to the intervention and completed both surveys. The surveys assessed burnout using the Professional Fulﬁllment Index and mind-body integration using the Multidimensional Assessment of Interoceptive Awareness scales. (3) Results. Two-thirds of HPs who were present at a staff meeting where the program was introduced went on to do a meditation on their own at least once. Citation: Heeter, C.; Allbritton, M.; Lehto, R.; Miller, P.; McDaniel, P.; Paletta, M. Feasibility, Acceptability, and Outcomes of a Yoga-Based Meditation Intervention for Hospice Professionals to Combat Burnout. Int. J. Environ. Res. Public Health 2021, 18, 2515. https://doi.org/10.3390/ ijerph18052515 Academic Editors: Paul B. Tchounwou, Emilia Inmaculada De la Fuente-Solana, Guillermo A. Cañadas-De la Fuente, Luis Albendín-García and José Luis Gómez-Urquiza Received: 28 December 2020 Accepted: 23 February 2021 Published: 3 March 2021 Academic Editors: Paul B. Tchounwou, Emilia Inmaculada De la Fuente-Solana, Guillermo A. Cañadas-De la Fuente, Luis Albendín-García and José Luis Gómez-Urquiza Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: meditation; burnout; interoception Article Feasibility, Acceptability, and Outcomes of a Yoga-Based Meditation Intervention for Hospice Professionals to Combat Burnout Half of HPs expressed a desire to continue with access to the meditations after the 6-week program ended. Due to COVID-19 work from home restrictions, three-fourth of HPs did a meditation at home, 29% in a car between patient visits (not while driving), and 23% at the ofﬁce. Higher interoceptive awareness was signiﬁcantly related to lower burnout, particularly lower work exhaustion. Meditation frequency was signiﬁcantly related to higher interoceptive awareness but not to burnout. Interpersonal disengagement was rare and temporary. (4) Conclusions. Findings showed that the yoga-based meditation intervention was feasible and acceptable and associated with higher interoceptive awareness. The results point to a role for interoceptive awareness in reducing the risk for burnout. International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health 1. Introduction The ﬁeld research presented in this article examined feasibility, acceptability and out- comes of delivering a yoga-based meditation intervention for hospice professionals (HPs) at a large non-proﬁt hospice organization to combat burnout, a syndrome characterized by mental and physical exhaustion and workplace negativity. Interventions are critically needed to combat the threat of burnout among HPs in order to support the quality of care they are able to provide and to support their personal health and wellbeing. The intervention adapted the tools of yoga to design meditations that promote mind–body integration and reduce the risk of burnout and for hospice workers. We offer a rationale 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/). Int. J. Environ. Res. Public Health 2021, 18, 2515. https://doi.org/10.3390/ijerph18052515 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, 2515 2 of 27 for why yoga-based meditation helps combat burnout and we provide intervention and delivery strategy details, utilization data and outcomes. This thorough explication lays the groundwork for hospice organizations to consider their own intervention strategies and enables burnout and meditation researchers to build upon this study. for why yoga-based meditation helps combat burnout and we provide intervention and delivery strategy details, utilization data and outcomes. This thorough explication lays the groundwork for hospice organizations to consider their own intervention strategies and enables burnout and meditation researchers to build upon this study. In the ﬁrst four sections, we elaborate on the problem of burnout for HPs. We review prior research on meditation interventions to combat burnout in healthcare professionals and HPs. We explain what we mean by yoga-based meditation. We apply theory and research on mechanisms of effect of yoga to explain why yoga-based meditations might improve mind-body integration and combat burnout. Then, in the methods section, we clarify important details of the meditation interven- tion, explain how the intervention delivery strategy was designed to leverage organiza- tional structure and employee work patterns, and discuss study design and instruments used to assess feasibility, accessibility, and outcomes. We report participant demographics. In the results section, we present feasibility and accessibility ﬁndings, including exposure to the meditation intervention and use of the meditations among HPs who were introduced to the program. We report data on meditation use context (home, ofﬁce and car). 1. Introduction Finally, overall pre- and post-intervention data are reported for the burnout and interoceptive awareness scales followed by analyses of the relationship between meditation usage frequency and target outcomes (reducing burnout and increasing mind- body integration). In the discussion section, we reﬂect on contributions of the study, including implica- tions for theory, research, and the hospice industry. We also consider study limitations and offer recommendations for future research on yoga-based meditation burnout interventions. The conclusions section summarizes the main achievements of the study. 2. HP Burnout and Mind-Body Interventions Given the demands of the original MBSR program, MBSR-like interventions for health care professionals have attempted to shorten the program, including variations of 1–3 full days [14], and 4 weeks of regular synchronous online sessions with homework [12,15]. g y MBSR is the most-studied MBI, but it is actually a lengthy amalgam of disparate secu- larized Buddhist meditations, mindful yoga, and cognitive behavioral practices. Studies of MBSR interventions are unable to separate which of the many techniques are responsible for stress reduction. The mindfulness meditation app, Headspace, developed by a former Buddhist monk, has been studied as a brief intervention to reduce healthcare provider burnout and stress. There are thousands of meditation apps. Headspace consistently ranks among the top ﬁve meditation apps [16–18] and the company actively supports external research and conducts its own research. Unlike MBSR, using Headspace as an intervention requires minimal time and has no group component or live sessions. Additionally, unlike MBSR, Headspace uses a single consistent meditation modality (mindfulness meditations) rather than an amalgam of techniques. In research using Headspace with healthcare professionals, participants downloaded the app and did the 10-day, 10 min a day introduction to mindfulness meditation. Some took up to one month to complete the 10 sessions [19,20]. The introductory series purports to teach how to meditate (by sitting in a quiet place, closing the eyes, calming the mind, focusing on the breath, and letting thoughts and feelings come and go [21]). Novice pediatric nurses who used the Headspace app’s 10 day sequence showed marginally more compassion satisfaction and marginally less burnout than nurses who did traditionally delivered meditation training [19]. Among medical school students, perceived stress decreased and general wellbeing increased among participants in the Headspace meditation app group, compared to a control group, which listened to 10 min autobio- graphical audio segments about the Headspace founder’s experiences with Buddhism [20]. g p g p p The MBI intervention for HPs studied in this article is grounded in yoga-based medi- tation, a form of somatic, movement-based meditation. As will be discussed in subsequent sections, the process, mechanisms and effects of this meditation approach differ from mindfulness meditation. The current research builds upon a study of an earlier, app-based version of the same yoga-based meditation program for HPs [22]. 2. HP Burnout and Mind-Body Interventions Prior research shows that burnout is a serious and pervasive challenge for health- care professionals overall, including nurses, physicians, and medical school students [1]. Burnout in HPs has also been identiﬁed to be a serious problem, compounded by workload stressors and administrative demands [2]. Hospice work is characterized by a multidisci- plinary team approach, where interprofessional communication and seamless coordination are essential to ensure optimal delivery of care. There has been careful attention paid to the stressors that working in a hospice and/or palliative care environment places on these essential health care workers over time. Much of this research has been conducted using sur- vey methodology and has depicted the prevalence of burnout in the HP population [3–6]. Surveys of more than 3500 palliative care and HPs in the US spanning 5 years estimate average burnout rates of 38.7% [7]. Burnout can be addressed through both personal and organizational interventions. Organizational burnout interventions seek to modify workplace values, processes and practices. It is critical to address organizational factors that contribute to burnout. Personal interventions do not replace the need for organization change. These are not mutually exclusive. One of the recommended steps organizations can take is to provide resources to promote HP self-care by offering personal interventions [8]. Mind–body interventions (MBIs) have been evaluated as methods to improve the self-management of stress since the introduction of Mindfulness-Based Stress Reduction (MBSR) by Kabat-Zinn in the 1990s [9]. MBIs can include all types of meditation, relaxation and breathing techniques, yoga, tai chi, qigong, hypnosis, biofeedback, and more [10]. In healthcare, MBIs have been tested as interventions to help professional providers manage work-related stressors [11]. MBIs are often time consuming and a poor ﬁt for busy healthcare professionals. Several healthcare professional studies have been conducted that incorporated the Mindfulness- Based Stress Reduction (MRBR) program [11,12]. MBSR, in its origins, was a group-based 8-week program that incorporated both informal and formal practices. The program in- cluded meeting for 2 h per week as a group, an all-day retreat, and daily homework [9]. Int. J. Environ. Res. Public Health 2021, 18, 2515 3 of 27 3 of 27 Attrition rates in the 8-week MBSR program with a sample size of 38 healthcare profession- als were reported to be 44%, even with a self-selected sample [13]. 3. Yoga-Based Meditations as a Burnout Intervention In this section we explain what we mean by yoga-based meditation and offer a rationale as to why practicing yoga-based meditations designed to promote mind–body integration might help prevent or alleviate the symptoms of HP burnout. To support the rigorous interpretation of meditation research ﬁndings, researchers must precisely deﬁne the characteristics of the meditation intervention and the expertise of the intervention developers [27]. The meditation intervention in this study was de- signed by study authors Allbritton and Heeter, who are experts in meditation and trained researchers with PhDs. Our experience and background in meditation come from the perspective of Viniyoga and Yoga Therapy, originating from Sri Krishnamacharya and TKV Desikachar [28–31]. Allbritton has studied Viniyoga for over 15 years and is a practicing C-IAYT clinical yoga therapist. Heeter is a professor of user experience and serious game design and an RYS 200 certiﬁed yoga and meditation teacher who has been studying and researching meditation for 9 years. g y Meditation is a popular form of self-care. A 2017 national study of complimentary care found that 14.2% of U.S. adults reported using meditation [32]. Approaches to meditation that together comprise that 14.2% include mantra meditation, mindfulness meditation, transcendental meditation, guided imagery, progressive relaxation, and spiritual medi- tation as well as meditation that is part of yoga, tai chi, and qi-gong [33]. Each speciﬁc meditation approach is associated with a small fraction of overall meditation practition- ers. By comparison, 14.3% of US adults reported doing yoga, including 19.8% of adult women [32]. Different MBI approaches engage the human system in different ways [34]. Yoga- based meditation practices draw upon the tools of yoga (such as attention, breath, physical movement, and meditation objects [35]). The selection and sequencing of steps in a practice are informed by goals for the practice, needs and characteristics of who will be doing the practice, and principles of yoga [36–38]. Although the range of possible practices is endless, yoga-based practices typically involve directing attention inward to breath, body, controlled movement, and other internal bodily sensations and feelings [31]. Theories of mechanisms of the effects of yoga suggest that yoga-based meditation supports psychological and physical well-being through combined effects on high level and low-level brain network functioning. 2. HP Burnout and Mind-Body Interventions In the initial research, one third of HPs at a medium sized hospice organization who were invited to take part in the program downloaded the app and did a meditation at least two times per week for six weeks. The entire organization participated at the same time. Participants were rewarded USD 20 for each week in which they meditated at least twice. The pre-post study (n = 36) found that burnout was already very low (below 26 on the Professional Quality of Life instrument [23]). Signiﬁcant though small pre-post survey improvements were found in burnout and compassion fatigue. Signiﬁcant large pre-post survey improvements were found in the attention regulation, emotional awareness, self-regulation, body noticing, body listening, body trusting dimensions of interoceptive awareness [22]. The study showed that the yoga-based meditation helped HPs cultivate heightened interoceptive awareness and improved mental focus—factors that can change how stressful events are experienced and responded to [22]. Focus group interviews detailed experiential changes that HPs attributed to doing the meditations and afﬁrmed the importance of social support that came from the entire staff going through the program at the same time [24]. The current study tested a revised version of the MBI and an intervention delivery strategy tailored to the needs and affordances of a large hospice organization. HPs were not compensated for doing the meditations. Thus, the uptake of the intervention served as an indicator of its acceptability. Research on MBI interventions for hospice and palliative care employees tend to show short-term improvements in wellbeing, but many of the studies have low quality of treatment ﬁdelity, small samples, high attrition, or a lack of theoretical consideration of what are the essential “ingredients” that underpin successful interventions [11,25,26]. Int. J. Environ. Res. Public Health 2021, 18, 2515 4 of 27 4 of 27 The meditation components are rarely described in detail and interventions varied in terms of delivery, length, schedule, type of instructor, practice requirements, resources provided, and context. Our study advances the understanding of yoga-based meditation and burnout, informs consideration of MBIs to address HP burnout, and addresses some of the weaknesses of prior MBI research. 3. Yoga-Based Meditations as a Burnout Intervention (See Figure 1.) In yoga-based meditation, high-level executive function directs attention toward interoceptive signals as they are occurring in the body during breathing, movement and focus on a meditation object while withholding or redirection attention away from mind wandering. Mind–body integration is supported by engaging interoception [14,39,40], the complex iterative process of noticing and appraising and responding to signals originating within the body [34,41]. Slow, controlled movement and breathing and attention to present moment interoceptive bodily sensations and feelings can help quiet the mind, calm the human system and build capacity for interoception [39,42]. Over time, practicing yoga-based meditation supports the inhibition of negative cog- nitive, emotional, and behavioral responses to stressful events, initially through conscious restraint [14,39,40]. For example, by noticing and self-regulating negative emotions and quieting self-talk. Over time the inhibition becomes automatic. Similarly, autonomic physi- ological responses to stress during yoga-based meditation gradually transfer beyond the meditation practice period, reducing inﬂammation, chronic stress response, lowering heart rate, and reducing reactivity [14,39,40]. Yoga-based meditation improves high-level and low-level brain network responses to stressors. Int. J. Environ. Res. Public Health 2021, 18, 2515 Int. J. Environ. Res. Public Health 2021, 18, x FOR 5 of 27 5 of 28 Figure 1. Yoga-based meditation facilitates mind–body integration. Figure 1. Yoga-based meditation facilitates mind–body integration. Figure 1. Yoga-based meditation facilitates mind–body integration Figure 1. Yoga-based meditation facilitates mind–body integration. Over time, practicing yoga-based meditation supports the inhibition of negative cog- nitive, emotional, and behavioral responses to stressful events, initially through conscious restraint [14,39,40]. For example, by noticing and self-regulating negative emotions and quieting self-talk. Over time the inhibition becomes automatic. Similarly, autonomic phys- iological responses to stress during yoga-based meditation gradually transfer beyond the meditation practice period, reducing inflammation, chronic stress response, lowering heart rate, and reducing reactivity [14,39,40]. Yoga-based meditation improves high-level and low-level brain network responses to stressors. The best way to experientially understand the meditations in the intervention is to d ll d h k l d h h l bl l The best way to experientially understand the meditations in the intervention is to spend 12 min actually doing the week 1 calming meditation, which is available as Supplementary Video S1 of this article. Each of the steps in all of the yoga-based meditations that comprise the intervention engage interoceptive awareness and help reduce mind wandering and rumination. 3. Yoga-Based Meditations as a Burnout Intervention The yoga-based meditations in the current study were designed with a long-term goal of helping to inhibit unhealthy responses to stress by training interoception and promoting mind–body integration. There are other general and speciﬁc goals of each meditation but activating and training interoception is a prominent and omnipresent function. The meditations are described in detail in Section 5.1. spend 12 min actually doin mentary Video S1 of this ar 4. Intervention Outcomes y p y g comprise the intervention engage interoceptive awareness and help reduce mind wander- ing and rumination. The yoga-based meditations in the current study were designed with a long-term goal of helping to inhibit unhealthy responses to stress by training interocep- In this section, we introduce two categories of outcomes of the meditation program (burnout and mind–body integration) and discuss why doing yoga-based meditations might bring about desirable changes for HPs. tion and promoting mind–body integr each meditation but activating and trai 4.1. Burnout and Yoga-Based Meditation g g p p p function. The meditations are described in detail in Section 5.1. 4. Intervention Outcomes In this section, we introduce two categories of outcomes of the meditation program (burnout and mind body integration) and discuss why doing yoga-based meditations To assess burnout, the study used the brief Stanford Professional Fulﬁlment Index (PFI) to capture elements of HP distress and dimensions of wellbeing over the last two weeks. The 16-item PFI measure is suited to assessing changes over time in work exhaustion, interpersonal disengagement and professional fulﬁlment [43]. The index has high reliability and convergent validity. (burnout and mind–body integration) and discuss why doing yoga-based meditations might bring about desirable changes for HPs. 4.1. Burnout and Yoga-Based Meditation To assess burnout, the study used the brief Stanford Professional Fulfilment Index (PFI) to capture elements of HP distress and dimensions of wellbeing over the last two weeks. The 16-item PFI measure is suited to assessing changes over time in work exhaus- tion, interpersonal disengagement and professional fulfilment [43]. The index has high reliability and con ergent alidity g y Work exhaustion combines feeling physically and emotionally exhausted at work, lacking enthusiasm for work, and feeling a sense of dread about the work one has to do. Interpersonal disengagement combines feeling less empathetic to one’s patients and to one’s colleagues, being less interested in talking with patients, less connected with patients and less sensitive to others’ feelings/emotions. Work exhaustion and interpersonal disengagement can be merged to measure overall burnout. The third dimension of the PFI, professional fulﬁlment, measures feeling satisfaction, feeling worthwhile, meaningfulness and having a sense that one is making a professional contribution. y g y Work exhaustion combines feeling physically and emotiona 4.2. Mind–Body Integration, Interoception, and Yoga-Based Meditation y g y Work exhaustion combines feeling physically and emotiona 4.2. Mind–Body Integration, Interoception, and Yoga-Based Meditation lacking enthusiasm for work, and feeling a sense of dread about the work one has to do. Interpersonal disengagement combines feeling less empathetic to one’s patients and to one’s colleagues, being less interested in talking with patients, less connected with pa- tients and less sensitive to others’ feelings/emotions. Work exhaustion and interpersonal disengagement can be merged to measure overall burnout. The third dimension of the PFI, As discussed in Section 3, theories of the mechanisms underpinning the beneﬁcial effects of yoga-based meditation predict (1) beneﬁcial impacts of regular practice on high level and low-level brain functions and (2) better integration of mind and body through interoception. Interoceptive awareness is one aspect of interoception, which theories of yoga point to as central to mind–body integration and to changes in how the human system Int. J. Environ. Res. Public Health 2021, 18, 2515 6 of 27 responds to stress. The Multidimensional Assessment of Interoceptive Awareness (MAIA) is a survey instrument designed to measure the effects of meditation on multiple dimen- sions of interoceptive awareness [12,44,45]. The scale was developed by a diverse team of scientists and practitioners with expertise in different meditation traditions, including yoga, mindfulness, qigong, somatic massage, and mindfulness. The yoga-based meditations in this study intervention were designed to activate and train interoceptive awareness most closely related to six of the eight MAIA subscales. (The other two subscales, non-distracting and not-worrying, ask about reacting to sensations of discomfort or pain, which our intervention does not address.) Below, we present MAIA’s explanation of the six subscales and offer two example questions from each subscale [45]: • Self-regulation (ability to regulate distress by attention to bodily sensations). Example questions: When I bring awareness to my body, I feel a sense of calm; When I am caught up in thoughts, I can calm my mind by focusing on my body/breathing. • Emotional awareness (awareness of the connection between body sensations and emotional state). Example statements: I notice how my body changes when I am angry; I notice that my body feels different after a peaceful experience. • Attention regulation (ability to sustain and control attention to body sensations). Example statements: I can maintain awareness of my inner bodily sensations even when there is a lot going on around me; I can return awareness to my body if I am distracted. • Body noticing (awareness of uncomfortable, comfortable, and neutral bodily sensa- tions). 5. Methods The methods section is organized in detail about the yoga-based meditation interven- tion, the intervention delivery strategy, and survey instruments. Next, sample size and demographics are presented. y g y Work exhaustion combines feeling physically and emotiona 4.2. Mind–Body Integration, Interoception, and Yoga-Based Meditation Example statements: When I am tense I notice where the tension is located in my body; I notice changes in my breathing, such as whether it slows down or speeds up. • Body listening (active listening to the body for insight). Example statements: I listen for information from my body about my emotional state; When I am upset, I take time to explore how my body feels. p y y • Body trusting (experience of trusting one’s body as safe and trustworthy). Example statements: I feel my body is a safe place; I trust my bodily sensations. The categories, summaries, and example questions illustrate what each subscale measures, and suggest ways that interoceptive awareness contributes to wellbeing, stress management, and combatting burnout. 5.1. Details about the Intervention To support future meta-analyses and rigorous research, meditation researchers [37] and yoga researchers [46,47] advocate for a thorough description of the process and in- tended outcomes for meditation interventions used in research. We begin by describing the overall structure of the intervention and then go into detail about the content for each week. The intervention offered one new 12-min yoga-based meditation recording per week for six weeks, half guided by a male meditation teacher (Allbritton) and half by a female meditation teacher (Heeter). HPs could access the meditations by logging in to the program website or by opening an app on the tablets and smartphones they used for hospice work. The program website had a page for each week with the meditation video (audio guidance and video of stick ﬁgures illustrating each step), a “how and why this works” video explaining principles of yoga-based meditation that have implications for daily life (see Figure 2), and additional messaging to motivate regular meditation practice. The internal app only included meditation audios. Yoga-based meditations consist of a series of steps that draw upon the tools of yoga (such as movement, breath, and meditation objects). Tables 1–3 lists the speciﬁc movements, Int. J. Environ. Res. Public Health 2021, 18, 2515 7 of 27 7 of 27 breathing, and attentional focus used in each meditation to enable readers expert in yoga to understand the intervention. These steps were selected and sequenced to help bring about the intended changes in the human system. For the meditations in the intervention, there were many design constraints. The duration had to be under 12 min. The practices, including clear instructions and amount of time in any step, had to be accessible to begin- ners who have never done yoga before. The practices were done seated comfortably in a chair. Movements needed to be gentle and participants were reminded to move only as far as is comfortable, to avoid potential stress or strain. Each meditation had unique goals. For example, the steps and sequencing of steps in the calming meditation were chosen with the goal of relaxing the body and quieting the mind. Aligning gentle movements with breath gave the mind something to do other than think about itself. Early steps involved larger movements (such as bringing the arms up from the front toward the ceiling). 5.1. Details about the Intervention Public Health 2021, 18, 2515 8 of 27 8 of 27 Attention to and regulation of breath are also tools of yoga-based meditations. Table 2 shows that, as previously discussed, every meditation includes aligning movement with breath. Different yoga breathing techniques (extending exhale, pausing after exhale, vi- sualization linked to inhale and exhale), were used to support the focus of particular meditations. The ﬁrst two breath techniques (free observed breath and aligning movement with breath) were part of all of the meditations. Table 2. Breath and Inhale:Exhale Ratios of the Yoga-Based Meditations. Breathing and Inhale:Exhale Ratios Meditations Calming Peaceful Feeling Nature Stability Releasing Cleansing Waves Free observed breath √ √ √ √ √ √ Align movement with breath √ √ √ √ √ √ Extend exhale √ √ Pause after exhale √ √ On exhale, release what you no longer need √ On inhale, visualize a wave washing onto the shore. On exhale the wave goes back out, cleansing the shore. √ Open hands on inhale, close hands on exhale √ Table 2. Breath and Inhale:Exhale Ratios of the Yoga-Based Meditations. Breathing and Inhale:Exhale Ratios Meditations Calming Peaceful Feeling Nature Stability Releasing Cleansing Waves Free observed breath √ √ √ √ √ √ Align movement with breath √ √ √ √ √ √ Extend exhale √ √ Pause after exhale √ √ On exhale, release what you no longer need √ On inhale, visualize a wave washing onto the shore. On exhale the wave goes back out, cleansing the shore. √ Open hands on inhale, close hands on exhale √ Table 2. Breath and Inhale:Exhale Ratios of the Yoga-Based Meditations. Meditation objects are another tool used in yoga-based meditation. A meditation object is an object the mind focuses on. Table 3 shows meditation objects for the intervention meditations. Meditation objects can help the meditator strengthen their connection to a the object or quality (such as calm, peaceful, stable [48]). In the nature meditation, participants called to mind the experience of being in a favorite place in nature, activating interoceptive feelings of spending time in nature and of being in a good-feeling place. The releasing meditation brought forward the idea of letting go of things the HP was holding on to that were no longer needed (perhaps tension or tightness, perhaps events from a stressful day). 5.1. Details about the Intervention As the meditation progressed, the mind and body became calmer, allowing movements to be more subtle. As the meditation progressed, participants were guided to move a little slower on exhale. Additionally, in the later steps, to pause for a moment or two at the end of each exhale. These exhale techniques support calming. The general intended direction of effect for each meditation is implied by its name: calming, peaceful feeling, nature, stability, releasing, and cleansing waves. g p g y g g All of the meditations in the intervention shared objectives of engaging interoception and promoting mind–body integration. Theoretical models and reviews of research ex- plain how and why yoga-based meditations promote mind–body integration [14,34,35,39]. For example, synchronizing slow, controlled gentle movements with inhale and exhale requires coordination, tracking the body’s location in space, and attention to both breath and movement. Executive function is involved in learning each movement and breath sequence. Interoceptive attention is engaged with kinesthetic, proprioceptive and spatial sensations [39]. Table 1 lists all of the gentle movements were part of the intervention, noting which of the meditations that movement appeared in. The ﬁrst two movements and postures were part of all six meditations. Others were unique to particular meditations. For example, arm extension out from the chest, palms facing outward, supports releasing. Leaning forward and back and ﬁnding a position that feels most stable supports stability. The movements in yoga-based meditations were chosen to support the function of that meditation. Table 1. Movements and Postures of the Yoga-Based Meditations. Movements, Postures Meditations Calming Peaceful Feeling Nature Stability Releasing Cleansing Waves Upright seated in chair, eyes closed √ √ √ √ √ √ Arm extension up in front √ √ √ √ √ √ Arms up from front then out to side √ Arm extension up in front alternating arms √ √ √ Raise arms up from sides a little ways, a bit higher with each breath √ Arm extension from chest down to side √ Arm extension out from chest √ √ Arm extension out from chest, palms facing outward √ Hands on chest, arm extension down by sides Hands on opposite shoulders, arm extension down to legs √ Seated forward bend √ √ Move hands over thighs toward knees √ Lean forward and back, ﬁnding position that feels most stable √ Table 1. Movements and Postures of the Yoga-Based Meditations. Int. J. Environ. Res. 5.1. Details about the Intervention Additionally, the cleansing waves meditation brought forward the idea of cleansing and the calming rhythm of breath as gentle waves. Table 3. Meditation Objects/Mental Focus of the Yoga-Based Meditations. Meditation Objects Meditations Calming Peaceful Feeling Nature Stability Releasing Cleansing Waves Notice bodily sensations √ √ √ √ √ √ Notice level of mental activity √ √ √ √ √ √ Call to mind a peaceful feeling √ Imagine peaceful feeling as a handful of seeds. Pick up seed and drop onto one of your relationships √ Think of favorite place in nature √ Bring feeling of favorite place inside √ Feel self in favorite place in nature √ Connect with feeling of stability √ Release tension, tightness, what you no longer need √ Visualize a sandy beach with gentle waves √ Table 3. Meditation Objects/Mental Focus of the Yoga-Based Meditations. Int. J. Environ. Res. Public Health 2021, 18, 2515 9 of 27 9 of 27 The program web site page content and videos for each week emphasized a learning principle that showed how what HPs were doing in the meditation can translate into daily life. Table 4 lists the principle for each week. These principles apply to all six meditations, but are introduced one week at a time, in logical order that is consistent with the particular meditation for that week and with the users’ growing experience with this meditation modality. Doing the week 1 calming meditation reinforced the principle that “yoga-based meditations are tools you can use to help change how you feel,” because this aligned with their experience of doing the meditation. At the beginning and end of each meditation, HPs were guided to notice how they feel. To check in with themselves. The principle for week 2 was that “checking in with yourself” is a skill, in meditation and in life. Regular practice of these yoga-based meditations built a capacity to check in with oneself. The “how and why this works” video for week 2 is included as Supplementary Video S2 with this article. Table 4. Meditations and Principles in the 6-Week Program. Week Meditation Principle 1 Calming Yoga-based meditations are tools you can use to help change how you feel. 2 Peaceful Feeling Checking in with yourself is a skill. 3 Place in Nature Moving only as far as is comfortable is a skill. 5.2. Meditation Program Delivery The impact of a burnout intervention for HPs depends upon both the design of the meditation program itself and also on the implementation approach and resulting uptake of the meditations by HPs. In this section, we explain the program delivery decisions and the organizational strategies intended to motivate and facilitate social support for doing the meditations. 5.2.1. Adapting Program Delivery to the Needs of Clinical HPs 5.1. Details about the Intervention 4 Stability Breath is an indicator and an inﬂuencer of the state of your system. 5 Releasing Keep your cool by responding rather than reacting to events. 6 Cleansing Waves Regular practice of meditation supports self-care. Table 4. Meditations and Principles in the 6-Week Program. 5.2.1. Adapting Program Delivery to the Needs of Clinical HPs There were three venues where HPs could do the meditations: at the office, at home, and in their cars (not while driving!) before or after patient visits (see Figure 2). and playing the meditation audio, with no need for a log-in or internet access. There were three venues where HPs could do the meditations: at the ofﬁce, at home, and in their cars (not while driving!) before or after patient visits (see Figure 2). site. HPs could also do the meditations by opening the app on their tablet or smartphone and playing the meditation audio, with no need for a log-in or internet access. There were three venues where HPs could do the meditations: at the office, at home, and in their cars (not while driving!) before or after patient visits (see Figure 2). Figure 2. Ways HPs Could Access the Meditations. 5.2.2. Leveraging Organizational Structure to Foster Community and Encourage Adop- tion and Use Figure 2. Ways HPs Could Access the Meditations. 5.2.2. Leveraging Organizational Structure to Foster Community and Encourage Adoption and Use Figure 2. Ways HPs Could Access the Meditations Figure 2. Ways HPs Could Access the Meditations. 5.2.2. Leveraging Organizational Structure to Foster Community and Encourage Adop- tion and Use 5.2.2. Leveraging Organizational Structure to Foster Community and Encourage Adoption and Use 5.2.2. Leveraging Organizational Structure to Foster Community and Encourage Adop- tion and Use 5.2.2. Leveraging Organizational Structure to Foster Community and Encourage Adoption and Use Here we discuss program delivery decisions informed by the structure of the large hospice organization to help HPs value the program, motivate them to do the meditations, and harness peer and management involvement for social support. Here we discuss program delivery decisions informed by the structure of the large hospice organization to help HPs value the program, motivate them to do the meditations, and harness peer and management involvement for social support. and harness peer and management involvement for social support. The 6-week intervention was offered in the context of clinical teams, so the interven- tion would be a shared experience among co-workers. The non-profit hospice we studied served dying patients in 62 counties across the state. The hospice was organized into ge- ographic service regions, and within those regions into local clinical teams that deliver hospice care support directly to patients and their families. 5.2.1. Adapting Program Delivery to the Needs of Clinical HPs Each team was led by an op- erations manager who coordinated the work of nurses, social workers, hospice aides, and chaplains. (There were other services and teams across the hospice organization, but the focus of our research was on the clinical teams ) The 6-week intervention was offered in the context of clinical teams, so the intervention would be a shared experience among co-workers. The non-proﬁt hospice we studied served dying patients in 62 counties across the state. The hospice was organized into geographic service regions, and within those regions into local clinical teams that deliver hospice care support directly to patients and their families. Each team was led by an operations manager who coordinated the work of nurses, social workers, hospice aides, and chaplains. (There were other services and teams across the hospice organization, but the focus of our research was on the clinical teams). focus of our research was on the clinical teams.) The implementation approach elicited leadership buy-in to demonstrate the insti- tutional valuing of the importance of the intervention. Yoga-based meditation program coordinators introduced the program at an online meeting to the hospice organization’s Executive Leadership team, and the executives did the week 1 meditation (calming) to- gether, then discussed plans for the program. Then, the program was introduced at an online meeting to the clinical directors, and they did the week 1 meditation together. Next, online introduction sessions were held with each clinical director and the clinical operations managers of the teams she supervised, and that group did the week 1 calming meditation together. Finally, for each clinical operation manager’s team at their regularly scheduled monthly team all-staff meeting, a yoga-based meditation program coordinator joined the meeting online, introduced the program to the team, and helped them locate the app on their tablets or smartphones. p The six-week program was deployed separately to each team tied to their monthly all-staff meeting schedules. Every participant who was able to attend their team’s monthly online all-staff meeting was introduced to the 12-min week 1 meditation during the meeting. The operations manager forwarded an email to their team each week throughout the program, introducing the new meditation for the week, pointing out the principle for the week, and encouraging use. 5.2.1. Adapting Program Delivery to the Needs of Clinical HPs The practice of clinical hospice work has implications for how to deliver a yoga-based meditation intervention. Clinical HPs are a mobile workforce who deliver onsite service to dying patients and their families and offer virtual care support by phone. The mix of in person and phone support varies by role, with hospice aides and nurses providing the most in-home care and chaplains and social workers engaging in more phone support. All patient care involves continuous need to access and update Electronic Medical Records (EMR). The hospice organization provides clinicians with secure tablets and smartphones to use for EMR and internal communication systems. y The original plan was to offer the yoga-based meditation program by providing HPs with login accounts to the mobile friendly program web site. Depending on the location of patient homes, internet access can be unreliable, especially in remote, low population density regions. To make access to the meditations as easy as possible and to eliminate the need for internet access, the hospice IT department created an internal app that included audio of all six meditations, which IT “pushed out” to all workplace tablets and smartphones. Thus, we took advantage of omnipresent secure tablets and smartphones that were deeply integrated into HP workﬂow, making it easy to access the meditations. MP3 audios of the meditations were used in the app instead of the videos because video ﬁles would have taken more storage space on workplace mobile devices. Since the meditations were done with eyes closed, the only difference was that HPs could not open their eyes to see a diagram of a movement if they were uncertain what to do. Thus, the yoga-based meditation intervention was accessible via the program web site. HPs could also do the meditations by opening the app on their tablet or smartphone Int. J. Environ. Res. Public Health 2021, 18, 2515 tations w eyes to 10 of 27 pen their 10 of 27 pen their and playing the meditation audio, with no need for a log-in or internet access. There were three venues where HPs could do the meditations: at the ofﬁce, at home, and in their cars (not while driving!) before or after patient visits (see Figure 2). site. HPs could also do the meditations by opening the app on their tablet or smartphone and playing the meditation audio, with no need for a log-in or internet access. • Do meditations when teams are together throughout the 6 weeks. 5.2.1. Adapting Program Delivery to the Needs of Clinical HPs At the introductory online session after the team did the calming meditation, the re- searcher encouraged HPs to do the meditation on their own and showed a slide suggesting ways HPs could use the meditations: • Do meditations when teams are together throughout the 6 weeks. Int. J. Environ. Res. Public Health 2021, 18, 2515 11 of 27 11 of 27 • Try to do the meditation on at least one (ideally more) other days each week on your own. • Try to do the meditation on at least one (ideally more) other days each week on your own. • In the car (while parked, not while driving!). • In the car (while parked, not while driving!). • When you ﬁrst get up. • Before you go to bed. • After or before seeing a client. • After or before seeing a client. Thus, HPs could do the 12 min meditations during working hours or on their own time. The research protocol did not reward HPs for meditation frequency because we wanted to measure acceptability and adoption of the intervention on its own merit. Teams were rewarded with a team gift card based on the percent of team members who submit- ted surveys. 5.3.1. Meditation Use, Feasibility, and Acceptability 5.3.1. Meditation Use, Feasibility, and Acceptability A primary aim of this research was to assesses the feasibility and acceptability of the yoga-based meditation intervention. In this section, we deﬁne and operationalize feasibility and acceptability. A consequence of making the meditations available on a local app on workplace smartphones and tablets was that the researchers had to rely on self-report instead of automatically tracking meditation usage. The program web site did track all meditation use for each logged in user, but almost all HP meditation use ended up being via the local app. Usage tracking of the custom app was beyond the scope of time and effort available to the project. p j Feasibility refers to the practicality and viability of the of the team-based intervention delivery strategy as a means to reach clinical HPs with the meditation program [49]. Feasibility was assessed using a self-report on the post-survey. The post-survey asked whether the HP had done any of the meditations. Since the program was introduced to the team, all-staff meetings in which the ﬁrst week meditation was played, doing at least one meditation showed that the HP had been reached by the intervention. Acceptability refers to how recipients react to the intervention [49]. Acceptability was assessed using self-report on the post-survey. Respondents were asked how many times they did a meditation, what platforms they used (tablet, phone, computer), where they did meditations (home, car, ofﬁce), and whether they would be interested in continuing to have access to the meditations now that the 6-week program was over. 5.3. Measures Here we explain how feasibility, acceptability and intervention outcomes were mea- sured. 5.4.1. Protocol Here we describe in detail the study protocol including how participation was orga- nized within regional teams, team-based rewards for participation, IRB approvals, consent procedures and the study schematic. Clinical care delivery at the hospice we studied is managed by 20 different regional teams, each with its own operations manager, nurses, hospice aides, social workers, and chaplains/spiritual care professionals. The intervention was deployed to all clinical care teams, one team at a time. Teams were rewarded with a gift card where the amount was determined by the proportion of team members who submitted a pre-survey (up to USD 100) and the propor- tion of team members who submitted a post-survey (also up to USD 100). There were no individual or team incentives for doing the meditations. The online pre-post survey research design and intervention delivery protocols used in the research were reviewed by the Michigan State University IRB and received an exempt determination (category 3i(b)), STUDY00003891. The pre- and post-surveys began with an online consent form. Consenting HPs then moved to the survey questions. As we stated in the Conﬂict of Interest section for this article, the meditation interven- tion is a commercial product and the ﬁrst two authors are associated with the company. The consent form included the following statement: We want to disclose that Yoga Mind Tools is a commercial program, and the creators (Carrie and Marcel) may personally beneﬁt from future sales if this trial is successful. The meditation intervention was delivered to 11 clinical care delivery teams in spring (Wave 1) and 7 teams in fall (Wave 2). Two other teams scheduled for fall participation had to postpone starting the program and were not included in the research. A mandatory statewide COVID-19 shut down began near the end of Wave 1, upending data collection. Before COVID-19, clinical HPs worked either on the road or at the ofﬁce. COVID-19 restrictions forced an immediate shift to work from home instead of working from the ofﬁce. In-home patient visits still happened, but with new safety precautions. Some services previously offered in person occurred virtually. Our university IRB shut down all research, and the hospice organization was occupied with the sudden, immediate need to pivot to employees working from home instead of the ofﬁce and establishing home visit procedures to protect HPs, patients, and their families from exposure to COVID-19. 5.3.2. Outcomes (Burnout and Mind–Body Integration) A secondary research aim of this study was to examine whether the intervention resulted in changes in burnout or mind–body integration among participating HPs. In this section, we introduce the instruments used to measure these outcomes. Details about these survey items were discussed earlier in Section 4, Intervention Outcomes. Burnout was assessed on the pre- and post-surveys using the Professional Fulﬁll- ment Index which measures professional fulﬁlment, work exhaustion, and interpersonal disengagement [43]. This index was described in Section 4.2. Mind–body integration was assessed on the pre- and post-surveys using six dimen- sions of interoceptive awareness from the MAIA scale: Self-regulation, attention regulation, emotional awareness, body noticing, body listening, and body trusting [44,45]. These scales were described in Section 4.2. Int. J. Environ. Res. Public Health 2021, 18, 2515 12 of 27 5.4. Study Protocol, Sample Characteristics, and Overall Burnout and Interoceptive Awareness Levels 5.4.1. Protocol Therefore, post-surveys from the ﬁrst 11 teams could not be collected. We did continue sending teams weekly emails introducing the new meditation for the week, and some operation managers contacted the PI to report that the meditations were helpful in coping with challenging times. The researchers continued to send the HP teams weekly emails introducing the new meditation for the week. To give the hospice organization time to normalize to COVID-19 changes, we waited 6 months to start the second wave of deployment to hospice teams. Due to ongoing COVID- 19 safety measures throughout the Wave 2 research period, HPs logged in to the all-staff meetings from home instead of meeting in person together as they had before COVID-19. Three teams use Microsoft Teams, and 4 teams use Zoom for their all-staff meetings. Figure 3 shows the Wave 2 study schematic. 13 of 27 staff 13 of 27 staff Int. J. Environ. Res. Public Health 2021, 18, 2515 COVID- meeting Figure 3. Study Schematic. Figure 3. Study Schematic. hematic. Figure 3. Study Schematic. hematic. Figure 3. Study Schematic. 5.4.2. Sample Characteristics H l i th 5.4.2. Sample Characteristics Here sample size on the pre- and post-survey response rates and sample characteris- tics are reported. Survey results describe team characteristics and professional roles of HP study participants. Demographics are estimated based on an earlier survey conducted within the hospice organization. Pre-surveys were submitted by 127 clinical HP team members and post-surveys were Here sample size on the pre- and post-survey response rates and sample characteristics are reported. Survey results describe team characteristics and professional roles of HP study participants. Demographics are estimated based on an earlier survey conducted within the hospice organization. Pre-surveys were submitted by 127 clinical HP team members and post-surveys were submitted by 116 HPs. Ninety-two pre-post surveys were able to be matched using par- ticipant email addresses. This means that 61% (n = 92) of the 151 unique participants sub- mitted both a pre-survey and a post-survey. Pre-surveys were submitted by 127 clinical HP team members and post-surveys were submitted by 116 HPs. Ninety-two pre-post surveys were able to be matched using participant email addresses. This means that 61% (n = 92) of the 151 unique participants submitted both a pre-survey and a post-survey. The teams participating in the study served patients and families in home hospices. Several teams covered large, sparsely populated counties. Others served medium sized cities and surrounding areas. The size of the seven teams ranged from 7 to 30 members. g g HP role was only asked on the presurvey. By role, 43% of pre-survey respondents were nurses and 22% were hospice aides (see Table 5). The remaining roles (social worker, chaplain/spiritual care, manager, administration, and other) each comprised fewer than 10% of respondents. Table 5. HP Roles (from the pre-survey). Role Percent Nurse 43% Aide 22% Social Worker 7% Chaplain/Spiritual Care 7% Manager 5% Administration 8% Other 9% n 126 Table 5. HP Roles (from the pre-survey). Role Percent Nurse 43% Aide 22% Social Worker 7% Chaplain/Spiritual Care 7% Manager 5% Administration 8% Other 9% n 126 Table 5. HP Roles (from the pre-survey). 6.1.1. Feasibility Among the 116 post-survey respondents, 22% never experienced meditation. (They must have missed the all-staff meeting where the program was introduced, and the ﬁrst meditation was played.) Thus, the intervention delivery strategy of introducing the pro- gram at each team’s monthly all-staff meeting successfully reached 78% (n = 91) of HPs on those teams. While running the program introduction sessions, it became clear to researchers that locating the app with the meditation audio on tablets and smartphones was not obvious or intuitive. It took time and many HPs attending the introductory online session relied on colleagues who had found the app to show them how to get to it. As will be seen in the next section on acceptability, the majority of meditation usage occurred via smartphone or tablet, not from logging on to the meditation program web site. A conclusion is that it was necessary for HPs to be at the program launch meeting so that they would know how to ﬁnd the meditations and be able to participate in the intervention. 6.1. Feasibility and Acceptability Feasibility was measured as the percent of HPs who were able to attend their team’s all-staff meeting where the program was introduced, and the week 1 meditation was played. Acceptability was assessed by examining details of how often, on what platform, and where the movement meditations were done by HPs who were exposed to the intervention. Desire to continue to have access to the meditations after the 6-week intervention ended was another indicator of acceptability. Table 5. HP Roles (from the pre-survey). Table 5. HP Roles (from the pre-survey). n Int. J. Environ. Res. Public Health 2021, 18, 2515 14 of 27 Demographics were not asked on the surveys, due to a need to keep the survey under 12 min while including the PFI and MAIA items. As a measure of comparison with other hospices, we offer organization-wide demographics from comprehensive survey of all clinical employees at the hospice organization that was conducted in 18 months earlier. That survey showed that 90% of the hospice organization’s clinical employees were female, 90% white, and 90% age 30 or older. 6. Results Results are organized into feasibility and acceptability ﬁndings, overall levels of pre- and post-survey burnout and interoceptive awareness, and analysis of the outcomes of meditation use. 6.1.2. Acceptability An important measure of acceptability is whether the yoga-based movement medita- tions appealed enough to participating HPs that they made time to do the meditations on their own. Table 6 shows the frequency of meditation use. HPs were asked on the post-survey how many times they did a meditation. Response categories were 0; 1; 2; 3 to 5; 6 to 10; more than 10. Since only one HP did meditations more than 10 times, that response was collapsed into a 6 to 10+ times category. Column 3 is the percentage of all post-survey respondents across all ﬁve categories of meditation use. Column 4 includes only HPs who were exposed to the intervention at their team’s all-staff meetings and, therefore, did at least the week 1 meditation at least one time. Results show that about one third (34%) experienced one meditation, presumably as a group at the online all staff meeting, and never made time to do a meditation again. Conversely, two-thirds of HPs who were exposed to the week 1 meditation did go on to do at least one meditation on their own. Int. J. Environ. Res. Public Health 2021, 18, 2515 15 of 27 Table 6. Meditation Frequency. Times Played n Among All HPs Among HPs Exposed to the Intervention 0 25 22% 1 31 27% 34% 2 22 19% 24% 3 to 5 29 25% 32% 6 to 10+ 9 8% 9% 116 91 Table 6. Meditation Frequency. About one-fourth (24%) of HPs who were exposed to the intervention made the effort to do a meditation one time on their own in addition to the all-staff meeting, for a total of two meditation sessions. Nearly one-third (32%) experienced program meditations 3 to 5 times. Nine percent did meditations between 6 and 10 times. The intervention was acceptable enough that two thirds of HPs who were exposed to the week 1 meditation went on to meditate again on their own. However, only 9% meditated six or more times. Analysis of outcomes in later sections of this report will examine the question of whether enough meditation experience occurred to result in a measurable change in burnout or interoceptive awareness. g p Findings about where and how HPs did the meditations conﬁrm the importance of making the intervention directly and easily available on hospice tablets and smartphones in addition to the program website. 6.1.2. Acceptability Three-fourths of post-survey respondents who played a meditation at least one time (n = 91) did so at home. About one-fourth (23%) played at least one meditation at the ofﬁce. Additionally, 29% did a yoga-based meditation in their car (not while driving). These add to more than 100% because some participants did meditations in more than one location. Among post-survey respondents who played a meditation more than at the initial all staff meeting (n = 38), 57.9% indicated that they did so using their workplace tablet, and 38.9% used their company smartphone. There were six different meditations, with a new meditation introduced each week. Participants could do any meditation as often as they wanted. Sixty-two percent of HPs only experienced one of the six meditations; 20% experienced two different meditations; 10% experienced three different meditations; 8% experienced four to six of the six meditations. A promising indicator of acceptability of the intervention is that, among HPs who did a meditation at least once (n = 91), 48% indicated they would like to continue to have access to the meditations after the 6-week program ended. (HPs who did not do any meditations were not asked whether they would like continued access.) Meditation use and interest in having continued access varied across the seven teams. The percent of post-survey respondents on each of the seven teams who were exposed to at least one meditation ranges from 60% to 87%, contributing to an average exposure of 79% across all teams. Desire to continue to have access to the meditations after the 6-week program ended was signiﬁcantly different by team (chi square = 13,449, df = 6, p = 0.036) with the percent of team members who wanted continued to have access ranging from a low of 0% to a high of 86% across different teams. This wide variation suggests something related to the team inﬂuenced meditation use. 6.2.1. Overall Pre- and Post-Survey Burnout 6.2.1. Overall Pre- and Post-Survey Burnout In this section, baseline pre-survey and post-survey PFI scores on professional fulﬁl- ment, work exhaustion, and interpersonal disengagement are reported. The data provide a general sense of the level of burnout among participating HPs in the 2 weeks prior to launching the meditation intervention and one week after the intervention ended. Al- though all respondents in the results were exposed to the intervention, the tables here do not differentiate HPs who went on to do meditations on their own from those who only experienced the initial meditation at the introductory all-staff session. Burnout was measured in order to examine effects of participating in the meditation program. Here we report overall pre- and post-survey burnout across all respondents, re- gardless of meditation use. Table 7 reports overall professional fulﬁlment, work exhaustion, and interpersonal disengagement data across all respondents. The table includes average score, standard deviation, the percent of employees whose scores qualiﬁed as “very high”, and number of respondents. Table 7. Overall Pre- and Post- Survey Burnout Scores. Pre-Survey Post-Survey Mean s.d. % High n Mean s.d. % High n Professional Fulﬁlment 2.95 0.67 39% 76 2.78 0.75 32% 76 Work Exhaustion 1.30 0.84 38% 76 1.34 0.90 44% 75 Interpersonal Disengagement 0.71 0.68 21% 75 0.56 0.61 13% 72 Burnout 1.00 0.71 24% 75 0.94 0.69 25% 72 Table 7. Overall Pre- and Post- Survey Burnout Scores. Responses on the scale can be between a low of 0 and a high of 4. Based on research, PFI developers have determined cut points for what is considered “high” on each of the three subscales [50]. For professional fulﬁlment, the cut point for classifying respondents as experiencing professional fulﬁlment is >3.0. For work exhaustion, interpersonal disen- gagement, and their combination, the cut point classifying respondents as experiencing these forms of burnout is 1.33 [50]. The pre-survey results show that 39% of Wave 2 HPs re- ported high professional fulﬁllment, 45% reported high work exhaustion and 14% reported experiencing high interpersonal disengagement. p g g p g g Pre- and post-survey PFI scores were compared using paired t-tests. Pre-survey profes- sional fulﬁllment was signiﬁcantly better (higher) than post-survey professional fulﬁllment (t (76) = 2.713, p = 0.008). Pre-survey interpersonal disengagement was signiﬁcantly worse (higher) than post-survey interpersonal disengagement (t (71) = 2.003, p = 0.049). 6.2. Overall Pre-Post Burnout and Interoceptive Awareness Here, overall pre-post survey levels of burnout and interoceptive awareness are reported. As described in Section 5.4.2, there were 127 pre-survey responses and 116 post- survey responses. Only 61% (91) of HPs completed both a pre-survey and a post-survey and, as shown in the feasibility and acceptability section, 84% (76) of HPs who completed both surveys were exposed to the intervention. The next two results sections focus on only those 76 participants who completed both surveys and were exposed to the intervention at least once. Pre-post comparisons provide a sense of levels of HP burnout and interoceptive awareness in week 0 and week 7. Int. J. Environ. Res. Public Health 2021, 18, 2515 16 of 27 16 of 27 6.2.1. Overall Pre- and Post-Survey Burnout Workplace exhaustion and overall burnout were not different between pre- and post-intervention surveys. The measurement of professional fulﬁllment and burnout 7 weeks apart (before and after the intervention period) provided a detailed view of how stable or changeable those dimensions of burnout were for this HP study population. Table 8 shows both percent and raw number of HPs classiﬁed as high on each PFI scale. The rows represent pre-survey states and the columns represent post survey classiﬁcations. p p y Professional fulﬁllment was the same in week 0 and week 7 for 72% of HPs. Fifty-nine percent of HPs on the pre-survey did not report high professional fulﬁlment, while 41% did report high professional fulﬁllment. On the post-survey, 71% did not and 29% did report high professional fulﬁllment. Professional fulﬁlment ﬂipped for 21 individual HPs, with 15 shifting out of high professional fulﬁllment and 6 shifting into high professional fulﬁllment. Sixteen HPs reported high professional fulﬁllment in both week 0 and week 7. Forty-three percent of HPs who reported high professional fulﬁllment in either survey reported high professional fulﬁllment in both surveys. 17 of 27 Int. J. Environ. Res. Public Health 2021, 18, 2515 17 of 27 Work exhaustion was the same in week 0 and week 7 for 76% of HPs. Sixty-one percent of HPs on the pre-survey did not report high work exhaustion, while 39% did report high work exhaustion. On the post-survey, 56% did not and 44% did report high work exhaustion. Work exhaustion ﬂipped for 17 individual HPs, with 6 shifting out of high work exhaustion and 11 shifting into high work exhaustion. Twenty-one HPs reported high work exhaustion in both week 0 and week 7. Fifty-ﬁve percent of HPs who reported high work exhaustion in either survey reported high work exhaustion in both surveys. Interpersonal disengagement was the same in week 0 and week 7 for 73% of HPs. Seventy-eight percent of HPs on the pre-survey did not report high interpersonal disen- gagement, while 22% did report high interpersonal disengagement. On the post-survey, 88% did not and 13% did report high professional fulﬁllment. Interpersonal disengagement ﬂipped for 19 individual HPs, with 13 shifting out of high interpersonal disengagement and 6 shifting into high interpersonal disengagement. Only three HPs reported high in- terpersonal disengagement in both week 0 and week 7. 6.2.1. Overall Pre- and Post-Survey Burnout Only 13% percent of HPs who reported high interpersonal disengagement in either survey reported high interpersonal disengagement in both surveys. g g y Of the three PFI scales, work exhaustion was the most persistent across the two time periods, whereas self-reported levels of interpersonal disengagement changed the most. Table 8. Crosstab Comparisons of Pre- and Post- Survey Burnout. Professional Fulﬁllment (43% Stable) Post-Survey Pre-survey No Yes Total No 51% (n = 39) 8% (n = 6) 59% Yes 20% (n = 15) 21% (n = 16) 41% total 71% 29% n = 76 Work Exhaustion (55% Stable) Post-Survey Pre-survey No Yes Total No 47% (n = 34) 15% (n = 11) 61% Yes 9% (n = 6) 29% (n = 21) 39% total 56% 44% n = 75 Interpersonal Disengagement (13% Stable) Post-Survey Pre-survey No Yes Total No 69% (n = 50) 8% (n = 6) 78% Yes 18% (n = 13) 4% (n = 3) 22% total 88% 13% n = 72 ll d d d Table 8. Crosstab Comparisons of Pre- and Post- Survey Burnout. able 8. Crosstab Comparisons of Pre- and Post- Survey Burnout. 6.2.2. Overall Pre- and Post-Survey Mind–Body Integration Here we report overall pre- and post-survey interoceptive awareness across all respon- dents, regardless of intervention exposure or meditation use. Similar to the burnout data in the previous section, mind–body integration data provide a general sense of the level of the six dimensions of interoceptive awareness among participating HPs prior to launching the meditation intervention and after the intervention ended. Table 9 reports overall self-regulation, attention regulation, emotional awareness, body noticing, body listening, and body trusting levels and composite overall MAIA scores across all respondents. Overall, MAIA is a composite variable computed by averaging the sum of the scores for each of the six subscales. Table 9 includes average score, standard deviation, and number of respondents. The scores for each MAIA subscale can range from a low of 1 to a high of 6. Paired t-tests of pre- and post-survey MAIA scores showed no signiﬁcant differences. Int. J. Environ. Res. Public Health 2021, 18, 2515 18 of 27 Table 9. Overall Pre- and Post- Survey Interoceptive Awareness Scores. Table 9. Overall Pre- and Post- Survey Interoceptive Awareness Scores. Pre-Survey Post-Survey Mean s.d. n Mean s.d. 6.2.1. Overall Pre- and Post-Survey Burnout n Self-Regulation 3.74 1.03 74 3.97 1.10 68 Attention Regulation 3.99 1.03 67 3.79 1.09 71 Emotional Awareness 4.71 1.02 69 4.70 1.04 75 Body Trusting 4.48 1.16 75 4.57 1.03 72 Body Noticing 4.23 1.04 70 4.42 1.02 69 Body Listening 3.69 1.35 75 3.73 1.21 72 Overall MAIA 4.15 0.79 64 4.24 0.96 61 6.3. Outcomes Public Health 2021, 18, 2515 19 of 27 19 of 27 Three of the six MAIA subscales (self-regulation, body trusting, and body listening) were signiﬁcantly lower among HPs who reported high burnout. These ﬁndings support the theoretical premise that interoceptive awareness plays a role in mitigating burnout. HPs who had better self-regulation, body trusting, and body listening reported less burnout. They were able to actively listen to their body for insight, to direct attention to bodily sensations, to notice how they were feeling, and to use that internal interoceptive focus as a way to regulate distress. Table 11. Post-Burnout t-Tests. Scale Burned out Mean s.d. Signiﬁcance Self-Regulation NO 4.22 1.03 t = 3.541 (65), p = 0.001 YES 3.19 0.96 Body Trusting NO 4.85 0.98 t = 3.984 (68), p =0.000 YES 3.72 1.16 Body Listening NO 3.94 1.20 t = 2.230 (68), p = 0.023 YES 3.19 1.13 Table 11. Post-Burnout t-Tests. Table 12 reports signiﬁcant independent sample t-test differences in post-intervention work exhaustion and interoceptive awareness between HPs experiencing high and low work exhaustion (a subcomponent of overall burnout). HPs who reported high work exhaustion were less likely to report experiencing professional fulﬁllment (only 24% of work exhausted HPs reported high professional fulﬁllment, compared to 55% who were not work exhausted). Interpersonal disengagement occurred exclusively among HPs who also reported high work exhausted HPs. Forty-four percent of HPs who were work exhausted also reported high interpersonal disengagement, but only one of the HPs who was not work exhausted reported interpersonal disengagement. p p g g Three of the six MAIA subscales (self-regulation, attention regulation, and body listening) were signiﬁcantly lower among HPs who reported high work exhaustion. Table 12. Post-Work Exhaustion t-Tests. Scale Exhausted Mean s.d. Signiﬁcance Self-Regulation NO 4.28 1.05 t = 2.823 (66), p = 0.006 YES 3.56 1.03 Attention Regulation NO 4.24 1.19 t = 2.035 (65), p = 0.046 YES 3.63 1.22 Body Listening NO 4.03 1.15 t = 2.376 (70), p = 0.020 YES 3.36 1.20 Pre-Professional Fulﬁllment (% high) NO 44% 50% t = 2.763 (73), p = 0.006 YES 24% 44% Pre-Interpersonal Disengagement (% high) NO 44% 50% t = 4.51 (72), p = 0.001 YES 5% 22% Table 12. Post-Work Exhaustion t-Tests. Table 13 shows signiﬁcant independent sample t-test differences in PFI and interocep- tive awareness between HPs experiencing high and low interpersonal disengagement (a subcomponent of overall burnout). 6.3. Outcomes Here, we examine whether the intervention reduced burnout—speciﬁcally, whether meditation frequency was associated with lower burnout. We also look at interrelationships among the four professional fulﬁllment PFI indices and the six MAIA dimensions of interoceptive awareness. 6.3.1. Meditation, Interoceptive Awareness, and Burnout Independent sample t-test mean comparisons were used to test whether HPs classiﬁed as high on the PFI scales differed in meditation frequency or interoceptive awareness. No relationship was found between meditation frequency and professional fulﬁllment, workplace exhaustion, or interpersonal disengagement. Table 10 reports signiﬁcant differences based on independent sample t-test mean comparisons for post-intervention professional fulﬁllment. The PFI indices were interde- pendent. Considering the relationships among the three PFI scales, HPs who reported high post-intervention professional fulﬁllment were less likely to report experiencing pre- intervention work exhaustion (only 14% of professionally fulﬁlled HPs had been reporting high work exhaustion, compared to 48% who had not reported professional fulﬁllment) (t = 2.928 (df = 74), p = 0.005). None of the HPs who reported high post-intervention profes- sional fulﬁllment had reported pre-intervention interpersonal disengagement, whereas 30% of HPs who were not experiencing professional fulﬁllment had reported high interpersonal disengagement. (t-tests could not be conducted because one of the two averages was 0). g g g One of the six MAIA subscales, body trusting, was signiﬁcantly higher among HPs who were high in professional fulﬁllment (x = 5.28, s.d. = 0.70) than among those reporting low professional fulﬁllment (x = 4.30, s.d. = 1.14, t = 3.579 (df = 70), p = 0.005). (Body trusting refers to experiencing one’s body as safe and trustworthy). Table 10. Post-Interpersonal Disengagement t-Tests. Fulﬁlled Mean s.d. Signiﬁcance Body Trusting NO 4.30 1.14 t = 3.579 (70), p = 0.005 YES 5.28 0.70 Pre-Work Exhaustion NO 48% 50% t = 2.928 (74), p = 0.005 YES 14% 35% Pre-Interpersonal Disengagement NO 30% 46% (t-tests could not be conducted) YES 0% 0% Table 10. Post-Interpersonal Disengagement t-Tests. Table 10. Post-Interpersonal Disengagement t-Tests. Table 10. Post-Interpersonal Disengagement t-Tests. Table 11 reports signiﬁcant independent sample t-test differences in post-intervention professional fulﬁllment and interoceptive awareness indices comparing HPs experiencing high and low overall burnout. HPs who reported high burnout were signiﬁcantly less likely to report post-intervention professional fulﬁllment. Only 6% of HPs with high burnout were high in professionally fulﬁllment, whereas 39% of HPs who were not experiencing burnout had high professional fulﬁllment. Int. J. Environ. Res. 6.3.2. Meditation and Interoceptive Awareness To test the impact of the intervention on interoceptive awareness, one-way ANOVAs were run to compare frequency of doing the meditations with MAIA scores. For these analyses, all HPs who completed a post-survey and were exposed to the intervention were included (n = 79). Three groups of HPs were compared: those who were exposed to a meditation at all-staff meeting but never did a meditation on their own (34%), HPs who did a meditation on their own one time in addition to the all-staff meeting (22%), and those who did a meditation two or more times on their own (44%). Table 14 shows the number of participants and average meditation frequency of these three groups. By deﬁnition the average times meditating for the ﬁrst group is 1, and the average times meditated for the second group is 2. Group 3 average times meditating was 4.95 times. Doing a yoga-based meditation once can help change how an HP feels while doing the meditation. However, repeated experiences with doing meditation are needed to effect systemic changes. We would expect group three to be more likely to show burnout and interoceptive awareness effects than groups 1 and 2. Table 14. Overall Meditation Frequency by Group. Times Meditated n Mean s.d. once 27 1 0 twice 17 2 0 more 35 4.95 1.72 Table 14. Overall Meditation Frequency by Group. Two of the six MAIA subscales were signiﬁcantly different based on meditation fre- quency. Table 15 shows means and ANOVA statistics with three categories of meditation frequency as the independent variable and self-regulation and attention regulation as independent variables, both of which were signiﬁcantly higher in group 3 (the HPs who did meditations more than twice). Planned comparisons showed that differences in intero- ceptive awareness were only detected among HPs who meditated more than twice. Simply attending the introduction webinar, and even doing a meditation once on their own, was not sufﬁcient to impact interoceptive awareness. The meditations in the intervention were designed in part with a goal of activating and training interoceptive awareness. HPs who did the meditations more often reported directing attention to interoception more often, speciﬁcally, being signiﬁcantly more likely to direct attention to bodily sensations as means of self-regulation and as a way to regulate attention. Table 15. Self-Regulation and Attention Regulation by Meditation Frequency. Times Meditated n Self-Regulation Attention Regulation Mean s.d. Mean s.d. 6.3. Outcomes Consistent with earlier reporting, high interpersonal disengagement was associated with less professional fulﬁllment. (t-tests could not be conducted because one of the two averages was 0). Work exhaustion was signiﬁcantly more likely to be present among HPs reporting high interpersonal disengagement (78%) than among HPs not reporting interpersonal disengagement (37%). g p g p g g None of the six MAIA subscales was signiﬁcantly different based on level of interper- sonal disengagement. Int. J. Environ. Res. Public Health 2021, 18, 2515 20 of 27 Table 13. Post-Interpersonal Disengagement t-Tests. PFI Index Disengaged Mean s.d. Signiﬁcance Post-Professional Fulﬁllment (% high) NO 35% 48% (t-tests could not be conducted) YES 0% 0% Post-Work Exhaustion (% high) NO 78% 44% t = 2.410 (70), p = 0.019 YES 37% 49% Table 13. Post-Interpersonal Disengagement t-Tests. 7. Discussion This article was written for different audiences including designers of meditation interventions, and hospice organizations looking for ways to address HP burnout, and research scientists who study interoception or burnout or meditation or yoga or meditation interventions for health. 6.3.2. Meditation and Interoceptive Awareness once 27 3.59 1.25 3.57 1.26 twice 19 3.78 0.98 3.69 1.05 more 35 4.24 0.88 4.27 1.12 F = 4.519 (2, 78), p = 0.018 F = 4.218 (2, 76), p = 0.048 Table 15. Self-Regulation and Attention Regulation by Meditation Frequency. Doing the meditations more often was associated with higher self-regulation and attention regulation. Although a direct relationship between doing the meditations and Int. J. Environ. Res. Public Health 2021, 18, 2515 21 of 27 burnout was not detected, meditation frequency was associated with higher interoceptive awareness and higher interoceptive awareness was linked to lower burnout. burnout was not detected, meditation frequency was associated with higher interoceptive awareness and higher interoceptive awareness was linked to lower burnout. 7.1. Understanding Yoga-Based Meditation It is important that scientists who study meditation and hospice organizations con- sidering burnout interventions pay attention to distinguishing characteristics of whatever speciﬁc meditation approach they are working with. Meditation is a diverse umbrella term. In addition to yoga-based meditation, there are other meditation approaches associ- ated with contemplative traditions and underlying philosophies (such as Buddhism and Qigong). There are secularized adaptations of these practices (such as mindfulness medi- tation and MBSR). Additionally, there are all manner of other meditative and meditation experiences, teachers, and apps. To provide clarity about the nature of the meditations used in the intervention, this article detailed the components of the six meditations in the intervention. We included the week 1 calming meditation video in the Supplementary Materials and explained how and why doing these meditations regularly might be expected to support mind–body integration. Meditation is fundamentally experiential. Reading a short explanation of a meditation does not convey what it is like to practice that form of meditation. Doing a meditation once provides much deeper insight into the nature of a meditation intervention than reading about it, although meditating once does not begin to convey the experience of meditating regularly over a long period of time. 7.2. Accessibility and Feasibility of the Intervention and Delivery Approach 7.2. Accessibility and Feasibility of the Intervention and Delivery Approach Doing yoga-based meditation requires time and attention; meditation interventions for busy HPs need to be palatable, easy to do, and worthwhile. The meditation program and intervention delivery approach presented here were designed to encourage HPs throughout the hospice organization to do the meditations, with goals of promoting interoceptive awareness and combating burnout. g Feasibility and acceptability ﬁndings were encouraging. Half of the participants who experienced at least one meditation wanted to continue to have access to the meditations after the 6-week program ended. This suggests that they found the meditations valuable and had a desire to use them in the future. Two thirds of HPs who experienced at least one meditation went on to do a meditation on their own, a ﬁnding that suggests that HPs valued having access to these meditations. We know from focus group interviews with HPs that they did not want company-sponsored personal burnout interventions to be something time consuming that HPs were expected to do on their own time [2]. For example, some HPs mentioned not wanting to have to use personal time to attend company picnics or to complete online training modules. To be responsive to not burdening personal time, the intervention meditations were optional, under 12 min and could be done at home or at work. That the majority of meditation use occurred using the app on the HP’s workplace tablet or smartphone shows the importance of providing that option. p p p g p Introducing the meditation intervention at each team’s all-staff meeting worked for HPs who attended that meeting but left out HPs who missed that meeting. The internal app was also hard to ﬁnd, so much so that, although it was available on every workplace tablet and smartphone, HPs did not discover it unless they were told it existed and shown exactly where and how to ﬁnd it. A more prominent and easier to ﬁnd app could encourage use even among HPs who miss the introductory session. Int. J. Environ. Res. Public Health 2021, 18, 2515 22 of 27 22 of 27 Huge differences were found in interest in long term access to them meditations based on teams, ranging for a low of 0% interest to a high of 86% interest. 7.3. Interoceptive Awareness and Burnout The ﬁndings supported the proposition that interoceptive awareness helps mitigate burnout. Here, we speculate on some of the ways that interoceptive awareness might work to reduce burnout in HPs. The ﬁndings show that burnout is more malleable than permanent and that the three burnout factors are interdependent. We measured burnout at the beginning and end of the 7-week study period. Interpersonal disengagement was the most variable, such that only 13% of HPs who reported high interpersonal disengagement in either timeframe did so in both. Work exhaustion was the most stable burnout factor, with 55% of HPs reporting high work exhaustion in either time period, doing so for both. Professional fulﬁllment was fairly stable, with 43% of HPs reporting high professional fulﬁllment in either time period doing so in both. This malleability is good news for hospice organizations and intervention developers. The changes observed cannot be attributed to the intervention, but they do show that burnout is far from a permanent, unchangeable state. p g The interdependence of burnout factors is also good news for mitigating burnout. Addressing one factor could help with the others. None of the HPs who scored high on professional fulﬁllment reported experiencing interpersonal disengagement. None of the HPs who scored low on work exhaustion reported experiencing interpersonal disengagement. Additionally, HPs who were low on work exhaustion were more likely to be high on professional fulﬁllment. Reducing work exhaustion could help increase professional fulﬁllment and reduce interpersonal disengagement. Theories about how doing yoga-based practices improves the ways the human system responds to stress posit that interoception facilitates mind–body integration. High level brain processes (cognition) are better connected to bodily sensations and feelings, helping to improve conscious decision making. Additionally, low-level brain networks (the autonomic nervous system), become conditioned to calmer and less reactive physiological responses. Why might interoceptive awareness help combat work exhaustion? When a HP is more aware of how she is feeling, she might take self-care steps such as taking a break, getting enough sleep, or going for a walk. She might make different choices, perhaps saying no to an assignment or approaching work tasks differently. A third possibility is that doing the meditations reduces reactivity to workload, mitigating the feeling of being overwhelmed. Why might interoceptive awareness help combat interpersonal disengagement? Doing the meditations involved focusing attention on breath and body, which helps quiet the mind and relax the body. 7.2. Accessibility and Feasibility of the Intervention and Delivery Approach The team- based approach to delivery can provide opportunities for social support and may have encouraged use, especially if the operations manager or some of the team members were enthusiastic. Conversely, an unenthusiastic operations manager or a vocal nay-sayer could have the opposite effect. More research is needed on intervention implementation approaches to understand team-related factors that impact meditation adoption, as well as whether team-based or organization-wide deployment is preferable. 7.5. Study Limitations The decision to make the meditation available via audio on an app automatically installed on hospice tablets and smartphones clearly facilitated access but introduced the limitation of relying on post-survey self-report rather than being able to automatically track meditation usage. g This ﬁeld research was conducted in the context of busy HPs at a large hospice organi- zation. The research team consisted of scientists who were external to the organization, and we were careful to make minimal time demands on clinical directors, operations managers, and HP clinical staff. Time and budget limitations precluded conducting interviews with the operations managers and focus group interviews with HPs who participated in the intervention would have yielded valuable qualitative data. 7.4. Meditation, Interoceptive Awareness, and Burnout Meditation frequency matters. Meditating more than twice was associated with increased interoceptive awareness, speciﬁcally with signiﬁcantly higher post-survey levels of self-regulation and attention regulation. Although the data did not show signiﬁcant relationship between burnout and meditation frequency, research with a larger sample and an intervention delivery strategy that engaged more frequent meditation may ﬁnd a relationship. p Future research should examine in detail how and when HPs choose to do the yoga- based meditations to manage how they feel. Research on the long term effects of meditation consistently show that salutatory effects increase over time with repeated meditation practice [51], but HPs in the study appeared to use the meditations when needed rather than regularly. HPs know that, due to their profession, they are at high risk of burnout. Focus group interviews with HPs at the same organization as the current study found that when HPs start noticing that they are feeling burned out, they take self-care steps such as mental health days to recover [2]. HPs indicated that workload and administrative demands were the major source of burnout, and that dealing with death and dying would periodically start to feel overwhelming, either because of a particularly distressing event or just unrelenting buildup [2]. The meditations in the intervention are tools HPs can use when life events present a need, such as before or after a stressful patient visit. Meditation can be done regularly, such as in the mornings before starting work. Alternatively, meditation can be done during particularly stressful times. 7.3. Interoceptive Awareness and Burnout Being more connected with oneself can reduce reactivity in challenging interpersonal situations. Furthermore, the mediation objects in the intervention meditations were selected to be helpful in coping with challenging situations and people. In the “peaceful feeling” meditation, the participant supercharges seeds with a peaceful feeling, then drops a seed onto a relationship. The “releasing” meditation helps HPs release what they are holding on to (such as tension or tightness, or something upsetting someone said) that they no longer need. In the “cleansing waves” meditation, gentle waves wash up onto shore and then go back out. All of these practices can be helpful with difﬁcult interpersonal situations. Why might interoceptive awareness help increase professional fulﬁllment? When the mind is less agitated, we can see more clearly. Regular practice of the meditations may Int. J. Environ. Res. Public Health 2021, 18, 2515 23 of 27 lessen focus on day-to-day problems and facilitate a big picture perspective more of the time. The bigger picture perspective allows for more effective identiﬁcation of solutions to challenges or problems. lessen focus on day-to-day problems and facilitate a big picture perspective more of the time. The bigger picture perspective allows for more effective identiﬁcation of solutions to challenges or problems. 7.4. Meditation, Interoceptive Awareness, and Burnout 7.6. Effects of COVID-19 on the Research Process and Outcomes The global pandemic severely impacted our research. Statewide COVID-19 restrictions interrupted data collection in midstream when the spring teams were nearing their 6th week. Research was halted (and, therefore, no post-surveys could be collected), drastically reducing our eventual sample size. Unusual stressful external events during the study period not directly related to hospice work could have increased HP burnout and diminished professional fulﬁllment levels. Pre-surveys were collected in August 2020, and post-surveys in October. That time period covered the end of summer, the start of K-12 schools in hybrid or online only modalities due to COVID-19, increasing COVID-19 cases across the state, and the upcoming November 3 presidential elections. Working from home, with children, partners, pets, and other interrupting factors may make it harder to ﬁnd 12 uninterrupted minutes to do a meditation than while working at the ofﬁce. Before COVID-19, HPs delivered care in person in patient homes, and worked from the regional ofﬁce when not traveling to make patient visits. COVID-19 forced HPs to instantly shift from working at the ofﬁce to working from their homes. The current Int. J. Environ. Res. Public Health 2021, 18, 2515 24 of 27 24 of 27 research cannot answer the question of whether working from home during quarantine reduced meditation frequency. research cannot answer the question of whether working from home during quarantine reduced meditation frequency. 7.7. Directions for Future Research Given the positive feasibility and acceptability results and signiﬁcant outcomes of the current study, future research with this intervention is warranted under more normal conditions after the global pandemic has ended. Future research should collect more detailed data about when, how often, and why HPs choose to do the meditation. The yoga-based meditation program was administered within the hospice organi- zational structure. Clinical team operations managers arranged for the program to be presented at a monthly all-staff meeting, and then send emails to their teams each week throughout the program, introducing the new meditation for the week. The meditations were accessible on workplace tablets and smartphones without the need for internet access. p p The 12-min meditations could be done during working hours or on an individual basis. g g The particular meditations were designed with HPs in mind. The duration and format as well as the speciﬁc and general intended outcomes were chosen to be accessible to and useful for HPs. There are myriad apps and web sites offering access to hundreds of yoga and meditation practices. Instead, this intervention offered only 6. As a result, uncertainty is reduced, and no time needs to be spent searching for the right meditation solution. Future research could compare adoption and use of access to generic meditations with a targeted intervention like the one studied here. g The intervention was intended to help HPs with managing stress. It may be more fruitful for hospice organizations to incorporate yoga-based meditations as tools in a sus- tained collection of resources for HPs to combat burnout, rather than a 6-week intervention that ends. Perhaps the meditations should always be available, with period events and re- minders to re-animate use. Studies of long-term meditators consistently ﬁnd that long term daily or frequent meditation has deeper and more systemic effects than the kinds of novice exposure the current research and most MBI intervention research studies [24,37,51,52]. Most HPs will not become daily meditators, but many may ﬁnd these meditations to be helpful tools to support navigating the challenges of hospice work. Developing organiza- tional approaches to sustain and increase engagement could amplify the value of MBIs in combatting HP burnout. 8. Conclusions Strategies to extend the intervention beyond six weeks and to engage in more meditation usage would likely increase beneﬁts for HPs. Supplementary Materials: The following are available online at https://www.mdpi.com/1660-4 601/18/5/2515/s1, Video S1: Yoga Mind Tools calming meditation, s2, Video S2: Yoga Mind Tools Week 2 Principle: Checking in With Yourself is an Important Skill. Author Contributions: Conceptualization, C.H., M.A. and R.L.; methodology, software, formal analysis, data curation, C.H.; study administration, C.H. and P.M. (Patricia McDaniel); writing— original draft preparation, C.H., M.A. and R.L.; writing—review and editing, P.M. (Patrick Miller), P.M. (Patricia McDaniel), and M.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was partially funded by a grant from the Michigan Health Endowment Fund. Institutional Review Board Statement: The online pre-post survey research design and intervention delivery protocols used in the research were reviewed by the Michigan State University IRB and received an exempt determination (category 3i(b)), STUDY00003891. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The dataset is available on Figshare, doi:10.6084/m9.ﬁgshare.14130590 (accessed on 27 November 2020). Data Availability Statement: The dataset is available on Figshare, doi:10.6084/m9.ﬁgshare.14130590 (accessed on 27 November 2020). Acknowledgments: Yoga Mind Tools LLC contributed organization-wide access to its Movement Meditation for Hospice Professionals program. This article is dedicated to Jeffrey Forman, who served as the principal investigator (PI) on the burnout survey and focus group components of the grant that funded this research until he passed away in spring 2020. A radiation oncologist, scientist, clinician, philanthropist, former faculty member and chairperson of radiation oncology at Wayne State University School of Medicine with various roles at Karmanos Cancer Institute, He was the PI on numerous clinical trials and authored more than 400 publications, book chapters and abstracts. Forman was passionate about improving care for hospice patients and reducing burnout in healthcare professionals. Tom Day, when he was a doctoral research assistant in the Department of Media and Information at Michigan State University, helped with study administration, including coordinating and running some of the online introductions of the program to HPs. Conﬂicts of Interest: In addition to being a university professor, C.H. is Director of Yoga Mind Tools LLC and M.A. 8. Conclusions This study validated the feasibility and acceptability of the yoga-based meditation intervention and delivery approach and conﬁrmed predicted relationships between yoga- based meditation, interoceptive awareness, and burnout. The yoga-based meditations were valued and used by HPs. Half of those exposed to at least one meditation expressed a desire to continue to have access to the meditations after the 6-week program ended. Nearly one-ﬁfth of HPs who were exposed to the intervention at the all-staff meeting made the effort to do a meditation one time on their own, for a total of two meditation sessions. Nearly one-third experienced program meditations 3 to 5 times. Nine percent did meditations between 6 and 10 times. Making the meditations available in an app that was placed on the workplace tablets and smartphones HPs use throughout the day for EMR updates and workplace communi- cation, accessible without a need for internet ended up being the primary way HPs did the meditations. Fifty-eight percent of HPs who did a meditation on their own did so using their workplace tablet and 38% did so using their workplace smartphone. Due to covid-19 work from home restrictions, three-fourth of HPs did a meditation at home, 29% in a car between patient visits (not while driving), and 23% at the ofﬁce. Introducing the 6-week program to individual clinical teams and doing the week 1 meditation together at their all-staff meeting gave HPs direct experience with what yoga- based meditations are like. Another beneﬁt of deploying within a team was the social factor Int. J. Environ. Res. Public Health 2021, 18, 2515 25 of 27 25 of 27 of being able to talk with co-workers about the experiences. A drawback of deploying at all-staff meetings was that HPs who missed the all-staff launch missed the intervention. of being able to talk with co-workers about the experiences. A drawback of deploying at all-staff meetings was that HPs who missed the all-staff launch missed the intervention. Higher interoceptive awareness was signiﬁcantly related to lower burnout, partic- ularly lower work exhaustion. Meditation frequency did not have measurable effects on burnout. Meditation frequency was linked to signiﬁcantly higher self-regulation and attention regulation. Higher scores on four of six interoceptive awareness subscales were signiﬁcantly related to lower burnout in HPs. g y These ﬁndings afﬁrm a role for interoceptive awareness training address burnout and provide validation of the yoga-based meditation intervention. 1. Cavanaugh, K.J.; Lee, H.Y.; Daum, D.; Chang, S.; Izzo, J.G.; Kowalski, A.; Holladay, C.L. 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https://openalex.org/W4393185955	https://www.researchsquare.com/article/rs-4114432/latest.pdf	English	null	MULTIMORBIDITY, MORBIDITIES, AND LONG COVID – findings of the Sulcovid longitudinal study	Research Square (Research Square)	2,024	cc-by	7,210	MULTIMORBIDITY, MORBIDITIES, AND LONG COVID – findings of the Sulcovid longitudinal study Lucas Souza Ventura Federal University of Rio Grande Yohana Pereira Vieira Federal University of Rio Grande Juliana Quadros Santos Rocha Federal University of Rio Grande Lorrany da Silva Nunes Fehlberg Federal University of Pelotas Cristiane de Souza Federal University of Pelotas Suele Manjourany Silva Duro Federal University of Pelotas Mirelle de Oliveira Saes Federal University of Rio Grande Federal University of Rio Grande Yohana Pereira Vieira Federal University of Rio Grande Juliana Quadros Santos Rocha Federal University of Rio Grande Lorrany da Silva Nunes Fehlberg Federal University of Pelotas Cristiane de Souza Federal University of Pelotas Suele Manjourany Silva Duro Federal University of Pelotas Mirelle de Oliveira Saes Federal University of Rio Grande Results In total, 2,919 people were interviewed. The most prevalent morbidities were anxiety (26.3%), hypertension (25.3%), and depression (19.4%). In addition, 17.8% reported two previous morbidities and 22.6% had three or more comorbidities. Individuals with depression (PR = 1.17 95% CI 1.05–1.30), anxiety (PR = 1.33 95% CI 1.21–1.47), two or more morbidities (PR = 1.22 95% CI 1.07–1.39), and three or more morbidities (PR = 1.40; 95% CI 1.24–1.57) were more likely to have long COVID. A linear trend was observed, where individuals with two and three or more morbidities were 1.22 (95% CI 1.07–1.39) and 1.40 (95% CI 1.24–1.57) times more likely to develop long COVID than those with no or one morbidity. Conclusions The findings of this study reinforce that individuals with morbidities and multimorbidities prior to infection had greater vulnerability to long COVID. Keywords: DOI: https://doi.org/10.21203/rs.3.rs-4114432/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Page 1/17 Methods Baseline data were obtained from the longitudinal study, Sulcovid, conducted on individuals diagnosed with COVID-19 through RT-PCR testing from December 2020 to March 2021, who were symptomatic and living in a city in southern Brazil. Long COVID was assessed based on the affirmative response to at least one of the 18 symptoms investigated and categorized as musculoskeletal, neurological, respiratory, sensory, or digestive. Morbidities were assessed based on the presence of at least one of nine self-reported diseases. Data were analyzed using the Stata 15.0 statistical package. Crude and adjusted analyses were performed using Poisson regression to assess the relationships between morbidity, multimorbidity, and long COVID. Background The aim of this study was to evaluate the association between long COVID, morbidities, and multimorbidity in adults and older adults six to nine months after infection with the SARS-CoV-2 virus in Southern Brazil. Introduction Long COVID is defined as a multisystem condition of persistent symptoms that occurs in individuals with a history of SARS-CoV-2 infection [1, 2]. The estimated global prevalence of long COVID lasting 28 days or more after acute infection is approximately 43.0% [3]. A meta-analysis that evaluated the long-term effects of COVID-19 over 12 months or more found a 57.0% prevalence of at least one symptom of long COVID, with the most common symptoms being dyspnea on exertion (34.0%), difficulty concentrating (32.0%), and fatigue (31.0%) [4]. Page 2/17 Currently, the literature presents several mechanisms to explain the pathophysiology of long COVID, including mitochondrial dysfunction, persistence of SARS-CoV-2 viral RNA and proteins in various tissues, immune dysregulation, microbiota disruption, autoimmunity, coagulation and endothelial abnormality, dysfunctional neurological signaling, Epstein-Barr virus, human herpesvirus 6, and severe acute respiratory syndrome related to SARS-CoV-2 [5, 6, 7]. Multimorbidity, or the coexistence of two or more chronic non-communicable diseases (NCDs) in an individual, has a prevalence of 29.4–58.6% in Brazil and is related to the occurrence of long COVID, corroborating it as a risk factor for the development of the disease [8, 9, 10, 11]. The prevalence of pre- COVID-19 morbidities was 62.1% in adults and older adults. Particularly, hypertension (34.5%), diabetes mellitus (17.6%), chronic kidney disease (14.2%), and cancer (12.8%) [12] were most prevalent. Although multimorbidity has been shown to increase the probability of complications, hospitalizations, and deaths from COVID-19, it is still necessary to understand the mechanisms of the relationship between morbidities and multimorbidity in long COVID, since morbidities and multimorbidity should be included in the strategic plan for decision-making in the management of patients with long COVID [13]. Due to the high prevalence of morbidities and multimorbidity in Brazil, a country with a continental dimension, and because its population has a higher risk of developing long COVID, the present study aimed to evaluate the association between long COVID, morbidities, and multimorbidity in individuals following SARS-CoV-2 infection in Southern Brazil. Methods This cross-sectional study used baseline data from the Sulcovid-19 longitudinal study, with the objective of monitoring individuals infected with SARS-CoV-2 in the port city of Rio Grande in the extreme south of Brazil. The municipality has a land area of 2,682.867 km² and a population of 191,900 [14]. This study was approved by the Health Research Ethics Committee (CEPAS) of the Federal University of Rio Grande (FURG) (CAAE:39081120.0.0000.5324), and more details about the design and methods of the study can be found in the literature [15]. To delimit the sample, the epidemiological health surveillance department of the municipality was contacted to identify adults and older adults with COVID-19 during the study period, and to create a list of 4,014 individuals with positive RT-PCR results and their respective data (name, address, telephone number, and presence of symptoms). After excluding individuals without telephone contact or addresses, 3,822 individuals were selected for the study. Trained interviewers collected data via telephone. To reduce losses and refusals from individuals who were fearful or had discomfort in answering the survey via telephone contact, a home visit was offered for the application of the face-to-face instrument. In cases where an individual could not be found after five attempts to contact them by phone, a standard message briefly explaining the study and requesting for the best time to contact them was sent to those who had Whatsapp. For those who could not be contacted via Whatsapp, received home visits. Individuals who could not be located after five attempts at telephone contact and three home visits were considered lost. Page 3/17 Page 3/17 Data were collected from July to October 2021, six to nine months after infection with SARS-CoV-2. Questionnaires were electronically collected (tablet) using the REDCap program and smartphones were used for telephone calls. To ensure the safety of the researcher and interviewees, calls were recorded through a free mobile application (Callmarter) and stored in a specific e-mail account. The time taken to answer the questionnaire was approximately 20 min. The outcome of long COVID was investigated based on the answer to the following question: "Which of these symptoms did you have after COVID-19?" and "If yes" > "At this moment, do you still have this symptom?". Methods A total of 18 symptoms of long COVID were investigated, namely headache, shortness of breath, dry cough, cough with phlegm, pain during breathing, loss of taste, loss of smell, change in sensitivity, tiredness or fatigue, sore throat, runny nose, nasal obstruction, diarrhea, nausea, joint pain, muscle pain, memory loss, and loss of attention. Each symptom was questioned individually and operationalized in a dichotomous way (yes/no). The presence of long COVID was considered an affirmative response to at least one of the symptoms investigated. The symptoms were also grouped under one of the following categories: musculoskeletal (muscle pain, joint pain, and fatigue), neurological (headache, memory loss, and loss of attention), respiratory (shortness of breath, dry cough, cough with phlegm, pain to breathe, sore throat, runny nose, and nasal obstruction), sensory (changes in sensitivity, loss of smell, and loss of taste), and digestive (nausea and diarrhea). The presence of at least one symptom in a category (yes/no) was considered positive. Exposure to morbidities was collected from the following question: "At some point in your life, has any physician ever said that you have XX morbidity?", with a dichotomous answer option (yes or no). Nine self- reported morbidities were assessed: depression, anxiety, respiratory diseases (asthma/bronchitis/emphysema/chronic obstructive pulmonary disease), osteoporosis (or weak bones), rheumatic diseases (arthritis/arthrosis/rheumatism), systemic arterial hypertension (SAH), diabetes mellitus, heart disease (heart failure, weak heart, and large heart), and cancer. Mental health morbidities (depression and anxiety) require diagnosis by a physician or health professional (psychiatrist or psychologist). Exposure to morbidities was collected from the following question: "At some point in your life, has any physician ever said that you have XX morbidity?", with a dichotomous answer option (yes or no). Nine self- reported morbidities were assessed: depression, anxiety, respiratory diseases (asthma/bronchitis/emphysema/chronic obstructive pulmonary disease), osteoporosis (or weak bones), rheumatic diseases (arthritis/arthrosis/rheumatism), systemic arterial hypertension (SAH), diabetes Exposure to morbidities was collected from the following question: "At some point in your life, has any physician ever said that you have XX morbidity?", with a dichotomous answer option (yes or no). Nine self- reported morbidities were assessed: depression, anxiety, respiratory diseases Multimorbidity was assessed considering the morbidities mentioned above, and was operationalized in the ordinal form of "0 or 1,” "2," and "≥3," and the presence of two or more diseases was considered a multimorbidity. Methods Sex (female or male), age (18–59 years or 60 years or older), skin color (white, yellow, black, brown, and indigenous), marital status (married, living with a partner/single, separated, and widowed), education (first grade, high school, or higher education), economic class (A, B1, B2, C1, C2, D, E) [16], health insurance (yes/no), body mass index - BMI, physical activity – 150 min per week or more (no/yes), and hospitalization due to COVID-19 (no/yes) were used to adjust variables. Descriptive data were presented as proportions and 95% confidence intervals (95% CI). Crude and adjusted analyses were performed using Poisson regression with robust variance adjustment. All associations with a 95% confidence interval (CI) with no overlap between categories were considered statistically significant. Data were analyzed using the Stata 15.0 statistical package. Page 4/17 Page 4/17 Results Among the 3,822 individuals eligible for the study, 2,919 adults and older adults were interviewed after 631 were lost and 272 refused to participate. The sample consisted mostly of females (58.6%) with white skin (77.9%), aged between 18 and 59 years (59.6%), and were married or living with a partner (60.6%). Among the 3,822 individuals eligible for the study, 2,919 adults and older adults were interviewed after 631 were lost and 272 refused to participate. The sample consisted mostly of females (58.6%) with white skin (77.9%), aged between 18 and 59 years (59.6%), and were married or living with a partner (60.6%). Approximately 40.0% had an income between R$1,001.00 and R$2,000. Regarding BMI, most were overweight or obese (73.3%), approximately 25.0% were smokers, and among those investigated, only 3.7% required hospitalization [15]. Approximately 45.0% of the interviewees had at least one preexisting morbidity, the most prevalent being anxiety (26.3%; 95% CI 24.7–27.9), SAH (25.3%; 95% CI 23.8–27.0), and depression (19.4%; 95% CI 18.0– 20.9), whereas 17.8% (95% CI 16.5–19.3) had two diseases and 22.6% (95% CI 21.1–24.2) had three or more previous morbidities. The prevalence of long COVID in the sample without any previous illness was 48.3% (95% CI 46.5–50.1). In those who reported having previous illness to infection, symptoms of long COVID were present with a higher prevalence in participants with osteoporosis (68.0%; 95% CI: 59.2–75.7), followed by rheumatic diseases (63.7%; 95% CI 58.4–68.6), anxiety (62.9%; 95% CI 59.3–66.3), and depression (62.3%; 95% CI 58.2–66.2) (Table 1). Page 5/17 Table 1 Long COVID and symptoms according to the presence of previous morbidities and multimorbidity (n = 2,919). Table 1 Long COVID and symptoms according to the presence of previous morbidities and multimorbidity (n = 2,919). Results Previous illnesses Musculoskeletala Neurologicalb Respiratoryc Sensoryd Digestivese Long COVID % (IC95%) % (IC95%) % (IC95%) % (IC95%) % (IC95%) % (IC95%) No previous illness 25.5 (23.9–27.1) 26.8 (25.2– 28.5) 15.7 (14.4– 17.1) 17.4 (16.0- 18.8) 2.1 (1.7– 2.8) 48.3 (46.5– 50.1) Depression 35.9 (32.1–40.0) 40.8 (36.8– 44.9) 21.5 (18.3– 25.1) 23.7 (20.3– 27.4) 4.2 (2.9– 6.3) 62.3 (58.2– 66.2) Anxiety 34.7 (31.3–38.1) 39.0 (35.5– 42.5) 21.1 (18.3– 24.1) 24.1 (21.2– 27.2) 3.7 (2.5– 5.3) 62.9 (59.3– 66.3) Diseases Respiratory 35.5 (31.4–39.9) 36.6 (32.4– 41.0) 27.3 (23.5– 31.5) 20.9 (17.5– 24.8) 4.7 (3.1- 7.0) 59.9 (55.5– 64.2) Osteoporosis 45.6 (37.0-54.5) 41.6 (33.2– 50.5) 33.6 (25.8– 42.4) 21.6 (15.2– 29.8) 6.4 (3.2– 12.4) 68.0 (59.2– 75.7) Rheumatic 40.9 (35.8–46.2) 37.8 (32.8– 43.0) 26.7 (22.2– 31.6) 23.8 (19.6– 28.6) 4.6 (2.8– 7.4) 63.7 (58.4– 68.6) HAS 32.7 (29.4–36.2) 31.7 (28.4– 35.2) 20.3 (17.6– 23.4) 20.0 (17.3– 23.1) 3.1 (2.1– 4.7) 55.8 (52.1– 59.3) Diabetes 31.5 (26.4–37.1) 28.6 (23.7– 34.1) 22.6 (18.1– 27.8) 18.8 (14.7– 23.8) 3.8 (2.1– 6.7) 53.8 (48.0- 59.5) Cardiopathy 33.1 (27.6–39.1) 34.4 (28.8– 40.4) 24.3 (19.5– 30.0) 20.8 (16.2– 26.2) 3.8 (2.1- 7.0) 54.7 (48.5– 60.7) Cancer 23.5 (14.8–35.3) 20.9 (12.6– 32.6) 20.6 (12.4– 32.2) 16.2 (9.0- 27.2) 2.9 (0.7– 11.4) 49.3 (37.3– 61.3) Morbidity 0–1 18.4 (16.6–20.3) 20.5 (18.7– 22.5) 10.6 (9.2– 12.1) 14.2 (12.6– 16.0) 1.7 (0.7– 1.7) 39.7 (37.4– 42.1) 2 30.3 (26.4–34.5) 29.1 (25.2– 33.3) 15.3 (12.4– 18.7) 17.7 (14.6– 21.3) 2.0 (1.1– 3.4) 54.2 (49.7– 58.5) Previous illnesses Musculoskeletala Neurologicalb Respiratoryc Sensoryd Digestivese Long COVID 3 or more 38.5 (34.7–42.3) 39.6 (39.5– 43.5) 27.1 (23.8– 30.7) 23.4 (20.3– 26.8) 4.2 (2.9– 6.1) 63.4 (59.6– 67.1) Previous illnesses Musculoskeletala Neurologicalb Respiratoryc Sensoryd Digestivese Long COVID 3 or more 38.5 (34.7–42.3) 39.6 (39.5– 43.5) 27.1 (23.8– 30.7) 23.4 (20.3– 26.8) 4.2 (2.9– 6.1) 63.4 (59.6– 67.1) amuscle pain, joint pain, fatigue, bheadache, memory loss, loss of attention, cshortness of breath, dry cough, cough with phlegm, pain in breathing, sore throat, runny nose, nasal obstruction, dchanges in sensation, loss of smell and loss of taste, enausea and diarrhoea. Respiratory diseases: asthma/bronchitis/emphysema/COPD; Rheumatic diseases: arthritis/arthrosis/rheumatism; Heart disease: heart failure, weak heart, large heart. Results Previous illnesses Musculoskeletala Neurologicalb Respiratoryc Sensoryd Digestivese Long COVID 3 or more 38.5 (34.7–42.3) 39.6 (39.5– 43.5) 27.1 (23.8– 30.7) 23.4 (20.3– 26.8) 4.2 (2.9– 6.1) 63.4 (59.6– 67.1) amuscle pain, joint pain, fatigue, bheadache, memory loss, loss of attention, cshortness of breath, dry cough, cough with phlegm, pain in breathing, sore throat, runny nose, nasal obstruction, dchanges in sensation, loss of smell and loss of taste, enausea and diarrhoea. Respiratory diseases: asthma/bronchitis/emphysema/COPD; Rheumatic diseases: arthritis/arthrosis/rheumatism; Heart disease: heart failure, weak heart, large heart. When assessing the presence of at least one symptom of long COVID, regardless of the symptom, it was found that the occurrence can be up to 20 percentage points (p.p.) higher among those with previous morbidities compared to those without previous diseases. Individuals with multimorbidity also had a higher prevalence of long COVID symptoms, with a dose-response effect, leading to a higher prevalence among those with three or more previous morbidities (Table 1). Table 2 presents the crude analysis of long COVID, previous morbidities, and multimorbidities. It was observed that all previous diseases that were evaluated, except for cancer, increased the probability of presenting with at least one symptom of long covid, with emphasis on anxiety, osteoporosis, rheumatic diseases, and depression. The presence of two or more previous illnesses increased the probability of long COVID symptoms by 36.0% (95% CI 1.23–1.51) and 60.0% (95% CI 1.47–1.73), respectively (Table 2). Page 7/17 Table 2 Crude analysis of long COVID and previous morbidities and multimorbidity (n = 2,919). ry cough, cough with phlegm, pain in breathing, sore throat, runny nose, nasal Results Previous morbidities Musculoskeletala Neurologicalb Respiratoryc Sensoryd Digestivese Long COVID % (IC95%) % (IC95%) % (IC95%) % (IC95%) % (IC95%) % (IC95%) Depression 1.56 (1.37–1.78) 1.73 (1.53– 1.98) 1.50 (1.24– 1.80) 1.49 (1.25– 1.77) 2.49 (1.51– 4.10) 1.39 (1.28– 1.50) Anxiety 1.56 (1.37–1.76) 1.73 (1.54– 1.95) 1.54 (1.29– 1.83) 1.60 (1.36– 1.88) 2.38 (1.45– 3.91) 1.46 (1.35– 1.57) Diseases 1.51 (1.31–1.73) 1.47 (1.29– 1.69) 2.04 (1.71– 2.43) 1.24 (1.03– 1.51) 2.84 (1.71– 4.69) 1.30 (1.20– 1.43) Respiratory Osteoporosis 1.86 (1.52–2.28) 1.59 (1.28– 1.97) 2.25 (1.73– 2.93) 1.25 (0.89– 1.77) 3.21 (1.56– 6.59) 1.44 (1.26– 1.63) Rheumatic 1.76 (1.52–2.03) 1.50 (1.28– 1.74) 1.91 (1.56– 2.33) 1.46 (1.18– 1.80) 2.55 (1.46– 4.45) 1.39 (1.27– 1.52) HAS 1.42 (1.25–1.61) 1.26 (1.11– 1.44) 1.43 (1.20– 1.71) 1.22 (1.02– 1.45) 1.74 (1.04– 2.89) 1.22 (1,13- 1.32) Diabetes 1.27 (1.06–1.52) 1.08 (0.89– 1.31) 1.51 (1.20– 1.90) 1.09 (0.85– 1.41) 1.89 (0.99– 3.57) 1.13 (1.01 (1.27) Cardiopathy 1.34 (1.11–1.61) 1.32 (1.10– 1.58) 1.65 (1.30– 2.08) 1.22 (0.94– 1.57) 1.94 (1.00- 3.78) 1.15 (1.02– 1.29) Cancer 0.92 (0.56–1.42) 0.77 (0.48– 1.24) 1.32 (0.82– 2.12) 0.92 (0.53– 1.60) 1.36 (0.34– 5.46) 1.02 (0.80– 1.30) Mobirdade 0–1 Ref Ref Ref Ref Ref Ref 2 1.65 (1.40–1.95) 1.41 (1.20– 1.67) 1.45 (1.13– 1.85) 1.25 (0.99– 1.56) 1.86 (0.86– 3.99) 1.36 (1.23– 1.51) 3 or more 2.10 (1.82–2.41) 1.93 (1.69– 2.20) 2.56 (2.11– 3.09) 1.65 (1.37– 1.98) 3.96 (2.19– 7.13) 1.60 (1.47– 1.73 ) * Poisson regression amuscle pain, joint pain, fatigue, bheadache, memory loss, loss of attention, Table 2 * Poisson regression amuscle pain, joint pain, fatigue, bheadache, memory loss, loss of attention, cshortness of breath, dry cough, cough with phlegm, pain in breathing, sore throat, runny nose, nasal g p , j p , g , , y , , cshortness of breath, dry cough, cough with phlegm, pain in breathing, sore throat, runny nose, nasal Musculoskeletala Neurologicalb Respiratoryc Sensoryd Digestives In the adjusted analysis, it was found that symptoms of anxiety, respiratory diseases, rheumatic diseases, and depression increased the likelihood of long COVID by up to 33.0%. There was a linear trend, where individuals with either two, and three morbidities or more were 1.22 (95% CI 1.07–1.39) and 1.40 (95% CI 1.24–1.57), respectively, times more likely to develop long COVID. Regarding the grouped symptoms of long covid, an increase in the probability of musculoskeletal symptoms of long covid was observed in individuals with two, three, or more previous diseases. ry cough, cough with phlegm, pain in breathing, sore throat, runny nose, nasal Discussion The results of this study revealed that the prevalence of long COVID symptoms was higher among individuals with previous morbidities, with an emphasis on the significant association between depression, anxiety, respiratory diseases, previous rheumatic diseases, and long COVID symptoms. The presence of multimorbidity was associated with the occurrence of long COVID symptoms, in a dose-response manner. Regarding the symptoms of long COVID, in a meta-analysis that covered 11,598 patients with persistent symptoms of COVID-19, it was highlighted that the prevalence of fatigue was 29.2% (95% CI 21.5–39.4); muscle pain was 13.3% (95% CI 7.4–23.6); joint pain was 28.2% (95% CI 14.7–54.0), loss of smell or taste and headaches were 14.7% and 10.4%, respectively, dyspnea was 21.4% (95% CI 14.3–21.2), cough was 17.8% (95% CI 13.3–23.8), and gastrointestinal problems was 6.2% (95% CI 4.6–8.3) over a period of four to eight months, which is in line with the results of our study [17]. Regarding mental health, several studies have pointed to a relationship between depression, anxiety, and stress and the number of symptoms of long COVID [18, 19]. In a study conducted in Mexico, the main symptoms were headache (62.1%); memory problems (58.6%); diarrhea (48.3%); mental confusion (48.3%); and dyspnea, arthralgia, and myalgia (41.4%) [18]. There is evidence of a range of long-term adverse effects of other viral infections, such as SARS-CoV-1 and MERS-CoV, and studies have verified the presence of chronic fatigue and long-term mental health changes 31 to 50 months after these infections [19–23]. Although the mechanisms of interaction between mental health morbidities and symptoms of long COVID are currently not well understood, there are some hypotheses on how COVID-19 may affect the nervous system, including neuroinflammation caused by the involvement of the respiratory system by the immune response to SARS-CoV-2 by increasing cytokines, chemokines, and immune cells in the brain, inducing reactive states in brain cells; an autoimmune response against the nervous system; the reactivation of viruses such as the Epstein-Barr virus, which can lead to neuropathology; ischemia of neural cells due to interruption of cerebral blood flow caused by cerebrovascular and thrombotic diseases; and lung and multi- organ dysfunction during the severe acute phase of the disease that can impair the functioning of neural cells [24]. Regarding respiratory morbidities, some studies corroborated our findings, finding an association between COPD/asthma and long COVID [25, 26, 27]. Previous morbidities Respiratory diseases: asthma/bronchitis/emphysema/COPD; Rheumatic diseases: arthritis/arthrosis/rheumatism; Heart disease: heart failure, weak heart, large heart. Adjusted for: gender, age, skin color, marital status, education, economic class, health insurance, and physical activity. Results Adjusted for: gender, age, skin color, marital status, education, economic class, health insurance, and physical activity. Previous morbidities Musculoskeletala Neurologicalb Respiratoryc Sensoryd Digestivese Long COVID obstruction, dchanges in sensation, loss of smell and loss of taste, enausea and diarrhoea. Respiratory diseases: asthma/bronchitis/emphysema/COPD; Rheumatic diseases: arthritis/arthrosis/rheumatism; Heart disease: heart failure, weak heart, large heart. Adjusted for: gender, age, skin color, marital status, education, economic class, health insurance, and physical activity. Results For the other grouped symptoms of long COVID, there was a higher probability among those with three or more previous illnesses, to present with respiratory (PR:2.09; 95% CI 1.57–2.78) and digestive (PR:4.34; 95% CI 1.94–9.70) symptoms of long covid (Table 3). Page 9/17 Table 3 Adjusted analysis of long COVID, previous morbidities, and multimorbidity (n = 2,919). Previous morbidities Musculoskeletala Neurologicalb Respiratoryc Sensoryd Digestivese Long COVID % (IC95%) % (IC95%) % (IC95%) % (IC95%) % (IC95%) % (IC95%) Depression 1.31 (1.09–1.57) 1.42 (1.20– 1.68) 1.21 (0.92– 1.59) 1.08 (0.85– 1.37) 1.79 (0.85– 3.76) 1.17 (1.05– 1.30) Anxiety 1.42 (1.20–1.68) 1.44 (1.24– 1.69) 1.41 (1.11– 1.79) 1.38 (1.12– 1.70) 1.92 (1.00- 3.69) 1.33 (1.21– 1.47) Diseases 1.44 (1.20–1.73) 1.34 (1.12– 1.61) 1.70 (1.31– 2.19) 1.08 (0.84– 1.40) 2.48 (1.21– 5.07) 1.23 (1.10– 1.37) Respiratory Osteoporosis 1.23 (0.88–1.72) 1.08 (0.75– 1.55) 1.42 (0.88– 2.30) 0.91 (0.52– 1.58) 2.82 (0.73– 10.8) 1.02 (0.81– 1.30) Rheumatic 1.31 (1.05–1.63) 1.21 (0.97– 1.52) 1.68 (1.23– 2.29) 1.26 (0.93– 1.71) 2.91 (1.28– 6.62) 1.18 (1.02– 1.35) HAS 1.14 (0.94–1.38) 1.01 (0.83– 1.23) 1.10 (0.84– 1.44) 1.13 (0.89– 1.45) 1.62 (0.80– 3.26) 1.09 (0.97– 1.22) Diabetes 1.11 (0.87–1.42) 1.09 (0.83– 1.42) 1.25 (0.87– 1.81) 1.05 (0.73– 1.49) 1.93 (0.72– 5.18) 1.12 (0.96– 1.31) Cardiopathy 1.22 (0.94–1.59) 1.30 (1.01– 1.67) 1.63 (1.17– 2.27) 1.28 (0.92– 1.79) 1.97 (0.78- 5.00) 1.08 (0.91– 1.28) Cancer 1.20 (0.74–1.93) 0.75 (0.40– 1.43) 1.23 (0.62– 2.44) 1.15 (0.61– 2.17) 4.41 (2.66– 7.30) 1.04 (0.75– 1.43) Morbidity 0–1 Ref Ref Ref Ref Ref Ref 2 1.36 (1.09–1.70) 1.14 (0.92– 1.42) 1.10 (0.77– 1.56) 1.02 (0,76 − 1,36) 1.68 (0.64– 4.41) 1.22 (1.07– 1.39) 3 or more 1.75 (1.44–2.13) 1.60 (1.33– 1.94) 2.09 (1.57– 2.78) 1.41 (1,08 − 1,82) 4.34 (1.94– 9.70) 1.40 (1.24– 1.57) * Poisson regression amuscle pain, joint pain, fatigue, bheadache, memory loss, loss of attention, Table 3 * Poisson regression amuscle pain, joint pain, fatigue, bheadache, memory loss, loss of attention, cshortness of breath, dry cough, cough with phlegm, pain in breathing, sore throat, runny nose, nasal g p , j p , g , , y , , cshortness of breath, dry cough, cough with phlegm, pain in breathing, sore throat, runny nose, nasal Previous morbidities Musculoskeletala Neurologicalb Respiratoryc Sensoryd Digestivese Long COVID obstruction, dchanges in sensation, loss of smell and loss of taste, enausea and diarrhoea. Respiratory diseases: asthma/bronchitis/emphysema/COPD; Rheumatic diseases: arthritis/arthrosis/rheumatism; Heart disease: heart failure, weak heart, large heart. Discussion In one study [25], there was an association between COPD (RR = 1.55; 95% CI 1.47; 1.64), asthma (RR = 1.15; 95% CI 1.12; 1.18) and long COVID. In another study [26], there was an association between longer duration of long COVID symptoms lasting ≥ 28 days (15.8%) and ≥ 56 days (18.0%) and asthma [25]. Page 11/17 In a survey conducted in Suriname, 26.4% of the respondents had SAH, 13.2% had a previous diagnosis of heart disease, 56.6% experienced mild COVID-19, and 39.6% experienced at least one persistent symptom after recovery from acute SARS-CoV-2 infection [28]. In a survey conducted in Suriname, 26.4% of the respondents had SAH, 13.2% had a previous diagnosis of heart disease, 56.6% experienced mild COVID-19, and 39.6% experienced at least one persistent symptom after recovery from acute SARS-CoV-2 infection [28]. One of the most distinguished mechanisms in the literature for the emergence of long COVID is mitochondrial dysfunction, which can have cytopathic effects on the central nervous, respiratory, circulatory, immune, renal, and digestive systems [29]. After infection with SARS-CoV-2, the binding between a viral protein and mitochondrial complexes can lead to mitochondrial dysfunction, which in turn increases oxidative stress and causes immune cells to overreact, this exacerbated reaction leads to inflammation and potentially persistent symptoms of COVID-19 [5, 6]. Corroborating the results of the present study [30], was found an association between rheumatologic diseases and long COVID, wherein 74.0% had this outcome [30]. For musculoskeletal diseases such as osteoporosis, which was highlighted in our study, some suggestions for the involvement of the musculoskeletal system, COVID-19, and long COVID have been proposed, including the interaction of the SARS-CoV-2 spike protein with angiotensin-converting enzyme 2 (ACE2), which, in addition to being present in lung tissue, is present in other tissues such as smooth muscle, cartilage, and kidneys, which are also affected by the cytokine inflammation cascade, hypoxia, and muscle catabolism [31]. Some studies have demonstrated the impact of the coronavirus pandemic on the management of osteoporosis, both for the performance of control and diagnostic tests, as well as in drug therapy, including significant reductions in the performance of bone densitometry examinations (–49.0%) when compared to the first semesters of 2019 and 2020. Discussion There was also a reduction in the contact of health professionals for recommended treatment, with a range of only 29.0%, and 51.7% of professionals dealing with individuals with osteoporosis reported delays in starting treatment during the COVID-19 pandemic [32, 33, 34]. Regarding cancer [35], they found that 60.0% of people with neoplasms reported symptoms of long COVID, with an average duration of 7–14 months after infection; fatigue (82%), sleep disturbances (78%), myalgia (67%), and gastrointestinal symptoms (61%), followed by dyspnea (47%), and cough (46%) were the most reported symptoms [35]. Regarding multimorbidity, a study [8] conducted with older adults in Canada showed that having two or more morbidities had a 1.90 (95% CI 1.02–3.49) times higher risk of developing long COVID [8]. In a multicenter, population-based survey conducted in China with 2,712 patients with a history of mild-to-severe COVID-19, a relationship was found between the presence of three or more morbidities (odds ratio [OR] = 2.71, 95% CI 1.54–4.79) and long COVID [36]. Hypotheses point to the mechanism of long COVID through long-term tissue damage to organs such as the lungs, brain, and heart, and a pathological inflammation due to viral persistence, immune dysregulation, and autoimmunity, among others, which may likely contribute to the hyperactivation of monocyte-derived macrophages in the acute and post-acute phases of the disease [7, 37–44]. Finally, the present study had some limitations. The diagnoses of morbidities and symptoms of long COVID were self-reported, which may have underestimated the occurrence of data due to recall bias. The lack of Page 12/17 inclusion of some important variables for adjustment, such as behavioral factors, generates residual confusion. Notably, 19 diseases included in the list of multimorbidities were not investigated; however, morbidities with the highest prevalence in the literature were investigated. The strengths of the study include the fact that this is a population-based study with a representative sample that were mostly non-hospitalized, which reveals the relationship between morbidities and long COVID in individuals who did not develop the severe form of the disease, the mechanism of which is still poorly understood. Thus, the findings of this study reinforce the greater vulnerability of individuals with morbidities and multimorbidity to long COVID, and the importance of considering that morbidities and multimorbidity should be monitored and included in strategic health plans for the population, especially in those with COVID-19. Availability of data and materials The datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request. Discussion It is concluded that the existence of previous morbidities and infection by the SARS-CoV-2 virus, may predispose individuals to long-term symptoms of long COVID, and this experience is accentuated according to the number of preexisting morbidities. Thus, the interaction of long COVID with prior morbidities requires active investigation so that all individuals diagnosed with long COVID can access targeted health strategies. The data from this study can help us to understand the mechanisms that explain the strong relationship between morbidities/multimorbidity and long COVID. Consent to publication Not applicable. Ethical approval and consent to participate This research involved human participants and was conducted in accordance with the pertinent guidelines and regulations of the Declaration of Helsinki and this study protocol was approved by the Health Research Ethics Committee of the Federal University of Rio Grande (Certificate of Submission for Ethical Evaluation No. 39081120.0 .0000.5324). This study complied with the specific resolution of the National Health Council (466/2012) and informed consent was obtained from all participants in accordance with the resolution of the Free and Informed Consent Form of the National Health Council. Competitive Interests The authors declare that they have no competing interests. Page 13/17 This study was conducted with the financial support of FAPERGS – Research Support Foundation of Rio Grande do Sul, Brazil (Scholarship number 21/2551-0000107-0 Research Program for the SUS: shared management in health, PPSUS). Recognitions The authors thank the Foundation for Research Support of the State of Rio Grande do Sul. The authors thank the Foundation for Research Support of the State of Rio Grande do Sul. References 1. Huerne K, Filion KB, Grad R, Ernst P, Gershon AS, Eisenberg MJ. Epidemiological and clinical perspectives of long COVID syndrome. Am J Med Open. 2023 Jun;9:100033. 1. Huerne K, Filion KB, Grad R, Ernst P, Gershon AS, Eisenberg MJ. Epidemiological and clinical perspectives of long COVID syndrome. Am J Med Open. 2023 Jun;9:100033. 2. Soriano JB, Murthy S, Marshall JC, Relan P, Diaz JV; WHO Clinical Case Definition Working Group on Post-COVID-19 Condition. A clinical case definition of post-COVID-19 condition by a Delphi consensus. Lancet Infect Dis. 2022 Apr; 22(4):e102-e107. 2. Soriano JB, Murthy S, Marshall JC, Relan P, Diaz JV; WHO Clinical Case Definition Working Group on Post-COVID-19 Condition. A clinical case definition of post-COVID-19 condition by a Delphi consensus. 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Identifying multimorbidity clusters among Brazilian older adults using network analysis: Findings and perspectives. PLoS One. 2022 Jul 20; 17(7):e0271639. 10. Bof de Andrade F, Thumé E, Facchini LA, Torres JL, Nunes BP. Education and income-related inequalities in multimorbidity among older Brazilian adults. PLoS One. 2022 Oct 13; 17(10):e0275985. 10. Bof de Andrade F, Thumé E, Facchini LA, Torres JL, Nunes BP. Education and income-related inequalities in multimorbidity among older Brazilian adults. PLoS One. 2022 Oct 13; 17(10):e0275985. Page 14/17 11. Pereira CC, Pedroso CF, Batista SRR, Guimarães RA. Prevalence and factors associated with multimorbidity in adults in Brazil, according to sex: a population-based cross-sectional survey. Front Public Health. 2023 Jun 2;11:1193428. 12. de Miranda DAP, Gomes SVC, Filgueiras PS, Corsini CA, Almeida NBF, Silva RA, Medeiros MIVARC, Vilela RVR, Fernandes GR, Grenfell RFQ. Long COVID-19 syndrome: a 14-months longitudinal study during the two first epidemic peaks in Southeast Brazil. Trans R Soc Trop Med Hyg. 2022 Nov 1; 116(11):1007- 1014. 13. Downer MB, Sinha SK. Multimorbidity and COVID-19 in Canada: How the Pandemic Has Highlighted a Key and Yet Underappreciated Risk Factor. Can Geriatr J. 2022 Dec 1; 25(4):404-406. 14. AREAS of the Municipalities. IBGE, Geosciences Directorate. Rio de Janeiro: IBGE, 2023. Available at: https://www.ibge.gov.br/geociencias/organizacao-do-territorio/estrutura-territorial/15761-areas-dos- municipios.html. Accessed in: Oct. 2023. 15. Saes, M. de O., Rocha, J. Q. S., Rutz, A. A. M., Silva, C. N. da, Camilo, L. dos S., Oliveira, B. C. de, Ritta, M. de C., Nunes, L. da S., Gonçalves, C. de S., Pereira Vieira, Y., Neves, R. G., & Duro, S. M. S. (2023). Methodological aspects and results of the baseline health monitoring of adults and elderly people infected with covid-19 (Sulcovid-19) . In SciELO Preprints. https://doi.org/10.1590/SciELOPreprints.5334 16. Brazilian Association of Research Companies (ABEP). Brazil Criteria: Brazil 2022 Economic Classification Criteria - PNADC 2020 [Internet]. 2022 [accessed 2023 Sep 12]. Available at: http://www.abep.org/criterio-brasil. 16. Brazilian Association of Research Companies (ABEP). Brazil Criteria: Brazil 2022 Economic Classification Criteria - PNADC 2020 [Internet]. 2022 [accessed 2023 Sep 12]. Available at: http://www.abep.org/criterio-brasil. 17. References 2009 Dec 14; 169(22):2142-7. Page 15/17 Page 15/17 24. Monje M, Iwasaki A. The neurobiology of long COVID. Neuron. 2022 Nov 2; 110(21):3484-3496. 24. Monje M, Iwasaki A. The neurobiology of long COVID. Neuron. 2022 Nov 2; 110(21):3484-3496. 25. Subramanian A, Nirantharakumar K, Hughes S, Myles P, Williams T, Gokhale KM, Taverner T, Chandan JS, Brown K, Simms-Williams N, Shah AD, Singh M, Kidy F, Okoth K, Hotham R, Bashir N, Cockburn N, Lee SI, Turner GM, Gkoutos GV, Aiyegbusi OL, McMullan C, Denniston AK, Sapey E, Lord JM, Wraith DC, Leggett E, Iles C, Marshall T, Price MJ, Marwaha S, Davies EH, Jackson LJ, Matthews KL, Camaradou J, Calvert M, Haroon S. Symptoms and risk factors for long COVID in non-hospitalized adults. Nat Med. 2022 Aug; 28(8):1706-1714. 26. Notarte KI, de Oliveira MHS, Peligro PJ, Velasco JV, Macaranas I, Ver AT, Pangilinan FC, Pastrana A, Goldrich N, Kavteladze D, Gellaco MML, Liu J, Lippi G, Henry BM, Fernández-de-Las-Peñas C. Age, Sex and Previous Comorbidities as Risk Factors Not Associated with SARS-CoV-2 Infection for Long COVID- 19: A Systematic Review and Meta-Analysis. J Clin Med. 2022 Dec 9; 11(24):7314. 27. Jia X, Cao S, Lee AS, Manohar M, Sindher SB, Ahuja N, Artandi M, Blish CA, Blomkalns AL, Chang I, Collins WJ, Desai M, Din HN, Do E, Fernandes A, Geng LN, Rosenberg-Hasson Y, Mahoney MR, Glascock AL, Chan LY, Fong SY; CLIAHUB Consortium; Chan Zuckerberg Biohub; Phelps M, Raeber O; Stanford COVID-19 Biobank Study Group; Purington N, Röltgen K, Rogers AJ, Snow T, Wang TT, Solis D, Vaughan L, Verghese M, Maecker H, Wittman R, Puri R, Kistler A, Yang S, Boyd SD, Pinsky BA, Chinthrajah S, Nadeau KC. Anti-nucleocapsid antibody levels and pulmonary comorbid conditions are linked to post-COVID-19 syndrome. JCI Insight. 2022 Jul 8; 7(13):e156713. 28. Krishnadath I, Harkisoen S, Gopie F, van der Hilst K, Hollum M, Woittiez L, Baldew SS. Prevalence of persistent symptoms after having COVID-19 in a cohort in Suriname. Rev Panam Salud Publica. 2023 May 16; 47:e79. 28. Krishnadath I, Harkisoen S, Gopie F, van der Hilst K, Hollum M, Woittiez L, Baldew SS. Prevalence of persistent symptoms after having COVID-19 in a cohort in Suriname. Rev Panam Salud Publica. 2023 May 16; 47:e79. 29. References Gonzalez-Garcia P, Fiorillo Moreno O, Zarate Peñata E, Calderon-Villalba A, Pacheco Lugo L, Acosta Hoyos A, Villarreal Camacho JL, Navarro Quiroz R, Pacheco Londoño L, Aroca Martinez G, Moares N, Gabucio A, Fernandez-Ponce C, Garcia-Cozar F, Navarro Quiroz E. From Cell to Symptoms: The Role of SARS-CoV-2 Cytopathic Effects in the Pathogenesis of COVID-19 and Long COVID. Int J Mol Sci. 2023 May 5; 24(9):8290. 30. Rivera J, Rodríguez T, Pallarés M, Castrejón I, González T, Vallejo-Slocker L, Molina-Collada J, Montero F, Arias A, Vallejo MA, Alvaro-Gracia JM, Collado A. Prevalence of post-COVID-19 in patients with fibromyalgia: a comparative study with other inflammatory and autoimmune rheumatic diseases. BMC Musculoskelet Disord. 2022 May 19; 23(1):471. 31. Evcik D. Musculoskeletal involvement: COVID-19 and post COVID 19. Turk J Phys Med Rehabil. 2023 Feb 28; 69(1):1-7. 31. Evcik D. Musculoskeletal involvement: COVID-19 and post COVID 19. Turk J Phys Med Rehabil. 2023 Feb 28; 69(1):1-7. 32. Fuggle NR, Singer A, Gill C, Patel A, Medeiros A, Mlotek AS, Pierroz DD, Halbout P, Harvey NC, Reginster JY, Cooper C, Greenspan SL. How has COVID-19 affected the treatment of osteoporosis? An IOF-NOF- ESCEO global survey. Osteoporos Int. 2021 Apr; 32(4):611-617. 33. Messina C, Buzzoni AC, Gitto S, Almolla J, Albano D, Sconfienza LM. Disruption of bone densitometry practice in a Northern Italy Orthopedic Hospital during the COVID-19 pandemic. Osteoporos Int. 2021 Jan; 32(1):199-203. 34. Peeters JJM, van den Berg P, van den Bergh JP, Emmelot-Vonk MH, de Klerk G, Lems WF, Winter EM, Zillikens MC, Appelman-Dijkstra NM. Osteoporosis care during the COVID-19 pandemic in the Page 16/17 Page 16/17 Netherlands: A national survey. Arch Osteoporos. 2021 Jan 7; 16(1):11. 35. Dagher H, Chaftari AM, Subbiah IM, Malek AE, Jiang Y, Lamie P, Granwehr B, John T, Yepez E, Borjan J, Reyes-Gibby C, Flores M, Khawaja F, Pande M, Ali N, Rojo R, Karp DD, Chaftari P, Hachem R, Raad II. Long COVID in cancer patients: preponderance of symptoms in majority of patients over long time period. Elife. 2023 Feb 7; 12:e81182. 36. Wong MC, Huang J, Wong YY, Wong GL, Yip TC, Chan RN, Chau SW, Ng SC, Wing YK, Chan FK. Epidemiology, Symptomatology, and Risk Factors for Long COVID Symptoms: Population-Based, Multicenter Study. JMIR Public Health Surveill. 2023 Mar 7; 9:e42315. 37. Sherif ZA, Gomez CR, Connors TJ, Henrich TJ, Reeves WB; RECOVER Mechanistic Pathway Task Force. References Pathogenic mechanisms of post-acute sequelae of SARS-CoV-2 infection (PASC). Elife. 2023 Mar 22; 12:e86002. 38. Yong SJ. Long COVID or post-COVID-19 syndrome: putative pathophysiology, risk factors, and treatments. Infect Dis (Lond). 2021 Oct; 53(10):737-754. 38. Yong SJ. Long COVID or post-COVID-19 syndrome: putative pathophysiology, risk factors, and treatments. Infect Dis (Lond). 2021 Oct; 53(10):737-754. 39. Christofoletti M, Duca GFD, Benedetti TRB, Malta DC. Sociodemographic determinants of multimorbidity in Brazilian adults and older adults: a cross-sectional study. Sao Paulo Med J. 2022 Jan-Feb; 140(1):115- 122. 39. Christofoletti M, Duca GFD, Benedetti TRB, Malta DC. Sociodemographic determinants of multimorbidity in Brazilian adults and older adults: a cross-sectional study. Sao Paulo Med J. 2022 Jan-Feb; 140(1):115- 122. 40. de Souza ASS, Braga JU. Trends in the use of health services and their relationship with multimorbidity in Brazil, 1998-2013. BMC Health Serv Res. 2020 Nov 25; 20(1):1080. 40. de Souza ASS, Braga JU. Trends in the use of health services and their relationship with multimorbidity in Brazil, 1998-2013. BMC Health Serv Res. 2020 Nov 25; 20(1):1080. 41. Hayhoe BW, Powell RA, Barber S, Nicholls D. Impact of COVID-19 on individuals with multimorbidity in primary care. Br J Gen Pract. 2021 Dec 31; 72(714):38-39. 41. Hayhoe BW, Powell RA, Barber S, Nicholls D. Impact of COVID-19 on individuals with multimorbidity in primary care. Br J Gen Pract. 2021 Dec 31; 72(714):38-39. 42. Honda H, Takamatsu A, Miwa T, Tabuchi T, Taniguchi K, Shibuya K, Tokuda Y. Prolonged Symptoms after COVID-19 in Japan: A Nationwide Survey of the Symptoms and Their Impact on Patients' Quality of Life. Am J Med. 2023 May 24:S0002-9343(23)00331-5. 43. Nunes BP, Souza ASS, Nogueira J, Andrade FB, Thumé E, Teixeira DSDC, Lima-Costa MF, Facchini LA, Batista SR. Multimorbidity and population at risk for severe COVID-19 in the Brazilian Longitudinal Study of Aging. Cad Saude Publica. 2020 Nov 20; 36(12):e00129620. 44. Skou ST, Mair FS, Fortin M, Guthrie B, Nunes BP, Miranda JJ, Boyd CM, Pati S, Mtenga S, Smith SM. Multimorbidity. Nat Rev Dis Primers. 2022 Jul 14; 8(1):48. Page 17/17
https://openalex.org/W2946965314	https://figshare.com/ndownloader/files/15282635	English	null	The effectiveness of surgical vs conservative interventions on pain and function in patients with shoulder impingement syndrome. A systematic review and meta-analysis	PloS one	2,019	cc-by	1,497	PRISMA 2009 Checklist ary 2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number. N 3 Describe the rationale for the review in the context of what is already known. 4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS). stration 5 Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number. 6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale. es 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched. 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis). ocess 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators. 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made. ividual 12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis. res 13 State the principal summary measures (e.g., risk ratio, difference in means). lts 14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I2) for each meta-analysis. Pages 1 - 2 Page - 1 Page 4 Pages 4 - 5 Page 5 Page - 5 Pages 5 - 6 Page 5 - 6 page - 6 Page 6 Page - 6 Page - 7 Page - 7 Pages 7 - 8 1 Identify the report as a systematic review, meta-analysis, or both. PRISMA 2009 Checklist PRISMA 2009 Checklist PRISMA 2009 Checklist # Checklist item Reported on page # 1 Identify the report as a systematic review, meta-analysis, or both. 2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number. 3 Describe the rationale for the review in the context of what is already known. 4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS). on 5 Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number. 6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale. 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched. 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis). s 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators. 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made. al 12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis. 13 State the principal summary measures (e.g., risk ratio, difference in means). 14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I2) for each meta-analysis. Pages 1 - 2 Page - 1 Page 4 Pages 4 - 5 Page 5 Page - 5 Pages 5 - 6 Page 5 - 6 page - 6 Page 6 Page - 6 Page - 7 Page - 7 Pages 7 - 8 1 Identify the report as a systematic review, meta-analysis, or both. PRISMA 2009 Checklist ary 2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number. N 3 Describe the rationale for the review in the context of what is already known. 4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS). stration 5 Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number. 6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale. es 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched. 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis). ocess 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators. 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made. dividual 12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis. res 13 State the principal summary measures (e.g., risk ratio, difference in means). lts 14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I2) for each meta-analysis. Page 1 of 2 Pages 1 - 2 Page - 1 Page 4 Pages 4 - 5 Page 5 Page - 5 Pages 5 - 6 Page 5 - 6 page - 6 Page 6 Page - 6 Page - 7 Page - 7 Pages 7 - 8 Page 1 of 2 PRISMA 2009 Checklist # Checklist item Reported on page # dies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies). PRISMA 2009 Checklist 16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified. 17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations. ies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12). udies 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot. 21 Present results of each meta-analysis done, including confidence intervals and measures of consistency. dies 22 Present results of any assessment of risk of bias across studies (see Item 15). 23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression [see Item 16]). 24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers). 25 Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias). 26 Provide a general interpretation of the results in the context of other evidence, and implications for future research. 27 Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review. Tetzlaff J, Altman DG, The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 6(7): e1000097. 00097 For more information, visit: www.prisma-statement.org. P 2 f 2 Page - 7 Pages 7 - 8 Page - 8 Pages 8 - 9 Page 9 Table - 1 Pages 10-13 Page - 9 Pages 10-13 Pages 13-14 Page 15 Page - 15 Page - 15 Page 2 of 2
https://openalex.org/W4392311927	https://inlibrary.uz/index.php/arabic-language/article/download/28747/29587	Kirghiz, Kyrgyz	null	Eron xalq ertaklari: fors folklor sh unosligida “هناسفا” (“Ertak”) so‘zining ilmiy atamasi	null	2,023	cc-by	1,854	“Arab tili globallashuv davrida: innovatsion yondoshuvlar va o‘qitish metodikasi” “Arab tili globallashuv davrida: innovatsion yondoshuvlar va o‘qitish metodikasi” “Arab tili globallashuv davrida: innovatsion yondoshuvlar va o‘qitish metodikasi” mavzusidagi xalqaro ilmiy-amaliy anjuman * Мазкур луғат Эронда 1899, 1921, 1925, 1926, 1938 ва 1957 йилларда чоп этилган 1952-1856 йилларда турли ислоҳлар, яъни кириш сўзи, батафсил иқтибослар, сўзларнинг ўзакларига шарҳлар, муаллифнинг баъзи хатолари тузатилган ҳолда 5 томдан иборат нашри Муҳаммад Муин томонидан чоп этилган // Қаранг: ،ﻣﺤﻤﺪ حسين ﺑن خﻠف ﺗبﺮﻳزی (ﺑﺮﻫﺎﻥ)، فﺮﻫﻨگ لﻐﺎﺕ فﺎرﺳی ﺑه فﺎرﺳی، وﺑﺮﺍﺳﻨﺎر ﻣﺤﻤﺪ ﻣعين، ﻣﻮﺳسۀ ﺍنتﺸﺎرﺍﺕ ﺍﻣيﺮ کبيﺮ1335 (چﺎپ ،)ﺍول1391 )(چﺎپ ﻫفتﻢ ) )ﻢ ( * Муҳаммад бен Али бен Муҳаммад Заҳирий Самарқандий VI аср охири VII аср бошларида Афросиёб сулоласининг энг сўнгги подшоҳи Қўлаж Иамғожхон Иброҳим саройида яшаган етакчи адиблардан бири бўлиб, 600 йилда вафот этган// ،ﻣﺤﻤﺪ ﺑن عﻠی ﺑن ﻣﺤﻤﺪ ظﻬيﺮی ﺳﻤﺮ ﻗﻨﺪی، ﺳﻨﺪﺑﺎد نﺎﻣه، ﺑه کﻮﺷﺶ ﻣﺤﻤﺪ ﺑﺎﻗﺮ کﻤﺎل لﺪﻳﻨی، ﺗﻬﺮﺍﻥ1381 .. ص94 ، 97 ، 124 . ERON XALQ ERTAKLARI: FORS FOLKLORShUNOSLIGIDA “افسانه” (“ERTAK”) SO‘ZINING ILMIY ATAMASI O‘.A.Umarov, Sh.M.Muxamedov O‘zDJTU ikkinchi chet tili kafedrasi katta o‘qituvchisi Nizomiy nomidagi TDPUning dosenti umarov.u@mail.ru Annotasiya. Mazkur maqolada Eron xalq ertaklaridagi hayvonlar, jumladan tulki va bo‘ri xarakterlariga xos ijobiy va salbiy jihatlar xususida so‘z yuritiladi. Ushbu hayvonlarning xatti-harakati orqali jamiyatdagi ba’zi insonlarga xos bo‘lgan fazilat va illatlar ta’riflanadi. f f Kalit so‘zlar: ertak, matal, cho‘pchak, hikoya. Abstract. This article talks about the positive and negative aspects of the characters of animals, including foxes and wolves, in Iranian folk tales. Through the behavior of these animals, the virtues and vices characteristic of some people in the society are described. y Key words: fairy tale, folk tale, fable, story. Bizga ma’lumki, tadqiqotchilar tomonidan ertak haqida va ularning o‘ziga xos xususiyatlari haqida ko‘pgina ilmiy ishlar amalga oshirilgan hamda umumiy ma’noda qadim zamonlardan to hozirgi kungacha ertaklar mavjudligi ko‘rsatib kelingan. Shu o‘rinda, masalan, rossiyalik olim Ye.M.Meletinskiy shunday yozadi: “Ertak – og‘zaki san’atning eng qadimgi turlaridan biridir. Bu xalq ijodiyotining ildizi sinfiy jamiyat yuzaga kelmasdan oldingi davrga borib taqaladi va hozirga qadar odamlarning hamrohi bo‘lib qolib, ularga juda katta badiiy zavq berib keladi. Ertakning yuqori poetik qadriyati ma’lum jihatdan yuz yilliklar davomida to‘plangan fikrlar, tasavvurlar va xalq hissiyotlarini ifodalashi bilan izohlanadi. Ertak – barcha xalqlarning eng mashhur va eng demokratik turdagi og‘zaki san’atidir. Ertak atamasi Mahmud Qashg‘ariyning XI asrda yozilgan “Divonu lug‘atit turk” asarida etuk shaklida uchraydi va biror voqyeani og‘zaki tarzda hikoya qilish ma’nosini bildirib, bu hikoyalar “o‘tmish voqyealardan xabar beruvchi... hikoyalarda faqat o‘tganlar haqida aytilishi shart emas”, deb izohlaydi. Vatanimizda “ertak” so‘zi folklor atama sifatida qubul qilingan bo‘lsa-da, turli viloyatlardagi aholi o‘rtasida matal, ushuk, cho‘pchak, hikoya, afsona, o‘tirik, tutal kabi ertak so‘zi sinonimlari ham ishlatiladi. Eron folklorshunosligida ham shunday holatni kuzatamiz. Fors tilidagi “افسانه” (“ertak”) so‘zining ilmiy atamasi va uning sinonimlarining qo‘llanishi haqidagi masala mutaxassislar o‘rtasida doimo baxs va tortishuvlarga sabab bo‘lgan. Albatta, bu tabiiy 702 “Arab tili globallashuv davrida: innovatsion yondoshuvlar va o‘qitish metodikasi” рﺮ (چ پ ،)ﺍول1391 (چﺎپ ﻫف mavzusidagi xalqaro ilmiy-amaliy anjuman mavzusidagi xalqaro ilmiy-amaliy anjuman holatdir, chunki eronliklar va forsiy zabon xalqlar o‘rtasida bu janr turli nomlarga ega va bu masalani o‘raganishda turlicha atamalar qo‘llaniladi. Shuni ham aytib o‘tish lozimki, eronlik folklorshunoslar dastlab, tegishli ravishda og‘zaki nasrga nisbatan ilmiy atamalarni qo‘llash haqida fikrlar bildirmaganlar va bir-biriga o‘xshash janrlar o‘rtasidagi farqlarni tavsiflamaganlar. Bu masala haqidagi murakkablik yana shu bilan bog‘liqki, lug‘atlarda va yozuvchilarning asarlarida, xalq va mutaxassislar o‘rtasida ishlatiladigan ba’zi atamalar boshqa turli janrlarni bildirsa-da, barcha atamalardan sinonim sifatida foydalanib kelganlar. y g Ma’lumki, dastlab, adabiyot tarixi bo‘yicha yozma adabiyot janrlari ham turlarga bo‘linmagan edi. Vaqt o‘tishi bilan ularning chegaralarini olimlar belgilay boshladilar. Eron folklorshunosligida bu ilm-fanning “yoshligini” e’tiborga olgan holda, oxirigi yillarda xalq og‘zaki ijodiyoti tadqiqotchilari bu janrlarning vazifalarini qadam baqadam chegaralarini aniqlashtirib, ularning o‘ziga xos xususiyat va farqlarini belgilash bo‘yicha fikrlar bildira boshladilar. So‘z “ertak” haqida ketar ekan, biz avvalo, “ﺍفسﺎنه” (“ertak”) so‘zi atamasini ilk o‘rta asrlarda yaratilgan kitoblar va og‘zaki manbalar bo‘yicha nafaqat tarixiy yo‘lini, balki Eron folklorshunoslari tomonidan bildirilgan fikr va mulohazalarni tahlil qilishga harakat qildik. Eng qadimgi lug‘atlardan birining muallifi, tahallusi Burhon bo‘lgan Muhammad ben Xalaf Tabriziy tomonidan yaratilgan “Foschadan forschaga lug‘at”da “ﺍ فسﺎنه” so‘zi shunday izohlangan: “ﺍفسﺎنه” “afsona” ommabop va og‘zaki adabiyotdagi juda qadimgi va mashhur hikoyadir. Eron afsonalari ko‘pincha nasrda va ba’zida qofiyali tarzda hikoya qilinadi. Fors adabiyotida “ﺍفسﺎنه” “afsona” yoki “rivoyat” ko‘pgina ma’noga ega bo‘lib, ulardan eng asosiylari qissa, hikoyat, tamsil, doston, sargo‘zasht, o‘tmish haqidagi ostura, yolg‘on va asoslanmagan so‘zlar hamda ma’lum bir aytilgan fikrlar hisoblanadi”. VI asr oxiri VII asr boshida yashagan adib Muhammad bin Ali bin Muhammad Zahiriy Samarqandiyning asarida “fors adabiyoti matnlarida afsona so‘zi barcha nasriy asar shakllariga sinonim sifatida ishlatiladi, ba’zida uni o‘rniga qissa, hikoyat, sargo‘zasht va hokazo atamalarni ishlatadilar. Aslini olganda bu atamalar ma’lum jihatdan bir ma’nodadir”, deb yozgan. Albatta ba’zi holatlarda afsona yolg‘on, uydirma qissa, g‘alati va g‘ayrioddiy hikoyat ma’nosini bildirib kelgan. Shunday qilib, Eron og‘zaki ijodiyoti va yozma adabiyotda qadim zamonlardan boshlab “ertak” atamasi turli xil va variantlarda qo‘llanilgan: Masalan, “ﺍفسﺎنه” (“ertak”), “ﻗصه” (“qissa”) , “ﺍﺳﻄﻮرﻩ” (“mif”, “afsona”), “دﺍﺳتﺎﻥ” 703 oyat”), “ﻣتﻞ” (“m hokazolar. ini Eronning nafa oqlarida ham tur turli-tuman she gi madaniy va et etishi uchun ar yaratilgan. Biz arqlarni ko‘rsati miz. miyatga molik Yaxshilik va “Arab tili globallashuv davrida: innovatsion yondoshuvlar va o‘qitish metodikasi” mavzusidagi xalqaro ilmiy-amaliy anjuman mavzusidagi xalqaro ilmiy-amaliy anjuman (“doston”), “روﺍﻳت” (“rivoyat”), “حکﺎﻳت” (“hikoyat”), “ﻣتﻞ” (“matal”), “نقﻞ” (“naq “og‘zaki hikoya”), “ﺳﺮگذﺷت” “sargo‘zasht”) va hokazolar. mavzusidagi xalqaro ilmiy-amaliy anjuman mavzusidagi xalqaro ilmiy-amaliy anjuman Rivoyatlar asosini voqyealar va tarixiy shaxslar, geografik nomlar va hayovonot olami tashkil qiladi va o‘z shakliga ko‘ra rivoyatlar afsonalarga o‘xshamaydi. Eronda hanuzgacha alohida rivoyatlar chop etilgan to‘plamlar mavjud emas. Lug‘atlarda esa, bu janrga nisbatan aniq nuqtai nazar yo‘q. Lekin afsona va rivoyat o‘rtasidagi farqlarga qaramay, ular o‘rtasida umumiylik mavjud bo‘lib, bu sohada ham aniq ajratishlar hali amalga oshirilmagan. Rossiyalik sharqshunos I. Braginskiyning yozishicha, “yozma adabiyotni tashkil qiluvchi afsona va rivoyatlarni hikoyachilar, va’zchilar o‘z usullarida gapirib beradilar. Shunday qilib, yozma adabiyot va og‘zaki ijodiyot o‘rtasida o‘ziga xos og‘zaki shakl mavjud bo‘lgan. Yozma adabiyotda mavjud bo‘lgan bunday og‘zaki shakl asosini turli hajmdagi – kichik latifalardan to katta romanlargacha bo‘lgan badiiy matnlar tashkil qilgan. Biroq bu matnlar keng kitobxonlar doirasi uchun emas, faqat tinglovchilar uchun mo‘ljallangan. Bu matnlarni aksariyat hollarda hikoyachilarning o‘zlari yozib, keyin aytib berganlar va vaqt o‘tishi bilan hikoyachilarning bu yozganlari yozma adabiyotga aylanib, ularning qo‘lyozmalarini bilimli odamlar o‘qishni boshlaganlar”. Avvalgi yuz yilliklarda biz olimlarning tarixiy va falsafiy asarlarida ko‘pgina ertaklarni uchratamiz. Bunday asarlarda ertaklar “hikoyat” yoki “rivoyat” nomi bilan keltirilgan. O‘z fikrlarini tasdiqlash yoki ifoda etilayotgan voqyealarning hissiyotli ta’sirini o‘stirish uchun Nizom ul-mulk o‘zining “Siyosatnoma” asarida, Muhammad G‘azzoliy o‘zining “Nasihat ul-muluk” va “Ajoyib ud-dunyo” asarlarida ertak va rivoyatlardan mahorat bilan foydalanganlar. Eron hikoyatlarining asosiy manbalari qadimgi Eron hikoyatlari – Bahrom- Go‘r, Anushervon hamda hindlarning Kalila va Dimna, To‘tinoma, Gresiyaning Aristotel, Sokrat va Aleksandr va hayvonlar haqidagi, arablarning Hotamtoy haqidagi va ko‘plab boshqa xalqlarning hikoyatlari hisoblanadi. Undan tashqari, parilar, devlar, jinlar haqidagi hikoyatlar ham shular jumlasiga kiradi. Matallar bolalar adabiyotida o‘yin-qo‘shiq shaklida tarqalgan bo‘lib, unda nasr va nazm uyg‘unlashgan. Matallar qadim zamonlardan mavjud va unda kulgili o‘yinlar, yoqimli hikoyalar bolalarda katta qiziqish orttiradi. Matallar hozirgi vaqtda bizning tushunchamizdagi kumulyativ ertaklar hisoblanadi. Ohangdorligi, harakatlarning qaytarilishi tufayli bolalar xotirasida oson saqlanib qoladi. g q y y q q Hozirgi davrga qadar “نقﻞ” (“naql”, “og‘zaki hikoya”) janr sifatida fors folklorida o‘z ifodasini topmagan va ilmiy adabiyotlarda ma’qullanmagan. Bitta dona ham to‘plam nashr etilmagan. “نقﻞ” o‘z shakli va mazmuni bo‘yicha “afsona”dan keskin farq qiladi. “Naql” hikoyaga ancha yaqin, lekin o‘z badiiy xususiyatlari tufayli hikoyaga sal yetib bormaydi. Lekin shu bilan birga, afsona va naql o‘rtasida ma’lum bir bog‘liqlik mavjud. Masalan, ijrochi ertak bayon etish jarayonida matndan uzoqlashib, unga bir naqlni kiritadi va faqat shundan keyin ertakni boshlaydi. Ba’zida hikoyachi katta badiiy yoki ilmiy ahamiyatga molik bo‘lgan bir naqlga yo ertakning boshida, yoki ertakning oxirida murojaat etadi. Shunisi qiziqki, “ﺍفسﺎنه” “afsona” atamasini Eronning nafaqat shahar, viloyat va mintaqalarida, balki hatto, alohida qishloqlarida ham turlicha tushunadilar. Bunga sabab, birinchidan, forslarning turli-tuman sheva va lahjalarda so‘zlashishlari va ikkinchi tomondan, Erondagi madaniy va etnik xilma-xillik har bir hudud aholisi ertak ma’nosini ifoda etishi uchun maxsus atamadan foydalanishlariga majbur etgan. Eron mifologiyasi haqida ko‘pgina asarlar yaratilgan. Biz asarlar haqida so‘z yuritmay, faqat afsona va ertak o‘rtasidagi farqlarni ko‘rsatib o‘tish maqsadida ularning ba’zilari ustida qisman to‘xtalib o‘tamiz. Albatta, Eron mifologiyasining ahamiyatga molik manbalaridan biri “Avesto” hisoblanadi. Mazkur yodgorlikda Yaxshilik va Yomonlik xudolari “Axuramazda va Axrimandan boshlanib, boshqalar, ya’ni yer yuzining barcha suvlarini boshqaradigan va o‘zida pokizalikni mujassamlantirgan Anaxita xudosi, tinchlik uchun kurashuvchi va devlarga qarshi bo‘lgan Virisranna, odamlarni Haqiqat yo‘liga yo‘naltiruvchi Mitra xudosi, urush axtaruvchi Vayu, yomg‘ir xudosi Tatariya, Axuramazdaning o‘g‘li Atar, salomatlik, oziq-ovqat, arzonchilik xudosi Havma va hokazo mifologik qahramonlar haqida juda ko‘p ma’lumotlar mavjud. Mifologiya haqida mukammal material beruvchi Firdavsiyning yirik va mashhur kitobi “Shohnoma” asarida ham juda ko‘p afsonalar mavjud. Firdavsiy mazkur asarida – Qayumars, Hushang, Tahmuras, Jamshid, Zahhok va Faridun kabi afsonaviy qahramonlarni birin-ketin bayon etgan. Hozirga qadar Eronning xalq mifologiyasini folklor janri sifatida o‘rganishga katta ahamiyat qaratilmoqda va ko‘plab kitoblar nashr etilmoqda. Ertak bu ermak, ko‘ngil-ochish bo‘lsa, mifologiya qahramonlari harakatiga ishonch, ulardan ma’naviy ozuqlanish munosabatini keltirib chiqaradi. Bundan shunday xulosa chiqarish mumkinki, ertaklarning paydo bo‘lishiga afsonalar asos bo‘lgan. Eron yozuvchisi, adabiyotshunos Jamol Mirsodiqiy dostonga quyidagicha izoh beradi: “Doston umumiy ma’noda yozma yoki og‘zaki, real yoki xayoliy tarzda o‘tmishni ketma-ketlikda bayon etish. Boshqacha qilib aytganda, doston – real va tarixiy voqyealarni, xayoliy va ijodiy fikrlarni ketma-ketlikda berishdir”. Doston turlari haqida so‘z yuritgan J.Mirsodiqiy yana shunday fikrlarni bildiradi: “Fors tilida doston so‘zi qissa, hikoyat, afsona va sargo‘zasht kabilarni belgilashda ishlatiladi va adabiy atama sifatida, bir tomondan, qissalarning turli shakllarini, ikkinchi tomondan, badiiy adabiyotning turli qismlarini, jumladan, doston, roman, uzun doston va shu kabi ijodiy adabiyot qismlarini o‘zida mujassam etgan. Bu atama Ali Akbar Dehxudo lug‘atida biror kimsaning so‘zlarini yoki xabarlarini aytib berish ma’nosiga ega, Muhammad Muyin lug‘atida ham, xuddi shunday ma’noga ega va bu xabar va hadislar Payg‘ambar (s.a.v.) yoki Imomlar haqida bo‘lishi ham mumkin. 704 “Arab tili globallashuv davrida: innovatsion yondoshuvlar va o‘qitish metodikasi” “Arab tili globallashuv davrida: innovatsion yondoshuvlar va o‘qitish metodikasi” Afsuski, hozirga qadar folklorshunoslar masalaning mana shu tomoniga kam e’tibor qaratilgan. 705 “Arab tili globallashuv davrida: innovatsion yondoshuvlar va o‘qitish metodikasi” mavzusidagi xalqaro ilmiy-amaliy anjuman Ali Akbar Dehxudoning lug‘atida “نقﻞ” so‘zi biron bir begona kishining so‘zlarini qayta aytib berish deb izohlangan. Undan tashqari, “نقﻞ” mashhur yoki badiiy mazmunga ega asarni yangi asarga aylantirib bayon qilish demakdir. Shunday qilib, Eron folklorshunosligida “afsona” so‘zining ko‘pgina sinonimlari bo‘lib, “afsona” va “qissa” atamalari fors ilmiy adabiyotlarda ko‘proq uchraydi. “Qissa”, “rivoyat”, “naql”, “asotir”, “hikoyat”, “doston”, “sargo‘zasht” kabi atamalar Eron ertaklariga nisbatan qo‘llanilsa-da, ularning “afsona” atamasi bilan yaqindan bog‘liqdir. “Afsona” so‘zi ertak yaratilgan shahar, qishloq lahjasiga qarab, turlicha nomlanishi ham mumkin. Ertaklarning turli-tuman atamalar bilan ifodalanishini eronlik tadqiqotchilarning ishlarida ham ko‘rishimiz mumkin. Aslini olganda, og‘zaki adabiyotning bu muhim turini belgilash uchun yagona atama ular orasida hanuz umumiy tarzda qabul qilinmagan. Foydalanilgan adabiyotlar ro‘yxati: Foydalanilgan adabiyotlar ro‘yxati: 1. Shomuhamedov Sh. Eron xalq ertaklari. Toshkent. 1959. 2. ersidskiye narodnыye skazki. Perevod i sostavitel A.A. Romaskevich M- L. 1934. 3. Persidskiye narodnыye skazki. Perevod i sostavitel A.Z. Rozenfeld. «Nauka». Moskva. 1958. 4. Persidskiye skazki. Perevod A.Aliyeva, A. Bertelsa, N. Osmanova. Izd-vo Vostochnaya literatura. Moskva. 1958. 5. Persidskiye narodnыye skazki.. Tashkent. 1959. 5. Persidskiye narodnыye skazki.. Tashkent. 1959. 6. Persidskiye narodnыye skazki. Sost. M.N. Osmanov. Predisl. D.S.Komissarova. Moskva. Izd-vo “Nauka”.Moskva. 1987. 706
https://openalex.org/W2965604290	https://europepmc.org/articles/pmc6723862?pdf=render	English	null	Aeration, Agitation and Cell Immobilization on Corncobs and Oak Wood Chips Effects on Balsamic-Styled Vinegar Production	Foods	2,019	cc-by	11,418	Received: 15 June 2019; Accepted: 22 July 2019; Published: 1 August 2019 Abstract: Optimum fermentor conditions are essential for desired microbial growth and activity in fermentations. In balsamic vinegar fermentation systems, the microorganisms used must endure several stressful conditions including high sugar concentration, low water activity, high osmotic pressure and high acetic acid concentration. Consequently, the present study was aimed at improving the performance of a microbial consortium of non-Saccharomyces yeast and acetic acid bacteria during balsamic-styled vinegar fermentation. Cell immobilization via adsorption on corncobs and oak wood chips in combination with aeration and agitation eﬀects, have never been tested during balsamic-styled vinegar fermentation. Therefore, fermentations were initially conducted under static conditions without aeration with successive fermentations also being subjected to low (0.15 vvm min−1) and high (0.3 vvm min−1) aeration. The results showed improved acetiﬁcation rates when cells were immobilized on corncobs under static conditions. Low aeration showed better acetiﬁcation rates (1.45–1.56 g·L·day−1), while only free-ﬂoating cells were able to complete fermentations (1.2 g·L·day−1) under high aeration conditions. Overall, cells immobilized on corncobs showed higher acetiﬁcation rates of 1.56 and 2.7 g·L·day−1 under low aeration and static fermentations, respectively. Oak wood chips were determined to be less eﬃcient adsorbents due to their relatively smooth surface, while the rough surface and porosity of corncobs led to improved adsorption and, therefore, enhanced acetiﬁcation rates. Keywords: aeration; adsorption; balsamic-styled vinegar; cell immobilization; corncobs; non-Saccharomyces yeast; oak wood chips foods foods Article Ucrecia F. Hutchinson 1,2,3,* , Sivuyile Gqozo 1, Neil P. Jolly 1, Boredi S. Chidi 1,2, Heinrich W. Du Plessis 1 , Maxwell Mewa-Ngongang 1,2,3 and Seteno K. O. Ntwampe 2,3 Ucrecia F. Hutchinson 1,2,3,* , Sivuyile Gqozo 1, Neil P. Jolly 1, Boredi S. Chidi 1,2, Heinrich W. Du Plessis 1 , Maxwell Mewa-Ngongang 1,2,3 and Seteno K. O. Ntwampe 2,3 1 Post-Harvest and Agro-Processing Technologies, ARC Infruitec-Nietvoorbij (The Fruit, Vine and Wine Institute of the Agricultural Research Council), Private Bag X5026, Stellenbosch 7599, South Africa 2 Bioresource Engineering Research Group (BioERG), Department of Biotechnology, Cape Peninsula University of Technology, P.O. Box 652, Cape Town 8000, South Africa 3 Department of Chemical Engineering, Cape Peninsula University of Technology, P.O. Box 652, Cape Town 8000, South Africa * Correspondence: Hutchinsonu@arc.agric.za; Tel.: +27-21-809-3442 * Correspondence: Hutchinsonu@arc.agric.za; Tel.: +27-21-809-3442 foods foods www.mdpi.com/journal/foods Keywords: aeration; adsorption; balsamic-styled vinegar; cell immobilization; corncobs; non-Saccharomyces yeast; oak wood chips 1. Introduction Similar to other vinegars, balsamic vinegar is a food ﬂavoring agent or condiment, which contains acetic acid as its main ingredient [1]. Balsamic vinegar is characterized by its sour and sweet taste [2] and can be used as a salad dressing, and for soothing sore throats among other uses [3]. Balsamic-styled vinegar (BSV) is a term adopted from Traditional Balsamic vinegar (TBV) of Modena and Reggio-Emilia Italy. BSV is a new type of Balsamic vinegar which has potential for being produced in South Africa using Chenin blanc wine grapes, since they are the most cultivated grape cultivar in South Africa [4]. Cell immobilization generally refers to a technique, which is performed to prevent cells from being freely suspended in the fermentation medium [5–7]. Cell immobilization improves biomass growth, Foods 2019, 8, 303; doi:10.3390/foods8080303 www.mdpi.com/journal/foods 2 of 15 Foods 2019, 8, 303 increases cell stability, protects cells from the toxic environment, and provides a reusability option [8]. Most fermentations generally employ the free-ﬂoating cell (FFC) method, which is eﬀective in less stressful fermentation systems. However, during balsamic vinegar fermentation, there are multiple factors that have antagonistic eﬀects on the microorganisms involved in the fermentation. For instance, the fermentation medium (cooked grape must) used for Balsamic-styled vinegar has a very high sugar content, low water activity and high osmotic pressure [9–11]. Furthermore, the high concentrations of acetic acid during fermentations can also have a negative impact on the microbial consortium used [12,13]. These aforementioned environmental stresses can cause the microbial consortia to enter the viable but non-culturable state, resulting in reduced microbial activity [14,15]. Consequently, to counteract these eﬀects, cell immobilization is an ideal approach, which can minimize the impact of stressful conditions on the microorganisms used. g Various methods are used to perform cell immobilization. These include entrapment in a gel matrix, immobilization on a solid surface and mechanical containment behind a barrier [16]; however, to date, numerous studies focus on cell immobilization via gel entrapment or immobilization on a solid surface (adsorption) [5]. In Balsamic-styled vinegar fermentation, cell immobilization has previously been studied using the gel entrapment technique [17], which is generally an expensive technique. However, cell immobilization by adsorption in combination with aeration and agitation eﬀects has never been tested for balsamic vinegar production. 2.1. Preparation of Fermentation Medium The study commenced with the boiling of Chenin blanc grape must (22 ◦Brix) using a double jacketed steam pot (S.W.18, Aluminium Plant and Vessel Co. Ltd., London, UK) until a sugar concentration of 30 ◦Brix was obtained. The grape must was aliquoted into 3 L Erlenmeyer ﬂasks, covered with cotton wool stoppers, subsequent to autoclaving at 120 ◦C for 20 min. After autoclaving, the grape must was analyzed chemically for sugar (◦B), pH, alcohol (% v/v) and total acidity (g·L−1) using a density meter (Density meter DMA 35, Anton Paar, Graz, Austria), pH meter (Metrohm pH meter 632, Herisau, Switzerland), alcolyzer (Anton Paar, Graz, Austria) and minititrator (Hanna instruments minititrator HI 84502, Johannesburg, South Africa); respectively. 1. Introduction Therefore, in this current study, cell immobilization on corncobs and oak wood chips was investigated using the adsorption technique, which is commonly classiﬁed as a simple and often cost-eﬀective method depending on the support material used [8]. Corncobs are an inexpensive and abundant agricultural by-product obtained from the corn-milling process. They are currently used as animal feed, with 80% of their dry matter being comprised of cellulose and hemicellulose [18–22]. These aforementioned characteristics impart unique attributes that brand corncobs as an ideal material for cell immobilization. On the other hand, oak wood chips are abundantly used as an alternative to wooden barrel aging in the wine industry for economic reasons. They are known to improve the sensorial qualities of the ﬁnal product due to the ﬂavors they impart to wines [23–25]. For this reason, the current study selected oak wood chips, based on both their cell immobilization properties and potential impartation of organoleptic characteristics to the BSV. Nevertheless, oak wood chips are usually imported to South Africa and they cost more than corncobs. Consequently, this study investigated readily available inert materials which can be obtained at varying cost for cell immobilization, in order to allow producers to make an informed decision in achieving the desired BSV quality. Furthermore, the study explored the capabilities of the selected materials for cell aﬃnity to the adsorbents selected and fermenter performance in both aerated and agitated cultures systems. 2.2. Inoculum Preparation Cryopreserved non-Saccharomyces yeast (n = 5) (Table 1) and acetic acid bacteria (n = 5) (Table 2) cultures were used for this study. The yeast and bacteria cultures were individually grown in 400 mL 3 of 15 Foods 2019, 8, 303 YPD (Merck, Modderfontein, South Africa) and in 400 mL GM broth (glucose 0.8%, mannitol 1.7%, peptone 0.3%, yeast extract 0.5%) (Merck, Modderfontein, South Africa), respectively. The yeast and bacteria inoculums were incubated at 28 ◦C for 48 and 72 h respectively, prior to inoculation. Table 1. Non-Saccharomyces yeast used in the study. Non-Saccharomyces Yeast Identity ARC Accession Numbers Origin Candida pulcherrima Y0839 Chardonnay grapes Candida zemplinina Y1020 Chardonnay grapes Hanseniaspora guilliermondii Y0558 Cabernet Sauvignon grapes Kloeckera apiculata C48V19 Chardonnay grapes Zygosaccharomyces bailii C45V69 Chardonnay grapes ARC: Agricultural Research Council of South Africa. Table 1. Non-Saccharomyces yeast used in the study. Table 2. Acetic acid bacteria used in the study. Acetic Acid Bacteria Identity ARC Accession Numbers NCBI Accession Numbers Origin Acetobacter pasteurianus 171/19 CP 021922.1 Healthy grapes Acetobacter malorum 172/36 KU 686765.1 Shiraz wine Kozakia baliensis 179/48 CP 014681.1 Grape pomace Gluconobacter cerinus 126/34 KX 578017.1 Shiraz wine Gluconobacter oxydans 172/36 LN 884063.1 Grape pomace NCBI: National Center for Biotechnology Information. Table 2. Acetic acid bacteria used in the study. The non-Saccharomyces yeasts were selected based on a previous screening investigation. The yeast used in this study were selected due to high acid formation on calcium carbonate agar plates, desired aroma of the ﬁnal fermented product, osmophilic characteristics and the ﬁnal concentration of the alcohol produced. Similarly, acetic acid bacteria were obtained from previous isolation procedures on various sources; namely, grape pomace, healthy grapes and Shiraz wine. The bacteria used for this study were selected based on their ethanol oxidation rate when inoculated in diluted grape juice or their sugar utilization abilities in autoclaved grape juice. 2.5. Quantification of Cells Adsorbed on Corncobs and Oak Wood Chips Prior- and Post-Fermentation 2.5. Quantiﬁcation of Cells Adsorbed on Corncobs and Oak Wood Chips Prior- and Post-Fermentation 2.5. Quantification of Cells Adsorbed on Corncobs and Oak Wood Chips Prior- and Post-Fermentation 2.5. Quantiﬁcation of Cells Adsorbed on Corncobs and Oak Wood Chips Prior- and Post-Fermentation The number of cells adsorbed on the CC and OWC for individual yeasts and bacteria were quantified using the dry cell weight method adapted from Stone et al. [26] and Nguyen et al. [27], with minor modifications. Prior to this, the yeast and bacterial cell concentration (Table 3) in liquid suspension were individually quantified following the procedure described in Hutchinson et al. [28]. Furthermore, the yeast and bacteria were individually studied to assess the variations in cell adsorption capabilities. Yeast and bacteria were grown individually following the procedure described in Section 2.2. Subsequently, the yeast and bacteria cells were individually adsorbed onto the CC and OWC following the procedure described in Section 2.4. CC and OWC were removed from the broth and dried in an oven set at 40 °C. The adsorbents were weighed daily until a stationery The number of cells adsorbed on the CC and OWC for individual yeasts and bacteria were quantiﬁed using the dry cell weight method adapted from Stone et al. [26] and Nguyen et al. [27], with minor modiﬁcations. Prior to this, the yeast and bacterial cell concentration (Table 3) in liquid suspension were individually quantiﬁed following the procedure described in Hutchinson et al. [28]. Furthermore, the yeast and bacteria were individually studied to assess the variations in cell adsorption capabilities. Yeast and bacteria were grown individually following the procedure described in Section 2.2. Subsequently, the yeast and bacteria cells were individually adsorbed onto the CC and OWC following the procedure described in Section 2.4. CC and OWC were removed from the broth and dried in an oven set at 40 ◦C. The adsorbents were weighed daily until a stationery weight was reached. the broth and dried in an oven set at 40 C. The adsorbents were weighed daily until a stationery weight was reached. To assess the quantity of cells adsorbed post fermentation, the CC and OWC were transferred into grape must and allowed to ferment for 20 days. Subsequently, the adsorbents were extracted and dried at 40 °C until a stationery weight was reached. 2.5. Quantification of Cells Adsorbed on Corncobs and Oak Wood Chips Prior- and Post-Fermentation 2.5. Quantiﬁcation of Cells Adsorbed on Corncobs and Oak Wood Chips Prior- and Post-Fermentation To determine the number of cells adsorbed for To assess the quantity of cells adsorbed post fermentation, the CC and OWC were transferred into grape must and allowed to ferment for 20 days. Subsequently, the adsorbents were extracted and dried at 40 ◦C until a stationery weight was reached. To determine the number of cells adsorbed for the procedures, the diﬀerence of the weight of the adsorbents prior and post adsorption was computed. 2.3. Sterilization of Corncobs and Oak Wood Chips Corncobs (CC) were dried in the oven prior to cutting into smaller pieces using a cutting tool (sizes are shown in Table 3 and Figure 1). French oak wood chips (OWC) were used for this study. The OWC did not require any drying. Subsequently, the CC and OWC were separately transferred into 4 × 3 L Erlenmeyer ﬂasks prior to autoclaving at 121 ◦C for 20 min. Autoclaving was repeated (n = 2) to maximize sterility. Table 3. Size of corncobs and oak wood chips used in the study. Adsorbents Length (cm) Width/Diameter (cm) Circumference/ Perimeter (cm) Surface Area of One Piece (cm2) Surface Area of all Adsorbents Used (cm2) Corncobs 6.00 ± 1.05 4.00 ± 0.66 12.57 100.53 402 (4 pieces) Oak woodchips 2.90 ± 0.65 1.80 ± 0.53 9.60 19.64 392 (20 chips) Length and width results are the average of repeats ± standard deviations. Table 3. Size of corncobs and oak wood chips used in the study. Length and width results are the average of repeats ± standard deviations. 4 of 15 Foods 2019, 8, 303 Figure 1. Corncobs and oak wood chips used in the study, (A) corncobs after autoclaving (B) oak wood chips in cooked grape must. A B Figure 1. Corncobs and oak wood chips used in the study, (A) corncobs after autoclaving (B) oak wood chips in cooked grape must. A A B Figure 1. Corncobs and oak wood chips used in the study, (A) corncobs after autoclaving (B) oak wood chips in cooked grape must. Figure 1. Corncobs and oak wood chips used in the study, (A) corncobs after autoclaving (B) oak wood chips in cooked grape must. Yeast and bacteria were immobilized separately due to their difference in cell size. Furthermore, the differences in turbidity of the broth after yeast and bacteria cell growth was assumed to be a factor that might affect cell adsorption between the yeasts and bacteria. Yeast and bacteria were immobilized separately due to their diﬀerence in cell size. Furthermore, the diﬀerences in turbidity of the broth after yeast and bacteria cell growth was assumed to be a factor that might aﬀect cell adsorption between the yeasts and bacteria. 2.4. Cell Immobilization on Corncobs and Oak Wood Chips 2.4. Cell Immobilization on Corncobs and Oak Wood Chips 2.4. Cell Immobilization on Corncobs and Oak Wood Chips 2.4. Cell Immobilization on Corncobs and Oak Wood Chips After yeast and bacteria were fully-grown, the inoculums were mixed resulting in a 2 L consortium of yeast and bacteria (Tables 1 and 2) (NB: yeast and bacteria consortiums were initially in separate flasks). Subsequently, 1 L of the yeast consortium was transferred into 3 L Erlenmeyer flasks, one containing CC and another one containing OWC. The same procedure was performed using the bacterial consortium. The yeast and bacterial cells were allowed to adsorb onto the surface of CC and OWC (serving as a solid support surface/bed) overnight at 28 °C. After yeast and bacteria were fully-grown, the inoculums were mixed resulting in a 2 L consortium of yeast and bacteria (Tables 1 and 2) (NB: yeast and bacteria consortiums were initially in separate ﬂasks). Subsequently, 1 L of the yeast consortium was transferred into 3 L Erlenmeyer ﬂasks, one containing CC and another one containing OWC. The same procedure was performed using the bacterial consortium. The yeast and bacterial cells were allowed to adsorb onto the surface of CC and OWC (serving as a solid support surface/bed) overnight at 28 ◦C. 2.7. Data Handling Data was analyzed and computed using Microsoft Excel v2016 (Microsoft, Redmond, Washington, DC, USA). Substrate (S) (sugar) consumption rates and product (P) (acetic acid) formation rates were calculated using Equations (1) and (2) respectively, with an assumption that the initial reaction rate determines the overall fermentation rate. NB: change in time (t) was expressed as dt for both equations. Furthermore, relative diﬀerences as employed in other studies [28,29] were calculated using Equations (3) and (4) in order to show the signiﬁcance of the diﬀerences observed under the diﬀerent conditions studied. All results were the average of three biological repeats accounting for standard deviations which were calculated using Microsoft Excel v2016. rs = dS dt (1) rp = dP dt (2) (1) Absolute diﬀerence = Amount of increase −Reference amount Relative diﬀerence = Absolute diﬀerence Reference amount × 100 (4) Relative diﬀerence = Absolute diﬀerence Reference amount × 100 (4) (4) 2.6.2. Phase 2: Eﬀect of Aeration Air pumps (Resun® AC 9906, Longgang, Shenzhen, China) were used to sparge air into the Erlenmeyer ﬂasks at diﬀerent airﬂow rates, i.e., low aeration (LA = 0.15 vvm min−1) and high aeration (HA = 0.30 vvm min−1). Erlenmeyer ﬂasks were covered with loose cotton wool stoppers and fermentations were conducted in triplicate. 2.6.1. Phase 1: Static vs. Agitated Fermentations 2.6.1. Phase 1: Static vs. Agitated Fermentations Static and agitated fermentations were both incubated at 28 ◦C with agitated fermentations at 135 rpm. Agitated fermentations were conducted using an orbital shaker (FMH 200, FMH Instruments, Cape Town, South Africa). Under both static and agitated conditions, CC, OWC and FFC fermentations were conducted in triplicate. 3. Results and Discussions 3.1. Phase 1: Non-Aerated Fermentations 3.1.1. Static vs. Agitated Fermentations the procedures, computed 2.6. Inoculation p 2.6. Inoculation After the adsorption process, the OWC and CC were extracted from the broth and allowed to dry for 4 h. A 3 L fermentation volume was used for this study. Four pieces of corncobs (2 pieces yeast 2 pieces Acetic Acid Bacteria) (Table 2) were used for inoculation in one flask Twenty pieces After the adsorption process, the OWC and CC were extracted from the broth and allowed to dry for 4 h. A 3 L fermentation volume was used for this study. Four pieces of corncobs (2 pieces yeast, 2 pieces Acetic Acid Bacteria) (Table 2) were used for inoculation in one ﬂask. Twenty pieces of oak wood chips (Figure 1B) were used for inoculation in another ﬂask (10 pieces yeast, 10 pieces AAB) (Table 2). 5 of 15 Foods 2019, 8, 303 3.1.1. Static vs. Agitated Fermentations Furthermore, most vinegar production systems generally employ agitation and aeration simultaneously [34,35]; therefore, it is important to consider that agitation might only be beneﬁcial under such settings. 2A,D). However, ethanol formation/consumption rates were not calculated, since alcohol is an intermediate product/substrate which is produced and consumed simultaneously. Furthermore, acetic acid production was unsatisfactory for all treatments under agitated conditions (Figure 2F). Agitation resulted in acetification rates ranging from 0.11 to 0.13 g·L−1·day−1, with the highest acetic acid concentration of 10.7 g·L−1 being achieved at day 35. However, for a successful BSV fermentation, an acetic acid concentration of at least 50 g·L−1 is usually required [10]. Hutchinson et al. [17] made similar observations when using cell immobilization with calcium alginate beads for BSV production. It is unclear if these observations were species (acetic acid bacteria) dependent or due to the agitation settings used. In light of these observations, other vinegar studies have successfully applied agitation speeds between 100 [31] and 200 rpm [32], with industrial spirit vinegars successfully employing maximum agitation up to 900 rpm [30,33,34]. This suggested that the current agitation setting could not have negatively impacted the microbial activity in the fermentations. Furthermore, most vinegar production systems generally employ agitation and aeration simultaneously [34,35]; therefore, it is important to consider that agitation might only be beneficial under such settings. Figure 2C shows successful acetiﬁcation/alcohol oxidation proﬁles for all treatments under static fermentations. Total acid formation rates ranged from 1.46 and 2.70 g·L−1·day−1 for all of the treatments studied. The relative diﬀerences for total acid formation between agitated and static fermentations were 93%, 92% and 95% for FFC, OWC and CC, respectively. Furthermore, the low substrate (sugar) consumption rates (2.68–6.10 g·L−1·day−1) resulted in much lower alcohol formation rates (Figure 2B) but high total acid formation rates (Figure 2C). These observations could mean that the AAB microbial activity was ideal at low alcohol concentrations, suggesting that the agitation speed should be optimized with the aim of reducing alcohol formation rates, in order to beneﬁt the AAB used. Furthermore, if agitation is not suitable for BSV fermentations, regardless of the speed, this might beneﬁt producers, because agitation is energy intensive and the cost input might escalate, not only due to the energy usage but also due to the mechanical equipment required for agitation. p g g y g Figure 2C shows successful acetification/alcohol oxidation profiles for all treatments under static fermentations. 3.1.1. Static vs. Agitated Fermentations TBV fermentation is normally carried-out under static conditions using batteria. This process is slow and inexpensive [1,9]; however, a rapid fermentation period for TBV is not a fundamental objective, since TBV is matured for a minimum of 12 years after fermentation [1], while BSV can be sold without ageing. Rapid vinegar production is a major goal for industrial processes. BSV can also be produced rapidly if the conditions are ideal for the microorganisms used. For the purposes of this study, it was important to evaluate the eﬀects that agitation could have on BSV production in contrast to stationary fermentations. Agitation is also recommended for spirit industrial vinegar production to generally shorten the fermentation period [30]. In the current study, the sugar consumption rates for all treatments under agitated conditions ranged between 6.36 and 7.12 g·L−1·day−1 whereas 2.68 and 6.10 g·L−1·day−1 were observed under static conditions. The relative diﬀerences for sugar consumption between FFC and immobilized cells were calculated to be 14% (OWC and FFC) and 41% (CC and FFC) under static conditions, while the relative diﬀerences under agitated conditions were calculated to be 5% (OWC and FFC) and 9% (CC and FFC) under static conditions. The ethanol production step of the process was more eﬀective under agitated conditions, as compared to static conditions 6 of 15 6 f 1 Foods 2019, 8, 303 (Figure 2A,D). However, ethanol formation/consumption rates were not calculated, since alcohol is an intermediate product/substrate which is produced and consumed simultaneously. Furthermore, acetic acid production was unsatisfactory for all treatments under agitated conditions (Figure 2F). Agitation resulted in acetiﬁcation rates ranging from 0.11 to 0.13 g·L−1·day−1, with the highest acetic acid concentration of 10.7 g·L−1 being achieved at day 35. However, for a successful BSV fermentation, an acetic acid concentration of at least 50 g·L−1 is usually required [10]. Hutchinson et al. [17] made similar observations when using cell immobilization with calcium alginate beads for BSV production. It is unclear if these observations were species (acetic acid bacteria) dependent or due to the agitation settings used. In light of these observations, other vinegar studies have successfully applied agitation speeds between 100 [31] and 200 rpm [32], with industrial spirit vinegars successfully employing maximum agitation up to 900 rpm [30,33,34]. This suggested that the current agitation setting could not have negatively impacted the microbial activity in the fermentations. 3.1.1. Static vs. Agitated Fermentations Total acid formation rates ranged from 1.46 and 2.70 g·L−1·day−1 for all of the treatments studied. The relative differences for total acid formation between agitated and static fermentations were 93%, 92% and 95% for FFC, OWC and CC, respectively. Furthermore, the low substrate (sugar) consumption rates (2.68–6.10 g·L−1·day−1) resulted in much lower alcohol formation rates (Figure 2B) but high total acid formation rates (Figure 2C). These observations could mean that the AAB microbial activity was ideal at low alcohol concentrations, suggesting that the agitation speed should be optimized with the aim of reducing alcohol formation rates, in order to benefit the AAB used. Furthermore, if agitation is not suitable for BSV fermentations, regardless of the speed, this might benefit producers, because agitation is energy intensive and the cost input might escalate, not only due to the energy usage but also due to the mechanical equipment required for agitation. Static Fermentations Agitated Fermentations 100 150 200 250 300 350 0 10 20 30 40 Sugar (g.L-1) Time (days) A 100 150 200 250 300 350 0 10 20 30 40 Sugar (g.L-1) Time (days) D Figure 2. Cont. Static Fermentations g 100 150 200 250 300 350 0 10 20 30 40 Sugar (g.L-1) Time (days) D 100 150 200 250 300 350 0 10 20 30 40 Sugar (g.L-1) Time (days) A D A Figure 2. Cont. Foods 2019, 8, 303 Foods 2019, 8, x F 7 of 15 7 of 15 0 7 of 15 7 of 15 0 0 20 40 60 80 100 120 0 10 20 30 40 Alcohol (g.L-1) Time (days) B 0 20 40 60 80 100 120 0 10 20 30 40 (g ) Time (days) B Alcohol (g.L-1) 0 10 20 30 40 Time (days) B 0 20 40 60 80 100 120 Alcohol (g.L-1) Figure 2. Chemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), alcohol (B,E) and total acid (C,F) developments during fermentation. Oak wood chips, Corncobs, Free-floating cells. accounting for standard deviatio 2 Effect of the Adsorbents Used ffect of the Adsorbents Used 3.1.2. Eﬀect of the Adsorbents Used Additional reasons attributed to the uccessful agitated fermentations, which were characterized by low acetification rates (0.11 to 0.13 −1·day−1) may be due to the interference of cell adsorption by agitation conditions, which eventually ulted in freely suspended cells Since agitate achieving 60 g·L− fermentations tha unsuccessful agita g·L−1·day−1) may b resulted in freely Moderately y and surface chemistry. nce agitated fermentations did not meet the required performance for BSV production, i.e., ng 60 g·L−1 acetic acid, a more comprehensive discussion is provided in this section on the tations that were only conducted under static conditions. Additional reasons attributed to the essful agitated fermentations, which were characterized by low acetification rates (0.11 to 0.13 ay−1) may be due to the interference of cell adsorption by agitation conditions, which eventually d in freely suspended cells Since agitated fermentations did not meet the required performance for BSV production, i.e., achieving 60 g·L−1 acetic acid, a more comprehensive discussion is provided in this section on the fermentations that were only conducted under static conditions. Additional reasons attributed to the unsuccessful agitated fermentations, which were characterized by low acetiﬁcation rates (0.11 to 0.13 g·L−1·day−1) may be due to the interference of cell adsorption by agitation conditions, which eventually resulted in freely suspended cells. g day ) ay be due to t e i te e e ce o ce adso ptio by agitatio co ditio s, w ic eve tua y resulted in freely suspended cells. Moderately higher sugar consumption rates of 8.14 and 5.14 g·L−1·day−1 for OWC and CC respectively, were observed at the initial stages (day 0–14) of the fermentation process (Figure 2A) Alcohol formation rates were also moderately higher for CC and OWC fermentation than FFC fermentations (Figure 2B). While, total acid formation for cells immobilized on CC was consistently higher throughout the fermentation process, with total acid formation rates of 2.7 g·L−1·day−1 being achieved in the shortest fermentation period (20 days). Comparatively, FFC and OWC fermentation resulted in lower total acid formation rates of 1.64 and 1.46 g·L−1·day−1 with a longer fermentation period of 33 and 37 days, respectively. Furthermore, the relative differences between FFC and ulted in freely suspended cells. Moderately higher sugar consumption rates of 8.14 and 5.14 g·L−1·day−1 for OWC and CC, pectively, were observed at the initial stages (day 0–14) of the fermentation process (Figure 2A). accounting for standard deviatio 2 Effect of the Adsorbents Used ffect of the Adsorbents Used 3.1.2. Eﬀect of the Adsorbents Used 3.1.2. Effect of the Adsorbents Used The adsorption technique for cell immobilization has been studied using several inert material for various vinegar production systems [31,32,36,37]. In the current study, CC were compared t OWC for cell affinity and influence on BSV production. These materials differ in terms of thei biological composition as they have different physical and structural attributes, as well as adsorptio The adsorption technique for cell immobilization has been studied using several inert materials various vinegar production systems [31,32,36,37]. In the current study, CC were compared to WC for cell affinity and influence on BSV production. These materials differ in terms of their ogical composition as they have different physical and structural attributes, as well as adsorption acity and surface chemistry The adsorpti for various vineg OWC for cell aff biological compo capacity and surf Since agitate he adsorption technique for cell immobilization has been studied using several inert materials ious vinegar production systems [31,32,36,37]. In the current study, CC were compared to for cell affinity and influence on BSV production. These materials differ in terms of their cal composition as they have different physical and structural attributes, as well as adsorption y and surface chemistry The adsorption technique for cell immobilization has been studied using several inert materials for various vinegar production systems [31,32,36,37]. In the current study, CC were compared to OWC for cell aﬃnity and inﬂuence on BSV production. These materials diﬀer in terms of their biological composition as they have diﬀerent physical and structural attributes, as well as adsorption capacity and surface chemistry. biological composition as they have different physical and structural attributes, as well as adsorption capacity and surface chemistry. Since agitated fermentations did not meet the required performance for BSV production, i.e achieving 60 g·L−1 acetic acid, a more comprehensive discussion is provided in this section on th fermentations that were only conducted under static conditions. Additional reasons attributed to th unsuccessful agitated fermentations, which were characterized by low acetification rates (0.11 to 0.1 g·L−1·day−1) may be due to the interference of cell adsorption by agitation conditions which eventually acity and surface chemistry. Since agitated fermentations did not meet the required performance for BSV production, i.e., ieving 60 g·L−1 acetic acid, a more comprehensive discussion is provided in this section on the mentations that were only conducted under static conditions. 3.1.1. Static vs. Agitated Fermentations Results are the average of three biological repeats i f d d d i i 0 20 40 60 80 100 120 0 10 20 30 40 Alcohol (g.L-1) Time (days) B 0 20 40 60 80 100 120 0 20 40 Alcohol (g.L-1) Time (days) E 0 20 40 60 0 10 20 30 40 Total acidity (g.L-1) Time (days) C 0 20 40 60 0 20 40 Total acidity (g.L-1) Time (days) F Figure 2. Chemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), alcohol (B,E) and total acid (C,F) developments during fermentation. igure 2. Chemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), lcohol (B,E) and total acid (C,F) developments during fermentation. Oak wood chips, Corncobs, Free-floating cells. Results are the average of three biological repeats ccounting for standard deviation. 0 20 40 60 80 100 120 0 10 20 30 40 Alcohol (g.L ) Time (days) B 0 20 40 60 80 100 120 0 20 40 Alcohol (g.L-1) Time (days) E 0 20 40 60 0 10 20 30 40 Total acidity (g.L-1) Time (days) C 0 20 40 60 0 20 40 Total acidity (g.L-1) Time (days) F Oak wood chips, Figure 2. alcohol (B C accountin 3 1 2 Effect of 0 20 40 60 80 100 0 Alcohol (g.L-1) 0 20 40 60 0 Total acidity (g.L-1) Corncobs, re 2. Chemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), ol (B,E) and total acid (C,F) developments during fermentation. Oak wood chips, Corncobs, Free-floating cells. Results are the average of three biological repeats unting for standard deviation. 0 0 0 0 0 0 0 0 10 20 30 40 Time (days) B 0 20 40 60 80 100 120 0 20 40 Alcohol (g.L-1) Time (days) E 0 0 0 0 0 10 20 30 40 Time (days) C 0 20 40 60 0 20 40 Total acidity (g.L-1) Time (days) F Free-ﬂoating cells. Results are the average of three biological repeats accounting for standard deviation. 3.1.1. Static vs. Agitated Fermentations 0 20 40 60 80 100 120 0 20 40 Alcohol (g.L-1) E 0 20 40 60 80 00 20 0 20 40 Time (days) E 0 20 40 60 80 100 0 Alcohol (g.L-1) 0 20 40 Time (days) E B B E 0 20 40 60 0 10 20 30 40 Total acidity (g.L-1) Time (days) C 0 0 0 0 0 10 20 30 40 Time (days) C Total acidity (g.L-1) 10 20 30 40 Time (days) C 0 20 40 60 Total acidity (g.L-1) 0 20 40 60 0 20 40 Total acidity (g.L-1) Time (days) F 0 0 0 0 0 20 40 Time (days) F Figure 0 20 40 60 Total acidity (g.L-1) 20 40 Time (days) F F Figure 2. Chemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D) alcohol (B,E) and total acid (C,F) developments during fermentation. Oak wood chips Corncobs, Free-floating cells. Results are the average of three biological repeat Figure 2. Chemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), alcohol (B,E) and total acid (C,F) developments during fermentation. ure 2. Chemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), hol (B,E) and total acid (C,F) developments during fermentation. Oak wood chips, Corncobs, Free-floating cells. Results are the average of three biological repeats ounting for standard deviation. Oak wood chips, alcohol ( C accountin 3 1 2 Eff Corncobs, 2. Chemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), (B,E) and total acid (C,F) developments during fermentation. Oak wood chips, Corncobs, Free-floating cells. Results are the average of three biological repeats ting for standard deviation. Free-ﬂoating cells. Results are the average of three biological repeats accounting for standard deviation. accounting for standard deviatio 2 Effect of the Adsorbents Used Effect of the Adsorbents Used 3.1.2. Eﬀect of the Adsorbents Used accounting for standard deviatio 2 Effect of the Adsorbents Used ffect of the Adsorbents Used 3.1.2. Eﬀect of the Adsorbents Used ohol formation rates were also moderately higher for CC and OWC fermentation than FFC mentations (Figure 2B). While, total acid formation for cells immobilized on CC was consistently her throughout the fermentation process, with total acid formation rates of 2.7 g·L−1·day−1 being ieved in the shortest fermentation period (20 days). Comparatively, FFC and OWC fermentations ulted in lower total acid formation rates of 1.64 and 1.46 g·L−1·day−1 with a longer fermentation iod of 33 and 37 days, respectively. Furthermore, the relative differences between FFC and mobilized cells were 11% (OWC and FFC) and 39% (CC and FFC) Therefore, it was deduced that o e a e y respectively, were Alcohol formatio fermentations (Fig higher throughou achieved in the sh resulted in lower period of 33 and immobilized cells d in freely suspended cells. oderately higher sugar consumption rates of 8.14 and 5.14 g·L−1·day−1 for OWC and CC, ively, were observed at the initial stages (day 0–14) of the fermentation process (Figure 2A). l formation rates were also moderately higher for CC and OWC fermentation than FFC tations (Figure 2B). While, total acid formation for cells immobilized on CC was consistently throughout the fermentation process, with total acid formation rates of 2.7 g·L−1·day−1 being ed in the shortest fermentation period (20 days). Comparatively, FFC and OWC fermentations d in lower total acid formation rates of 1.64 and 1.46 g·L−1·day−1 with a longer fermentation of 33 and 37 days, respectively. Furthermore, the relative differences between FFC and ilized cells were 11% (OWC and FFC) and 39% (CC and FFC). Therefore, it was deduced that Moderately higher sugar consumption rates of 8.14 and 5.14 g·L−1·day−1 for OWC and CC, respectively, were observed at the initial stages (day 0–14) of the fermentation process (Figure 2A). Alcohol formation rates were also moderately higher for CC and OWC fermentation than FFC fermentations (Figure 2B). While, total acid formation for cells immobilized on CC was consistently higher throughout the fermentation process, with total acid formation rates of 2.7 g·L−1·day−1 being achieved in the shortest fermentation period (20 days). Comparatively, FFC and OWC fermentations resulted in lower total acid formation rates of 1.64 and 1.46 g·L−1·day−1 with a longer fermentation period of 33 and 37 days, respectively. Furthermore, the relative diﬀerences between FFC and immobilized cells were 11% (OWC and FFC) and 39% (CC and FFC). accounting for standard deviatio 2 Effect of the Adsorbents Used ffect of the Adsorbents Used 3.1.2. Eﬀect of the Adsorbents Used A comparison of the performance of immobilized cells in other studies. Table 4 also shows that agitation speeds of 100 rpm, including reciprocating shaking speed of 60 rpm were employed eﬀectively in other studies. These ﬁndings illustrate that agitation may also be dependent on several other factors, such as the microorganisms used, the type of vinegar being produced and other fermentor conditions. 3.2. Phase 2: Aerated Fermentations accounting for standard deviatio 2 Effect of the Adsorbents Used ffect of the Adsorbents Used 3.1.2. Eﬀect of the Adsorbents Used Therefore, it was deduced that Foods 2019, 8, 303 8 of 15 cells adsorbed on CC fermentations led to the highest microbial activity because of the adsorbents’ attributes, i.e., rough surface and porosity, which improved the adsorption of cells. As a result, the microbial consortium was able to form a highly eﬀective community when adsorbed onto the CC. Contrarily, the OWC are relatively smoother than the porous CC, which consequently led to the poor adsorption of cells. Cell immobilization on CC was also studied for the production of tea vinegar, with acetiﬁcation rates of 2.88 g·L−1·day−1 being obtained [38]. Several studies often use wood shavings for cell immobilization instead of OWC. For example, Thiripurasundari and Usharani [39] and Kocher and Dhillon [40] reported acetiﬁcation rates of 0.24 g·L−1·day−1 and 5.76–22.8 g·L−1·day−1 when using wood shavings for the production of cashew apple vinegar and sugar cane vinegar, respectively. It is not clear as to the reasons why the acetiﬁcation rates for these two studies were diﬀerent; however, other factors such as substrate availability, bacteria strain used and other fermentor conditions might have played a role. Furthermore, wood shavings and OWC may not be comparable materials due to their diﬀerentiated physical properties, and thus, diﬀerentiated adsorption capabilities. Table 4 shows a comparison of other studies which investigated the eﬀect of cell immobilization by adsorption in other vinegar production systems. The current work established a direct relationship between cell immobilization and higher acetiﬁcation rates. Similarly, other studies [41] also reported a proportional relationship between cell immobilization and acetiﬁcation rates, with the ﬁbrous bed being reported to show a conspicuous increase in acetiﬁcation compared to FFC (Table 4). Furthermore, when a loofa sponge was used, it led to increased acetiﬁcation rates [42] which were similar to the rates observed in the current study at static conditions. Table 4. A comparison of the performance of immobilized cells in other studies. Product Adsorbent Agitation Acetiﬁcation Rate (g·L−1·day−1) Reference Balsamic-styled vinegar Corncobs Oak wood chips 135 rpm FFC: 0.11 CC: 0.13 OWC: 0.11 Current study Acetic acid from lactose and milk permeate Fibrous-Bed/matrix 100 rpm FFC (lactose): 1.44 FFC (milk permeate): 1.92 IC (Lactose): 12.96 IC (milk permeate) 7.2 [31] Rice wine vinegar Loofa sponge 1 Hz reciprocating shaking rate = 60 rpm IC: 1.68–2.4 [42] IC: immobilized cells; CC: Corncobs; FFC: free-ﬂoating cell; OWC: oak wood chips. Table 4. 3.2. Phase 2: Aerated Fermentations 3.2.1. Eﬀect of Aeration Rates Aeration is generally a fundamental aspect in most vinegar fermentation systems [43,44]. Aeration systems vary between transferring pure oxygen or air, with pure oxygen systems being noted as expensive due to the cost of technical grade oxygen [45] and deleterious eﬀects such as hyperoxia which leads to cellular death. For this study, aeration with air only was investigated as it was deemed cost eﬀective. It was observed that both high aeration (HA) and low aeration (LA) led to similar alcohol formation/consumption proﬁles (Figure 3B,E). The observations were surprising since alcoholic fermentation is often performed under anaerobic conditions or low dissolved oxygen conditions [46,47]. Consequently, yeast performance was expected to also be negatively aﬀected by HA, albeit that 9 of 15 rising xygen Foods 2019, 8, 303 to similar alcoh l h l f yeasts are generally characterized as facultative anaerobes and some yeasts can still survive or grow under aerobic conditions [48–51]. Since only air (with 21% oxygen) was used, it was also plausible that diﬀerent alcohol consumption proﬁles could have been observed if technical grade oxygen was tested—a condition that can exacerbate hyperoxia. conditions [46,47]. Consequently, yeast performance was expected to also be negatively affected by HA, albeit that yeasts are generally characterized as facultative anaerobes and some yeasts can still survive or grow under aerobic conditions [48–51]. Since only air (with 21% oxygen) was used, it was also plausible that different alcohol consumption profiles could have been observed if technical grade oxygen was tested—a condition that can exacerbate hyperoxia. under aerobic conditions [48–51]. Since only air (with 21% oxygen) was used, it was also plausible that diﬀerent alcohol consumption proﬁles could have been observed if technical grade oxygen was tested—a condition that can exacerbate hyperoxia. urvive or grow under aerobic conditions [48–51]. Since only air (with 21% oxygen) was used, it was lso plausible that different alcohol consumption profiles could have been observed if technical grade oxygen was tested—a condition that can exacerbate hyperoxia. 3.2.1. Eﬀect of Aeration Rates Low Aeration High Aeration 0 50 100 150 200 250 300 350 0 10 20 30 40 50 Sugar (g.L-1) Time (days) A 0 50 100 150 200 250 300 350 0 10 20 30 40 50 Sugar (g.L-1) Time (days) D OR PEER REVIEW 7 of 15 Foods 2019, 8, x FOR PEER RE PEER REVIEW 7 of 15 High Aeration Low Aeration Low Aeration 0 50 100 150 200 250 300 350 0 10 20 30 40 50 Sugar (g.L-1) Time (days) A EER REVIEW R REVIEW 0 50 100 150 200 250 300 350 0 10 20 30 40 50 Sugar (g.L-1) Time (days) D 7 of 15 Foods 2019, 8, x FOR PEER RE 7 of 15 D A Time (days) 7 of 15 7 of 15 0 20 40 60 0 10 20 30 40 50 Alcohol (g.L-1) Time (days) B 10 20 30 40 Ti (d ) B 0 20 40 60 80 100 120 0 Alcohol (g.L-1) 0 20 30 40 Ti (d ) B 0 20 40 60 80 100 120 0 Alcohol (g.L-1) 0 20 40 60 0 10 20 30 40 50 Alcohol (g.L-1) Time (days) E 20 40 Time (days) E 0 20 40 60 80 100 0 10 Alcohol (g.L-1) T 20 40 Time (days) E B E L-1) 0 20 40 60 0 10 20 30 40 50 Total acidity (g.L-1) Time (days) C 10 20 30 40 Time (days) C 0 20 40 60 0 Total acidity (g.L-1) 0 20 30 40 Time (days) C 0 20 40 60 0 Total acidity (g.L-1) 0 20 40 60 0 10 20 30 40 50 Total acidity (g.L-1) Time (days) F 20 40 Time (days) F Figure 2 Chemical d 0 20 40 60 0 10 Total acidity (g.L-1) T 20 40 Time (days) F Figure 3. Chemical developments under low (A–C) and high (D–F) aeration. Sugar (A,D), alcohol (B,E) and total acid (C,F) developments during fermentation. mical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), nd total acid (C,F) developments during fermentation. Oak wood chips, bs, Free-floating cells. Results are the average of three biological repeats t d d d i ti Oak wood chips, alcohol (B,E) and t Corncobs, accounting for stand Corncobs, l developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), total acid (C,F) developments during fermentation. 3.2.1. Eﬀect of Aeration Rates Low Aeration High Aeration 0 50 100 150 200 250 300 350 0 10 20 30 40 50 Sugar (g.L-1) Time (days) A 0 50 100 150 200 250 300 350 0 10 20 30 40 50 Sugar (g.L-1) Time (days) D 0 20 40 60 0 10 20 30 40 50 Alcohol (g.L-1) Time (days) B 0 20 40 60 0 10 20 30 40 50 Alcohol (g.L-1) Time (days) E 0 20 40 60 0 10 20 30 40 50 Total acidity (g.L-1) Time (days) C 0 20 40 60 0 10 20 30 40 50 Total acidity (g.L-1) Time (days) F Figure 3. Chemical developments under low (A–C) and high (D–F) aeration. Sugar (A,D), alcohol (B,E) and total acid (C,F) developments during fermentation. R PEER REVIEW 7 of 15 hemical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), E) and total acid (C,F) developments during fermentation. Oak wood chips, rncobs, Free-floating cells. Results are the average of three biological repeats for standard deviation. 10 20 30 40 Time (days) B 0 20 40 60 80 100 120 0 20 40 Alcohol (g.L-1) Time (days) E 10 20 30 40 Time (days) C 0 20 40 60 0 20 40 Total acidity (g.L-1) Time (days) F Oak wood chips, Foods 2019, 8, x FOR PEER REVI Figure 2. Chemical de alcohol (B,E) and tota Corncobs, accounting for standard 3.1.2. Effect of the Adsorb 0 20 40 60 80 100 120 0 10 Alcohol (g.L-1) Tim 0 20 40 60 0 10 Total acidity (g.L-1) Tim Corncobs, EER REVIEW 7 of 15 mical developments under static (A–C) and agitated (D–F) conditions. Sugar (A,D), and total acid (C,F) developments during fermentation. Oak wood chips, obs, Free-floating cells. Results are the average of three biological repeats standard deviation. 10 20 30 40 Time (days) B 0 20 40 60 80 100 120 0 20 40 Alcohol (g.L-1) Time (days) E 10 20 30 40 Time (days) C 0 20 40 60 0 20 40 Total acidity (g.L-1) Time (days) F Free-ﬂoating cells. Results are the average of three biological repeats ± standard deviation. 3.2.2. Performance of Adsorbents Used Under Aerated Conditions When cells adsorbed on the diﬀerent materials were evaluated at the initial stages of fermentation, sugar consumption showed similar proﬁles with sugar consumption rates between 20.15 to 27.2 g·L−1.day−1 under both aeration (HA and LA) settings for all treatments (Figure 3A,D). After 7 days, sugar consumption at LA was higher for OWC fermentations with rates between 3.32 g·L−1·day−1 followed by FFC and CC with a sugar consumption rate of 1.80 and 0.11 g·L−1·day−1 respectively (Figure 3A). On the other hand, HA, CC and OWC fermentations led to relatively similar results, as well as the highest sugar consumption rates (2.70–3.46 g·L−1·day−1) compared to FFC (1.28 g·L−1·day−1) (Figure 3D). The lowest sugar consumption rates were observed in FFC fermentations under HA with trends that we cannot compare with any previously available literature. However, it is possible that the activity of freely suspended yeast cells under HA was reduced by AAB FFC that eventually led to a higher production of acetic acid. Overall, sugar consumption rates were 4.14, 5.78 and 7.4 g·L−1·day−1 under LA and 7.62, 4.82 and 7.23 under HA for CC, FFC and OWC, respectively. The relative diﬀerences for sugar consumption between LA and HA fermentations were 2%, 17% and 46% for OWC, FFC and CC; respectively. Furthermore, alcoholic fermentation was successful on all immobilized and FFC fermentations for both aeration settings. Alcohol formation/consumption proﬁles were moderately similar for all treatments studied under both aeration settings. Total acid formation provided an insight on the productivity of the process evaluated. It was evident, that CC fermentations had the highest total acid formation at LA, followed by FFC and then OWC fermentations. The diﬀerences observed were minor, since the total formation rates were 1.56, 1.46 and 1.42 g·L−1·day−1 with a fermentation period of 31, 33 and 34 days for CC, FFC and OWC fermentations, respectively. Under HA, only FFC fermentations completed the fermentation with an acetiﬁcation rate of 1.2 g·L·day−1 in 38 days, while CC and OWC fermentations initiated by showing an increase in total acid subsequent to its decrease from day 14 to 42. It was not well understood as to how the FFC fermentations were able to reach the required levels of total acid concentration. A credible assumption could be that HA may have disrupted the adsorbed cells, thus causing and maintaining a low microbial activity. Foods 2019, 8, 303 10 of 15 Nevertheless, total acid formation, which is a key step in BSV production was higher (1.42–1.56 g·L−1·day−1) at LA (Figure 3C) as compared to HA (0.14–1.2 g·L−1·day−1) (Figure 3F) for cell immobilized treatments. However, FFC completed the fermentations under both aeration settings with acetiﬁcation rates of 1.27 and 1.46 g·L−1·day−1 for HA and LA, respectively. Additionally, over oxidation has been reported as a common challenge in most vinegar production systems [10]. It seems unlikely that the excessive/increased oxidation rates may have aﬀected the fermentations in this study; since the alcohol proﬁles under HA and LA conditions (Figure 3B,E) were similar to some degree. Other studies reported higher acetiﬁcation rates compared to the current study when aeration was employed. Rubio-Fernández et al. [44] reported acetiﬁcation rates of 17.24 and 32.4 g·L−1·day−1 for wine vinegar production when air and oxygen rich air (0.06 vvm) were used respectively. Additionally, Qi et al. [52] reported acetiﬁcation rates of 43.44 g·L−1·day−1 for industrial vinegar production when oxygen (0.13 vvm m−1) was used. It is important to mention that the observations between the current study and other studies are inﬂuenced by several factors, which include media composition and the AAB strains used. The most obvious inﬂuential factor is that BSV fermentation involves high sugar concentration/high osmotic pressures with the simultaneous involvement of non-Saccharomyces yeasts and AAB. Furthermore, spirit vinegar production is generally faster than the production of other vinegars. It is unfortunate that there are no similar BSV studies with which to compare the current data, due to the traditional techniques used for Balsamic vinegar production. 3.2.1. Eﬀect of Aeration Rates Oak wood chips, Free-floating cells. Results are the average of three biological repeats d d d i ti Free-ﬂoating cells. Results are the average of three biological repeats ± standard deviation. Foods 2019, 8, 303 3.3.1. Individual Yeast Adsorption on Corncobs and Oak Wood Chips 3.3.1. Individual Yeast Adsorption on Corncobs and Oak Wood Chips The phenomenon of adsorption eﬃciency is critical to the current study. The adsorption by diﬀerent microorganisms varied due to cell aﬃnity diﬀerences to the adsorbents surface used. However, it is possible that microorganisms employed will adsorb diﬀerently when tested as a consortium. With regards to yeast adsorption on CC, it was observed that C. zemplinina had the lowest number of cells adsorbed prior and post fermentation. Z. bailii initially exhibited low cell adsorption before fermentation; however, a relatively higher cell adsorption (92.93%) after fermentation was observed. Yeasts species such as C. pulcherrima, H. guilliermondii and K. apiculata showed a noticeably high cell adsorption prior and post fermentation (Table 5). Table 5. Evaluation of yeast cells adsorbed on corncobs and oak wood chips. Yeast Cells Adsorbed on Corncobs Yeast Cells Adsorbed on Oak Wood Chips Identity Cell Concentration YPD Broth (Cells·mL−1) Before Fermentation (g·g−1) After Fermentation (g·g−1) Relative Diﬀerence (%) Before Fermentation (g·g−1) After Fermentation (g·g−1) Relative Diﬀerence (%) Candida pulcherrima 2.34 × 105 0.0371 1.1580 96.80 0.0214 0.1751 87.78 Candida zemplinina 7.40 × 105 0.0006 0.0290 97.93 0.0903 0.0566 −37.32 Hanseniaspora guilliermondii 7.70 × 105 0.0110 0.7906 98.61 0.1001 0.1032 3.00 Kloeckera apiculata 1.95 × 106 0.0283 1.6955 98.33 0.0926 0.1804 48.67 Zygosaccharomyces bailii 3.97 × 105 0.0400 0.5661 92.93 0.1785 0.1393 −21.96 Table 5. Evaluation of yeast cells adsorbed on corncobs and oak wood chips. Furthermore, yeast cell adsorption was diﬀerent for both OWC and CC. Some yeast species such as C. zemplinina (59.54%) and Z. bailii (28%) showed a relative decrease in cell adsorption post fermentation (Table 5). The trends showed that C. zemplinina and Z. bailii might have low cell aﬃnity to the smooth ‘easy-to wash-oﬀ’ OWC surface during the fermentation compared to CC surfaces. On the other hand, C. pulcherrima (87.78%), H. guilliermondii (3.00%) and K. apiculata (48.67%) showed a relative increase in cell adsorption capacity during the fermentation, with H. guilliermondii showing the lowest adsorption. Overall, the cells adsorbed (g·g−1) on CC and OWC were not comparable since they have diﬀerent densities, including surface chemistry properties. 3.3.2. Individual Bacteria Adsorption on Corncobs and Oak Wood Chips 3.3. Variations in Cell Adsorption Capabilities among the Yeast/Bacterial Species Used 3.3. Variations in Cell Adsorption Capabilities among the Yeast/Bacterial Species Used 3.3. Variations in Cell Adsorption Capabilities among the Yeast/Bacterial Species Used 3.2.2. Performance of Adsorbents Used Under Aerated Conditions Overall, based on the results obtained, it was evident that agitation and HA did not favor the production of BSV, where cell immobilization by adsorption was used. Foods 2019, 8, 303 11 of 15 3.3. Variations in Cell Adsorption Capabilities among the Yeast/Bacterial Species Used 4. Conclusions According to all the results obtained, it is evident that cell immobilization improves acetiﬁcation rates during BSV fermentation. However, the highest acetiﬁcation rates were obtained under static and non-aerated conditions. Corncobs were observed to be the most suitable material for cell immobilization presumably due to their physical structure. Consequently, the shortest fermentation period was 20 days when cells were immobilized on corncobs under static fermentation conditions. A study to investigate the concurrent optimization of agitation and aeration as well as the eﬀect of corncobs and oak wood chips on organoleptic properties is recommended. The current study therefore, serves as a foundation for cell immobilization by adsorption on materials during balsamic-styled vinegar production. Author Contributions: Conceptualization, U.F.H. and N.P.J.; methodology, U.F.H., S.G., B.S.C., N.P.J., M.M.-N. and H.W.D.P.; formal analysis, U.F.H., M.M.-N., N.P.J., B.S.C., H.W.D.P., and S.K.O.N.; investigation, U.F.H., S.G. and M.M.-N.; resources, N.P.J., H.W.D.P. and S.K.O.N.; data curation, U.F.H., S.G., B.S.C., H.W.D.P. and S.K.O.N.; writing—original draft preparation, U.F.H., review and editing, U.F.H., S.G., N.P.J., B.S.C., H.W.D.P., M.M.-N., and S.K.O.N.; Supervision, N.P.J., H.W.D.P. and S.K.O.N.; funding acquisition, N.P.J. and S.K.O.N. Author Contributions: Conceptualization, U.F.H. and N.P.J.; methodology, U.F.H., S.G., B.S.C., N.P.J., M.M.-N. and H.W.D.P.; formal analysis, U.F.H., M.M.-N., N.P.J., B.S.C., H.W.D.P., and S.K.O.N.; investigation, U.F.H., S.G. and M.M.-N.; resources, N.P.J., H.W.D.P. and S.K.O.N.; data curation, U.F.H., S.G., B.S.C., H.W.D.P. and S.K.O.N.; writing—original draft preparation, U.F.H., review and editing, U.F.H., S.G., N.P.J., B.S.C., H.W.D.P., M.M.-N., and S.K.O.N.; Supervision, N.P.J., H.W.D.P. and S.K.O.N.; funding acquisition, N.P.J. and S.K.O.N. Funding: Financial support was provided by the Agricultural Research Council (ARC) and the Cape Peninsula University of Technology (South Africa). Funding: Financial support was provided by the Agricultural Research Council (ARC) and the Cape Peninsula University of Technology (South Africa). Acknowledgments: The authors would acknowledge the Bioresource Engineering Research Group (BioERG), Department of Biotechnology, Cape Peninsula University of Technology (RK 16) and the Agricultural Research Council (ARC). Acknowledgments: The authors would acknowledge the Bioresource Engineering Research Group (BioERG), Department of Biotechnology, Cape Peninsula University of Technology (RK 16) and the Agricultural Research Council (ARC). Conﬂicts of Interest: The authors hereby declare that they have no conﬂict of interest. 3.3.2. Individual Bacteria Adsorption on Corncobs and Oak Wood Chips However, the data also displayed an unstable cell adsorption proﬁle on OWC, since a decrease in cell adsorption was observed for some yeast and bacterial species. The data further suggested a more sustainable approach on the reusability of immobilized cells on CC and OWC treatments. Corncobs Bacteria Cells Adsorbed on Oak Wood Chips Overall, these results showed a successful adsorption on CC and OWC for both yeasts and bacteria, but with varying adsorption eﬃciency. However, the data also displayed an unstable cell adsorption proﬁle on OWC, since a decrease in cell adsorption was observed for some yeast and bacterial species. The data further suggested a more sustainable approach on the reusability of immobilized cells on CC and OWC treatments. Overall, these results showed a successful adsorption on CC and OWC for both yeasts and bacteria, but with varying adsorption eﬃciency. However, the data also displayed an unstable cell adsorption proﬁle on OWC, since a decrease in cell adsorption was observed for some yeast and bacterial species. The data further suggested a more sustainable approach on the reusability of immobilized cells on CC and OWC treatments. 3.3.2. Individual Bacteria Adsorption on Corncobs and Oak Wood Chips The adsorption of bacteria onto CC and OWC was also evaluated (Table 6). A. pasteurianus and A. malorum appeared to have the highest (0.1461 and 0.095 g·g−1, respectively) cell adsorption eﬃciency on CC before fermentation (Table 6). K. baliensis, G. cerinus and G. oxydans had relatively similar adsorption eﬃciency on CC before fermentation, with relatively low variations (97.76%, 99.26%, 96.12%, respectively) observed post fermentation. Similar decreases in adsorption eﬃciency patterns on OWC were observed between A. malorum and K. baliensis (Table 6). A. pasteurianus had the lowest initial cell adsorption (0.0020 g·g−1) before fermentation; however, it had the highest percentage cell increase (98.87%) after fermentation. G. cerinus and G. oxydans showed a similar pattern before and after fermentation, with 52.40% and 56.33% in cell adsorption after fermentation. Other studies have investigated the cell adsorption on CC and wood shavings; however, these studies did not report the number of cells adsorbed on the adsorbents used, which leads to the lack of comparative data. 12 of 15 Foods 2019, 8, 303 Table 6. Evaluation of bacterial cells adsorbed on corncobs and oak wood chips. Table 6. Evaluation of bacterial cells adsorbed on corncobs and oak wood chips. Bacteria Cells Adsorbed on Corncobs Bacteria Cells Adsorbed on Oak Wood Chips Identity Cell Concentration GM Broth (Cells·mL−1) Before Fermentation (g·g−1) After Fermentation (g·g−1) Relative Diﬀerence (%) Before Fermentation (g·g−1) After Fermentation (g·g−1) Relative Diﬀerence (%) Acetobacter pasteurianus 3.97 × 105 0.1461 1.1968 87.79 0.0020 0.1772 98.87 Acetobacter malorum 1.25 × 106 0.0948 1.0882 91.29 0.1851 0.1545 −16.53 Kozakia baliensis 3.13 × 105 0.0260 1.1589 97.76 0.1224 0.1122 −8.33 Gluconobacter cerinus 2.22 × 106 0.0116 1.5618 99.26 0.1498 0.3147 52.40 Gluconobacter oxydans 6.38 × 105 0.0426 1.0977 96.12 0.1008 0.2308 56.33 p Bacteria Cells Adsorbed on Corncobs Bacteria Cells Adsorbed on Oak Wood Chips Identity Cell Concentration GM Broth (Cells·mL−1) Before Fermentation (g·g−1) After Fermentation (g·g−1) Relative Diﬀerence (%) Before Fermentation (g·g−1) After Fermentation (g·g−1) Relative Diﬀerence (%) Acetobacter pasteurianus 3.97 × 105 0.1461 1.1968 87.79 0.0020 0.1772 98.87 Acetobacter malorum 1.25 × 106 0.0948 1.0882 91.29 0.1851 0.1545 −16.53 Kozakia baliensis 3.13 × 105 0.0260 1.1589 97.76 0.1224 0.1122 −8.33 Gluconobacter cerinus 2.22 × 106 0.0116 1.5618 99.26 0.1498 0.3147 52.40 Gluconobacter oxydans 6.38 × 105 0.0426 1.0977 96.12 0.1008 0.2308 56.33 Overall, these results showed a successful adsorption on CC and OWC for both yeasts and bacteria, but with varying adsorption eﬃciency. 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https://openalex.org/W4321390851	https://zenodo.org/records/7656922/files/ESiWACE2_D2.9_Final_Model_Container.pdf	English	null	Final Report on Porting Earth System Models to Containers - Deliverable D2.9	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	4,981	Final Report on Porting Earth System Models to Containers Deliverable D2.9 Final Report on Porting Earth System Models to Containers Deliverable D2.9 The project Centre of Excellence in Simulation of Weather and Climate in Europe Phase 2 (ESiWACE2) has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No 823988. The project Centre of Excellence in Simulation of Weather and Climate in Europe Phase 2 (ESiWACE2) has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No 823988. ESiWACE2 Deliverable D2.9 About this document Work package in charge: WP2 - Establish, evaluate and watch new technologies for the community Actual delivery date for this deliverable: 28 Feb. 2022 Dissemination level: PU (for public use) About this document Work package in charge: WP2 - Establish, evaluate and watch new technologies for the community Actual delivery date for this deliverable: 28 Feb. 2022 Dissemination level: PU (for public use) Lead authors: Swiss National Supercomputing Centre, ETH Zurich (ETHZ/CSCS): William Sawyer, Theofilos Manitaras Center for Climate System Modeling, ETH Zurich (ETHZ/C2SM): Jonas Jucker, Matthieu Leclair Project internal reviewer: Max Planck Institute for Meteorology (MPIM): Reinhard Budich Lead authors: Swiss National Supercomputing Centre, ETH Zurich (ETHZ/CSCS): William Sawyer, Theofilos Manitaras Center for Climate System Modeling, ETH Zurich (ETHZ/C2SM): Jonas Jucker, Matthieu Leclair Project internal reviewer: Max Planck Institute for Meteorology (MPIM): Reinhard Budich Max Planck Institute for Meteorology (MPIM): Reinhard Budich Contact details Project Office: esiwace@dkrz.de Visit us on: www.esiwace.eu Access our documents in Zenodo: https://zenodo.org/communities/esiwace Disclaimer: This material reflects only the authors view and the Commission is not responsible for any use that may be made of the information it contains. Contact details Project Office: esiwace@dkrz.de Visit us on: www.esiwace.eu Access our documents in Zenodo: https://zenodo.org/communities/esiwace Disclaimer: This material reflects only the authors view and the Commission is not responsible for any use that may be made of the information it contains. Disclaimer: This material reflects only the authors view and the Commission is not responsible for any use that may be made of the information it contains. 2 ESiWACE2 Deliverable D2.9 ESiWACE2 Deliverable D2.9 Contents 1. Abstract /publishable summary ....................................................................................................... 4 2. Conclusion & Results ........................................................................................................................ 4 3. Project objectives ............................................................................................................................. 5 4. Detailed report on the deliverable .................................................................................................. 5 4.1 EC-Earth ................................................................................................................................. 6 4.2 LFRic ....................................................................................................................................... 6 4.3 COSMO .................................................................................................................................. 8 4.4 ICON ....................................................................................................................................... 9 5. References ...................................................................................................................................... 11 6. Changes made and/or difficulties encountered, if any ................................................................. 12 8. 1. Abstract /publishable summary Atmosphere and ocean models are characterised among other things by complex dependencies, external configurations, and performance requirements. The containerisation of such a software stack helps to provide a consistent environment to ensure security, portability and performance. Since the container is built only once but then can be deployed on multiple platforms, productivity is increased. ETH Zurich has led a multi-year effort to containerise key models from the climate / numerical weather prediction community. This effort started in 2018 with a “hackathon” (programming session) to help ESiWACE2 community scientists create containers for their Earth-system models. The six teams made fast progress on their containers and their results were reported in the ESiWACE2 deliverable “D2.8 Summary of Container Hackathon Experiences”. Of these teams, three received extended ESiWACE2 funding to refine their containers : LFRic, EC-Earth, and COSMO. Even though they did not receive funding, the ICON team viewed their containerisation as strategic and has offered their results as part of this final deliverable. Therefore, we report here on four model containers – LFRic, EC-Earth, COSMO and ICON – rather than the originally foreseen three. The timing of the hackathon – just before the Covid-19 pandemic – was fortunate, for the teams were able to return home to refine their containers and put them into a production framework with only virtual interaction. ETH Zurich provided technical assistance to the teams, which became highly independent with time. The requirement for the final deliverable was not only the containerisation itself but also that the resulting model performance did not suffer adversely. The overall process and an overview of the containerization work is given in the subsequent sections, each of which references the public online documentation which the teams developed during their efforts. Final Report on Porting Earth System Models to Containers Deliverable D2.9 Sustainability .................................................................................................................................. 12 9. Dissemination, Engagement and Uptake of Results ...................................................................... 12 9.1 Target audience ....................................................................................................................... 12 9.2 Record of dissemination/engagement activities linked to this deliverable ............................ 13 9.3 Publications in preparation OR submitted............................................................................... 14 9.4 Intellectual property rights resulting from this deliverable .................................................... 14 3 3 ESiWACE2 Deliverable D2.9 5 1 Container Hackathon for Modellers, 3-5 December 2019, Lugano (CH): https://www.esiwace.eu/events/container- hackathon-for-modellers 2 Sawyer, William, & Benedicic, Lucas. (2020). Summary of Container Hackathon Experiences (D2.8). Zenodo. https://doi.org/10.5281/zenodo.4323261 2. Conclusion & Results Work on this deliverable took place intermittently during 2020-2022, with each of the three teams receiving roughly 3 PM for the implementation. ETH Zurich provided container assistance on request through Theofilos Manitaras, as well as the possibility to continue their efforts to deploy and benchmark their containers on the ETHZ/CSCS Piz Daint HPC platform. The efforts of at least two of the three teams were a success. Containers were successfully integrated into the production workflow for the ICON, LFRic, and COSMO models, and at least one publication is pending from these efforts. The risk assessed at the onset of ESiWACE2 was that significant performance might be lost when running large, complex models using message passing and even Graphics Processing Units (GPUs). Thanks to HPC-aware middleware, this problem did not materialise: results in the detailed report below indicate that the overhead of running in containers is negligible (<10%) and is considerably smaller than the variations in compute time due to changing load on the underlying HPC platform. We view the containerisation results as a clear indication that containers can increase productivity in the deployment of Earth system models running on HPC systems. 4 ESiWACE2 Deliverable D2.9 Macro-objectives Specific goals in the workplan Contribution of this deliverable? Boost European climate and weather models to operate in world-leading quality on existing supercomputing and future pre-exascale platforms Yes Establish new technologies for weather and climate modelling Yes Enhance HPC capacity of the weather and climate community Yes Improve the toolchain to manage data from climate and weather simulations at scale No Strengthen the interaction with the European HPC ecosystem Yes Foster co-design between model developers, HPC manufacturers and HPC centres Yes 3. Project objectives j j This deliverable contributes directly and indirectly to the achievement of all the macro-objectives and specific goals indicated in section 1.1 of the Description of the Action: Macro-objectives Contribution of this deliverable? (1) Enable leading European weather and climate models to leverage the available performance of pre-exascale systems with regard to both compute and data capacity in 2021. Yes (2) Prepare the weather and climate community to be able to make use of exascale systems when they become available. Yes Specific goals in the workplan Contribution of this deliverable? Boost European climate and weather models to operate in world-leading quality on existing supercomputing and future pre-exascale platforms Yes Establish new technologies for weather and climate modelling Yes Enhance HPC capacity of the weather and climate community Yes Improve the toolchain to manage data from climate and weather simulations at scale No Strengthen the interaction with the European HPC ecosystem Yes Foster co-design between model developers, HPC manufacturers and HPC centres Yes 4. Detailed report on the deliverable ETH Zurich (ETHZ) coordinated the containerisations by intermittent coordination and periodic This deliverable contributes directly and indirectly to the achievement of all the macro-objectives and specific goals indicated in section 1.1 of the Description of the Action: am, & Benedicic, Lucas. (2020). Summary of Container Hackathon Experiences (D2.8). Zenodo. 10.5281/zenodo.4323261 3 William Sawyer, Jonas Jucker, & Simon Wilson. (2022). Containers 2: Status of containerisation of Earth Science Models - Milestone MS5. Zenodo. https://doi.org/10.5281/zenodo.7462595 4 https://github.com/eth-cscs/ContainerHackathon/blob/master/EC-Earth/Readme.pdf 5 https://doi.org/10.1016/j.jpdc.2019.02.007 6 https://www.metoffice.gov.uk/research/foundation/dynamics/next-generation 7 https://github.com/stfc/PSyclone 4. Detailed report on the deliverable  Theofilos Manitaras  Jonas Jucker  Alberto Madonna  Christopher Bignamini  Andreas Jocksch  William Sawyer  Theofilos Manitaras  Jonas Jucker  Alberto Madonna  Christopher Bignamini  Andreas Jocksch  William Sawyer After the ESiWACE2 “Container Hackathon for Modellers”1, reported in deliverable D2.82, efforts were concentrated on 3 models: LFRic (led by University of Reading), EC-Earth (led by Barcelona 5 ESiWACE2 Deliverable D2.9 Supercomputing Center, BSC), and COSMO (led by MeteoSwiss and ETHZ). In mid-2022 the impending containerisation of ICON model by the ETH Zurich -- needed for the community’s production workflow -- led us to suggest this work also for the final deliverable. Supercomputing Center, BSC), and COSMO (led by MeteoSwiss and ETHZ). In mid-2022 the impending containerisation of ICON model by the ETH Zurich -- needed for the community’s production workflow -- led us to suggest this work also for the final deliverable. The following people made non-trivial contributions to the containerisation efforts The following people made non-trivial contributions to the containerisation efforts: The following people made non-trivial contributions to the containerisation efforts: Model Contributors EC-Earth Pablo Echevaria Julian Berlin (BSC) Uwe Fladrich (SMHI) ICON Jonas Jucker (ETHZ/C2SM) Theofilos Manitaras (ETHZ/CSCS) Andreas Fink (ETHZ/CSCS) William Sawyer (ETHZ/CSCS) LFRic Iva Kavcic (UKMetOffice) Simon Wilson (UKMetOffice) COSMO Jonas Jucker (ETHZ/C2SM) Matthieu Leclair (ETHZ/C2SM) Theofilos Manitaras (ETHZ/CSCS) 4.1 EC-Earth The BSC team made very quick progress on during the Container Hackathon (Dec. 2019, extensively reported in D2.8), containerising versions of two simplified EC-Earth configurations: one for the current stable version EC-Earth3, and another for the next generation EC-Earth4, which is currently under development. BSC considers the results of the hackathon as sufficiently successful to also satisfy the ESiWACE2 Milestone MS53 reporting requirement. Public online documentation for this effort is not available, but the final report4 is available. 4.2 LFRic LFRic5 is the new weather and climate modelling system being developed by the UK Met Office to replace the existing Unified Model (UM) in preparation for exascale computing in the 2020s. LFRic uses the GungHo6 dynamical core and runs on a semi-structured cubed-sphere mesh. One of the guiding design principles, imposed to promote performance portability, is the ”separation of concerns” between the science code and parallel code. An application called PSyclone7 developed at the STFC Hartree Centre, can generate the parallel code enabling deployment of a single source science code onto different machine architectures. 6 ESiWACE2 Deliverable D2.9 At the Container Hackathon (Dec. 2019), both a Docker and a Singularity container were developed The singularity container8 is being actively used for several projects. These include: ontainer Hackathon (Dec. 2019), both a Docker and a Singularity container were develo At the Container Hackathon (Dec. 2019), both a Docker and a Singularity container were developed. The singularity container8 is being actively used for several projects. These include: The singularity container8 is being actively used for several projects. These include:  Development of LFRic code on individuals’ laptops.  On ARCHER29, the UK's academic supercomputer, using the Cray interconnect: o A C1152 (8.5 km) resolution on 81 nodes and 324 nodes and a C2304 (4.3 km) resolution on 324 nodes. This is the highest resolution we have ever run LFRic (Gungho, dynamics only). Limited from running further by Archer2 config. o It was used as part of the XIOS benchmarking study for the Excalidata pro o It will form the basis of any future LFRic installations on ARCHER2.  On Microsoft's Azure: It has been run (so far) on 5 nodes in Azure with much larger runs planned.  On Microsoft's Azure: It has been run (so far) on 5 nodes in Azure with much larger runs planned.  On Jasmin11: As part of a proposed student's benchmarking project using OpenMP and the fast ethernet interconnect.  On Jasmin11: As part of a proposed student's benchmarking project using OpenMP and the fast ethernet interconnect.  On Dirac CSD3 system at Cambridge12: Preparation for the I/O using both network fabric and advanced burst buffers as part of the Excalidata project, using the Infiniband interconnect.  On Dirac CSD3 system at Cambridge12: Preparation for the I/O using both network fabric and advanced burst buffers as part of the Excalidata project, using the Infiniband interconnect. 8 https://github.com/NCAS-CMS/LFRic_container 9 https://www.archer2.ac.uk 10 https://excalibur.ac.uk/projects/excalidata 11 https://jasmin.ac.uk 12 https://www.hpc.cam.ac.uk/dirac-csd3 4.2 LFRic Figure 1: Strong scaling of the Gungho dynamical core for both the native (“bare-metal”) and Singularity container versions. Figure 1: Strong scaling of the Gungho dynamical core for both the native (“bare-metal”) and Singularity container versions. A performance comparison between natively compiled and containerised LFRic indicates that running in container introduces virtually no overhead, see Figure 1. Containers have thus proved to be an extremely useful tool when the long-term operation of software is required, beyond the longevity of the software stack, which is frequently updated on HPC platforms. 7 ESiWACE2 Deliverable D2.9 In its investigations, the LFRic team determined there was no meaningful loss in performance when running within a container, assuming the container run-time has the proper HPC support. In its investigations, the LFRic team determined there was no meaningful loss in performance when running within a container, assuming the container run-time has the proper HPC support. 4.3 COSMO COSMO has been in production at the German Weather Service (DWD) until 2015. At MeteoSuisse it is used for weather forecasting and will be supplanted by ICON in 2023. Although it was not represented at the Container Hackathon (Dec. 2019), it is an ideal candidate for containers since users require outdated configurations to run after COSMO is no longer officially supported. In this common case, containers can offer long term stability for a given software stack. Figure 2: The simulation run times of COSMO are depicted in the ETHZ/C2SM Continuous Figure 2: The simulation run times of COSMO are depicted in the ETHZ/C2SM Continuous Integration pipeline, which periodically runs both the bare-metal (top) and containerised (bottom) versions of COSMO in a testing mode. The significant run time variations of either version come from the dynamic load of the machine and, particularly, the file system. This dynamic variability is far greater than the difference (roughly 5%) in execution times of the bare- metal and container versions, which run contemporaneously to take the machine load into account. Figure 2: The simulation run times of COSMO are depicted in the ETHZ/C2SM Continuous Integration pipeline, which periodically runs both the bare-metal (top) and containerised (bottom) versions of COSMO in a testing mode. The significant run time variations of either version come from the dynamic load of the machine and, particularly, the file system. This dynamic variability is far greater than the difference (roughly 5%) in execution times of the bare- metal and container versions, which run contemporaneously to take the machine load into account. 8 ESiWACE2 Deliverable D2.9 The Center for Climate System Modeling (C2SM) at ETH Zurich took up the task of containerising COSMO in 2021. The container workflow is documented in https://github.com/C2SM/container (public access), which also contains the requisite Dockerfiles. The Center for Climate System Modeling (C2SM) at ETH Zurich took up the task of containerising COSMO in 2021. The container workflow is documented in https://github.com/C2SM/container (public access), which also contains the requisite Dockerfiles. The resulting COSMO-container is embedded in the Jenkins Continuous Integration (CI) system used by C2SM, where it runs in addition to the COSMO code built natively (without containers). 13 Samiento, Rafael, Sawyer, William, Kosukhin, Sergey, Dietlicher, Remo, & Walser, Andre. (2020). The Containerisation of the ICON model for quasi-biennial oscillation simulations. Zenodo. https://doi.org/10.5281/zenodo.4322638 14 https://spack.io/ 4.3 COSMO Although runtimes vary due to the load characteristics of the Piz Daint platform at CSCS, the overhead for the containerization is estimated to have less than 10% degradation in performance, as is illustrated in the timings for both the native and container runs of within periodic Jenkins testing, see Figure 2. It should be noted that there is a much larger variation in the timings due to system fluctuations over time (which are often related to file system load) than there are between the native and container timings running on the machine at the same time. Although it is now embedded into the C2SM workflow, the COSMO container is not currently exploited by users, for the simple reason that they prefer to compile and run with the existing software stack. Since COSMO is no longer maintained by the German Weather Service (DWD), it is highly likely that users will need the containers soon to run on outdated software stacks when COSMO no longer compiles with upgraded stacks. 4.4 ICON The Icosahedral Non-hydrostatic model is essentially the successor of COSMO for numerical weather forecasting but can also be used for climate simulations. A prototype container for ICON was already the subject of Milestone MS413 (Dec. 2020). While that prototype was largely successful, it was still a one-off: the ICON software and build system quickly moved on, and the Dockerfiles were no longer maintained. The maintainability question for containers is a central consideration and needs to be addressed to make containers useful in the long term. The success of inserting the COSMO into the production workflow (mentioned above) led C2SM to proposal a similar approach for ICON. This workflow requires that ICON is built using the Spack14 package manager to manage ICON’s many dependencies. Spack vastly simplifies the current ICON build system (which also includes the construction of the software stack), and thus would increase maintainability. Although this Spack-based build system is not yet in the official ICON release, the discussion about its inclusion is ongoing. The resulting Dockerfile becomes essentially a call to spack install. The Dockerfiles are currently maintained in https://github.com/C2SM/container, however the ICON team is in the process of incorporating these into the ICON repository, which will then become available in a public release. MeteoSuisse is in the process of completing its transition from COSMO to ICON as its production software, and the ICON containers will be fully incorporated into the workflow, allowing any ICON run to be executed through a container, even on Graphics Processing Units (GPUs), on which the 14 https://spack.io/ 9 ESiWACE2 Deliverable D2.9 production model typically runs. The containers are tested through the CSCS Alps Continuous Integration (CI) testing system, which validates the container workflow. production model typically runs. The containers are tested through the CSCS Alps Continuous Integration (CI) testing system, which validates the container workflow. MeteoSuisse has provided the benchmark mch_bench_r19b07_dev, which illustrates the current operational system under development. This benchmark was run within public release of the icon- exclaim repository (the latest benchmarks require the ICON version in the release candidate), and therefore it is possible to compare performance between the native-built and the container versions. The benchmark is designed to run on a 4xA100 GPUs on the MeteoSuisse production platform, but has been run for the sake of this comparison on the CSCS Daint system with single- P100 GPU nodes. 4.4 ICON As one can see in the subsequent strong scaling graph, the “bare-metal” (blue) timings are obscured by the red container timings, meaning that there is no major overhead in running the containerised code. The strong scaling is in Figure 3 below. Figure 3: The strong scalability of both the “bare-metal” (blue) and containerised (red) versions of the MeteoSwiss regional 2km benchmark are given, along with the “ideal” linear scalability (green) normalised for the 4-node run. The strong scaling is reasonable from 4 to 32 nodes, and the execution times of the two versions coincide, such that the red dots largely obscure the blue. In addition, the Max Planck Institute for Meteorology (MPI-M) has contributed the atm_qubicc_r2b7 benchmark, a medium (20 km) resolution test case for a “thick atmosphere” (191 0 100 200 300 400 500 600 700 0 5 10 15 20 25 30 35 Time (s.) Nodes mch_bench_r19b07_dev benchmark Bare-metal Container Ideal mch_bench_r19b07_dev benchmark Figure 3: The strong scalability of both the “bare-metal” (blue) and containerised (red) versions of the MeteoSwiss regional 2km benchmark are given, along with the “ideal” linear scalability (green) normalised for the 4-node run. The strong scaling is reasonable from 4 to 32 nodes, and the execution times of the two versions coincide, such that the red dots largely obscure the blue. In addition, the Max Planck Institute for Meteorology (MPI-M) has contributed the atm_qubicc_r2b7 benchmark, a medium (20 km) resolution test case for a “thick atmosphere” (191 In addition, the Max Planck Institute for Meteorology (MPI-M) has contributed the atm_qubicc_r2b7 benchmark, a medium (20 km) resolution test case for a “thick atmosphere” (191 10 ESiWACE2 Deliverable D2.9 vertical levels) designed for the investigation of the Quasi-Biennial Oscillation15. Due to its memory requirements, this benchmark must be run on more than 50 P100 nodes of the ETHZ/CSCS Piz Daint platform. Figure 4 depicts the strong scaling of both the “bare-metal” and containerised versions, along with the “ideal” run times normalised to the time for 64 nodes. As for the MeteoSwiss benchmark, the overhead for running in a container is minimal, and the absolute scaling in the realm 64-256 nodes is reasonable for both versions. vertical levels) designed for the investigation of the Quasi-Biennial Oscillation15. Due to its memory requirements, this benchmark must be run on more than 50 P100 nodes of the ETHZ/CSCS Piz Daint platform. 4.4 ICON Figure 4 depicts the strong scaling of both the “bare-metal” and containerised versions, along with the “ideal” run times normalised to the time for 64 nodes. As for the MeteoSwiss benchmark, the overhead for running in a container is minimal, and the absolute scaling in the realm 64-256 nodes is reasonable for both versions. Figure 4: The strong scalability of both the “bare-metal” (blue) and containerised (red) versions of the Max Planck Institute for Meteorology (MPI-M) Quasi-Biennial Oscillation (QBO) 20km benchmark are given, along with the “ideal” linear scalability (green) normalised for the 64-node run. The strong scaling is reasonable for both. The performance of blue and red is so close that the red square often obscures the blue diamond 0 20 40 60 80 100 120 140 160 0 50 100 150 200 250 300 Time (s.) Nodes Quasi-biennial oscillation 20km-global benchmark Bare-metal Container Ideal Figure 4: The strong scalability of both the “bare-metal” (blue) and containerised (red) versions of the Max Planck Institute for Meteorology (MPI-M) Quasi-Biennial Oscillation (QBO) 20km benchmark are given, along with the “ideal” linear scalability (green) normalised for the 64-node run. The strong scaling is reasonable for both. The performance of blue and red is so close that the red square often obscures the blue diamond. 0 20 40 60 80 100 120 140 160 0 50 100 150 200 250 300 Time (s.) Nodes Quasi-biennial oscillation 20km-global benchmark Bare-metal Container Ideal Quasi-biennial oscillation 20km-global benchmark Figure 4: The strong scalability of both the “bare-metal” (blue) and containerised (red) versions of the Max Planck Institute for Meteorology (MPI-M) Quasi-Biennial Oscillation (QBO) 20km benchmark are given, along with the “ideal” linear scalability (green) normalised for the 64-node run. The strong scaling is reasonable for both. The performance of blue and red is so close that the red square often obscures the blue diamond. 15 https://www.metoffice.gov.uk/weather/learn-about/weather/atmosphere/quasi-biennial-oscillation 11 15 https://www.metoffice.gov.uk/weather/learn-about/weather/atmosphere/quasi-biennial-oscillation 7. How this deliverable contributes to the European strategies for HPC 7. How this deliverable contributes to the European strategies for HPC The advantages of containers within HPC where the software stack is frequently upgraded are well known. Frequent the upgraded stack causes the models to fail or to give incorrect answers, while the stack which produced correct answers may no longer be supported on the operational HPC platform. Containers can incorporate the software stack which is known to work and give longevity to the working model version. This deliverable illustrates clearly that weather and climate applications can be built and run inside containers with little loss in performance and adding only minimal development overhead. We feel this is a crucial step forward for HPC strategy, not only in Europe but worldwide. 8. Sustainability As mentioned in Section 4, the container knowledge has been passed to the application teams at UK Met Office, ETHZ/C2SM, MeteoSuisse, BSC, SMHI, MPI-M and DWD, which have now partially or completely integrated containers into their HPC workflows. These teams are largely independent of the container specialists at ETHZ/CSCS, although the latter is still available to assist through user tickets. We at ETHZ/CSCS believe that the step to utilising containers for weather and climate on HPC platforms has now been enabled for the long term. We have learned that while some complexity is added by the containerisation, it is largely a porting exercise based on the existing build system. It is computationally expensive to build containers, but the effort can be automated to avoid the need for user oversight. 16 https://c2sm.ethz.ch/research/exclaim.html 5. References All references in this document have been added as footnotes. /www.metoffice.gov.uk/weather/learn-about/weather/atmosphere/quasi-biennial-oscillation 11 ESiWACE2 Deliverable D2.9 6. Changes made and/or difficulties encountered, if any The ICON container was added to the deliverable, since it requires three production-quality applications with real-world benchmarks, and the work on EC-Earth does not meet this standard. We were fortunate that the ICON container was foreseen within the EXCLAIM16 project in any event. All the teams seemed to agree that containerisation was conceptually easy, but there were many subtleties. Some of these were related to HPC system issues, such as security requirements and the management of SSH keys within the container. And a major challenge is debugging the container version, which requires expertise in entering the container. In the case of ICON, these issues could only be resolved with the help of container experts at ETHZ/CSCS. 9.1 Target audience As indicated in the Description of the Action, the audience for this deliverable is: 16 https://c2sm.ethz.ch/research/exclaim.html 12 ESiWACE2 Deliverable D2.9 A group specified by the consortium, including the Commission services (RE) This reports is confidential, only for members of the consortium, including the Commission services (CO) A group specified by the consortium, including the Commission services (RE) This reports is confidential, only for members of the consortium, including the Commission services (CO) This is how we are going to ensure the uptake of the deliverables by the targeted audience: This is how we are going to ensure the uptake of the deliverables by the targeted audience: The containerisation of LFRic, COSMO and ICON is documented in publicly accessible GitHub repositories17 18. The value of containers for climate and weather applications on HPC platforms has been underlined at conferences such as PASC2119. Furthermore, in the realm of ESiWACE2 Work Package 6, containers were a topic at the summer schools in ESiWACE2 summer schools on “Effective HPC for Weather and Climate” in 202020 and 202121. In the future ETHZ/CSCS will encourage its users to adopt containers as the mechanism of choice to run their applications on the new Alps22 HPC platform, offering educational opportunities to smooth the transition to containers, as well as extensive user documentation. In fact, containers are now a requirement to utilise the CSCS Continuous Integration system23 for the Alps architecture. 9.2 Record of dissemination/engagement activities linked to this deliverable Type of dissemination and communication activities Details Date and location of the event Type of audience Zenodo Link Estimated number of persons reached Container hackathon Hackathon (intensive programming session) Dec. 3-5, 2019 at CSCS in Lugano, Switzerland Public https://zenodo. org/record/3685 888 16 Summer school Summer School on Effective HPC for Weather and Climate Aug. 23-29, 2020, virtual due to Covid-19 Public https://zenodo. org/record/5795 447 90+ Summer school Summer School on Effective HPC for Weather and Climate Aug. 23-27, 2021, virtual due to Covid-19 Public https://zenodo. org/record/5795 447 65+ 9.2 Record of dissemination/engagement activities linked to this deliver 13 17 https://github.com/NCAS-CMS/LFRic_container 18 https://github.com/C2SM/container 19 Minisymposium “Sarus: Highly Scalable Docker Containers for HPC Systems”, PASC21, (July 201), https://pasc21.pasc- conference.org/program/schedule/index.html%3Fpost_type=page&p=10&id=msa125&sess=sess130.html 20 https://www.esiwace.eu/the-project/stories/summer-school-on-effective-hpc-for-climate-and-weather 21 https://www.esiwace.eu/events/summer-school-2021 22 https://www.cscs.ch/computers/alps/ 23 https://gitlab.com/cscs-ci/ci-testing/containerised_ci_doc 13 17 https://github.com/NCAS-CMS/LFRic_container 18 https://github.com/C2SM/container 19 Minisymposium “Sarus: Highly Scalable Docker Containers for HPC Systems”, PASC21, (July 201), https://pasc21.pasc- conference.org/program/schedule/index.html%3Fpost_type=page&p=10&id=msa125&sess=sess130.html 20 https://www.esiwace.eu/the-project/stories/summer-school-on-effective-hpc-for-climate-and-weather 21 https://www.esiwace.eu/events/summer-school-2021 22 https://www.cscs.ch/computers/alps/ 23 https://gitlab.com/cscs-ci/ci-testing/containerised_ci_doc ESiWACE2 Deliverable D2.9 9.3 Publications in preparation OR submitted In preparation OR submitted? Title All authors Title of the periodical or the series Is/Will open access be provided to this publication? In preparation Addressing portability and performance of a next generation weather and climate code using a Singularity container Christopher Maynard, Simon Wilson, Bryan N. Lawrence Concurrency and Computation: Practice and Experience Open Access 9.4 Intellectual property rights resulting from this deliverable All the docker files created for this deliverable are in the public domain on the websites specifically mentioned in Section 4. This is how we are going to ensure the uptake of the deliverables by the targeted audience: However, this does not imply that the weather and climate models themselves are also in the public domain since these clearly predate ESiWACE2. As such, no new IP rights have resulted from this deliverable. 9.3 Publications in preparation OR submitted In preparation OR submitted? Title All authors Title of the periodical or the series Is/Will open access be provided to this publication? In preparation Addressing portability and performance of a next generation weather and climate code using a Singularity container Christopher Maynard, Simon Wilson, Bryan N. Lawrence Concurrency and Computation: Practice and Experience Open Access 9.3 Publications in preparation OR submitted p p y g g All the docker files created for this deliverable are in the public domain on the websites specifically mentioned in Section 4. However, this does not imply that the weather and climate models themselves are also in the public domain since these clearly predate ESiWACE2. As such, no new IP rights have resulted from this deliverable. All the docker files created for this deliverable are in the public domain on the websites specifically mentioned in Section 4. However, this does not imply that the weather and climate models themselves are also in the public domain since these clearly predate ESiWACE2. As such, no new IP rights have resulted from this deliverable. 14 14
https://openalex.org/W3095070709	https://www.duo.uio.no/bitstream/10852/81578/2/The%2bRelationship%2bBetween%2bPerceived%2bSocial%2bSupport%2band%2bPTSD%2bSymptoms%2bAfter%2bExposure%2bto%2bPhysical%2bAssault%253B%2bAn%2b8%2bYears%2bLongitudinal%2bStudy.pdf	English	null	The Relationship Between Perceived Social Support and PTSD Symptoms After Exposure to Physical Assault: An 8 Years Longitudinal Study	Journal of interpersonal violence	2,020	cc-by	12,036	The Relationship Between Perceived Social Support and PTSD Symptoms After Exposure to Physical Assault: An 8 Years Longitudinal Study ttps://doi.org/10.1177/0886260520970314 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/0886260520970314 journals.sagepub.com/home/jiv Venke A. Johansen, 1,2 Anne Marita Milde, 3,4 Roy Miodini Nilsen, 2 Kyrre Breivik, 3 Dag Øystein Nordanger, 1,5 Kjell Morten Stormark, 3,4 and Lars Weisæth 6 4 and Lars Weisæth 6 Original Research ttps://doi.org/10.1177/0886260520970314 Journal of Interpersonal Violence 1 –28 © The Author(s) 2020 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/0886260520970314 journals.sagepub.com/home/jiv The Relationship Between Perceived Social Support and PTSD Symptoms After Exposure to Physical Assault: An 8 Years Longitudinal Study 1 Haukeland University Hospital, Bergen, Norway 2 Western Norway University of Applied Sciences (HVL), Bergen, Norway 3 NORCE Norwegian Research Centre AS, Bergen, Norway 4 University of Bergen, Norway 5 Oslo Metropolitan University, Oslo, Norway 6 University of Oslo, Norway Corresponding Author: Venke A. Johansen, Haukeland University Hospital, 5021 Bergen, Norway. E-mail: venke.a.johansen@helse-bergen.no ttps://doi.org/10.1177/0886260520970314 Journal of Interpersonal Violence 1 –28 © The Author(s) 2020 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/0886260520970314 journals.sagepub.com/home/jiv Keywords d perceived social support, PTSD, physical assault, cross-lagged, longitudinal, nondomestic violence Abstract C Consequences of nondomestic violence are known to be multifaceted with high rates of emotional and psychological problems in addition to physical injuries, and victims report many trauma related symptoms. This study explore if perceived social support (PSS) (Social Provisions Scale [SPS]) and post-traumatic stress disorder (PTSD) symptoms (Impact of Event Scale [IES- 22]) are interrelated among adult victims at four assessment points up until eight years after the exposure to physical assault; soon after the event (T1), three months after T1 (T2), one year after T1 (T3), and eight years after T1 University of Oslo, Norway Corresponding Author: Venke A. Johansen, Haukeland University Hospital, 5021 Bergen, Norway. E-mail: venke.a.johansen@helse-bergen.no Corresponding Author: Venke A. Johansen, Haukeland University Hospital, 5021 Bergen, Norway. E-mail: venke.a.johansen@helse-bergen.no Journal of Interpersonal Violence 2 (T4). One hundred and forty-three subjects participated at T1, 94 at T2, 73 at T3, and 47 accepted a follow-up at T4. At T1, 138 of 143 completed the questionnaires within 16 weeks after the incident. PTSD symptoms were highly correlated across time (p < .001); PSS were significantly correlated only between T1 and T2 (p < .001), T1 and T3 (p < .05), and between T2 and T3 (p < .05). Cross-lagged analyses showed an inverse relationship between prior PSS and later PTSD symptoms across all time points (ps < .05); not proved between prior PTSD symptoms and later PSS (ps > .1). PSS at T1 was an independent predictor of PSS one year and eight years after the incident. We conclude that higher perception of social support protects against the development of PTSD symptoms; diminished perception of social support increases the risk of developing PTSD symptoms. These findings suggest that PSS after experiencing a violent assault should be considered as an important factor in natural recovery in the long run, as well as essential alongside psychiatric treatment. Establishing psychosocial interventions for victims of physical violence in the acute phase may prevent prolonged trauma reactions. Introduction It is well documented that consequences of interpersonal violence for crime victims represent a significant public health problem (Kilpatrick et al., 2003; WHO, 2002). We have however sparse knowledge on the long-term conse- quences on mental health among adults exposed to physical assault by a per- petrator who is not an intimate partner, ex-partner, or close family. Acute distress, as well as prolonged post-traumatic stress disorder (PTSD) symp- toms predicts poor mental health across lifespan (Olatunji et al., 2010; Sareen, 2014). Exposure to physical assault combined with actual physical injury and perceived fear of being more seriously injured or killed during the event, are major risk factors for developing PTSD (Kilpatrick & Acierno, 2003; Sareen, 2014). Most of the affected victims do recover within weeks or months, but 10%–40% develop persistent PTSD lasting for years or even for life (Sareen, 2014). Exposure to intentional interpersonal violence is more likely to con- duct PTSD symptoms than accidents or disasters (Holbrook et al., 2001; Stein et al., 1997). Moreover, intentional or assaultive injury, among others, has shown to be a risk factor for the onset of PTSD (Sareen, 2014). Johansen et al. 3 Several studies have highlighted the relationship between social support (SS) and PTSD symptoms after trauma-exposure (Brewin et al., 2000; Ozer et al., 2003; Peleg & Shalev, 2006; Vogt et al., 2017). Most literature include perceived positive SS and empathy from significant others as protective fac- tors, while blaming and social exclusion are presented as risk factors (Maercker & Horn, 2013; Vogt et al., 2017). Two important metastudies con- cluded respectively that lack of SS and support of poor quality were among the most potent peri- and post-trauma risk factors for the development of PTSD (Brewin et al., 2000; Ozer et al., 2003). Defining SS in the aftermath of potential traumatic events (PTE) may be of challenge. The phenomenon of SS may be understood both in terms of its multifaceted, interactive nature, as well as how it is perceived (Pruitt & Zoellner, 2008). Though some studies focus upon actual versus perceived social support (PSS), the latter seem more frequently reported (Guay et al., 2011). The value of SS seem to be more dependent of the recipients’ percep- tion of the interaction, than of the intention from the other person trying to provide support (Pruitt & Zoellner, 2008). Introduction It can be health protective if the recipient perceives others as reliable during stressful experiences. Positive support can be understood as relationships offering information assistance, material or emotional help, and a sense of cohesion that are perceived as lov- ing or caring by the victim (Hobfoll & Stephens, 1990; Hollifield et al., 2016). The protective role of PSS from close others is well documented (Brewin et al., 2000; Vogt et al., 2017). Negative SS, on the other hand, includes blame, disbelief, taking control of the victim’s choice and with- drawal from the beneficiary. Absence of SS is defined as a lack of any reac- tion from others (Pruitt & Zoellner, 2008). It seems important to get a broader view of the individual interactions in different contexts (Vogt et al., 2017). Maercker and Horn (2013) have con- structed a sociointerpersonal model that organize the individual’s involve- ment within different levels of social contexts. The model includes three levels of contextual and interpersonal processes that arise after exposure to traumatic events. The first level, “Individual” (social-effective responses), consists of intrapersonal features or impairments. The second level “Close social relationships” occur during interactions between the victim and those who share a close relationship that in adulthood is typically represented by a romantic partner, close family members, and friends. The third level “Distant social contexts” (culture and society) is based on a shared culture, religion, or society, where the interactions are related to a specific group. The model reflects transactional or reciprocal relationships between the different levels. The interplay between SS and PTSD symptoms needs to be scrutinized, as SS includes many perspectives with a variety of relations to PTSD symptoms Journal of Interpersonal Violence 4 (Guay et al., 2011). In explaining the association between SS and PTSD symptoms, two main sets of theories are predominant in the literature field. One is the social causation theory that explains SS in terms of its antecedent effect on PTSD symptoms: negative/poor SS can lead to impaired mental health and increased psychopathology, while positive support protects against development of PTSD symptoms (Joseph et al., 1997; Mossakowski, 2014). The second way of annotation includes social selection theories (also known as social deterioration and social erosion theory) embracing that SS is affected by poor mental health (Wagner et al., 2016; Woodward et al., 2018). Introduction According to theories of social causation, favorable SS may have protec- tive effects, while adverse social conditions such as low SS, small networks, social isolation or low socioeconomic status, may increase the risk of PTSD (Mossakowski, 2014; Woodward et al., 2018). Emotional support, validation, and involvement with significant others in a noncritical context are often found to be protective and helpful (Ozer et al., 2003; Scarpa et al., 2006). Interactions with others, who intentionally give negative and unsupportive responses, for instance by blaming or excluding the victim, are found to have negative impacts. When relatives who are expected to be supportive, have opposite reactions to what is expected by the victim, for example by blaming or taking control, it can undermine the victims’ self-confidence (Scarpa et al., 2006; Ullman & Siegel, 1996). In sum, receiving SS may have a positive or caring effect, but while experienced as criticism, consequences may be induced or maintained distress both immediately after an adverse event or in the long run (Andrews et al., 2003; Guay et al., 2011). The social selection theories propose that PTSD symptoms such as inse- curity, mistrust, avoidance and social isolation, can unfold rejection and less SS from others (Mossakowski, 2014). In addition, PTSD symptoms can break down social relationships through an increased number of negative social interactions as well as through erosion of social resources in close relationships across time (Freedman et al., 2015; Wagner et al., 2016; Woodward et al., 2018). This perspective thus addresses that developing PTSD symptoms may increase the risk of experiencing negative dyadic com- munication, lesser networks, reduced support, in addition to cause an inabil- ity for the victim to improve poor SS (Freedman et al., 2015; Woodward et al., 2018). Further, caregiver burden and secondary traumatization can dis- turb such communication thus underline the complexity of social interaction (Lambert et al., 2012). Social causality and social selection theories has previously been under- stood as two competing explanations (Kaniasty & Norris, 2008). However, gradually these theories have been presented as being more complementary thus contributing to the discussion of a potent association between SS and Johansen et al. 5 PTSD symptoms and its implications for general health (Shallcross et al., 2016). Evidence from longitudinal studies support a bidirectional relation- ship between SS and PTSD symptoms in adults, involving processes from both theories (Kaniasty & Norris, 2008; Shallcross et al., 2016; Woodward et al., 2018). Introduction The majority of studies among adult crime- or community-victims exposed to physical assault have been cross-sectional (Kaniasty & Norris, 2008; Vogt et al., 2017), and among those, only a few report findings on both SS and PTSD symptoms (Jaycox et al., 2003; Scarpa et al., 2006; Yap & Devilly, 2004). Longitudinal studies including cross-lagged analysis on the relationship between PSS and PTSD symptoms in adult civilians seem scarce (Kaniasty & Norris, 2008; Woodward et al., 2018). Cross-lagged panel models can highlight and clarify the complex and dynamic relation- ship between interpersonal processes and PTSD symptoms. Such analyses demonstrates both bidirectional and unidirectional relations between PSS and PTSD symptoms across time (Woodward et al., 2018). In their study, Freedman and colleagues (2015) included recent civilian trauma survivors (n = 501) who sought emergency care. The authors show that changes in social relationship satisfaction in the early phases following exposure to traumatic events, contributed to changes in PTSD, rather than the other way around. They suggest that being satisfied with one’s relationship may be crucial to the natural recovery of trauma as well to be able to benefit from cognitive behavior therapy. Moreover, a postdisaster study among of 658 victims of the Hurricane Ike revealed a significant bidirectional relationship between emotional SS and PTSD symptoms in the early phase from 2–6 months, but no association between the two variables neither 5–9 nor 14–19 months after the incident (Platt et al., 2016). In a study of 557 natural disas- ter victims from Mexico, Kaniasty and Norris (2008) found that PSS low- ered PTSD symptoms in the earlier post disaster phase from 6–12 months (social causation). However, the bidirectional relationship was significant 12–18 months after the event, and at 18 months, were only PTSD symptoms diminished PSS over time (social selection). The relationship was thus altered due to time; PSS closer to the event had greater impact on PTSD symptoms, while the opposite was evident as time went by. Therefore, Kaniasty and Norris (2008) suggest elapsed time to be of importance. Moreover, Ozer and colleagues (2003) concluded that the time interval from exposure to violence affected the strength of the relationship between SS and PTSD symptoms. They argue that SS is a stronger predictor if more than three years have proceeded after the event, compared to less time. Such find- ings suggest of developmental trajectory to the interaction between SS and PTSD. Introduction Wagner and coworkers (2016) found an inverse relationship to PTSD, Journal of Interpersonal Violence 6 where the association seemed reinforced across time. In sum, research that analyses the relationship between SS and PTSD symptoms differ in method- ology, analytic approaches, and both phenomena are operationalized in sev- eral ways. Typically, surveyed populations are a mix of civilians and veterans, and consist of participants exposed to different types of traumatic events (Vogt et al., 2017; Wagner et al., 2016). In the presented sample of nondomestic victims, we have previously reported a prevalence of 30% (22/73) probable PTSD after one year, and 19% (9/47) after eight years (Johansen et al., 2013). Here, we are interested in add- ing knowledge to the association between perceived social support and PTSD symptoms across time since only a few longitudinal studies have utilized cross-lagged models testing a three or four assessment points relationship (Shallcross et al., 2016; Woodward et al., 2018). The main purposes of the present study were therefore to advance the inquiry into the relative importance of social causation or social selection between perceived social support and PTSD symptoms through a period of eight years, and to reflect and discuss clinical implications. We used crossed- lagged models to investigate the relation between PSS and PTSD symptoms in four assessment rounds within the eight-year period after exposure to a physical assault. Design This study is part of a larger prospective investigation utilizing within a one- group design of psychological trajectories in physically assaulted adult vic- tims of a single physical assault, defined as nondomestic violence. Four assessment rounds were performed throughout a period of eight years com- bined with semistructural interviews at the first round. Participants. The participants were all victims of physical assault vio- lence; 93% of the perpetrators were strangers and 7% were acquaintances or friends. The victims either sought medical aid, and received health care from a medical emergency unit, or they requested legal aid by submitting a police report at the local police department. With assistance of local police or medi- cal service staff, the participants were recruited from the two largest com- munities in Norway, the cities of Oslo and Bergen. Of 189 invited victims, 46 declined to participate or did not return the questionnaires, leaving a total sample of 143 (response rate 75.6%). Most participants (138/143) completed the initial questionnaires within 16 weeks after the assault, T1. The second assessment round T2, followed 3 months after T1, the third T3, 12 months 7 Johansen et al. after T1, and the last T4, eight years after T1. In total at T1, there were 143 participants, at T2 the response rate was 66% (n = 94), at T3 51% (n = 73). Written consents were obtained in connection with the conduct of the semis- tructured interview, either in person or by post. Those who completed at least 2 out of the first 3 assessment rounds (n = 97) were reinvited to participate at T4. The response rate at T4 was 48% (n = 47), thus representing 33% of the original sample (Figure 1). Fourteen of the 143 invited participants at T3, and 10 of the 97 at T4, could not be reached due to unknown addresses. Sample characteristics, such as gender, violence category (physical injury), educational level, and level of perceived threat during the assault, are presented for T1 and T4 (Table 1). Figure 1. Flowchart for inclusion of participants. Figure 1. Flowchart for inclusion of participants. Table 1. Descriptive Information of Participants at T1 (n = 143) and Those Participating at All Assessment Points (n = 43). Sample at T1 (n = 143) Sample Responding at All Assessment Points (n = 43) Sign. Diff. Design Dropoutsa n % n % Chi-square p Value Gender Male Female 114 29 79.7% 20.3% 35 8 81.4 18.6 0.11 .74 Marital statusb Married/registered partner Single Divorced/separated 25 101 16 17.6% 71.1% 11.3% 9 29 5 20.9% 67.4% 11.6% 0.51 .77 Educationb Elementary school Intermediate-level education Upper-secondary education Higher education, up to 4 years Higher education, more than 4 years 11 50 31 38 12 7.7% 35.2% 21.8% 26.8% 8.5% 2 12 5 18 6 4.7% 27.9% 11.6% 41.9% 14.0% 12.10 .02 Unemployed Yes No 16 127 11.2% 88.8% 3 40 7.0% 93.0% 1.10 .30 Violence categoryc Assault Inflicting bodily harm 45 98 31.5 68.5% 15 28 34.9 65.1% 0.33 .56 (continued) Sample at T1 (n = 143) Sample Responding at All Assessment Points (n = 43) Sign. Diff. Dropoutsa n % n % Chi-square p Value Victim’s perception of threat Felt life was at risk Fear of severe physical injury Understood danger afterward Did not perceive as dangerous 50 25 15 28 42.4% 21.2% 12.7% 23.7% 16 9 4 9 42.1% 23.7% 10.5% 23.7% 0.38 .95 Age (years at T1) Mean 31 SD Range 11.0 18–75 Note. aSignificance test of the difference between the sample who participated at all assessment points (n = 43) and dropouts at any time point (n = 100). bInformation is missing for one participant. cThe injuries of each participant were classified into these legal categories at T1 in cooperation with the police and in accordance with a judgment based on the level of physical injury and the intention of the perpetrator to cause harm (where physical injury is the most important criterion) (The Norwegian General Civil Penal Code (Straffeloven) Sections 228 and 229) . The assault category includes less serious physical injuries, often combined with threats of more severe physical injury. The victims of inflicted bodily harm include people with more serious physical injuries, ranging from near-fatal injuries to bone fractures or other substantial damage. Table 1. continued Sample at T1 (n = 143) Sample Responding at All Assessment Points (n = 43) Sign. Diff. Dropoutsa n % n % Chi-square p Value Victim’s perception of threat Felt life was at risk Fear of severe physical injury Understood danger afterward Did not perceive as dangerous 50 25 15 28 42.4% 21.2% 12.7% 23.7% 16 9 4 9 42.1% 23.7% 10.5% 23.7% 0.38 .95 Age (years at T1) Mean 31 SD Range 11.0 18–75 Note. Design aSignificance test of the difference between the sample who participated at all assessment points (n = 43) and dropouts at any time point (n = 100). bInformation is missing for one participant. cThe injuries of each participant were classified into these legal categories at T1 in cooperation with the police and in accordance with a judgment based on the level of physical injury and the intention of the perpetrator to cause harm (where physical injury is the most important criterion) (The Norwegian General Civil Penal Code (Straffeloven) Sections 228 and 229) . The assault category includes less serious physical injuries, often combined with threats of more severe physical injury. The victims of inflicted bodily harm include people with more serious physical injuries, ranging from near-fatal injuries to bone fractures or other substantial damage. Table 1. continued 10 Journal of Interpersonal Violence At T1, 80% of the participants were males and 20% females, ranging from 18 to 75 years of age (mean = 31 years, SD = 11.0). Facial and other head injuries were frequent. Approximately one-third of the sample had serious physical injuries that required specialist treatment beyond the emergency unit. Of those participating in all four assessment rounds, 23% (n = 10/43) had received psychiatric treatment afterwards, four by the public health sys- tem, four at their workplace, and two by private contacts. Several of the par- ticipants reported having sought treatment, without being prioritized within the public health services. Independent sample t-tests showed statistically significant differences in mean educational levels between respondents and dropouts (t = 2.46, p = .01, df = 140), where respondents had higher levels of education. No statistically significant differences were found between par- ticipants responding at all four assessment rounds (n = 43) and dropouts at any time (n = 100) in terms of age, gender, level of physical injury, prior experience of violence, cohabitation, marital status, employment, social sta- tus, perceived life threat, PTSD symptoms or PSS at T1. For detailed infor- mation about the sample, crime characteristics, experiences of prior violence, reported emotions during the assault and acute reactions see (Johansen et al., 2006, 2008). In total, 42% (18/43) who participated in all four-assessment rounds had been exposed to physical violence before recruitment, while 23% (10/43) of them were exposed to new incidents later. Design For information on exposure to subsequent occurrence of violence and other negative life events during the eight years, see (Johansen et al., 2013, p. 3). Ethical approval. The study was approved by the Regional Committee for Medical and Health Research Ethics, West (REK-West, No. 154.01), and by the Privacy Ombudsman, Norwegian Social Science Data Services (NSD, No. 8750). Instruments PTSD symptoms—Impact of Event Scale (IES-22) is a self-reported ques- tionnaire measuring three core dimensions of stress during the previous 7 days in response to a traumatic event: intrusion (8 items); avoidance (8 items), and arousal (6 items) (Weiss & Marmar, 1997). The items are scored on a 4-point Likert scale; 0 = not at all, 1 = rare, 3 = sometimes, and 5 = often, where a higher score represents higher level of symptom load. The scale, which is related to the psychiatric diagnostic systems ICD-10 (World Health Organization, WHO, 1992) and DSM-IV (American Psychiatric Association, APA, 2000) show excellent psychometric properties (Creamer et al., 2003). The sum score was used in the cross-lagged analysis as previ- ous research has found IES-R (revised), which contains the same items as 11 Johansen et al. IES-22, to be essentially unidimensional (Tiemensma et al., 2018). In sup- port, the Omega hierarchical was 0.84 in the present sample for the first assessment round. Perceived social support—The Social Provisions Scale (SPS) consists of a 24-item questionnaire (Cutrona & Russell, 1987; Perera, 2016) with six provisions (social factors): attachment, social integration, nurturance, reas- surance of worth, reliable alliance and guidance. According to Weiss (1974), who developed the provisions; “Attachment,” this is characterized as emotional closeness that attains a sense of security, usually arising from close relationships such as family members or friends. “Social integration” is belonging to a group that share same interests and is often experienced through friendships. “Guidance” consists of advice or information from parents, teachers, or others, while belonging to a family most often gives “Reliable alliance,” which is the need of security of tangible assistance. “Reassurance of self-worth,” described as recognition of competence, skills, and value by others, arise for instance from colleagues at work, while the provision “Opportunity for nurturance” is often described as the sense of others relying on you as one’s offspring or spouse (Cutrona & Russell, 1987; Weiss, 1974). The components of several interpersonal relationships are included in the SPS through these 6 provisions. Each item is scored on a 4-point Likert scale; 1 = strongly disagree, 2 = disagree, 3 = agree, and 4 = strongly agree, where the respondents indicate the extent to which the statements describe their current social relationships. Adding the six sub- scales together forms a total social provision score representing a general support index (Perera, 2016). Instruments Scores are ranged from 1 to 4 for the sub- scales, and 6 to 24 for the total SPS. The questionnaire has been shown to display high sensitivity and specificity (Perera, 2016). The sum-score for all six subscales was used in the cross-lagged analysis as an index including several social networks and relationships. All six provisions were mea- sured, and the results are presented to describe the contextual span, impor- tant for clinical practice. Statistical Analysis Initially, mean values and standard deviation (SD) were used to describe the basic features at the four assessment points of the total and the subscale scores of both PTSD symptoms measured by IES-22, and PSS measured by SPS (SPSS package 22). The relationship between PTSD symptoms and PSS across eight years was analyzed using an autoregressive cross-lagged panel model. As shown in Figure 2 (model), the cross-lagged path analyses investigates whether PTSD symptoms are associated with PSS (at a given Journal of Interpersonal Violence 12 time point t), after controlling for stability over time (PSS scores at a given time point t (T1, T2, or T3) regressed on the immediately preceding time point). These path analyses also modeled the opposite relationship: whether PSS (at time point t) is associated with PTSD symptoms after controlling for the stability of PTSD symptoms. The cross-lagged panel model was ana- lyzed by the use of the lavaan package in R version 3.5.1, including the full information maximum likelihood estimation estimator (FIML) (R Core Team, 2018). Consequently, available observations of participants who had completed the assessments at least on one time-point, were included in the analyses. This is a valid method of handling missing data that are completely missing at random (MCAR), or depends only on the observed data used in the analysis (missing at random; MAR) (Schafer & Graham, 2002). As edu- cational level was related to the probability of dropping out from the study, it was included as an auxiliary variable in the cross-lagged analyses to aid the plausibility of the MAR assumption (Enders, 2008). The overall fit of the models was assessed with: χ2 statistics with degrees of freedom and p val- ues, root mean square error of approximation (RMSEA) with 90% confi- dence intervals (CIs) and p values, comparative fit index (CFI), and Tucker–Lewis index (TLI) (Browne & Cudeck, 1992; Hu & Bentler, 1999). The cutoff for acceptable model fit has been suggested to be .95 or above for CFI and TLI, and from .06 to .08 or less for RMSEA (Browne & Cudeck, 1992; Hu & Bentler, 1999). Results Perceived Social Support (PSS) and Post-traumatic Stress Disorder (PTSD) Symptoms Perceived Social Support (PSS) and Post-traumatic Stress Disorder (PTSD) Symptoms Table 2 shows means and SD on total scales and subscales at all assessment points for PSS as measured by the Social Provision Scale (SPS), and PTSD symptoms as measured by the Impact of Event Scale-22 (IES-22). Mean val- ues for the three subscales (symptom clusters) intrusion, avoidance and arousal assessed by IES-22, were found to decline in the same way as total scores over time, but still being much higher than the general population after eight years (for more details see Johansen et al., 2013). Participants’ interpersonal relationships visualized by the subscores appear to be stable with high values across all assessment rounds. For exam- ple, the provision “Attachment” that expresses emotional closeness to for instance family and friends, appears to be stable as well as “Social integra- tion” that represent group affiliation. 13 Johansen et al. Table 2. Descriptive Information on Scales and Subscales at T1, T2, T3, and T4. T1 T2 T3 T4 Scale/subscales Mean SD Mean SD Mean SD Mean SD IES-22-tot 39.6 27.6 34.9 27.1 32.5 28.9 20.6 25.3 Intrusion 14.5 10.6 12.3 10.2 11.2 11.6 6.9 8.9 Avoidance 12.3 10.5 9.4 11.8 12.0 11.8 7.2 9.6 Arousal 10.4 8.7 9.7 8.8 9.5 8.9 6.5 8.9 SPS-tot 21.4 2.5 21.2 2.2 20.7 3.5 21.7 4.3 Attachment 3.6 0.5 3.5 0.6 3.4 0.7 3.6 0.6 Social integration 3.6 0.5 3.6 0.5 3.5 0.7 3.6 0.7 Guidance 3.6 0.6 3.6 0.6 3.5 0.7 3.5 0.8 Reassurance of worth 3.6 0.6 3.6 0.5 3.4 0.7 3.5 0.7 Opportunity to provide nurturance 3.2 0.7 3.2 0.7 3.3 0.6 3.3 0.8 Reliable alliance 3.7 0.5 3.7 0.5 3.6 0.6 3.7 0.6 Note. IES = Post-traumatic stress disorder symptoms as measured by the Impact of Event Scale–22 and perceived social support as measured by Social Provisions Scale (SPS). T1 = within four months after the assault (n = 143), T2 = 3 months after T1 (n = 94), T3 = 12 months after T1 (n = 73), T4 = 8 years after T1 (n = 47). Figure 2 (model) and Table 3 depict the analysis of the relationship between PSS and PTSD symptoms through the four assessment points. Perceived Social Support (PSS) and Post-traumatic Stress Disorder (PTSD) Symptoms PTSD = Post- traumatic stress disorder symptoms as measured by the Impact of Event Scale-22 (IES-22). T1= within four months after the assault, T2 = three months after T1, T3 = one year after T1 and T4 = eight years after T1. Figure 2. Cross-lagged model showing the relation between PSS and PTSD through four assessment points. through four assessment points. Note. p < .10. p < .05. *p < .001. The model shows standardized regression weights through four time points, and covariates at each measure point of PSS and PTSD. PSS = Perceived social support as measured by the Social Provisions Scale (SPS). PTSD = Post- traumatic stress disorder symptoms as measured by the Impact of Event Scale-22 (IES-22). T1= within four months after the assault, T2 = three months after T1, T3 = one year after T1 and T4 = eight years after T1. Cross-lagged relations. The relation between prior PSS and later PTSD symp- toms were found to be inverse and statistically significant through all assessment points (PSS-T1 → PTSD-T2 b = –0.33, p < .05; PSS-T2 →PTSD-T3, b* = –0.51, p < .05; PSS-T3 →PTSD T4 b* = –0.47, p <.05). The relation between prior PTSD symptoms and later level of PSS were also found to be inverse but not statistically significant (PTSD at T1→PSS-T2 b* = –0.02, p = .188; PTSD T2→PSS-T3 b* = –0.06, p = .352; PTSD T3→PSS-T4 b* = –0.12, p = .311). Perceived Social Support (PSS) and Post-traumatic Stress Disorder (PTSD) Symptoms The cross-lagged model had adequate fit to the data after allowing for PSS at T3 and T4 to be regressed on PSS at T1 (determined by modification indices); robust χ2 = 10.85; df = 10; p = .370; robust RMSEA = .027 90% CI = .00– .107; p < .001, robust CFI = .998; and robust TLI = .993. Stability of PTSD symptoms. The analysis showed a strong and stable association between the prior scores of PTSD symptoms and later PTSD symptoms through all assessment points. PTSD scores at all-time points were highly correlated (between 0.76 and 0.69, p < .001). Variability of perceived social support. Prior PSS scores and later PSS scores between T1→ T2 and T1→T3 were highly related (b* = 0.78, p < .001 and b* = 0.37, p < .05). The relation between T2→T3 was also statistically significant (b* = 0.34, p < .5). The relationship between T1→T4 was near significant (b* = 0.32, p = .054), and T3→T4 was not significant (b* = 0.27, p = .42). The added paths that was included in the modified model revealed that PSS at T1 was statistically significant related to PSS at T3 (b* = 0.37, p < .05) and nearly statistically significant related to PSS at T4 (b* = 0.32, p < .10) (see Figure 2 [model] and Table 3). Journal of Interpersonal Violence 14 Figure 2. Cross-lagged model showing the relation between PSS and PTSD through four assessment points. Note. p < .10. p < .05. *p < .001. The model shows standardized regression weights through four time points, and covariates at each measure point of PSS and PTSD. PSS = Perceived social support as measured by the Social Provisions Scale (SPS). PTSD = Post- traumatic stress disorder symptoms as measured by the Impact of Event Scale-22 (IES-22). T1= within four months after the assault, T2 = three months after T1, T3 = one year after T1 and T4 = eight years after T1. Figure 2. Cross-lagged model showing the relation between PSS and PTSD through four assessment points. Note. p < .10. p < .05. p < .001. The model shows standardized regression weights through four time points, and covariates at each measure point of PSS and PTSD. PSS = Perceived social support as measured by the Social Provisions Scale (SPS). Discussion The present study examined the longitudinal bidirectional relationship between PSS and PTSD symptoms in assault victims across an eight-year period. We find that higher levels of PSS protect against PTSD symptoms, and that lower levels of PSS can increase PTSD symptoms. The opposite, effects of PTSD symptoms on PSS levels, were not significant. However, especially as the relationship is heading in the expected direction, and the relatively small sample size of the study, we cannot rule out the possibility of such an association (Figure 2, model). 15 Johansen et al. Table 3. Regression Weights for the Cross-lagged Model of Perceived Social Support (PSS) and Post-traumatic Stress Disorder Symptoms (PTSD) Over Time. Model Standard All Estimate Standard Error z Value p Value PSS T1 → PSS T2 0.78 0.84 0.07 12.7 <.001 PSS T1 → PSS T3 0.37 0.53 0.24 2.3 .024 PSS T1 → PSS T4 0.32 0.60 0.31 1.9 .054* PSS T1 → PTSD T2 −0.11 −0.33 0.16 −2.1 .035 PTSD T1 → PTSD T2 0.79 0.76 0.07 11.6 <.001* PTSD T1 → PSS T2 −0.07 −0.02 0.02 −1.3 .188 PSS T2 → PSS T3 0.34 0.46 0.25 1.8 .071* PSS T2 → PTSD T3 −0.18 −0.51 0.25 −2.05 .041 PTSD T2 → PTSD T3 0.66 0.72 0.09 7.9 <.001* PTSD T2 → PSS T3 −0.12 −0.06 0.07 −0.9 .352 PSS T3 → PSS T4 0.27 0.34 0.43 0.8 .424 PSS T3 → PTSD T4 −0.24 −0.47 0.17 −2.7 .006 PTSD T3 → PTSD T4 0.75 0.69 0.08 8.6 <.001* PTSD T3 → PSS T4 −0.20 −0.12 0.12 −1.0 .311 Note. Standardized regression weights in the cross-lagged model presented in Figure 2. PSS = Perceived social support as measured by the Social Provisions scale (SPS); PTSD = Post- traumatic stress disorder symptoms as measured by the Impact of Event Scale-22 (IES-22); T1 = within 4 months after the assault, T2 = 3 months after T1, T3 = 12 months after T1, T4 = 8 years after T1. p < .10. p < .05. *p < .001. Table 3. Regression Weights for the Cross-lagged Model of Perceived Social Support (PSS) and Post-traumatic Stress Disorder Symptoms (PTSD) Over Time. Note. Standardized regression weights in the cross-lagged model presented in Figure 2. Discussion PSS = Perceived social support as measured by the Social Provisions scale (SPS); PTSD = Post- traumatic stress disorder symptoms as measured by the Impact of Event Scale-22 (IES-22); T1 = within 4 months after the assault, T2 = 3 months after T1, T3 = 12 months after T1, T4 = 8 years after T1. p < .10. p < .05. *p < .001. The relationship between prior levels of PSS and later levels of PTSD symptoms has a quite similar strength between T1 and T2, as well as for T2 and T3, and even stronger for the third association between one and eight years. Our findings correspond with Ozer and colleagues’ meta-analysis (2003) that revealed stronger associations between SS and PTSD in studies conducted more than three years after the traumatic event, compared to studies with a shorter postassessment period. The inverse influence we found of PSS on PTSD symptoms, across all assessment points supports the causation theo- ries previously introduced (Freedman et al., 2015; Pruitt & Zoellner, 2008). A study of disaster victims additionally give support for the selection model when investigating PSS and PTSD symptoms 12–18 months postexposure, while supporting the causation model when measuring the relationship 6–12 months and 18–24 months postexposure (Kaniasty & Norris, 1993). 16 Journal of Interpersonal Violence We note that in sum, quite a few studies support the selection model (Kaniasty & Norris, 2008). Among U.S. veterans, more severe PTSD symp- toms predict later worsening in SS, while levels of SS does not seem to affect future PTSD (Dworkin et al., 2018; King et al., 2006; Laffaye et al., 2008), also shown in torture victims from Iraq (Hall et al., 2014). The inconsistency of findings may be due to differences in the characteristics of the populations studied, i.e., civilian versus military personnel. We find PSS at T1 to be an independent predictor of PSS at T3 and T4, explained by the rather strong relation between the first PSS assessment close to the exposure to violence, and PSS after one and eight years. Ozer and col- leagues’ (2003) suggested a cumulative effect of SS over time, with a growing strength after several years after the exposure, alternatively, that SS function as a secondary prevention. Discussion Ullman (1996) found negative social reactions to be strongly associated with increased psychiatric symptoms in sexual assaulted victims. The only factor related to better adjustment was being believed in and being listened to by others. More, positive social reac- tions were unrelated to adjustment (Ullman, 1996). Further, studies among victims of the terror attack at Utøya, Norway, on the 22nd of July 2011, show that SS barriers are highly associated with post-traumatic stress symptoms (Thoresen et al., 2014). Our findings correspond to other longitudinal studies that underline how PSS plays an important role for adult victims by amelio- rating and/or protecting against the development of PTSD, however, none of these studies includes a reassessment eight years after the event (Kaniasty & Norris, 2008; Robinaugh et al., 2011; Vogt et al., 2017; Yap & Devilly, 2004). To feel supported, people seem to need involvement within different lev- els of social context, and to receive stability through different types of rela- tionships (Maercker & Horn, 2013; Weiss, 1974). Another discussion of relevance is whether PSS is related to the individual personality trait, or to the more dynamic personality state. The first interpretation, recognizes PSS as being consistent and long lasting, thus presupposes stability across time and events. PSS is then a strong personality component imbedded in more tradi- tional attachment theories. Our findings are more consistent with the second comprehension that personality is more dynamic as PSS varies and fluctuates due to recent experiences (Sarason et al., 1990; Yap & Devilly, 2004). Compared to individuals who report low levels of perceived support, those who report high levels of support seem to habit somewhat different coping strategies to overcome their emotional distress (Kaniasty & Norris, 2008). Discussion Several researchers now acknowledge that social net- work size or density of social contacts does not necessarily equal the actual support provided (Guay et al., 2011; Platt et al., 2014). The level of engage- ment in social groups and being socially active may have a greater personal impact on mental health than mere perception of the strength of SS. For instance, in their epidemiologic survey (n = 31,650), Platt and colleagues (2014) found that a diversity of social networks were protective against PTSD symptoms. Regarding negative or even lack of SS, several studies have suggested that this has a stronger explanatory power than positive sup- port in describing the relationship between PSS and PTSD symptoms (Brewin & Holmes, 2003; Scarpa et al., 2006). Ullman (1996) found negative social reactions to be strongly associated with increased psychiatric symptoms in sexual assaulted victims. The only factor related to better adjustment was being believed in and being listened to by others. More, positive social reac- tions were unrelated to adjustment (Ullman, 1996). Further, studies among victims of the terror attack at Utøya, Norway, on the 22nd of July 2011, show that SS barriers are highly associated with post-traumatic stress symptoms (Thoresen et al., 2014). Our findings correspond to other longitudinal studies that underline how PSS plays an important role for adult victims by amelio- rating and/or protecting against the development of PTSD, however, none of these studies includes a reassessment eight years after the event (Kaniasty & Norris, 2008; Robinaugh et al., 2011; Vogt et al., 2017; Yap & Devilly, 2004). be useful to illustrate the importance of PSS for both the specific victim and for the professionals. Several researchers now acknowledge that social net- work size or density of social contacts does not necessarily equal the actual support provided (Guay et al., 2011; Platt et al., 2014). The level of engage- ment in social groups and being socially active may have a greater personal impact on mental health than mere perception of the strength of SS. For instance, in their epidemiologic survey (n = 31,650), Platt and colleagues (2014) found that a diversity of social networks were protective against PTSD symptoms. Regarding negative or even lack of SS, several studies have suggested that this has a stronger explanatory power than positive sup- port in describing the relationship between PSS and PTSD symptoms (Brewin & Holmes, 2003; Scarpa et al., 2006). Discussion Their explanation is that as PTSD symptoms eventu- ally diminishes and no longer are considered as acute reactions after traumatic exposure, the preventive effect of SS also becomes clearer (Ozer et al., 2003). p , p ( , ) In addition, our results of the relationship between PSS at different time points show that the relative stability varied, with a high value between T1 and T2, and low values between T2 and T3 as well as between T3 and T4. This indicate that PSS after 3 months has quite low explanatory value when it comes to PSS one year after the event. Similarly, after one year, PSS has a low explanatory value for PSS after eight years. It is reasonable to suggest that PSS near exposure to violence is more positively expressed from close rela- tionships, such as family and friends, while later PSS has features of more distanced social acquaintances thus affecting the questionnaire scores. Social relations after trauma is a complex issue, and how we seek the benefit of rela- tionships affects the actual availability of social connections, which further influences how we think, feel and act (Bryant, 2016). To understand how post- traumatic stress reactions inflict on a social network context is of importance for illuminating many of the core mechanisms that may influence interper- sonal adjustments (Bryant, 2016). The combination of total PSS scores used in the cross-lagged model, and all six different provisions, give us a broad and inclusive understanding of the participants’ perception of SS. Usually, each provision reflects scores from one type of relationship only, but a person commonly receive several provi- sions at once (Cutrona & Russell, 1987). The total PSS score were stable with significant relationship through all assessment points as shown in Figure 2 (model), and the mean value of both total scores and subscales showed just small variations as displayed in Table 2. Probably, to get a better understand- ing of the victim’s need for SS after violence, the context of their involve- ment in various levels of relationships should be recognized. For example, the sociointerpersonal model introduced by Maerceker and Horn (2013) can 17 Johansen et al. be useful to illustrate the importance of PSS for both the specific victim and for the professionals. Clinical Implications In clinical contexts, the dynamic characteristics of PSS, as well as the phe- nomenon of victimization, must be considered. The experience of someone Journal of Interpersonal Violence 18 intentionally wanting to inflict injury makes exposure to such violence some- what different from other types of trauma. Perceived, intention of a harming act is a serious risk factor, which often increases the victim’s negative reac- tions (Kilpatrick & Acierno, 2003; Sareen, 2014). Exposure to violence as in physical assault, is characterized as interpersonal potential traumatic events (PTEs), and the prospective of preventing long-term mental health problems should be highlighted (Birur et al., 2017; Johansen et al., 2007; Kilpatrick & Acierno, 2003). Assault variables such as the severity of physical injury, characteristics of the assault scenery, and victims level of self-efficacy, may affect the development of PTSD symptoms as well as PSS (Kilpatrick & Acierno, 2003; Nygaard et al., 2017). Females are two to three times more likely to develop PTSD symptoms than males (Olff, 2017), which was also the case from our findings at T1. In total, 86% (24/28) of the female victims and 52% (57/110) of the male victims scored within probable or partial PTSD in the acute phase by IES-15 with values between 35 and 75 for probable, and 20 and 34 for partial (Johansen et al., 2006, Table 2). Andrews et al. (2003) found in a sample of victims of violent crime (118 males and 39 females), that the effect of support satisfaction or negative responses six-month after exposure were significantly enhanced for the females. Despite the well-doc- umented benefits of SS, there are barriers that prevent active use, especially among males. Women seem to adopt SS to a greater extent than men for cop- ing with major life events (Taylor et al., 2000). The use of social networks is an example of such gender difference, where females tend to show more active approach coping strategies compared to males (Larsen, 2011). The total sample consisting of 80% male participants had a high and stable rate of PTSD symptoms throughout all four assessment points (Table 2). Considering that the sample included several young males (mean age = 31, range = 18–75), being at the peak of their physical and psychological health, should indicate expectations of a high probability of recovery. Clinical Implications Instead, the likelihood of developing PTSD symptoms was found to be 48% among those who participated in all rounds (Johansen et al., 2013). The steady and high numbers of PTSD symptoms in a long-term perspective, show the impor- tance of providing sufficient measures to prevent or alleviate the sufferings, regardless of gender. It will be of most interest to gain increased knowledge on how SS can contribute within this context. Unfortunately, victims of nondomestic violence does not seem to be pri- oritized or integrated into Norwegian political and public health strategies (Norwegian Ministry of Justice and Public Security, 2014). Not to compete with the necessary attention towards follow-up victims of violence in close relationships, but the many incidents of nondomestic violence in society 19 Johansen et al. combined with a significant amount of long-term prevalence of PTSD symp- toms, addresses the need to focus upon these victims accordingly. combined with a significant amount of long-term prevalence of PTSD symp- toms, addresses the need to focus upon these victims accordingly. Our participants consisted of victims who did receive psychiatric treat- ment or none such treatment after the incident. Of those who completed the assessments at all four rounds, 23% (10/43) had received psychiatric treat- ment. One might speculate whether high scores on PSS should be considered an important long-term factor in natural recovery after being subjected to a violent assault, as well as representing importance for the outcomes of psy- chosocial follow-up or psychiatric treatment. Likewise, low PSS might be considered a risk factor among those who did not receive treatment as well as for those who did. A study of social relationship satisfaction associated with PTSD by Freedman et al. (2015), included both treated (98/501) and untreated (313/501) individuals, and the results show similar trend following traumatic events. Hence, it might be appropriate to prioritize interventions aiming at increasing positive SS, as a preventive measure. Further, that clinical practice systematically advise victims of violence and their closed ones to be aware of potentially emerging PTSD symptoms, to reinforce positive social interac- tions as well as attenuate negative interactions (Guay et al., 2006; Wagner et al., 2016). As held by the causation theories (Freedman et al., 2015; Pruitt & Zoellner, 2008), victims perceptions of available support may be disturbed, both in early stages and in the long run after exposure to PTE. Clinical Implications Within a clini- cal perspective, one should be attentive towards the avoidance effect emerg- ing from PTSD symptoms; avoidance and retraction behavior may drive family and friends away (Andrews et al., 2003). Not to ignore, family and friends may alienate an individual with PTSD, and this should equally be addressed (Ladd & Churchill, 2012). We believe that proper information to members of formal as well as infor- mal support networks on how both positive and negative SS may influence post-traumatic stress reactions after exposure to physical assault, may increase knowledge and understanding about their ability to respond support- ively. This might in turn encourage strategies of recovery making a crucial difference for the affected individual (Ullman & Peter-Hagene, 2016). Within this context, relations and interpersonal communication are typically at stake as time passes. The situation may include distress and uncertainty for the partner, with the potential of secondary traumatization, mistrust, or caregiver burden that might ruin the relationship (Lambert et al., 2012). Regardless of the trauma survivor’s perception of SS being accurate or mistaken, low levels of PSS experiences may render the victim less resistant to the negative psy- chological impact of significant others after the violence. An informed pro- fessional judgment of the victims PSS can be crucial to follow-up decisions (Yap & Devilly, 2004). If a family member displays PTSD symptoms, Journal of Interpersonal Violence 20 information about the importance of SS, common PTSD symptoms and potential challenges for all family members, may be beneficial as well as advice to seek help if it becomes particularly demanding. These kind of increased knowledge might prevent or mitigate development of symptoms as well as negative consequences for the family (Dyregrov & Dyregrov, 2008). g q y ( y g y g ) Partner accommodation is a relatively new and potentially important con- struct to consider in treatment planning for people with PTSD (Fredman et al., 2016). When individual perceptions of actual support are mistaken, inter- ventions should include components to change this cognitive scheme, to dis- courage the negative effects of reduced PSS, for example, by correcting the perception through exposure (Yap & Devilly, 2004). However, family mem- bers should be included in follow-up also when perceptions of low support seem correct. It might be warranted to increase support-seeking behavior as well (Kaniasty & Norris, 2008). Clinical Implications A better understanding of how PSS affects the course of PTSD in couples’ therapy may be particularly useful (Fredman et al., 2016). Importantly, victims of nondomestic violence do not necessarily seek medical assistance due to physical injuries, and even less psychiatric treatment (McCart et al., 2010). During the acute phase of emotional numb- ness and cognitive deterioration, it is sometimes difficult to comprehend sup- port from others, and to evaluate need of professional care. One should therefore implement routines at frontline medical units to contact the victims after the initial visit, identifying resources such as social networks and sup- port access. Other potentially harmful post-traumatic reactions such as sleep disturbances, nightmares, changes in eating habits, self-medication, sick leave, or other symptoms related to PTSD should be noted. Involving the partner in clinical practice can be particularly effective for couples where the partner is very aware of PTSD symptoms. Combined with other relevant sup- port measures to relieve such symptoms, it may prevent development of long-term weakening, and improve quality of life and well-being for both the inflicted individual and their significant other. Strengths and Limitations The main strength of our study is the longitudinal design. As far as we are aware, this is the first study being published that includes the assessment of PTSD symptoms and PSS across four different time points after a nondomes- tic violence event, the last measures conducted eight years later. The study design offers the possibility of investigating the effects of such violence in short, intermediate, and longer terms. By using cross-lagged autoregressive structural equation models, we minimized the undesirable effects of Johansen et al. 21 confounders, and were able to address the long-term bidirectional interaction between PTSD symptoms and PSS. Another strength is the homogeneity of the potential traumatic events; all the respondents were exposed to physical assault by a perpetrator other than a family member. In addition, restrictive inclusion criteria and legally defined definitions of physical injuries were applied, and the gender distribution at T1 was representative to the population experiencing violent crime (other than domestic assault) in Norway (Statistics Norway, 2009; Steen & Hunskaar, 2004). According to the review by Kaniasty & Norris (2008), only a handful of longitudinal studies display similar homogeneity. Naturally, we lack pre- violence (baseline) measures of PTSD symptoms and PSS. To improve the validity of our study by minimizing recall bias, we strived to perform the interviews at T1 as soon as possible after the event. Furthermore, knowledge of the exact date of exposure and the elapsed time from the exposure to the interview for all participants, strengthens the validity. One limitation to consider is generalization of the current findings to other groups exposed to serious violence. Our sample, mostly males, were recruited based on the experience of a single physical assault. Further, there is a chal- lenge associated with the complexity of the conceptual and methodological issues within this type of research. However, some individuals are clearly more susceptible to develop PTSD than others (Sareen, 2014; WHO, 2002). Trauma reactions have some specific symptom characteristics, and at the same time, there are numbers of individual variations (WHO, 2018). Understanding the individual trauma history, personal self-esteem and poten- tial SS will be important in terms of prevention and treatment, regardless of the specific event or gender. Another limitation is the relatively small sample size and a rather high degree of drop out across the four assessments were only 43 of 143 participants completed all four rounds up to the eight-year period. Conclusion Regardless of theoretical models, quality and availability of SS seems to play an important role by protecting the victims against developing PTSD after being subjected to a physical assault. Our results support the need to recog- nize that a single physical assault can have major impacts on the victim’s life for years to come. It is essential to widen this knowledge for the prevention and relief of PTSD symptoms. We also need to identify those at risk for developing serious psychopathology, and to provide relevant public informa- tion, victim support, and sufficient treatment. Acknowledgments We thank the local police and the local outpatient workers in Bergen and Oslo for assisting with the recruitment of victims of violence. The authors acknowledge Irmelin B. Christensen for her contribution to making contact with the participants again, after eight years. Most of all, we thank the participants. Strengths and Limitations High numbers of participants who drop out is an unfortunate but a common problem in longitudinal studies of assault victims, as motivation to take part diminishes as time passes (Andrews et al., 2003; Elklit & Brink, 2004; Peleg & Shalev, 2006). To include all available observation of all par- ticipants at any time and to reduce the drop out effect, we used FIML in the regression model. We used self-reported questionnaires to measure PTSD symptoms and PSS. Knowing the complexity of these phenomena, the fact that we only measured PSS must be considered a limitation as well. A more thorough understanding of SS and interpersonal interactions requires different types of assessment (Vogt et al., 2017). To compensate for not performing structured clinical interviews, two different questionnaires were used to assess PTSD symptoms at each assessment point, as described in previous articles 22 Journal of Interpersonal Violence (Johansen et al., 2013). We found IES-22 to be a valid measure of post-trau- matic stress symptoms. Despite these limitations, our findings seem solid. Both questionnaires, IES-22 and SPS, are commonly used for measuring PTSD symptoms and PSS, both are reported to be reliable and valid (Creamer et al., 2003; Cutrona & Russell, 1987; Perera, 2016). Future Research Interpersonal processes in close relationships, as well as influences from other social networks, represent an important research approach in under- standing the interaction between SS and PTSD symptoms (Maercker & Horn, 2013). Future studies should be designed to grasp deeper into the connection between the dimensions of PSS and post-traumatic stress symptoms, and how this connection might coexist for many years or even for life. Furthermore, we need to know more about how to prevent and reduce severe psychopathol- ogy for those exposed to nondomestic violence, in the long run. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. ORCID iDs Venke A. Johansen https://orcid.org/0000-0001-9556-7496 Kjell Morten Stormark https://orcid.org/0000-0001-8543-1878 Funding The author(s) received no financial support for the research, authorship, and/or publi- cation of this article. 23 Johansen et al. References Exploring the links between post- traumatic stress disorder and social support: Processes and potential research avenues. Journal of Traumatic Stress, 19(3), 327–338. Guay, S., Billette, V., & Marchand, A. (2006). Exploring the links between post- traumatic stress disorder and social support: Processes and potential research avenues. Journal of Traumatic Stress, 19(3), 327–338. Guay, S., Beaulieu-Prévost, D., Beaudoin, C., St-Jean-Trudel, E., Nachar, N., Marchand, A., & O’connor, K. P. (2011). 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Her research focuses on among other possible prevention and increased risk of post-traumatic stress reactions after exposure to violence or other potentially traumatic events, as well as consequences for quality of life. Anne Marita Milde, PhD, has a specialist degree in clinical psychology. She is work- ing as a senior researcher at Regional Centre for Child and Youth Mental Health and Child Welfare, NORCE. Her research focuses on among other traumatic stress and psychophysiological stress responses and effects on behavior, with experience from both animal and human studies, thus an interest in identifying mechanisms within a translational perspective. Roy Miodini Nilsen, PhD, is a professor at the Western Norway University of Applied Sciences, Norway. He is a biostatistician with a special focus on longitudinal modeling in epidemiological research. Currently, his research focuses on perinatal health outcomes in immigrant women using large national population-based data. Kyrre Breivik, PhD, is senior researcher at the Regional Centre for Child and Adolescent Mental Health and Welfare, NORCE, the Norwegian Research Centre. His current research interests focus on bullying, child and adolescent development, and children’s adjustment to divorce and measurement issues. Dag Øystein Nordanger, PhD, is professor in psychology at Oslo Metropolitan University and specialist in clinical child psychology at Resource Centre on Violence, Traumatic Stress and Suicide Western Norway (RVTS West). His research and pub- lications have circled around the impacts of trauma in general, with a particular focus the later years on the consequences of childhood adversities and on interventions to prevent and treat them. Kjell Morten Stormark, PhD, is a professor at Regional Centre for Child and Youth Mental Health and Child Welfare, NORCE. His research interests are on psychiatric epidemiology, health promotion and developmental trajectories in children and adolescents. Lars Weisæth, MR, PhD, is emeritus professor of psychiatry (traumatic stress) at the University of Oslo. Since the mid-1970s his research has focused on how humans respond before, during and after life threatening situations such as natural and man- made disasters, war, peacekeeping operations, accidents, terror, violence, and CBRN-incidents.
https://openalex.org/W2789646648	https://www.nature.com/articles/s41419-017-0089-1.pdf	English	null	Mitochondrial glutamine metabolism via GOT2 supports pancreatic cancer growth through senescence inhibition	Cell death and disease	2,018	cc-by	8,411	The Author(s) 2018 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/. A R T I C L E O p e n A c c e s s Abstract Abstract Cellular senescence, which leads to a cell cycle arrest of damaged or dysfunctional cells, is an important mechanism to restrain the malignant progression of cancer cells. Because metabolic changes underlie many cell-fate decisions, it has been suggested that cell metabolism might play key roles in senescence pathways. Here, we show that mitochondrial glutamine metabolism regulates senescence in human pancreatic ductal adenocarcinoma (PDAC) cells. Glutamine deprivation or inhibition of mitochondrial aspartate transaminase (GOT2) results in a profound induction of senescence and a suppression of PDAC growth. Glutamine carbon ﬂow through GOT2 is required to create NADPH and to maintain the cellular redox state. We found that elevated reactive oxygen species levels by GOT2 knockdown lead to the cyclin-dependent kinase inhibitor p27-mediated senescence. Importantly, PDAC cells exhibit distinct dependence on this pathway, whereas knockdown of GOT2 did not induce senescence in non-transformed cells. The essentiality of GOT2 in senescence regulation of PDAC, which is dispensable in their normal counterparts, may have profound implications for the development of strategies to treat these refractory cancers. Mitochondrial glutamine metabolism via GOT2 supports pancreatic cancer growth through senescence inhibition Seungyeon Yang1,2, Sunsook Hwang1,2, Minjoong Kim1,2, Sung Bin Seo1,2, Jeong-Hwa Lee1,2 and Seung Min Jeong1,2 Yang et al. Cell Death and Disease (2018) 9:55 DOI 10.1038/s41419-017-0089-1 Yang et al. Cell Death and Disease (2018) 9:55 DOI 10.1038/s41419-017-0089-1 Cell Death & Disease Cell Death & Disease Correspondence: Seung Min. Jeong (smjeong@catholic.ac.kr) 1Department of Biochemistry, Institute for Aging and Metabolic Diseases, College of Medicine, The Catholic University of Korea, 222, Banpo-daero, Seocho-gu, Seoul 06591, Republic of Korea 2Institute for Aging and Metabolic Diseases, College of Medicine The Catholic University of Korea, 222, Banpo-daero, Seocho-gu, Seoul 06591, Republic of Korea Edited by C. Munoz-Pinedo Introduction oncogene activation3,4. Senescence has been proposed to be an important tumor-suppressive mechanism. The premature senescence induction by tumor suppressors was observed in multiple cancer models5,6. Mutations or dysregulation of senescence regulators, including p53, p21, p16, and retinoblastoma protein (Rb), are frequently observed in many human cancers and strongly correlate with a worse prognosis6,7. Because accruing evidence suggests that metabolic regulation has a predominant role for determining cellular states, including proliferation and cell cycle arrest, it is not surprising that cell metabolism contributes to cellular senescence. Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is one of the most malignant cancers1. PDAC exhibits acute resistance to all form of therapy, including conventional chemotherapy, radiotherapy and targeted agents, which leads to the dis- mal prognosis of PDAC patients2. The profound resis- tance to treatments indicates that these refractory cancers may have altered cell survival pathways and aberrant metabolism. Thus, it is critical to identify new therapeutic targets for PDAC. Cellular senescence is a state of growth arrest in response to various cellular stress stimuli including replicative cell culture, oxidative stress, DNA damage and The dysregulation of cell metabolism is a deﬁning fea- ture of cancer cells. In PDAC, oncogenic KRAS, serving a critical role in PDAC initiation and maintenance, med- iates this reprogramming of cellular energy metabolism to support its growth and survival8. Interestingly, PDAC cells exhibit a distinct glutamine (Gln) metabolism. Whereas many cancer cells rely on deamination of Gln-derived glutamate to replenish mitochondrial carbon pool, PDAC © The Author(s) 2018 © The Author(s) 2018 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/. Ofﬁcial journal of the Cell Death Differentiation Association Yang et al. Cell Death and Disease (2018) 9:55 Page 2 of 10 Page 2 of 10 We ﬁrst noticed that Gln deprivation caused a pro- found increase in 8988T PDAC cells expressing senescence-associated β-galactosidase (SA-β-Gal) (Fig. 1a). The induction of senescence in these cells was also indicated by large and ﬂattened cellular morphol- ogy (Fig. 1b). Because proliferating cells use precursors derived from tricarboxylic acid (TCA) cycle, replen- ishment of the mitochondrial carbon pool is required for the maintenance of mitochondrial integrity and function12. Gln anaplerosis is essential to provide a carbon source to the TCA cycle. To assess the role of mitochondrial Gln metabolism in senescence, we trea- ted cells with 6-diazo-5-oxo-L-norleucin (DON), an inhibitor of glutaminase (GLS). GLS is the ﬁrst required enzyme for mitochondrial Gln anaplerosis. Notably, GLS inhibition signiﬁcantly induced senescence in 8988T PDAC cells (Fig. 1c and S1A). Consistent with these results, GLS knockdown by using short hairpin RNAs (shRNAs) also strongly elicited senescence (Fig. 1d and S1B). Thus, these results demonstrate that mitochondrial Gln metabolism is essential for senes- cence regulation in PDAC cells. use Gln-derived aspartate (Asp) to maintain the cellular redox state, which is essential for PDAC growth9. More- over, our previous work demonstrated that enhanced mitochondrial glutamine anaplerosis suppresses PDAC growth10. However, the importance of Gln metabolism in PDAC senescence is not well elucidated. Due to the pivotal role of Gln metabolism as an important regulator of cellular redox balance and multiple cell fate determination, we sought to speciﬁcally probe the role of mitochondrial Gln pathways in regulating pan- creatic cancer growth and senescence. Mitochondrial glutamine metabolism suppresses cellular senescence in PDAC Increasing evidence demonstrates that many cancer cells use Gln to support their energetic and synthetic needs11,12. However, the role of Gln metabolism in cel- lular senescence is not well determined. Recently, we and others reported that Gln is critical for PDAC growth and survival9,10. Thus, we sought to explore the functional role of Gln metabolism in PDAC senescence. A Control (-) Gln 0 10 20 30 * Senescence (% SA-β-gal+ cells) shGFP shGLS GLS Actin shGFP shGLS 0 10 20 30 Senescence (% SA-β-gal+ cells) Control 10 20 0 10 20 30 40 50 * * DON (μM) Senescence (% SA-β-gal+ cells) B C D (kDa) 50 37 Control (-) Gln Fig. 1 The Inhibition of mitochondrial glutamine metabolism induces senescence in PDAC cells. a, 8988T cells were plated in complete media which was replaced the following day with Gln-free medium. Percentages of SA-β-gal positive cells are shown. b, Representative images of SA β-gal staining for 8988T cells in the presence or absence of Gln. c, Percentages of SA-β-gal positive cells in 8988T cells treated with increasing concentrations of DON. d, Percentages of SA-β-gal positive cells in control (shGFP) and GLS knockdown (shGLS) 8988T cells (left). Western blot conﬁrmed knockdown of GLS expression (right). β-actin serves as a loading control. All error bars ± SEM. p < 0.01 and p < 0.001 A Control (-) Gln 0 10 20 30 Senescence (% SA-β-gal+ cells) B Control (-) Gln B ( ) shGFP shGLS GLS Actin shGFP shGLS 0 10 20 30 Senescence (% SA-β-gal+ cells) Control 10 20 0 10 20 30 40 50 * * DON (μM) Senescence (% SA-β-gal+ cells) C D (kDa) 50 37 D C Fig. 1 The Inhibition of mitochondrial glutamine metabolism induces senescence in PDAC cells. a, 8988T cells were plated in complete media which was replaced the following day with Gln-free medium. Percentages of SA-β-gal positive cells are shown. b, Representative images of SA β-gal staining for 8988T cells in the presence or absence of Gln. c, Percentages of SA-β-gal positive cells in 8988T cells treated with increasing concentrations of DON. d, Percentages of SA-β-gal positive cells in control (shGFP) and GLS knockdown (shGLS) 8988T cells (left). Western blot conﬁrmed knockdown of GLS expression (right). β-actin serves as a loading control. All error bars ± SEM. Mitochondrial glutamine metabolism suppresses cellular senescence in PDAC p < 0.01 and p < 0.001 Ofﬁcial journal of the Cell Death Differentiation Association A Control EGCG AOA 0 10 20 30 40 Senescence (% SA-β-gal+ cells) shGFP #1 #2 0 10 20 30 40 50 shGOT2 * * Senescence (% SA-β-gal+ cells) shGFP shGOT2 shGFP #1 #2 0.0 0.4 0.8 1.2 shGOT2 * ** Relative cell growth shGFP shGOT2 shGFP #1 #2 0 5 10 15 shGLUD1 Senescence (% SA-β-gal+ cells) shGFP shGLUD1 B C F D E G0/G1 S G2/M 0 20 40 60 80 shGFP shGOT2 * * P = 0.0559 % Cells shGFP #1 #2 0 4 8 12 shGOT2 * No. of PML-NBs per nucleus shGFP #2 #1 shGOT2 G shGFP #1 #2 shGFP #1 #2 0 10 20 30 shGOT2 shGOT2 ** GOT2-OE Vector Senescence (% SA-β-gal+ cells) Fig. 2 Mitochondrial transaminase GOT2 is responsible for the regulation of PDAC senescence. a, Senescence induction of 8988T cells treated with EGCG (50 μM) or AOA (0.5 mM). b and c, Senescence induction of 8988T cells expressing a control shRNA (shGFP) or two independent shRNAs targeting GLUD1 b, or GOT2 c. d, Relative proliferation of 8988T cells expressing a control shRNA or shRNAs to GOT2. e, Percentage of cells in G0/G1, S and G2/M phases in 8988T cells expressing a control shRNA or a shRNA to GOT2. f, Percentages of SA-β-gal positive cells in GOT2 overexpressed (GOT2-OE) 8988T cells expressing a control shRNA or shRNAs to GOT2. g, Numbers of promyelocytic leukemia nuclear bodies (PML-NBs; left) and immunoﬂuorescent staining using nuclear (DAPI; blue) and PML (green) in 8988T cells expressing a control shRNA or shRNAs to GOT2 (right).All error bars ± SEM. p < 0.05, p < 0.01 and *p < 0.001 Yang et al. Cell Death and Disease (2018) 9:55 Page 3 of 10 Page 3 of 10 Yang et al. Mitochondrial glutamine metabolism suppresses cellular senescence in PDAC of PML-NBs per nucleus G G Fig. 2 Mitochondrial transaminase GOT2 is responsible for the regulation of PDAC senescence. a, Senescence induction of 8988T cells treated with EGCG (50 μM) or AOA (0.5 mM). b and c, Senescence induction of 8988T cells expressing a control shRNA (shGFP) or two independent shRNAs targeting GLUD1 b, or GOT2 c. d, Relative proliferation of 8988T cells expressing a control shRNA or shRNAs to GOT2. e, Percentage of cells in G0/G1, S and G2/M phases in 8988T cells expressing a control shRNA or a shRNA to GOT2. f, Percentages of SA-β-gal positive cells in GOT2 overexpressed (GOT2-OE) 8988T cells expressing a control shRNA or shRNAs to GOT2. g, Numbers of promyelocytic leukemia nuclear bodies (PML-NBs; left) and immunoﬂuorescent staining using nuclear (DAPI; blue) and PML (green) in 8988T cells expressing a control shRNA or shRNAs to GOT2 (right).All error bars ± SEM. p < 0.05, p < 0.01 and p < 0.001 Ofﬁcial journal of the Cell Death Differentiation Association Mitochondrial glutamine metabolism suppresses cellular senescence in PDAC Cell Death and Disease (2018) 9:55 A Control EGCG AOA 0 10 20 30 40 Senescence (% SA-β-gal+ cells) shGFP #1 #2 0 10 20 30 40 50 shGOT2 * * Senescence (% SA-β-gal+ cells) shGFP shGOT2 shGFP #1 #2 0 5 10 15 shGLUD1 Senescence (% SA-β-gal+ cells) shGFP shGLUD1 B C shGFP #1 #2 0 5 10 15 shGLUD1 Senescence (% SA-β-gal+ cells) shGFP shGLUD1 B A Control EGCG AOA 0 10 20 30 40 * Senescence (% SA-β-gal+ cells) shGFP #1 #2 0 10 20 30 40 50 shGOT2 * * Senescence (% SA-β-gal+ cells) shGFP shGOT2 C A Control EGCG AOA 0 10 20 30 40 * Senescence (% SA-β-gal+ cells) S shGFP #1 #2 0 5 10 15 shGLUD1 Senescence (% SA-β-gal+ cells) shGFP shGLUD1 B C C B shGFP #1 #2 0.0 0.4 0.8 1.2 shGOT2 * ** Relative cell growth shGFP shGOT2 F D E G0/G1 S G2/M 0 20 40 60 80 shGFP shGOT2 * * P = 0.0559 % Cells shGFP #1 #2 shGFP #1 #2 0 10 20 30 shGOT2 shGOT2 GOT2-OE Vector Senescence (% SA-β-gal+ cells) D F E shGFP #1 #2 0 4 8 12 shGOT2 *** No. of PML-NBs per nucleus shGFP #2 #1 shGOT2 G GOT2 OE Vector Fig. 2 Mitochondrial transaminase GOT2 is responsible for the regulation of PDAC senescence. a, Senescence induction of 8988T cells treated with EGCG (50 μM) or AOA (0.5 mM). b and c, Senescence induction of 8988T cells expressing a control shRNA (shGFP) or two independent shRNAs targeting GLUD1 b, or GOT2 c. d, Relative proliferation of 8988T cells expressing a control shRNA or shRNAs to GOT2. e, Percentage of cells in G0/G1, S and G2/M phases in 8988T cells expressing a control shRNA or a shRNA to GOT2. f, Percentages of SA-β-gal positive cells in GOT2 overexpressed (GOT2-OE) 8988T cells expressing a control shRNA or shRNAs to GOT2. g, Numbers of promyelocytic leukemia nuclear bodies (PML-NBs; left) and immunoﬂuorescent staining using nuclear (DAPI; blue) and PML (green) in 8988T cells expressing a control shRNA or shRNAs to GOT2 (right).All error bars ± SEM. p < 0.05, p < 0.01 and p < 0.001 shGFP #1 #2 0 4 8 12 shGOT2 * No. of PML-NBs per nucleus shGFP #2 #1 shGOT2 G shGFP #1 #2 0 4 8 12 shGOT2 * No. Glutamine metabolism through GOT2 regulates senescence in PDAC mitochondrial carbon pool. To examine the mechanisms involved in the regulation of senescence in PDAC, we treated cells with either epigallocatechin gallate (EGCG), a GLUD1 inhibitor, or aminooxyacetate (AOA), a transa- minase inhibitor. Whereas EGCG treatment had no effect on senescence, AOA treatment markedly induced In mitochondria, Gln is metabolized via GLS to gluta- mate and ammonia (NH4 +) and further catabolized to the TCA cycle intermediate α-ketoglutarate (αKG) via gluta- mate dehydrogenase (GLUD1) or transaminases to fuel Ofﬁcial journal of the Cell Death Differentiation Association Page 4 of 10 Yang et al. Cell Death and Disease (2018) 9:55 A B C HPDE WI38 HEK293T 0 10 20 30 40 Senescence (% SA-β-gal+ cells) shGFP shGOT2 * * Panc1 PL45 Tu8902 0 10 20 30 40 Senescence (% SA-β-gal+ cells) shGFP shGOT2 * * * * * Panc1 PL45 Tu8902 0.0 0.4 0.8 1.2 Relative cell growth shGFP shGOT2 Fig. 3 GOT2 regulates senescence in PDAC cells but not in normal cells. a and b, Senescence induction a, and relative proliferation b, of PDAC cell lines (Panc1, PL45 and Tu8902) expressing a control shRNA (shGFP) or two independent shRNAs to GOT2. c, Percentages of SA-β-gal positive cells in HPDE, WI38 and HEK293T cells expressing a control shRNA (shGFP) or two independent shRNAs to GOT2. All error bars ± SEM. p < 0.01 and *p < 0.001 A * * Panc1 PL45 Tu8902 0 10 20 30 40 Senescence (% SA-β-gal+ cells) shGFP shGOT2 senescence (Fig. 2a and S2A). To further conﬁrm that mitochondrial transaminases regulate PDAC senescence, we reduced the levels of GLUD1 or mitochondrial aspartate transaminase (GOT2), highly upregulated in PDAC compared to other cancers13, by using shRNA constructs. Concordant with previous results, the levels of the SA-β-Gal positive cells upon GLUD1 knockdown were comparable with control (shGFP) (Fig. 2b and S2B). Conversely, GOT2 knockdown signiﬁcantly elicited senescence in PDAC cells (Fig. 2c) and also resulted in morphological changes (Fig. S2C), impaired PDAC growth (Fig. 2d) and accumulation of cells in the G1 phase (Fig. 2e). Next, we found that overexpression of human GOT2 in PDAC cells can rescue the induction of senes- cence by GOT2 knockdown (Fig. 2f and S2D). Moreover, the induction of senescence upon GOT2 knockdown was also indicated by the accumulation of promyelocytic leu- kemia protein nuclear bodies (Fig. 2g). Glutamine metabolism through GOT2 regulates senescence in PDAC However, we observed no obvious increase in cell death under these conditions (Fig. S2E). Finally, we examined the involve- ment of another mitochondrial glutamate-dependent transaminase, pyruvate transaminase (GPT2), in PDAC senescence. We found that GPT2 knockdown did not affect senescence in PDAC cells (Fig. S2F and G). Toge- ther, these data demonstrate that mitochondrial transa- minase GOT2 regulates senescence in PDAC cells. A B * * * * * Panc1 PL45 Tu8902 0.0 0.4 0.8 1.2 Relative cell growth shGFP shGOT2 B C HPDE WI38 HEK293T 0 10 20 30 40 Senescence (% SA-β-gal+ cells) shGFP shGOT2 Panc1 PL45 Tu8902 Fig. 3 GOT2 regulates senescence in PDAC cells but not in normal cells. a and b, Senescence induction a, and relative proliferation b, of PDAC cell lines (Panc1, PL45 and Tu8902) expressing a control shRNA (shGFP) or two independent shRNAs to GOT2. c, Percentages of SA-β-gal positive cells in HPDE, WI38 and HEK293T cells expressing a control shRNA (shGFP) or two independent shRNAs to GOT2. All error bars ± SEM. p < 0.01 and *p < 0.001 C HPDE WI38 HEK293T 0 10 20 30 40 Senescence (% SA-β-gal+ cells) shGFP shGOT2 C GOT2 is essential for sustaining growth and suppressing senescence in PDAC To further conﬁrm the essential role of GOT2, we assessed whether GOT2 regulates senescence in multiple PDAC cells. Indeed, GOT2 knockdown signiﬁcantly eli- cits senescence in all three tested PDAC lines (Fig. 3a and S3A). In line with these results, GOT2 inhibition also impaired the growth of these cells (Fig. 3b). Recent evi- dence suggests that PDAC cells have a greater reliance on Gln-derived Asp, whereas normal cells are dependent on GLUD1-mediated Gln pathway to fuel the TCA cycle9. Thus, we tested whether GOT2 regulates senescence in normal cells. Remarkably, in contrast to PDAC, GOT2 knockdown had only modest effects on senescence in non-transformed human pancreatic ductal cells (HPDEs) (Fig. 3c and S3B). Moreover, we observed similar results with human ﬁbroblasts (WI-38) and human embryonic kidney 293 (HEK293T) cells. Thus, these results demon- strate that the Gln pathway mediated by GOT2 plays an important role in regulation of senescence in PDAC but not in non-transformed cells. ig. 3 GOT2 regulates senescence in PDAC cells but not in Fig. 3 GOT2 regulates senescence in PDAC cells but not in normal cells. a and b, Senescence induction a, and relative proliferation b, of PDAC cell lines (Panc1, PL45 and Tu8902) expressing a control shRNA (shGFP) or two independent shRNAs to GOT2. c, Percentages of SA-β-gal positive cells in HPDE, WI38 and HEK293T cells expressing a control shRNA (shGFP) or two independent shRNAs to GOT2. All error bars ± SEM. p < 0.01 and *p < 0.001 knockdown of GOT2 results in signiﬁcant decreases in Asp and oxaloacetate (OAA) in PDAC cells (Fig. S4A). Because this Gln-derived Asp plays an essential role in the maintenance of cellular redox homeostasis in PDAC cells9, we next investigated whether GOT2 directly modulates cellular reactive oxygen species (ROS) pro- duction. Indeed, GOT2 knockdown cells exhibited sig- niﬁcantly increased ROS levels, whereas the antioxidant N-acetylcysteine (NAC) treatment completely abrogated the increased ROS production (Fig. 4a). Given that increased ROS levels can contribute to induction of Ofﬁcial journal of the Cell Death Differentiation Association GOT2 represses senescence by maintaining cellular redox balance Relative ROS levels (DCFDA Fluorescence) 0 10 20 30 40 50 * Senescence (% SA-β-gal+ cells) NAC: - + - + - shGFP shGOT2 #1 #2 B 0 10 20 30 40 50 * Senescence (% SA-β-gal+ cells) NAC: - + - + - shGFP shGOT2 #1 #2 B 0 10 20 30 40 50 * ** Senescence (% SA-β-gal+ cells) NAC: - + - + - shGFP shGOT2 #1 #2 B A Control NAC 0.0 0.5 1.0 1.5 2.0 shGFP shGOT2 * n.s. Relative ROS levels (DCFDA Fluorescence) B AOA: - + - + NAC: - + - + 0 10 20 30 *** Senescence (% SA-β-gal+ cells) C D OAA: - + - + - #1 #2 + shGFP shGOT2 0 1 2 3 4 5 * ** NADPH/NADP+ D OAA: - + - + - #1 #2 + shGFP shGOT2 0 1 2 3 4 5 * NADPH/NADP+ C D 0.0 0.5 1.0 1.5 Relative cell growth shGFP shGOT2 Asp: - + - - #1 #2 - + + - - OAA: - + - - + - - + - E F 0 10 20 30 40 50 * Senescence (% SA-β-gal+ cells) shGFP shGOT2 OAA: - + - + - #1 #2 + ig. 4 GOT2 suppresses PDAC senescence by regulating cellular ROS. a, Relative ROS levels in 8988T cells expressing a control shRNA (shGFP) or shRNA to GOT2 with or without NAC (10 mM). b, Senescence induction of 8988T cells expressing a control shRNA or two independent shRNAs to OT2 supplemented with or without NAC. c, Senescence induction in control or AOA treated 8988T cells cultured with or without NAC. d, NADPH/ E 0 10 20 30 40 50 ** * Senescence (% SA-β-gal+ cells) shGFP shGOT2 OAA: - + - + - #1 #2 + 0.0 0.5 1.0 1.5 Relative cell growth shGFP shGOT2 Asp: - + - - #1 #2 - + + - - OAA: - + - - + - - + - F E #2 Fig. 4 GOT2 suppresses PDAC senescence by regulating cellular ROS. a, Relative ROS levels in 8988T cells expressing a control shRNA (shGFP) or a shRNA to GOT2 with or without NAC (10 mM). b, Senescence induction of 8988T cells expressing a control shRNA or two independent shRNAs to GOT2 supplemented with or without NAC. GOT2 represses senescence by maintaining cellular redox balance In PDAC cells, the majority of aspartate (Asp) is con- verted from Gln and GOT2 knockdown markedly decreases Gln-derived Asp9. Indeed, we found that Ofﬁcial journal of the Cell Death Differentiation Association Yang et al. Cell Death and Disease (2018) 9:55 Page 5 of 10 A Control NAC 0.0 0.5 1.0 1.5 2.0 shGFP shGOT2 * n.s. Relative ROS levels (DCFDA Fluorescence) 0 10 20 30 40 50 * Senescence (% SA-β-gal+ cells) NAC: - + - + - shGFP shGOT2 #1 #2 AOA: - + - + NAC: - + - + 0 10 20 30 * Senescence (% SA-β-gal+ cells) 0.0 0.5 1.0 1.5 Relative cell growth shGFP shGOT2 Asp: - + - - #1 #2 - + + - - OAA: - + - - + - - + - B C D E F 0 10 20 30 40 50 * Senescence (% SA-β-gal+ cells) shGFP shGOT2 OAA: - + - + - #1 #2 + OAA: - + - + - #1 #2 + shGFP shGOT2 0 1 2 3 4 5 * ** NADPH/NADP+ Fig. 4 GOT2 suppresses PDAC senescence by regulating cellular ROS. a, Relative ROS levels in 8988T cells expressing a control shRNA (shGFP) or a shRNA to GOT2 with or without NAC (10 mM). b, Senescence induction of 8988T cells expressing a control shRNA or two independent shRNAs to GOT2 supplemented with or without NAC. c, Senescence induction in control or AOA treated 8988T cells cultured with or without NAC. d, NADPH/ NADP+ ratio in 8988T cells expressing a control shRNA or shRNAs to GOT2 supplemented with or without OAA (4 mM). e and f, Senescence induction e, and relative proliferation f, of 8988T cells expressing a control shRNA or two independent shRNAs to GOT2 supplemented with or without OAA (4 mM, or Asp (4 mM. All error bars ± SEM. p < 0.05, p < 0.01 and *p < 0.001 Yang et al. Cell Death and Disease (2018) 9:55 Page 5 of 10 A Control NAC 0.0 0.5 1.0 1.5 2.0 shGFP shGOT2 n.s. Ofﬁcial journal of the Cell Death Differentiation Association levels in control and GOT2 knockdown cells (Fig. 4b and S4B). Likewise, we also obtained similar results in AOA treatment-induced senescence (Fig. 4c and S4C). Gln-derived Asp is metabolized by cytosolic GOT1 into OAA and then converted into malate, which is oxidized by malic enzyme (ME1) to produce NADPH, providing GOT2 represses senescence by maintaining cellular redox balance c, Senescence induction in control or AOA treated 8988T cells cultured with or without NAC. d, NADPH/ NADP+ ratio in 8988T cells expressing a control shRNA or shRNAs to GOT2 supplemented with or without OAA (4 mM). e and f, Senescence induction e, and relative proliferation f, of 8988T cells expressing a control shRNA or two independent shRNAs to GOT2 supplemented with or without OAA (4 mM, or Asp (4 mM. All error bars ± SEM. p < 0.05, p < 0.01 and p < 0.001 senescence4,14, we reasoned that the mechanism by which GOT2 regulates PDAC senescence involves ROS. To test this idea, we treated cells with NAC in order to probe the model that suppressing ROS could block the senescence induction by GOT2 knockdown. Notably, we observed that NAC treatment reduced senescence to comparable levels in control and GOT2 knockdown cells (Fig. 4b and S4B). Likewise, we also obtained similar results in AOA treatment-induced senescence (Fig. 4c and S4C). Gln-derived Asp is metabolized by cytosolic GOT1 into OAA and then converted into malate, which is oxidized by malic enzyme (ME1) to produce NADPH, providing Ofﬁcial journal of the Cell Death Differentiation Association Page 6 of 10 Page 6 of 10 Yang et al. Cell Death and Disease (2018) 9:55 the reducing power to maintain redox homeostasis9,15. We found that GOT2 knockdown decreased the cellular NADPH/NADP+ ratio and the addition of OAA was able to rescue this ratio (Fig. 4d), suggesting that the GOT2- mediated conversion of Gln is important for the main- tenance of ROS levels in PDAC cells. As further con- ﬁrmation of the importance of this GOT2-mediated Gln pathways in PDAC senescence, we assessed the levels of SA-β-Gal positive cells in the presence of downstream metabolites of GOT2, such as Asp or OAA. Importantly, senescence induced by GOT2 knockdown was sig- niﬁcantly reduced upon supplementation with OAA or Asp (Fig. 4e, S4D,E and F). Moreover, the addition of OAA or Asp rescued the growth suppression of GOT2 knockdown cells (Fig. 4f). Taken together, these results illustrate that GOT2 has a critical role in maintaining redox homeostasis, which is associated with modulation of cellular senescence in PDAC cells. in p27 abundance following GOT2 knockdown is not due to increased p27 half-life (Fig. S5B). Given the function of GOT2 in the redox homeostasis, we speculated that GOT2 knockdown increased cellular ROS levels which may elicit p27-mediated senescence. p27 mediates GOT2 knockdown-induced senescence p27 mediates GOT2 knockdown-induced senescence Next, we investigated the molecular mechanism underlying senescence induction by GOT2 down- regulation. The tumor suppressors p53 and p16 have critical roles in cellular senescence3. First, to test whether GOT2 knockdown induces senescence through p53- mediated processes, we treated cells with piﬁthrin-α (PFT-α), a p53 inhibitor. Interestingly, the inhibition of p53 did not inﬂuence senescence induction by GOT2 knockdown in 8988T PDAC cells (Fig. 5a and S5A). Consistent with these results, we did not detect the change of messenger RNA (mRNA) levels of p21, a p53 target gene (Fig. 5b). Moreover, when we assessed the protein levels of p21 and p53, we did not observe a clear, coordinated change by GOT2 knockdown (Fig. 5c). Sti- muli that drive senescence can also engage the p16-pRb pathway and increased p16 expression contributes to the acquisition of senescence-related phenotypes3,16. How- ever, we found that GOT2 knockdown also had no sig- niﬁcant effect on p16 mRNA levels (Fig. 5b). Together, these data indicate that GOT2 knockdown may elicit senescence not through classical p53 or p16 pathways in PDAC cells. Ofﬁcial journal of the Cell Death Differentiation Association GOT2 represses senescence by maintaining cellular redox balance To test this idea, we ﬁrst investigated whether p27 expression is affected by cellular ROS levels. In the presence of exo- genous hydrogen peroxide (H2O2), p27 is highly induced (Fig. S5C). Next, to conﬁrm that the elevated p27 expression upon GOT2 knockdown was caused by increased ROS levels, we assessed p27 protein levels with NAC. Importantly, NAC treatment signiﬁcantly reduced p27 expression in GOT2 knockdown cells (Fig. 5e, S5D and E). Consistent with these results, we found that the increase of p27 upon GOT2 knockdown was also rescued by the addition of OAA or Asp (Fig. 5f, S5D and E). Lastly, to directly test whether p27 is required for the GOT2 knockdown-mediated senescence, we suppressed p27 expression using short interfering RNA (siRNA) in 8988T cells and assessed senescence upon GOT2 knock- down. Remarkably, silencing of p27 almost completely diminished senescence induction (Fig. 5g, S5F and G). Collectively, these results support our model that GOT2 knockdown increases ROS levels in PDAC cells, which elicits the p27-mediated cellular senescence (Fig. 5h). Discussion p < 0.01 and *p < 0.001 g ( ) g A 0 10 20 30 Senescence (% SA-ββ-gal+ cells) shGFP shGOT2 - + - + - #1 #2 PFT- α: + shGFP shGOT2 GOT2 p21 p16 p27 0 1 2 3 * Relative gene expression B B D shGFP shGOT2 p27 Actin GOT2 #1 #2 (kDa) 25 50 50 C shGFP shGOT2 p21 p53 #1 #2 (kDa) 20 50 Actin 50 C D C F shGFP shGOT2 p27 Actin - OAA Asp - 25 50 (kDa) E shGFP shGOT2 p27 Actin - NAC - 25 50 (kDa) F F E shGFP shGOT2 p27 siRNA Control siRNA shGFP #1 #2 shGFP #1 #2 0 10 20 30 40 Senescence (% SA-β-gal+ cells) G p27 GOT2 ROS GOT2 p27 ROS senescence H G H e for GOT2 knockdown-mediated senescence induction in PDAC cells. a, Percentages of SA-β-gal positives cells in ig. 5 p27 is responsible for GOT2 knockdown-mediated senescence induction in PDAC cells. a, Percentages of SA-β knockdown-mediated senescence induction in PDAC cells. Fig. 5 p27 is responsible for GOT2 knockdown-mediated senescence induction in PDAC cells. a, Percentages of SA-β-gal positives cells in 8988T cells expressing a control shRNA or two independent shRNAs to GOT2 supplemented with or without PFT-α. b, Relative mRNA levels of indicated genes in 8988T cells expressing a control shRNA or a shRNA to GOT2. c, p21 and p53 protein levels in whole-cell lysates from 8988T cells expressing a control shRNA or shRNAs to GOT2. β-actin serves as a loading control. d, The effect of GOT2 knockdown on p27 protein levels in 8988T cells expressing a control shRNA or shRNAs to GOT2. e and f, p27 protein levels in 8988T cells expressing a control shRNA or a shRNA to GOT2 supplemented with or without NAC e, OAA or Asp f. g, Percentages of SA-β-gal positive cells in control and GOT2 knockdown 8988T cells transfected with p27 or control siRNA as indicated. h, A proposed model depicting the regulation of PDAC senescence by GOT2. All error bars ± SEM. p < 0.01 and p < 0.001 Fig. 5 p27 is responsible for GOT2 knockdown mediated senescence induction in PDAC cells. a, Percentages of SA β gal positives cells in 8988T cells expressing a control shRNA or two independent shRNAs to GOT2 supplemented with or without PFT-α. Discussion In this study we demonstrate that mitochondrial Gln metabolism via GOT2 regulates senescence in PDAC cells. GOT2 is required to sustain PDAC growth and to repress senescence, probably through maintaining cellular redox balance. We ﬁnd that GOT2 knockdown results in an elevated ROS production, which leads to p27- dependent senescence. Consistent with previous work showing that PDAC cells are strongly dependent on Gln metabolism9,10, our study reveals GOT2 as an important regulator of senescence in PDAC. Metabolic regulation is intimately involved in deter- mining cell-fate decisions, such as quiescence and pro- liferation. Emerging evidence has suggested that metabolism plays key roles in senescence pathways. For example, dysregulation of mitochondrial functions causes both growth arrest and senescence-associated secretory phenotype18. In progeroid mice, which have mitochon- drial DNA mutations and aging phenotypes, senescent cells exhibits rapidly accumulated in inguinal adipose tissues. Recently, NAD+ and NADP+-linked malic enzymes have been reported to repress replicative senes- cence15. Downregulation of malic enzymes activates p53, leading to induction of senescence in IMR90 cells. As malic enzymes are involved in Gln-dependent NADPH production and ROS regulation, these results support our notion that GOT2-mediated redox balance contributes to senescence regulation. The cyclin-dependent kinase inhibitor p27, frequently downregulated in many human cancers, regulates cell cycle progression and also plays a crucial role in the induction and maintenance of senescence6,7. When GOT2 was knocked down, p27 mRNA and protein levels were robustly induced in PDAC cells (Figs. 5b, d), implying that p27 might be required for senescence induction by GOT2 knockdown. Because p27 also can be regulated by proteasome-dependent proteolytic path- way17, we tested whether GOT2 knockdown has a post- translational effect on p27 protein stability. However, cycloheximide (CHX) treatment revealed that the increase Ofﬁcial journal of the Cell Death Differentiation Association Page 7 of 10 Yang et al. Discussion Cell Death and Disease (2018) 9:55 Our ﬁndings are consistent with previous work showing that Gln metabolism is essential for the maintenance of NADPH production and ROS regulation in PDAC cells (Fig 4) The enhanced levels of cellular ROS and an A 0 10 20 30 Senescence (% SA-ββ-gal+ cells) shGFP shGOT2 - + - + - #1 #2 PFT- α: + shGFP shGOT2 GOT2 p21 p16 p27 0 1 2 3 * * Relative gene expression shGFP shGOT2 p27 siRNA Control siRNA shGFP #1 #2 shGFP #1 #2 0 10 20 30 40 Senescence (% SA-β-gal+ cells) p27 GOT2 ROS GOT2 p27 ROS senescence B C E F G D shGFP shGOT2 p27 Actin GOT2 #1 #2 (kDa) 25 50 50 shGFP shGOT2 p27 Actin - NAC - 25 50 (kDa) shGFP shGOT2 p27 Actin - OAA Asp - 25 50 (kDa) H shGFP shGOT2 p21 p53 #1 #2 (kDa) 20 50 Actin 50 Fig. 5 p27 is responsible for GOT2 knockdown-mediated senescence induction in PDAC cells. a, Percentages of SA-β-gal positives cells in 8988T cells expressing a control shRNA or two independent shRNAs to GOT2 supplemented with or without PFT-α. b, Relative mRNA levels of indicated genes in 8988T cells expressing a control shRNA or a shRNA to GOT2. c, p21 and p53 protein levels in whole-cell lysates from 8988T cells expressing a control shRNA or shRNAs to GOT2. β-actin serves as a loading control. d, The effect of GOT2 knockdown on p27 protein levels in 8988T cells expressing a control shRNA or shRNAs to GOT2. e and f, p27 protein levels in 8988T cells expressing a control shRNA or a shRNA to GOT2 supplemented with or without NAC e, OAA or Asp f. g, Percentages of SA-β-gal positive cells in control and GOT2 knockdown 8988T cells transfected with p27 or control siRNA as indicated. h, A proposed model depicting the regulation of PDAC senescence by GOT2. All error bars ± SEM. Discussion b, Relative mRNA levels of indicated genes in 8988T cells expressing a control shRNA or a shRNA to GOT2. c, p21 and p53 protein levels in whole-cell lysates from 8988T cells expressing a control shRNA or shRNAs to GOT2. β-actin serves as a loading control. d, The effect of GOT2 knockdown on p27 protein levels in 8988T cells expressing a control shRNA or shRNAs to GOT2. e and f, p27 protein levels in 8988T cells expressing a control shRNA or a shRNA to GOT2 supplemented with or without NAC e, OAA or Asp f. g, Percentages of SA-β-gal positive cells in control and GOT2 knockdown 8988T cells transfected with p27 or control siRNA as indicated. h, A proposed model depicting the regulation of PDAC senescence by GOT2. All error bars ± SEM. p < 0.01 and *p < 0.001 Our ﬁndings are consistent with previous work showing that Gln metabolism is essential for the maintenance of cellular redox state in PDAC9. This study identiﬁes the profound impact of GOT2 function on Gln-dependent NADPH production and ROS regulation in PDAC cells (Fig. 4). The enhanced levels of cellular ROS and an altered redox status have been observed in most cancer cells. Increased ROS levels facilitate cancer growth and Ofﬁcial journal of the Cell Death Differentiation Association Page 8 of 10 Page 8 of 10 Yang et al. Cell Death and Disease (2018) 9:55 Material and methods Cell culture malignant progression, while at the same time these can cause cellular toxicity. As ROS generation is also inti- mately linked with both the initiation of PDAC and Ras- induced tumor growth2,19, a delicate regulation of intra- cellular ROS levels is essential for PDAC development and progression. Thus, this may explain why GOT2 expression is increased in human pancreatic cancers13 and PDAC cells are markedly sensitive to GOT2 inhibi- tion, whereas it is dispensable in non-transformed cells. HEK293T, 8988T and other human PDAC cell lines were cultured in Dulbecco’s modiﬁed Eagle’s medium (Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (FBS, Biowest) and penicillin/streptomycin (Biowest). WI38 cells were cultured in MEM media (Hyclone, Logan, UT, USA) with 10% FBS and antibiotics. HPDE cells were cultured as described previously9. Previous studies suggest that pancreatic cancer develops through the successive accumulation of mutations2. In addition to KRAS, the key regulators of classical senes- cence pathways, such as p53 and p16, are the most fre- quently mutated proteins in human PDAC20. Likewise, genetically engineered mouse models of PDAC, which recapitulate the human disease, have Kras activation and combinations of inactivating mutation in p53 and p16 to develop pancreatic cancer2. Therefore, it is likely that mutations of these genes may permit PDAC cells to bypass the classical senescence pathways and contribute to the therapeutic resistance of PDAC. In support of this idea, GOT2 knockdown did not induce signiﬁcant dif- ferences in p53, p21, and p16 expression (Figs. 5b, c). Importantly, we ﬁnd that knockdown of GOT2 induces p27-dependent premature senescence in PDAC (Fig. 5g), highlighting the potential importance of this GOT2- mediated senescence pathway for developing therapeutic approaches for PDAC. Cell viability assay Cells were plated into 96-well plates at 1000 cells per well in 100 μl of growth media. The following day, growth media was replaced with that containing oxaloacetate (4 mM) or Aspartate (4 mM). Parallel plates were analyzed at 3 days by Cell Titer Glo analysis (Promega, Fitchburg, WI, USA), per the manufacturer’s instruction. Our studies demonstrate that elevated ROS in the inhibition of GOT2 induces p27 expression. pRb has been shown to play critical roles in cell cycle arrest in response to ROS and increases p27 protein levels by inhibiting Skp2-medaited protein degradation21,22. However, we observe that the induction of p27 following GOT2 knockdown primarily occurs through a transcriptional mechanism but not through proteasome-dependent degradation in PDAC cells (Fig. 5b and S5B). Several transcription factors are known to regulate p27 expres- sion. For example, transcription of p27 gene is activated in part by forkhead box class O family (FoxO) proteins21,23, a conserved family of transcription factors involved in metabolism, cell cycle progression and cellular redox homeostasis24,25. Interestingly, several groups have pro- vided evidence that FoxO transcriptional activity is enhanced by cellular oxidative stresses21,25–27. Thus, it will be interesting for future studies to examine whether FoxO transcription factors regulate growth and senescence of PDAC cells, in part via modulating p27 expression and/or cellular ROS levels. Constructs and reagents The following antibodies were used: GLS (Abcam, Cambridge, MA, USA), GOT2 (Cusabio biotech, Wuhan, China), p27 (BD biosciences, Franklin Lakes, NJ, USA), p53 (Santa cruz Biotechnology, Dallas, USA), p21 (Abcam) and β-actin (Sigma, St. Louis, MO, USA). NAC, DON, EGCG, AOA, PFT-α and hydrogen peroxide were purchased from Sigma. Information about all shRNA vectors were described previously9. The human GOT2 was cloned into retroviral pBabe vector and used to generate stable cell line. Western blotting Cells were lysed with lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5 and 0.5 % NP-40) supplemented with protease inhibitor cocktail (Roche, Diagnostics, Man- nheim, Germany) and phosphatase inhibitors (Sigma). Cell lysates were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting. Ofﬁcial journal of the Cell Death Differentiation Association Quantitative RT-PCR Total RNA was prepared with Ribospin kit (GeneAll, Seoul, Korea) according to the manufacturer’s instruc- tions. 0.5 μg of total RNA was reverse-transcribed using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Diluted cDNAs were analyzed by real-time PCR using SYBR Green master mix on a Light Cycler 480 (Roche). The level of gene expression was normalized to β-actin. The primer sequences were: GCCCTCCCCA GTCTCTCTTA and TCAAAACTCCCAAGCACCTC for p27; GGCAGACCAGCATGACAGATTT and GGCGGATTAGGGCTTCCTCT for p21; GTGGACCT GGCTGAGGAG and CTTTCAATCGGGGATGTCTG for p16; TTCCAGAAGGCACAGACATG and GGCTC AGTACTCTTTCACCAG for GLS1; AGGAATGA- CACCAGGGTTTG and TCAGACTCACCAACAGCAA In this study we demonstrate that mitochondrial GOT2 serves to control senescence in PDAC cells through regulation of ROS, suggesting that targeting the unique redox regulatory pathways of PDAC might be an effective strategy to treat these cancers. Page 9 of 10 Yang et al. Cell Death and Disease (2018) 9:55 TAC for GLUD1; GTTTGCCTCTGCCAATCATATG and GAGGGTTGGAATACATGGGAC for GOT2; CATGGACATTGTCTGAACC and TTACCCAG- GACCGACTCCTT for GPT2; and CTACGTCGCCC TGGACTTCGAGC and GATGGAGCCGCCGATCCA- CACGG for human actin. References B 1. Bryant, K. L., Mancias, J. D., Kimmelman, A. C. & Der, C. J. KRAS: feeding pancreatic cancer proliferation. Trend. Biochem. Sci. 39, 91–100 (2014). 1. Bryant, K. L., Mancias, J. D., Kimmelman, A. C. & Der, C. J. KRAS: feeding pancreatic cancer proliferation. Trend. Biochem. Sci. 39, 91–100 (2014). 2. Hidalgo, M. Pancreatic cancer. N. Engl. J. Med. 362, 1605–1617 (2010). 2. Hidalgo, M. Pancreatic cancer. N. Engl. J. Med. 362, 1605–1617 (2010). 3. Campisi, J. & d’Add adi Fagagna, F. Cellular senescence: when bad things happen to good cells. Nat. Rev. Mol. Cell. Biol. 8, 729–740 (2007). 3. Campisi, J. & d’Add adi Fagagna, F. Cellular senescence: when bad things happen to good cells. Nat. Rev. Mol. Cell. Biol. 8, 729–740 (2007). 4. van Deursen, J. M. The role of senescent cells in ageing. Nature. 509, 439–446 (2014). 4. van Deursen, J. M. The role of senescent cells in ageing. Nature. 509, 439–446 (2014). 5. Nardella, C., Clohessy, J. G., Alimonti, A. & Pandolﬁ, P. P. Pro-senescence therapy for cancer treatment. Nat. Rev. Cancer. 11, 503–511 (2011). 5. Nardella, C., Clohessy, J. G., Alimonti, A. & Pandolﬁ, P. P. 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Cell 149, 656–670 (2012). Immunoﬂuorescence Received: 7 June 2017 Revised: 25 September 2017 Accepted: 20 October 2017 Received: 7 June 2017 Revised: 25 September 2017 Accepted: 20 October 2017 Received: 7 June 2017 Revised: 25 September 2017 Accepted: 20 October 2017 Cells were grown on coverslips in a 6-well plate and were ﬁxed in methanol for 5 min at room temperature (RT). After PBS washing, cells were permeablized for 20 min on 0.5% PBST (PBS containing 0.5% Triton X-100). The permeabilized cells were washed with PBS twice and blocked in 0.1% PBST (PBS containing 0.1% Triton X- 100) with 5% normal goat serum (NGS) for 30 min. Then the cells were incubated with PML antibody (Santa Cruz) in 0.1% PBST with 5% NGS overnight at 4 °C. After washing in 0.1% PBST, cells were stained with FITC- conjugated secondary antibody for 1 h at RT. Finally, cell were washed ﬁve times in 0.1% PBST and mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). 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https://openalex.org/W2612568633	https://www.scielo.br/j/bdj/a/WydfzvdWsyCd3YCd6sJN9Ry/?lang=en&format=pdf	English	null	Selective COX-2 Inhibitor (Meloxicam) and Tooth-Supporting Bone Quality. A Histomorphometric Study in Rats	Brazilian Dental Journal	2,017	cc-by	3,630	Brazilian Dental Journal (2017) 28(2): 135-139 http://dx.doi.org/10.1590/0103-6440201701081 Brazilian Dental Journal (2017) 28(2): 135-139 http://dx.doi.org/10.1590/0103-6440201701081 ISSN 0103-6440 1Department of Dentistry, UFRN - Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil 2Private Practice, Delmiro Gouveia, AL, Brazil 3Department of Prosthodontics, School of Dentistry of Arcoverde, UPE – Universidade de Pernambuco, PE, Brazil 4Department of Pediatric Dentistry, Piracicaba Dental School, UNICAMP – Universidade Estadual de Campinas, Piracicaba, SP, Brazil 5Department of Prosthodontics and Periodontics, Piracicaba Dental School, UNICAMP – Universidade Estadual de Campinas, Piracicaba, SP, Brazil Correspondence: Prof. Dr. Bruno César de Vasconcelos Gurgel, Av. Salgado Filho, 1787, Lagoa Nova, 59056-000 Natal, RN, Brasil. Tel: +55-84-3215- 4103. e-mail: bcgurgel@yahoo.com.br Introduction NSAIDs are drugs able to block the potential co- stimulation of osteophytogenesis by mediators of inflammation (11) and prescribed primarily for chronic management of rheumatic conditions. In dentistry, NSAIDs are often used to control pain and inflammation after several procedures such as tooth extraction, orthodontic movement, orthognathic procedures and other oral surgeries (10). Among these NSAIDs, Meloxicam is prescribed worldwide, due to its analgesic and anti-inflammatory actions. Meloxicam, 4-hydroxy-2-methyl-N-(5-methyl-2- tiazolil)-2H-1,2-benzotiacina-3-carboxamide-1,1-dioxide, is a highly specific COX-2 inhibitor and offers high efficiency with very few side effects on the gastrointestinal and renal systems (6,12). Bone tissue is constantly renewed in a synchronized process mediated by cytokines and growth factors involved in bone resorption and deposition. The understanding of the mechanisms involved with bone homeostasis is crucial, especially to approach metabolic bone disorders leading to bone loss and consequently decreased quality of life (1). The effects of inflammatory mediators (such as prostaglandins) and growth factors on osteoclasts and osteoblasts may trigger the start of bone adaptive changes in response to mechanical (2) or endocrine signals (2,3). Prostaglandins are produced from membrane phospholipids by sequential actions of phospholipase A2 (PLA2) and cyclooxygenase (COX) (4). Two COX isoforms have been identified in human tissues: the regulatory COX-1 enzyme (which provides homeostatic levels of prostaglandins) and the inducible COX-2 (which can be stimulated by inflammation) (4). COX inhibitors are known as non-steroidal anti-inflammatory drugs (NSAIDs) and for years the effects of these drugs on bone quantity and quality have been investigated. Previous studies utilizing specific COX-2 inhibitors demonstrate that blocking of COX-2 activity may prevent bone resorption (1,4,5-7), but it may also delay healing in bone fractures (8,9) and during alveolar bone repair in rats (10). Previous animal studies showed that Meloxicam may prevent bone loss in experimental periodontitis (6,7,10,13), possibly by controlling COX-2 enzyme function, which is up-regulated in highly-inflamed periodontal tissues (12). Nowadays, it is proposed that alveolar bone quality is a critical factor for teeth and dental implant general health and long-term stability. Thus, animal models are successfully used to assess the impact of systemic conditions and hormone replacement therapy on alveolar bone quality (14). Because of the lack of information on the impact of Previous animal studies showed that Meloxicam may prevent bone loss in experimental periodontitis (6,7,10,13), possibly by controlling COX-2 enzyme function, which is up-regulated in highly-inflamed periodontal tissues (12). Selective COX-2 Inhibitor (Meloxicam) and Tooth-Supporting Bone Quality. A Histomorphometric Study in Rats Bruno César de Vasconcelos Gurgel1, Krysna Torres de Almeida2, Raniel Fernandes Peixoto3, Kenio Costa Lima1, Kamila Rosamilia Kantovitz4, Francisco Humberto Nociti-Junior5, Sergio de Toledo5 The effects of the non-steroidal anti-inflammatory drugs (NSAIDs) on bone quantity and quality were investigated for years. However, there is lack of information on the impact of NSAIDs on the quality of tooth-supporting alveolar bone in absence of periodontal inflammation. Thus, the aim of this study was to evaluate histometrically the influence of a selective COX-2 NSAID (Meloxicam) on the inter-radicular bone mineral density in rats. Forty-nine adult male Wistar rats were randomly divided into four experimental groups: Subcutaneous injection of 0.9% sterile saline for 15 days (G1; n=12) and 45 days (G2; n=11); and subcutaneous injection of Meloxicam for 15 days (G3; n=13) and 45 days (G4; n=13). Mineral density was histometrically determined in the inter-radicular area of the 1st mandibular molars and data analysis performed by two-way ANOVA (a=5%). Results showed no interaction between time and treatment (p>0.05) and that meloxicam did not affect the alveolar bone density. In contrast, it was found that inter-radicular alveolar bone density increased with time (91.88±3.08% and 92.86±2.38% for groups 15 and 45 days, respectively) (p<0.05). Within the limits of this study, daily administration of a selective COX-2 inhibitor (Meloxicam) did not affect the quality of the inter-radicular alveolar bone in absence of periodontal infection. Key Words: cyclooxygenase-2 inhibitor, meloxicam, alveolar bone density. Key Words: cyclooxygenase-2 inhibitor, meloxicam, alveolar bone density. Material and Methods Animals After excluding the first and the last sections in which the furcation region was evident, ten equally distant sections of each tooth were selected for histomorphometric analysis (Fig. 1). Using an image-analysis system (UTHSCSA ImageTool 3.0, San Antonio, TX, USA), the percentage of mineralized bone (alveolar bone density) in the inter-radicular area of the 1st mandibular molar was histometrically determined by a blinded and calibrated examiner. Forty-nine male Wistar rats (Rattus norvegicus albinus), weighing 250-350 g and aged 90 days at the beginning of the experiment were used. During the experiment, the animals were maintained in plastic cages (4-5 per cage) under the same environmental conditions, with solid food and water ad libitum throughout the study. The protocol was approved by the University of Campinas Institutional Animal Care and Use Committee (protocol number 265-2). B.C.V. Gurgel et al. B.C.V. Gurgel et al. Results Quantitative data of alveolar bone density determined by histomorphometric analyses are in Table 1 and Figure 2. There were no statistically significant differences between time and treatment (p=0.305), assuming that the effect of saline and Meloxicam was the same on the quality of the inter-radicular alveolar bone at 15 and 45 days. Interestingly, data analysis showed that inter- radicular bone density increased with time regardless of the treatment group (88.81±3.21%/91.88±3.08% and 87.78±4.49%/92.86±2.38% for saline and meloxicam administration at 15/45 days, respectively) (p<0.001). Statistical Analysis At the study start, the animals were randomly assigned to one of the following experimental groups: Mean values of alveolar bone density were determined for each group and the Kolmogorov-Smirnov test was used to determine data normality. Next, two-way ANOVA test was used to detect differences between groups according to time and treatment. All analyses were performed using a 5% significance level (α=0.05). Group 1: Daily (1x/day) Subcutaneous injection (SC) of sterile saline (1mL/kg) for 15 days (n=12); Group 2: Daily (1x/day) SC injection of sterile saline (1mL/kg) for 45 days (n=11); Group 3: Daily (1x/day) SC injection of Meloxicam (Movatec®, Boehringer Ingelheim do Brasil Química e Farmacêutica Ltda., Itapecerica da Serra, SP, Brazil) (3 mg/kg) (7) for 15 days (n=13); Group 4: Daily (1x/day) SC injection of Meloxicam (3 mg/kg) (7) for 45 days (n=13). Introduction Nowadays, it is proposed that alveolar bone quality is a critical factor for teeth and dental implant general health and long-term stability. Thus, animal models are successfully used to assess the impact of systemic conditions and hormone replacement therapy on alveolar bone quality (14). Because of the lack of information on the impact of Braz Dent J 28(2) 2017 NSAIDs on the quality of tooth-supporting alveolar bone in absence of periodontal inflammation, the aim of the present study was to assess the effect of daily NSAIDs administration to the inter-radicular alveolar bone in rats by histological analysis. before fixing in 4% neutral formalin (pH 7.2 – 7.4) for 48 h. Demineralization was performed in a 1:1 solution of 50% formic acid and 20% sodium citrate (Morse solution) for 60 days. Next, the demineralized bone was dehydrated in absolute alcohol, diaphanized in xylol and embedded in paraffin. Longitudinal serial sections (6 µm) were obtained in a mesial-distal direction, and stained with hematoxylin and eosin. Histological Analysis The animals were euthanized by deep anesthesia after the experimental period. The jaws were removed and divided at the mandibular symphysis in hemi-mandibles, Figure 1. Photomicrograph showing the inter-radicular region of the first mandibular molar where bone density was histometrically determined (yellow line). Mineralized regions circumscribed in black (original magnification: 6.25×). Table 1. Mean and standard deviation (SD) of the percentage of alveolar bone density for the control and test groups Period (days) Saline Mean (SD) Meloxicam Mean (SD) F p* 15 days 88.81% (3.21) 87.78% (4.49) 45 days 91.88% (3.08) 92.86% (2.38) Time 17.582 p<0.001 Treatment 0.000 p=0.983 Time-Treatment 1.074 p=0.305 * Two-way ANOVA. Table 1. Mean and standard deviation (SD) of the percentage of alveolar bone density for the control and test groups Figure 1. Photomicrograph showing the inter-radicular region of the first mandibular molar where bone density was histometrically determined (yellow line). Mineralized regions circumscribed in black (original magnification: 6.25×). 136 Braz Dent J 28(2) 2017 15 days (A) and 45 days (B) and Meloxicam for 15 days (C) and 45 days (D). Figure 3 illustrates the histological aspects of bone density observed after administration of sterile saline for Discussion p p Irrational use of NSAIDs, selective and nonselective, is associated with a range of potential adverse effects, including gastric mucosa damages and an increased risk of adverse cardiovascular effects. The risk of different events depends on the clinical context, medication and dose (23). In addition, long-term NSAIDs administration is employed for the treatment of specific diseases including, for example, the rheumatoid arthritis. In these cases, a secondary osteoporosis with bone loss in the joints is easily recognized and may increase the risk of fractures and accompanying co-morbidities in these subjects (24). Specifically in relation to Meloxicam, Bezerra et al. (6) examined the effect of this drug on gastric mucosa of rats and found that the dosage of up to 3 mg/kg did not produce significant gastric effects. Nevertheless, new therapeutic alternatives to the treatment of diseases involving bone metabolism like osteoporosis has been carried out for years and the phytotherapeutic agents showed promising results (25). The impact of meloxicam administration on the alveolar bone loss resulting from experimental periodontitis has been previously investigated (6,7) and the results suggested that meloxicam may significantly decrease periodontitis- resulting bone loss; these findings are in line with others (1,4,5,13). Fewer renal side effects have been reported for meloxicam compared to other NSAIDs (6,12), indicating that meloxicam treatment may be used for a longer time (17). Interestingly, aspirin was reported to potentiate the effect of selective COX-2 inhibitors, acting on prostaglandin production (dependent on arachidonic acid), as well as on nitric oxide (NO) and nuclear factor kappa-light-chain- enhancer of activated B (NF-kB) production (independent from arachidonic acid) in cells, leading to increased bone density at multiple skeletal sites in men and women (5). On the other hand, Shen et al. (18) observed that short-term administration of selective COX-2 inhibitors resulted in suppression of bone formation and increased bone resorption in rats. COX-2 function was reported as essential for fracture healing, since this enzyme is critically involved in bone repair and required for intramembrane and endochondral bone formation (8). After continuous administration, selective COX-2 inhibitors (meloxicam) may negatively influence bone healing in cortical and cancellous bone around titanium implants inserted in rats (19). In the present study, in both groups, saline and meloxicam, there was an increase in alveolar bone density with time, indicating that meloxicam alone did not affect tooth-supporting bone quality. Discussion Figure 2. Percentage of alveolar bone density after subcutaneous injections of sterile saline solution (15 days [G1] and 45 days [G2]) and meloxicam (15 days [G3] and 45 days [G4]). One asterisk indicate statistically significant difference (p<0.05) and two asterisks indicate no statistically significant difference (p>0.05). The present study demonstrated that meloxicam, a selective COX-2 inhibitor, did not affect alveolar bone density in the inter-radicular area of periodontally healthy mandibular 1st molars in rats. We additionally found that, regardless of treatment, bone density increased with time. Therefore, these findings suggest that, in physiological conditions, NSAIDs did not impact the quality of the tooth- supporting alveolar bone. Morton et al. (15) reported that certain NSAID classes may have different effect on bone metabolism. Selective COX-2 inhibitors may have greater effects on bone metabolism, as compared with COX-1 inhibitors, since production of prostaglandins in osteoclasts is primarily mediated by COX-2 (16). Thus, selective COX-2 inhibitors have been suggested to reduce bone remodeling and resorption, conserving trabecular bone mass and Figure 2. Percentage of alveolar bone density after subcutaneous injections of sterile saline solution (15 days [G1] and 45 days [G2]) and meloxicam (15 days [G3] and 45 days [G4]). One asterisk indicate statistically significant difference (p<0.05) and two asterisks indicate no statistically significant difference (p>0.05). Effect of meloxicam on bone mineral density 137 Figure 3. Photomicrograph showing bone density after subcutaneous injections of sterile saline for 15 days (A) and 45 days (B) and meloxicam for 15 days (C) and 45 days (D). Effect of meloxicam on bone min Figure 3. Photomicrograph showing bone density after subcutaneous injections of sterile saline for 15 days (A) and 45 days (B) and meloxicam for 15 days (C) and 45 days (D). h showing bone density after subcutaneous injections of sterile saline for 15 days (A) and 45 days (B) and meloxicam ys (D) 137 Braz Dent J 28(2) 2017 for 7 days after tooth extraction in rats. Taken together, these findings suggest that meloxicam may affect bone homeostasis in a VEGF-dependent manner, at least in part. architecture (4). Studies with bone marrow cultures from COX-2 knockout mice demonstrated a marked decrease in osteoclast responses to stimulators of bone resorption and that selective COX-2 inhibitors blocked osteoclast formation in this system (3). B.C.V. Gurgel et al. B.C.V. Gurgel et al. The role of vascular endothelial growth factor (VEGF) in bone density was investigated during the bone repair. VEGF, described as the most important molecule for regulating angiogenesis, plays a critical role in bone homeostasis (20). VEGF receptor activation induces endothelial cell mobilization, recruitment, differentiation and proliferation, as well as the enrollment and survival of mesenchymal progenitor cells, osteoblasts and osteoclasts (21,22). Although systemic therapies with selective COX- 2 inhibitors may affect VEGF expression in rats (13), the results of this study showed similar bone repair between saline and Meloxicam after 45 days and this finding suggests no direct relationship between Meloxicam and increased VEGF expression. Thus, the increased bone density observed in 45 days compared to 15 days may suggest other mechanisms, including those not depending on arachidonic acid. However, Arantes et al. (10) reported an alveolar bone repair delay following daily administration of meloxicam Within the limits of the present study, it was concluded that daily administration of a selective COX-2 inhibitor (Meloxicam) did not affect the quality of the inter-radicular alveolar bone in absence of periodontal infection. However, further pre-clinical and clinical studies should be considered in order to determine the relevance of long-term NSAIDs administration to the tooth-supporting alveolar bone quality and the potential involved mechanisms. Discussion Further controlled clinical studies are required to evaluate their long-term benefits and to search for less harmful alternative therapies (9). In addition, other analyses including immunohistochemical and molecular biology and other drugs, may be performed to clarify the effect of NSAIDs on cell and tissue behavior during the process of bone repair, because multiple mechanisms (not yet fully elucidated) may be related to inflammation and bone resorption and deposition processes, such as VEGF expression, receptor activator of nuclear fator kB, RANK/RANKL/receptor osteoprotegerin (OPG) and tumor necrosis factor (TNF). Thus, knowledge of the behavior and mechanisms of selective COX-2 inhibitors is required for future clinical applications. References 16. Cryer B, Feldman M. Cyclooxygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory drugs. Am J Med 1998;104:413-421. 1. Kasukawa Y, Miyakoshi N, Srivastava AK, Nozaka K, Maekawa S, Baylink DJ, et al.. The selective cyclooxygenase-2 inhibitor celecoxib reduces bone resorption, but not bone formation, in ovariectomized mice in vivo. Tohoku J Exp Med 2007;211:275-283. 1. Kasukawa Y, Miyakoshi N, Srivastava AK, Nozaka K, Maekawa S, Baylink DJ, et al.. The selective cyclooxygenase-2 inhibitor celecoxib reduces bone resorption, but not bone formation, in ovariectomized mice in vivo. Tohoku J Exp Med 2007;211:275-283. 17. Feldman M, McMahon AT. Do cyclooxygenase-2 inhibitors provide benefits similar to those of traditional nonsteroidal anti-inflammatory drugs, with less gastrointestinal toxicity? Ann Intern Med 2000;132:134-143. 2. Richards JB, Joseph L, Schwartzman K, Kreiger N, Tenenhouse A, Goltzman D, et al.. The effect of cyclooxygenase-2 inhibitors on bone mineral density: Results from the Canadian Multicentre Osteoporosis Study. Osteoporos Int 2006;17:1410-1419. 18. Shen CL, Yeh JK, Wang X. Short-term supplementation of COX-2 inhibitor suppresses bone turnover in gonad-intact middle-aged male rats. J Bone Miner Metab 2006;24:461-466. 3. Okada Y, Lorenzo JA, Freeman AM, Tomita M, Morham SG, Raisz LG, et al.. Prostaglandin G/H synthase-2 is required for maximal formation of osteoclast-like cells in culture. J Clin Invest 2000;105:823-832. 3. Okada Y, Lorenzo JA, Freeman AM, Tomita M, Morham SG, Raisz LG, et al.. Prostaglandin G/H synthase-2 is required for maximal formation of osteoclast-like cells in culture. J Clin Invest 2000;105:823-832. 19. Ribeiro FV, Nociti FH, Jr., Sallum EA, Casati MZ. Effect of aluminum oxide-blasted implant surface on the bone healing around implants in rats submitted to continuous administration of selective cyclooxygenase-2 inhibitors. Int J Oral Maxillofac Implants 2009;24:226-233. 4. Gregory LS, Kelly WL, Reid RC, Fairlie DP, Forwood MR. Inhibitors of cyclo-oxygenase-2 and secretory phospholipase A2 preserve bone architecture following ovariectomy in adult rats. Bone 2006;39:134- 142. 4. Gregory LS, Kelly WL, Reid RC, Fairlie DP, Forwood MR. Inhibitors of cyclo-oxygenase-2 and secretory phospholipase A2 preserve bone architecture following ovariectomy in adult rats. Bone 2006;39:134- 142. 20. Lee JH, Um S, Jang JH, Seo BM. Effects of VEGF and FGF-2 on proliferation and differentiation of human periodontal ligament stem cells. Cell Tissue Res 2012;348:475-484. 5. Carbone LD, Tylavsky FA, Cauley JA, Harris TB, Lang TF, Bauer DC, et al.. Association between bone mineral density and the use of nonsteroidal anti-inflammatory drugs and aspirin: impact of cyclooxygenase selectivity. Resumo Os efeitos dos fármacos anti-inflamatórios não esteroidais (AINEs) sobre a quantidade e qualidade óssea tem sido investigados ao longo dos anos.Entretanto, há falta de informação sobre o impacto dos AINEs na qualidade do osso alveolar de suporte na ausência de inflamação periodontal. Assim, o objetivo deste estudo foi avaliar, histometricamente, a influência de um AINE seletivo para COX-2 (Meloxicam) na densidade mineral óssea inter-radicular em ratos. Quarenta e nove ratos Wistar, machos e adultos foram divididos aleatoriamente em quatro grupos experimentais: injeções subcutâneas de 0,9% de solução salina estéril 138 Braz Dent J 28(2) 2017 drugs as preventive treatments of osteoarthritis in the rat. Arthritis Rheum 2010;62:2726-2735. por 15 dias (G1, n=12) e 45 dias (G2, n=11); e injeções subcutâneas de Meloxicam por 15 (G3, n=13) e 45 dias (G4, n=13). A densidade mineral foi determinada histometricamente na área inter-radicular dos primeiros molares mandibulares e a análise dos dados realizada por meio de ANOVA (a=5%). Os resultados mostraram nenhuma interação entre tempo e tratamento (p>0,05) e que o meloxicam não afetou a densidade óssea alveolar. Em contraste, foi encontrado que a densidade óssea alveolar inter-radicular aumentou ao longo do tempo (91,88±3,08% e 92,86±2,38% para os grupos 15 e 45 dias, respectivamente) (p<0,05). Dentro dos limites deste estudo, a administração diária de um inibidor seletivo para COX-2 (Meloxicam) não afetou a qualidade do osso alveolar inter-radicular na ausência de infecção periodontal. 12. Morton RS, Dongari-Bagtzoglou AI. Cyclooxygenase-2 is upregulated in inflamed gingival tissues. J Periodontol 2001;72:461-469. 13. Oliveira TM, Sakai VT, Machado MA, Dionisio TJ, Cestari TM, Taga R, et al.. 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https://openalex.org/W2164881750	https://europepmc.org/articles/pmc2678105?pdf=render	English	null	Adenomatous polyposis coli-mediated control of β-catenin is essential for both chondrogenic and osteogenic differentiation of skeletal precursors	BMC developmental biology	2,009	cc-by	10,540	This article is available from: http://www.biomedcentral.com/1471-213X/9/26 This article is available from: http://www.biomedcentral.com/1471-213X/9/26 This article is available from: http://www.biomedcen © 2009 Miclea 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. Open Ac Research article Adenomatous polyposis coli-mediated control of β-catenin is essential for both chondrogenic and osteogenic differentiation of skeletal precursors Razvan L Miclea†1, Marcel Karperien†2, Cathy AJ Bosch3, Geertje van der Horst4, Martin A van der Valk5, Tatsuya Kobayashi6, Henry M Kronenberg6, Georges Rawadi7, Pinar Akçakaya3, Clemens WGM Löwik8, Riccardo Fodde9, Jan Maarten Wit1 and Els C Robanus-Maandag3 Address: 1Department of Pediatrics, Leiden University Medical Centre, Leiden, the Netherlands, 2Department of Tissue Regeneration, Institute for Biomedical Technology, University of Twente, Enschede, the Netherlands, 3Department of Human Genetics, LUMC, Leiden, the Netherlands, 4Department of Urology, Leiden University Medical Centre, Leiden, the Netherlands, 5Department of Animal Pathology, The Netherlands Cancer Institute, Amsterdam, the Netherlands, 6Department of Medicine, Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA, 7Galapagos, Romainville, 93230, France, 8Department of Endocrinology and Metabolic Diseases, Leiden University Medical Centre, Leiden, the Netherlands and 9Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Centre, Rotterdam, the Netherlands mail: Razvan L Miclea - r.l.miclea@lumc.nl; Marcel Karperien - h.b.j.karperien@tnw.utwente.nl; Cathy AJ Bosch - eertje van der Horst - g.van_der_horst@lumc.nl; Martin A van der Valk - ma.vd.valk@nki.nl; j g Tatsuya Kobayashi - kobayash@HELIX.MGH.HARVARD.EDU; Henry M Kronenberg - hkronenberg@partners.org; Georges Rawadi - georges.rawadi@glpg.com; Pinar Akçakaya - pinarakcakaya@gmail.com; Clemens WGM Löwik - c.w.g.m.lowik@lumc.nl; Riccardo Fodde - r.fodde@erasmusmc.nl; Jan Maarten Wit - j.m.wit@lumc.nl; Els C Robanus-Maandag - e.c.robanus@lumc.nl * Corresponding author †Equal contributors Received: 4 December 2008 Accepted: 8 April 2009 BioMed Central BioMed Central BMC Developmental Biology Open Access BMC Developmental Biology 2009, 9:26 doi:10.1186/1471-213X-9-26 BMC Developmental Biology 2009, 9:26 doi:10.1186/1471-213X-9-26 Background It has been also demonstrated that β-catenin is required at an early stage to repress chondrocytic differentiation [15]. Upon conditional inactivation of β-catenin in the limb and head mesenchyme before or during early mesenchy- mal condensations, Prx1-expressing and Dermo1-express- ing skeletal precursors, respectively, differentiate into chondrocytes instead of osteoblasts [11,15]. Finally, results on both constitutively active and inactivated β-cat- enin in Osterix (Osx)-, Collagen 1a1 (Col1a1)- or Osteocal- cin (Osc)-expressing osteoblasts suggest that Wnt/β- catenin signaling coordinates bone formation by control- ling the differentiation and activity of both osteoblasts and osteoclasts in a sequential, stage-specific manner [16,17]. g During vertebrate embryogenesis, the axial and appendic- ular skeleton develop through endochondral bone forma- tion. In this process, mesenchymal cells aggregate to form a chondrocytic template that prefigures the shape of the future bone. At the periphery of this cartilaginous mold, osteoblasts differentiate to form the bone collar. The car- tilaginous mold is eventually replaced by bone in a step- wise program. Besides chondrocytes and osteoblasts, the skeleton also contains osteoclasts, which are of haemat- opoietic origin and play pivotal roles in both cartilage and bone resorption and remodelling [1-3]. Every step in the proliferation, differentiation, matura- tion, apoptosis, and resorption of both chondrocytes and osteoblasts is characterized by a specific transcriptional guideline [4]. Sox9, a high-mobility-group transcription factor, and Runx2, a Runt domain transcription factor, are both expressed in bi-potential skeletal precursor cells dif- ferentiating into either chondrocytes or osteoblasts [5-7]. Sox9 and Runx2 play leading roles in lineage commit- ment of these precursors: upregulation of Sox9 leads to chondrogenic differentiation [8], while activation of Runx2 is required for their osteogenic commitment [9]. Little is known about the mechanisms regulating β-cat- enin activity in skeletal precursors. Through its wide range of specific motifs and domains, APC is involved in multi- ple cellular processes such as signal transduction, cytoskeletal organization, apoptosis, cell adhesion and motility, cell fate determination, and chromosomal sta- bility [18]. However, biochemical and genetic evidence has been provided showing that APC's main tumor sup- pressing activity resides in its ability to bind to β-catenin and induce its degradation, thereby acting as a strong neg- ative regulator of the canonical Wnt pathway [19-21]. Recently, based on mouse models, the canonical Wnt/β- catenin signaling pathway was found to act upstream of Sox9 and Runx2. Background In this pathway, in the absence of a Wnt signal, cytosolic β-catenin is degraded by the ubiquitina- tion/proteasome system upon its phosphorylation at spe- cific Ser-Thr residues by a destruction complex consisting of scaffold proteins such as Axin1, Axin2 (also known as Conductin) and the adenomatous polyposis coli (APC) tumor suppressor, and two kinases, namely glycogen syn- thase kinase 3β (GSK3β) and casein-kinase 1α (CK1α). Binding of Wnt to a complex composed of the transmem- brane frizzled receptor and low-density lipoprotein recep- tor-related protein 5 or 6 (LRP5 or 6) co-receptor results in inactivation of the destruction complex and accumula- tion of cytoplasmic β-catenin. Upon its nuclear transloca- tion, β-catenin acts as transcriptional co-activator in complex with transcription factors of the TCF/LEF family, leading to transcriptional activation of Wnt target genes [10]. In wild type mouse embryos, high levels of β-catenin and activation of canonical Wnt signaling have been found in osteoblastic precursors in developing skull and limb bones [11]. Accumulating evidence suggests that increased levels of canonical Wnt/β-catenin signaling inhibit Sox9 expression and activity, and stimulate Runx2 expression, leading to decreased chondrocyte differentia- tion and increased osteoblast differentiation, respectively [12-15]. Similar results have been found in transgenic mice with Wnt14 overexpression in Collagen 2a1 (Col2a1)- expressing cells [11]. Familial adenomatous polyposis (FAP) patients hetero- zygous for an APC mutation frequently develop osteomas and dental anomalies [22]. Heterozygous Apc1638N mutant mice occasionally develop osteomas (R. Fodde, personal communication). Homozygosity for the severely trun- cated ApcMin and for the more hypomorphic Apc1638N allele in the mouse results in a failure of primitive ecto- derm development shortly after implantation, leading to lethality prior to gastrulation [23,24]. Mutant Apc dis- turbs the differentiation capacity of mouse embryonic stem (ES) cells in a quantitative and qualitative fashion depending on the dose of β-catenin signaling. Aberrant differentiation capacity of ES cells ranges from a strong differentiation blockade in case of two severely truncated ApcMin alleles, to more specific neuroectodermal, dorsal mesodermal, and endodermal defects (e.g., no differenti- ation in bone or cartilage) in case of two hypomorphic Apc1638N alleles [25,26]. Osteoblast-specific loss of Apc in the mouse leads to early onset of dramatically increased bone deposition and to lethality early in life [17]. How- ever, Apc has not yet been linked with a role in the differ- entiation of skeletal precursor cells. http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 Abstract Background: During skeletogenesis, protein levels of β-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of β-catenin turnover by down-regulating intracellular levels of β-catenin. Results: To investigate whether Apc is involved in lineage commitment of skeletal precursor cells, we generated conditional knockout mice lacking functional Apc in Col2a1-expressing cells. In contrast to other models in which an oncogenic variant of β-catenin was used, our approach resulted in the accumulation of wild type β-catenin protein due to functional loss of Apc. Conditional homozygous Apc mutant mice died perinatally showing greatly impaired skeletogenesis. All endochondral bones were misshaped and lacked structural integrity. Lack of functional Apc resulted in a pleiotropic skeletal cell phenotype. The majority of the precursor cells lacking Apc failed to differentiate into chondrocytes or osteoblasts. However, skeletal precursor cells in the proximal ribs were able to escape the noxious effect of functional loss of Apc resulting in formation of highly active osteoblasts. Inactivation of Apc in chondrocytes was associated with dedifferentiation of these cells. Conclusion: Our data indicate that a tight Apc-mediated control of β-catenin levels is essential for differentiation of skeletal precursors as well as for the maintenance of a chondrocytic phenotype in a spatio-temporal regulated manner. Page 1 of 14 (page number not for citation purposes) (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 Results Conditional Apc15lox mice and transgenic Col2a1-Cre mice Recently, we (ECR-M and RF) generated a novel mouse model carrying a conditional Apc15lox allele where exon 15, encoding the majority of the coding region of Apc, and the polyadenylation signal, is flanked by loxP sites. Mice heterozygous and homozygous for the conditional Apc15lox allele did not show any major abnormalities or susceptibility to tumors. ApcΔ15/+mice, heterozygous for the ApcΔ15mutant allele obtained by germline Cre-medi- ated deletion of exon 15, developed multiple intestinal tumors at an early age similar to ApcMin/+animals. These results indicate that Cre-mediated recombination of the Apc15lox allele leads to inactivation of the Apc protein and to the constitutive activation of Wnt/β-catenin signaling (Robanus-Maandag et al., in preparation). Col2a1-Cre;Rosaflox mice express Cre at sites of endochon- dral bone formation Figure 1 Col2a1-Cre;Rosaflox mice express Cre at sites of endochondral bone formation. (A-D) LacZ expression in Col2a1-Cre;Rosaflox embryos following Cre recombination, detected by whole-mount X-Gal staining. (A) Macroscopic picture of E12.5 Col2a1-Cre;Rosaflox embryo. (B) Transver- sal section of E9.5 embryo showing β-galactosidase-positive sclerotomal cells adjacent to the neural tube. (C) Transversal section of E12.5 embryo showing LacZ expression in verte- brae primordia. (D) Sagital section of E16.5 embryo showing LacZ expression in the femur. The boxed region in D is mag- nified in D' showing LacZ expression in the periosteum (arrow head), osteoblasts (red arrow) and osteocytes (black arrow). Ne, neuroepithelium; Sc, sclerotome; N, notochord; Fe, femur. Scale bars: 1 mm in A; 50 μm in B, D'; 100 μm in C, D. Col2a1-Cre;Rosaflox mice express Cre at sites of endochon- dral bone formation Figure 1 Col2a1-Cre;Rosaflox mice express Cre at sites of endochondral bone formation. (A-D) LacZ expression in Col2a1-Cre;Rosaflox embryos following Cre recombination, detected by whole-mount X-Gal staining. (A) Macroscopic picture of E12.5 Col2a1-Cre;Rosaflox embryo. (B) Transver- sal section of E9.5 embryo showing β-galactosidase-positive sclerotomal cells adjacent to the neural tube. (C) Transversal section of E12.5 embryo showing LacZ expression in verte- brae primordia. (D) Sagital section of E16.5 embryo showing LacZ expression in the femur. The boxed region in D is mag- nified in D' showing LacZ expression in the periosteum (arrow head), osteoblasts (red arrow) and osteocytes (black arrow). Ne, neuroepithelium; Sc, sclerotome; N, notochord; Fe, femur. Scale bars: 1 mm in A; 50 μm in B, D'; 100 μm in C, D. Background Here, we report that skeletal precursors of the axial and appendicular skeleton, when exposed to an uncontrolled rise of the β-catenin level due to conditional inactivation of Apc, lose their differentiation capacity to both the chon- Page 2 of 14 (page number not for citation purposes) Page 2 of 14 (page number not for citation purposes) BMC Developmental Biology 2009, 9:26 http://www.biomedcentral.com/1471-213X/9/26 Col2a1-Cre;Rosaflox mice express Cre at sites of endochon- dral bone formation Figure 1 Col2a1-Cre;Rosaflox mice express Cre at sites of endochondral bone formation. (A-D) LacZ expression in Col2a1-Cre;Rosaflox embryos following Cre recombination, detected by whole-mount X-Gal staining. (A) Macroscopic picture of E12.5 Col2a1-Cre;Rosaflox embryo. (B) Transver- sal section of E9.5 embryo showing β-galactosidase-positive sclerotomal cells adjacent to the neural tube. (C) Transversal section of E12.5 embryo showing LacZ expression in verte- brae primordia. (D) Sagital section of E16.5 embryo showing LacZ expression in the femur. The boxed region in D is mag- nified in D' showing LacZ expression in the periosteum (arrow head), osteoblasts (red arrow) and osteocytes (black arrow). Ne, neuroepithelium; Sc, sclerotome; N, notochord; Fe, femur. Scale bars: 1 mm in A; 50 μm in B, D'; 100 μm in C, D. drogenic and osteogenic lineage. Moreover, conditional Apc mutant ribs show enhanced osteoblast activity, while the mutant nasal septum displays chondrocyte dedifferen- tiation. These results provide the first genetic evidence that Apc plays a crucial role throughout mouse skele- togenesis by regulating the differentiation of skeletal pro- genitor cells and maintenance of chondrocytes. Results Next, we investigated the temporal and spatial expression pattern of Cre in transgenic Col2a1-Cre mice [27] using LacZ reporter mice ("Rosaflox") [28]. Col2a1-Cre;Rosa- flox embryos expressed Cre specifically at all sites of endo- chondral bone formation (Fig. 1A). In accordance with previous studies suggesting that Col2a1 is already expressed at E9.5 in the sclerotome of the somites [29], we detected Cre activity (based on positive LacZ staining) in mesenchymal condensations forming the sclerotome at E9.5 (Fig. 1B). At E12.5, LacZ-positive cells were identified in cartilage primordia later forming the vertebrae, long bones, sternum and cranial bones (Fig. 1C; data not shown). As reported in other Col2a1-Cre mouse lines [11,30], we found LacZ staining in the perichondrium at E14.5 (data not shown), and in the periosteum and pri- mary spongiosa of long bones at E16.5, sites where oste- oblasts normally differentiate (Fig. 1D, D'). The early onset (E9.5) of the LacZ expression in the sclerotome as well as its presence at later developmental stages (E14.5 and E16.5) in cells of the osteogenic lineage prompted us to conclude that the Col2a1-Cre-mediated recombination occurred in skeletal precursors characterized by both a chondrogenic and osteogenic differentiation potential. developmental stages (E12.5, E14.5, E16.5) displayed a normal spatio-temporal expression of all chondrogenic and osteogenic markers investigated (data not shown). developmental stages (E12.5, E14.5, E16.5) displayed a normal spatio-temporal expression of all chondrogenic and osteogenic markers investigated (data not shown). To study postnatal growth and bone acquisition, 18 Col2a1-Cre;Apc15lox/+ mice (7 males, 11 females) and 11 Apc15lox/+ mice (7 males, 4 females) were monitored for 12 weeks after birth. Mice of both genotypes were healthy, similar in appearance, size, body length/weight ratio and growth rate (data not shown). We next assessed bone architecture in these animals by micro-computed tomog- raphy (μCT) of the distal femora. No difference was detected between Col2a1-Cre;Apc15lox/+ mice and gender- matched Apc15lox/+ control littermates with respect to bone mineral density, trabecular bone volume fraction, trabec- ular number, trabecular thickness, and trabecular separa- tion (Fig. 2A–D; data not shown). We further wanted to study whether conditional heterozygous Apc inactivation would lead to skeletal anomalies later in life. For this pur- pose, 10 Col2a1-Cre;Apc15lox/+ mice (5 males and 5 females) and 5 Apc15lox/+ male mice were followed for 24 months. At the end of this period, animals were sacrificed and tissues were analyzed microscopically using hematox- iline/eosine-stained sections. No important abnormalities Heterozygous Apc15lox/+ mice do not show any skeletal defect upon Col2a1-driven Cre expression Heterozygous Apc15lox/+ mice do not show any skeletal defect upon Col2a1-driven Cre expression Apc15lox/15lox mice were bred with Col2a1-Cre mice to gen- erate conditional heterozygous Col2a1-Cre;Apc15lox/+ mice. Microscopical analysis performed on Col2a1- Cre;Apc15lox/+ and control Apc15lox/+ embryos at various f p p Apc15lox/15lox mice were bred with Col2a1-Cre mice to gen- erate conditional heterozygous Col2a1-Cre;Apc15lox/+ mice. Microscopical analysis performed on Col2a1- Cre;Apc15lox/+ and control Apc15lox/+ embryos at various Page 3 of 14 (page number not for citation purposes) Page 3 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 Skeletal development occurs normally in Col2a1-Cre;Apc15lox/ + mice Figure 2 Skeletal development occurs normally in Col2a1- Cre;Apc15lox/+ mice. (A-D) μCT analysis of the distal diaphy sis of the femur did not reveal significant differences between 12-week-old Col2a1-Cre;Apc15lox/+ mice and control litterma- tes in any of the parameters investigated: (A) trabecular bone volume [BV/TV (%)], (B) number of trabeculae [Tb.N (1/ mm)], (C) trabecular thickness [Tb.Th (μm)], and (D) trabec ular separation [Tb.Sp (mm)]. All data represent mean values ± s.d limb outgrowth compared to control littermates (Fig. 3A). At E14.5 and E16.5, Col2a1-Cre;Apc15lox/15lox embryos were much smaller in comparison to controls, displayed craniofacial abnormalities, short trunk, and an incom- plete closure of both thoracic and abdominal cavities (Fig. 3B, C). Gross analysis further indicated a severe trunca- tion of both upper and lower limbs. Already at E14.5, but more significantly at E16.5, Col2a1-Cre;Apc15lox/15lox embryos presented large skin blisters especially in the dorso-lumbar region (Fig. 3C). Skeletal preparations of mouse embryos stained with Alcian blue (chondrocyte matrix) and Alizarin red (min- eralized matrix) of embryos at E14.5 revealed a clear dif- ference in size between Col2a1-Cre;Apc15lox/15lox mutants and control littermates (Fig. 3D). All mutant structures were severely misshaped and fragmented. Mutants failed to develop a cartilaginous mold of both the mandibles and the occipital bone. The axial skeleton contained patchy and irregular cartilaginous structures that did not organize in vertebrae. All 13 rib pairs could be individu- ally distinguished, however, due to their inadequate ori- entation, size, and shape and due to lack of a sternum, no thoracic basket was formed (Fig. 4A, B). Distorted carti- lage rudiments were found where forelimbs should nor- mally arise (Fig. 4E, F), while no signs of bone formation were found in hindlimb rudiments. Furthermore, no car- tilaginous primordia of pelvic bones were observed. Homozygous Col2a1-Cre;Apc15lox/15lox mice die perinatally due to severe defects in skeletogenesis Col2a1-Cre;Apc15lox/+ mice were crossed with Apc15lox/15lox mice to generate conditional homozygous Col2a1- Cre;Apc15lox/15lox mice (1:4). None of these mice were found at one month of age among 77 liveborn offspring. Of 27 dead pups found within the first month after deliv- ery, only 5 pups on the day after delivery were Col2a1- Cre;Apc15lox/15lox. To further investigate the Col2a1- Cre;Apc15lox/15lox phenotype, embryonic litters at various developmental stages were isolated. Eight of 31 embryos isolated between E16.5 and E19.5 were Col2a1- Cre;Apc15lox/15lox (26%). We concluded that conditional homozygosity for this Apc mutant allele was perinatally lethal. At E12.5, Col2a1-Cre;Apc15lox/15lox embryos, although normal in size, displayed poor mandible and Heterozygous Apc15lox/+ mice do not show any skeletal defect upon Col2a1-driven Cre expression Sim- ilar observations were made in Col2a1-Cre;Apc15lox/15lox embryos at E16.5 (Fig. 3E). At this developmental stage however, distinct areas of mineralization were observed in most parts of the mutant skeleton. The mutant hind skull showed mineralized regions, whereas the control occipital and temporal bone primordias stained only with Alcian blue (Fig. 4I, J). Mutant proximal ribs in these Col2a1- Cre;Apc15lox/15lox embryos were much thicker and shorter in comparison to those in control embryos, and stained intensively with Alizarin red (Fig. 4C, D). In the mutant forelimb, a hypoplastic scapula could be identified, whereas more distal components were agenetic and replaced by an irregular cartilaginous structure (Fig. 4G, H). Skeletal de + mice Figure 2 Skeletal de + mice Figure 2 Skeletal development + mice Figure 2 Skeletal development occurs normally in Col2a1-Cre;Apc15 + mice Figure 2 Skeletal development occurs normally in Col2a1-Cre;Apc15 + mice Figure 2 Skeletal development occurs normally in Col2a1- p y ; p g Skeletal development occurs normally in Col2a1- p y p g Skeletal development occurs normally in Col2a1- p y Cre;Apc15lox/+ mice. (A-D) μCT analysis of the distal diaphy- sis of the femur did not reveal significant differences between 12-week-old Col2a1-Cre;Apc15lox/+ mice and control litterma- tes in any of the parameters investigated: (A) trabecular bone volume [BV/TV (%)], (B) number of trabeculae [Tb.N (1/ mm)], (C) trabecular thickness [Tb.Th (μm)], and (D) trabec- ular separation [Tb.Sp (mm)]. All data represent mean values ± s.d could be distinguished in the skull, ribs, vertebral column and long bones. We evenly detected in both groups signs of cartilage degradation, fibrosis, and osteochondritis, pathological findings which most likely were all age- related (data not shown). Altogether, we considered con- ditional heterozygous Apc mutant embryos as controls for the next experiments. Page 4 of 14 (page number not for citation purposes) Loss of functional Apc inhibits differentiation of skeletal precursors p Lack of functional Apc results in accumulation of cyto- plasmic β-catenin, which subsequently translocates into the nucleus. This process can be well detected by immu- nohistochemistry (IHC). To investigate endochondral bone formation in Col2a1-Cre;Apc15lox/15lox embryos, we analyzed vertebra formation at E12.5 and E14.5, and humerus development at E16.5 using IHC for β-catenin in combination with Alcian blue staining, and in situ hybrid- ization (ISH) for several chondrocyte- and osteoblast-spe- cific genes. Strongly elevated levels of β-catenin were seen at all sites of endochondral ossification in the Col2a1- Page 4 of 14 (page number not for citation purposes) Page 4 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 Skeletogenesis is severely impaired in Col2a1-Cre;Apc15lox/15lox embryos Figure 3 Skeletogenesis is severely impaired in Col2a1-Cre;Apc15lox/15lox embryos. (A-E) Greatly impaired skeletal development and growth arrest of Col2a1-Cre;Apc15lox/15lox embryos. Gross appearance (A-C) and Alcian blue and Alizarin red staining (D-E) of skeletal preparations of Col2a1-Cre;Apc15lox/15lox embryos and control littermates at indicated developmental stages. Condi- tional Apc mutants showed lack of mandible outgrowth (arrowheads), poor limb development (black arrows), and an open tho- racic and abdominal cavity (red arrows). Scale bars: 1 mm. g y p ; p y g Skeletogenesis is severely impaired in Col2a1-Cre;Apc15lox/15lox embryos. (A-E) Greatly impaired skeletal development and growth arrest of Col2a1-Cre;Apc15lox/15lox embryos. Gross appearance (A-C) and Alcian blue and Alizarin red staining (D-E) of skeletal preparations of Col2a1-Cre;Apc15lox/15lox embryos and control littermates at indicated developmental stages. Condi- tional Apc mutants showed lack of mandible outgrowth (arrowheads), poor limb development (black arrows), and an open tho- racic and abdominal cavity (red arrows). Scale bars: 1 mm. Cre;Apc15lox/15lox embryos at E12.5, E14.5 and E16.5, indi- cating efficient Col2a1-Cre-mediated Apc inactivation. Cre;Apc15lox/15lox embryos at E12.5, E14.5 and E16.5, indi- cating efficient Col2a1-Cre-mediated Apc inactivation. Col2a1) and mature chondrocyte markers, like Indian hedgehog (Ihh) and Collagen 10a1 (Col10a1), indicating a normal progression of endochondral ossification (Fig. 5F–J). Although somite formation was present, mutant vertebrae were heavily crumbled and failed to organize in a cartilaginous anlage. Occasionally Alcian blue-positive clusters of chondrocytes were seen, which lacked detecta- ble β-catenin immunostaining and were positive for chondrogenic marker expression. These cells were proba- bly derived from non-recombined cells due to mosaicism of Cre expression. Surrounding these cartilage islands, mesenchymal-like spindle-shaped cells were observed. Page 5 of 14 (page number not for citation purposes) Loss of functional Apc inhibits differentiation of skeletal precursors Comparable to the defects observed at E12.5, these cells expressed high levels of nuclear β-catenin due to Apc inac- tivation and lacked not only an Alcian blue-positive matrix but also expression of both chondrogenic and oste- ogenic markers (Fig. 5F–J; data not shown). At E12.5, transversal sections of control vertebral primor- dia showed normal mesenchymal cell condensation and subsequent chondrogenic differentiation (Fig. 5A–C). Chondrocytes stained negatively for β-catenin, started to deposit an Alcian blue-stained matrix, and expressed the nascent chondrocyte markers Sox9 and Col2a1. In marked contrast, mutant sclerotomal cells failed to condense into skeletal primordias. They showed strong nuclear β-cat- enin staining and displayed a mesenchymal-like spindle shape morphology. These cells expressed neither Sox9, nor Col2a1, implying that conditional loss of functional Apc in skeletal precursors inhibited mesenchymal cell conden- sation and chondrogenic differentiation. Next, we investi- gated whether these cells had switched their commitment to the osteogenic lineage due to the accumulation of β-cat- enin. Surprisingly, they did not express the early osteob- last markers Runx2 and Col1a1, suggesting that β-catenin accumulation due to Apc inactivation impaired osteogenic differentiation of skeletal precursors as well (Fig. 5D, E). At E16.5, chondrocytes of control proximal humeri did not express detectable β-catenin protein levels and were surrounded by a proteoglycan-rich matrix, which stained positively with both Alcian blue and Toluidine blue (Fig. 6A, B). They were organized in growth plates with a char- acteristic spatial expression pattern of the chondrogenic markers Sox9, Col2a1, Ihh, and Col10a1 (Fig. 6C–F). Young osteoblasts in the perichondrium, periosteum and At E14.5, chondrocytes in the control vertebrae did not stain positively for β-catenin, displayed an intensely Alcian blue-stained matrix and expressed both early (Sox9, Page 5 of 14 (page number not for citation purposes) Page 5 of 14 (page number not for citation purposes) BMC Developmental Biology 2009, 9:26 http://www.biomedcentral.com/1471-213X/9/26 Details of skeletal preparations Figure 4 Details of skeletal preparations. (A-D) Vertebral column of control and mutant littermates at E14.5 (A, B) and E16.5 (C D). Mutant vertebrae lacked structural integrity (arrowheads). At E16.5, mineralization was enhanced in the proximal part o the mutant rib (arrow). (E-H) Forelimb of control and mutant littermates at E14.5 (E, F) and E16.5 (G, H). At E16.5, only th scapula was identified (arrowhead), while more distal parts were represented by patchy cartilage aggregations (arrow). (I, J) Skull of control and mutant littermates at E16.5. Loss of functional Apc inhibits differentiation of skeletal precursors The mutant displayed mineral deposition in the back skull corresponding t the cartilaginous structure in the control (arrowheads). Scale bars: 3 mm in A-H; 1 mm in I, J. Details of Figure 4 Details of Figure 4 Details of skeletal preparations Figure 4 Details of skeletal preparations. (A-D) Vertebral column of control and mutant littermates at E14.5 (A, B) and E16.5 (C, D). Mutant vertebrae lacked structural integrity (arrowheads). At E16.5, mineralization was enhanced in the proximal part of the mutant rib (arrow). (E-H) Forelimb of control and mutant littermates at E14.5 (E, F) and E16.5 (G, H). At E16.5, only the scapula was identified (arrowhead), while more distal parts were represented by patchy cartilage aggregations (arrow). (I, J) Skull of control and mutant littermates at E16.5. The mutant displayed mineral deposition in the back skull corresponding to the cartilaginous structure in the control (arrowheads). Scale bars: 3 mm in A-H; 1 mm in I, J. Page 6 of 14 (page number not for citation purposes) BMC Developmental Biology 2009, 9:26 http://www.biomedcentral.com/1471-213X/9/26 Abnormal axial skeleton formation of Col2a1-Cre;Apc15lox/15lox embryos already detectable at E12.5 Figure 5 Abnormal axial skeleton formation of Col2a1-Cre;Apc15lox/15lox embryos already detectable at E12.5. (A) Immu- nostaining for β-catenin combined with Alcian blue (AB) staining, and (B-E) gene expression analysis by in situ hybridization with indicated probes on consecutive transversal sections of the sclerotome of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E12.5. (F-J) Similar analysis of vertebrae primordia at E14.5. β-Catenin-positive spindle-shaped cells lacked expres- sion of all indicated chondrogenic and osteogenic markers (arrowheads). Scale bars: 100 μm. Abnormal Figure 5 Abnormal axial skeleton formation of Col2a1 Cre;Apc embryos already detectable at E12.5 Figure 5 Abnormal axial skeleton formation of Col2a1-Cre;Apc15lox/15lox embryos already detectable at E12.5. (A) Immu- nostaining for β-catenin combined with Alcian blue (AB) staining, and (B-E) gene expression analysis by in situ hybridization with indicated probes on consecutive transversal sections of the sclerotome of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E12.5. (F-J) Similar analysis of vertebrae primordia at E14.5. β-Catenin-positive spindle-shaped cells lacked expres- sion of all indicated chondrogenic and osteogenic markers (arrowheads). Scale bars: 100 μm. primary spongiosa were surrounded by a mineralized osteoid as detected by von Kossa staining (Fig. 6B) and expressed Runx2 and Col1a1 (Fig. 6G, H). Mature osteob- lasts expressed Osc (Fig. 6I), while osteoclasts expressed Matrix metalloproteinase 9 (Mmp9) (Fig. 6J). In contrast, mutant humeri were completely misshaped and con- tained nuclear β-catenin-positive cells that were organized in clusters, showing a mesenchymal-like shape (Fig. 6A). Increased osteoblastogenesis in proximal ribs of Col2a1- Cre;Apc15lox/15lox embryos icantly thicker and shorter compared to those of control embryos (Fig. 4C, D and 7E–G). They consisted of a mas- sive mineralized bone matrix and a poorly developed bone marrow cavity, although osteoclast differentiation and activity were normal as assessed by ISH for Mmp9 and TRAP staining, respectively (Fig. 7K, L, L'). Interestingly, β- catenin-positive cells expressed all osteogenic markers analyzed (Runx2, Col1a1, and Osc), indicating that, unlike in the long bones and vertebrae, Apc inactivation in skele- tal precursors of the proximal ribs did not impair osteob- lastogenesis (Fig. 7H–J). Since Ihh is a critical regulator of osteoblastogenesis, we subsequently tested whether the increased ossification might be due to increased Ihh expression in the non-recombined neighbouring chondrocytes. The β-catenin-negative cells, however, matured normally expressing all chondrocyte markers investigated (Sox9, Col2a1, Ihh and Col10a1) at similar lev- els compared to control cartilage (data not shown). The abundant presence of a bone matrix combined with evi- dence of functional osteoclasts suggested that the β-cat- enin-positive osteoblasts were sclerotic. p y Despite the inhibitory effect of Apc inactivation on differ- entiation of skeletal precursors in long bones and verte- brae, proximal ribs of Col2a1-Cre;Apc15lox/15lox embryos at E16.5 showed clearly enhanced mineralization upon skel- etal staining (Fig. 4D). Therefore, we analyzed the devel- opment of these skeletal structures in more detail. The ribs develop through endochondral ossification from the paired lateral sclerotomic areas [31]. Formation of the proximal rib depends on the notochord and the ventral neural tube, whereas development of the distal part depends on the surface ectoderm [32]. At E14.5, proximal ribs of control embryos were cartilaginous and contained mature chondrocytes that did not stain for β-catenin (Fig. 7A–C). Mutant proximal ribs were severely misshaped and contained β-catenin negatively stained cartilage islands, accounting for the positive Alcian blue staining observed upon skeletal preparation (Fig. 4B, 7A). β-Cat- enin-positive cells were negative for chondrogenic and osteogenic markers (Fig. 7A–D). At E16.5, the β-catenin- negative proximal ribs of control embryos consisted of cartilage and mineralized bone matrix as indicated by combined von Kossa-Toluidine blue staining (Fig. 7E, F; data not shown). They contained chondrocytes, osteob- lasts, and osteoclasts as assessed by ISH (Fig. 7H–K; data not shown). In contrast, proximal ribs of mutant litterma- tes stained strongly positive for β-catenin and were signif- Details of Figure 4 Similar to our observations at E12.5 and E14.5, these cells expressed neither chondrogenic, nor osteogenic markers (Fig. 6C–I). In addition, no Mmp9 expression could be detected (Fig. 6J), suggesting that differentiation of bone- resorbing cells was impaired as well. These β-catenin-pos- itive cell clusters were surrounded by chondrocytes expressing Sox9 and Col2a and lacked positive staining for β-catenin. These cell most likely have not undergone a recombination event as observed at E14.5 (Fig. 6A, C, D). Page 7 of 14 (page number not for citation purposes) Page 7 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 No chondrogenic and osteogenic differentiation in the developing humerus due to lack of functional Apc Figure 6 No chondrogenic and osteogenic differentiation in the developing humerus due to lack of functional Apc. (A-B) Immunostaining for β-catenin combined with Alcian blue (AB) staining (A), combined von Kossa-Toluidine blue staining (B), and (C-J) gene expression analysis by in situ hybridization with indicated probes for (C-F) chondrocytes, (G-I) osteoblasts and (J) osteoclasts on consecutive transversal sections of the developing humerus of a Col2a1-Cre;Apc15lox/15lox embryo and control lit- termate at E16.5. β-Catenin-positive spindle-shaped cells organized in clusters and failed to express chondrogenic and osteo- genic markers (arrowheads). β-Catenin-negative cells at the periphery of these clusters expressed early chondrogenic markers only (arrows), probably due to lack of Cre-mediated loss of functional Apc. Scale bars: 100 μm. No chondrogenic and osteogenic differentiation in the developing humerus due to lack of functional Apc Figure 6 No chondrogenic and osteogenic differentiation in the developing humerus due to lack of functional Apc. (A-B) Immunostaining for β-catenin combined with Alcian blue (AB) staining (A), combined von Kossa-Toluidine blue staining (B), and (C-J) gene expression analysis by in situ hybridization with indicated probes for (C-F) chondrocytes, (G-I) osteoblasts and (J) osteoclasts on consecutive transversal sections of the developing humerus of a Col2a1-Cre;Apc15lox/15lox embryo and control lit- termate at E16.5. β-Catenin-positive spindle-shaped cells organized in clusters and failed to express chondrogenic and osteo- genic markers (arrowheads). β-Catenin-negative cells at the periphery of these clusters expressed early chondrogenic markers only (arrows), probably due to lack of Cre-mediated loss of functional Apc. Scale bars: 100 μm. Page 8 of 14 (page number not for citation purposes) Chondrocyte dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos The nasal septum is a midline vertical plate of hyaline car- tilage, which undergoes endochondral ossification in postnatal life [33]. Endochondral ossification of the cau- dal and dorsal borders of the septum, when combined Page 8 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 onal Apc inactivation enhances osteoblast formation and mineral deposition in the developing proximal rib 7 ional Apc inactivation enhances osteoblast formation and mineral deposition in the developing proximal L) Immunostaining for β-catenin combined with Alcian blue (AB) staining (A, E), combined von Kossa-Toluidine blue Conditional Apc inactivation enhances osteoblast formation and mineral deposition in the developing proximal rib Figure 7 Conditional Apc inactivation enhances osteoblast formation and mineral deposition in the developing proximal rib. (A-L) Immunostaining for β-catenin combined with Alcian blue (AB) staining (A, E), combined von Kossa-Toluidine blue staining (F), hematoxylin/eosin staining (G), gene expression analysis by in situ hybridization with indicated probes for chondro- genic (B, C), osteogenic (D, H-J) and osteoclastogenic differentiation (K) on consecutive transversal sections of the developing proximal rib of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E14.5 (A-D) and E16.5 (E-K). The double-headed arrows in G indicate the thickness of the rib. (L) Tartrate-resistant acid phosphatase (TRAP) staining of the developing proxi- mal rib of a Col2a1-Cre;Apc15lox/15lox embryo at E16.5. The boxed region in L is magnified in L' showing multinucleated osteo- clasts (arrowheads) staining positive for TRAP. Scale bars: 100 μm in A-L; 50 μm in L'. p p p g p g Conditional Apc inactivation enhances osteoblast formation and mineral deposition in the developing proximal rib. (A-L) Immunostaining for β-catenin combined with Alcian blue (AB) staining (A, E), combined von Kossa-Toluidine blue staining (F), hematoxylin/eosin staining (G), gene expression analysis by in situ hybridization with indicated probes for chondro- genic (B, C), osteogenic (D, H-J) and osteoclastogenic differentiation (K) on consecutive transversal sections of the developing proximal rib of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E14.5 (A-D) and E16.5 (E-K). The double-headed arrows in G indicate the thickness of the rib. (L) Tartrate-resistant acid phosphatase (TRAP) staining of the developing proxi- mal rib of a Col2a1-Cre;Apc15lox/15lox embryo at E16.5. The boxed region in L is magnified in L' showing multinucleated osteo- clasts (arrowheads) staining positive for TRAP. Scale bars: 100 μm in A-L; 50 μm in L'. Conditional homozygous loss of functional Apc severely disrupts mouse skeletogenesis via stabilized β-catenin According to most of the transgenic mouse studies reported, levels of β-catenin, the effector of the canonical Wnt ligands, need to be downregulated in skeletal precur- sor cells to enable chondrogenic differentiation, whereas elevated β-catenin levels promote differentiation into Dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos at E16.5 Figure 8 Dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos at E16.5. (A, E) Hematoxylin/eosin stain- ing, (B, F, H) immunostaining for β-catenin combined with Alcian blue (AB) staining, and (C-D, G, I) gene expression analysis by in situ hybridization with indicated probes for chondrogenic differentiation on consecutive transversal sections of the develop- ing nasal septum of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E16.5. (F', G', H', I') High magnification pictures of the boxed regions in F, G, H, and I, respectively. Mesenchymal-like β-catenin-positive cells (arrow in F, H) were present between crumbled cartilage islands. Within these cartilage islands, although displaying chondrocytic morphology and an Alcian blue stained matrix, most of the β-catenin-positive cells did not express Sox9 (arrowheads in G') or Col2a1 (arrows in I'). Scale bars: 100 μm in A, E, F, H; 5 μm in F', H'. Dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos at E16.5 Figure 8 Dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos at E16.5. (A, E) Hematoxylin/eosin stain- ing, (B, F, H) immunostaining for β-catenin combined with Alcian blue (AB) staining, and (C-D, G, I) gene expression analysis by in situ hybridization with indicated probes for chondrogenic differentiation on consecutive transversal sections of the develop- ing nasal septum of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E16.5. (F', G', H', I') High magnification pictures of the boxed regions in F, G, H, and I, respectively. Mesenchymal-like β-catenin-positive cells (arrow in F, H) were present between crumbled cartilage islands. Within these cartilage islands, although displaying chondrocytic morphology and an Alcian blue stained matrix, most of the β-catenin-positive cells did not express Sox9 (arrowheads in G') or Col2a1 (arrows in I'). Scale bars: 100 μm in A, E, F, H; 5 μm in F', H'. p ; p y g Dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos at E16.5. Chondrocyte dedifferentiation in the nasal septum of Col2a1-Cre;Apc15lox/15lox embryos Page 9 of 14 (page number not for citation purposes) Page 9 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 with interstitial expansion, has the effect of displacing the facial skeleton away from the neurocranium and thus enlarging the skull [34]. At E16.5, chondrocytes forming the nasal septum of control mice did not stain for β-cat- enin, were surrounded by an Alcian blue-positive matrix, and expressed Sox9 and Col2a1 (Fig. 8A–D). In the mutant nasal cartilage we distinguished crumbled chondrogenic islands surrounded by β-catenin-positive cells with an undifferentiated mesenchymal-like phenotype (Fig. 8F– H). The chondrogenic islands consisted of round cells embedded in chondrons surrounded by extracellular matrix (ECM) (Fig. 8E). Interestingly, molecular analysis of these chondrogenic islands revealed the presence of two cell populations: β-catenin-negative and β-catenin- positive cells. The former expressed chondrogenic markers like Sox9 and Col2a1, and their ECM stained positive with Alcian blue, whereas the latter did not express any chon- drogenic or osteogenic markers, while their ECM stained significantly less with Alcian blue (Fig. 8F–I; data not shown). The presence of β-catenin-positive cells in the chondro- genic islands suggested that these cells, due to mosaicism of Cre expression, had initiated normal chondrocyte dif- ferentiation before undergoing Apc inactivation. Subse- quently, the increased level of β-catenin triggered the loss of expression of the early chondrogenic markers and initi- ated degradation of the ECM. These observations were indicative of dedifferentiated chondrocytes. Similar obser- vations were made in cartilaginous rudiments at other sites of endochondral bone formation, but the effect was most pronounced in the nasal septum (data not shown). http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 osteoblasts [11,12,15,35,36]. This theory is partly based on observations in heterozygous gain-of-function models in which Cre-mediated recombination results in the expression of oncogenic β-catenin. The cellular mecha- nisms controlling the biological effects of oncogenic β- catenin in the presence of wild type β-catenin are largely unknown. In addition, there are no reports on the role of Apc in regulation of skeletal precursor differentiation via control of β-catenin in the mouse. Here, we have focused on this important role of the multifunctional protein Apc, binding to and downregulating β-catenin. We have selec- tively inactivated one or both alleles of Apc in murine Col2a1-expressing cells. Our data indicate that the Col2a1 promoter is suitable for this study, since Cre-mediated recombination starts very early (E9.5) in skeletal precur- sor cells that have not yet committed to the chondrogenic or the osteogenic lineage, consistent with previous find- ings in other Col2a1-Cre lines [11,30]. cytic and osteoblastic fates. This key regulating role in lineage commitment has been attributed to β-catenin. Indeed, conditional gain-of-function mutation of β-cat- enin leads to decreased chondrocyte differentiation in Prx1-expressing and Col2a1-expressing cells [12,15]. How- ever, corresponding increased osteoblast differentiation has not been observed in these models, instead, a decreased osteoblast marker expression has been seen in case of Prx1-expressing cells, suggesting that activation of β-catenin negatively affects skeletogenesis [12,15]. In addition, conditional loss-of-function mutation of β-cat- enin in Prx1-expressing cells leads to increased expression of not only chondrocyte but also early osteoblast markers [15]. These data strongly suggest that β-catenin negatively regulates the differentiation of mesenchymal cells into a common skeletal precursor [38]. We report here that in the vast majority of endochondral skeletal elements, precursor cells lacking functional Apc express strong nuclear β-catenin staining and fail to differ- entiate into both chondrogenic and osteogenic lineages. These data are in line with the inability of mouse embry- onic stem cells carrying specific bi-allelic Apc mutations to differentiate into bone and cartilage [25]. Our data are also consistent with those based on conditional stabiliza- tion of β-catenin in mesenchymal skeletal precursors which had an undifferentiated appearance [15]. This con- sistency strongly suggests that, notwithstanding the multi- ple functions of Apc, its β-catenin-controlling role is the most important during skeletogenesis. Loss of functional Apc in skeletal precursors of the proximal rib stimulates osteogenesis Although in the vast majority of the endochondral skele- ton both chondrogenic and osteogenic differentiation is inhibited due to loss of functional Apc in skeletal precur- sors, we find a different phenotype in the proximal ribs. Notwithstanding the cartilaginous structure at E14.5, proximal ribs of Col2a1-Cre;Apc15lox/15lox mutants at E16.5 show abundant bone matrix deposited by osteoblasts, invariably expressing high levels of nuclear β-catenin. Since osteoblasts do not express Col2a1, these cells are most likely derived from Col2a1-expressing skeletal pre- cursors lacking functional Apc. This implies that, in con- trast to other skeletal elements, skeletal precursors of the proximal ribs are able to escape from the noxious effects of strongly elevated β-catenin levels on differentiation of precursor cells by an as yet unknown mechanism. Since Ihh expression is normal in the non-recombined neigh- bouring chondrocytes, we speculate that Ihh may be a prime target for inducing osteoblastogenesis in the recom- http://www.biomedcentral.com/1471-213X/9/26 We conclude that Apc plays a crucial role in differentiation of skeletal pre- cursors in vertebrae and long bones: it enables the differ- entiation into both skeletal lineages by decreasing the level of β-catenin. Conditional heterozygous inactivation of Apc does not result in a detectable level of its target β-catenin as deter- mined by IHC. Moreover, heterozygous Col2a1-Cre- mediated Apc inactivation does not interfere with embry- onic skeletal development, postnatal growth or bone acquisition up to 24 months of age, as determined by his- tological and μCT analysis. Our data imply that the level of Apc protein produced by a single functional Apc allele is sufficient to mediate appropriate β-catenin degradation. This is in agreement with normal body weight, size, and growth of young ApcMin/+ mice [37]. In marked contrast, conditional inactivation of Apc results in a strongly elevated level of (wild type) β-catenin in skel- etal precursors, leading to greatly impaired embryogenesis and perinatal lethality. The significantly reduced size and the vast range of skeletal malformations in these embryos is most likely due to the specific Col2a1-Cre activity in skeletal primordias at a very early embryonic stage starting at E9.5 resulting in massive β-catenin accumulation in the developing endochondral skeleton. Probably several fac- tors, like the open rib cage and the severe malformation, from E14.5 on have led to the perinatal lethality. The loss of the multiple β-catenin-independent functions of the Apc protein might have contributed to the gravity and complexity of the skeletal phenotype observed in Col2a1- Cre;Apc15lox/15lox mice as well [18]. Moreover, since Col2a1 expression is not completely restricted to skeletal tissues during mouse embryogenesis [29], we can not exclude that the severity of the phenotype might have been partly due to loss of functional Apc in other Col2a1-Cre-express- ing cell types. Conditional homozygous loss of functional Apc severely disrupts mouse skeletogenesis via stabilized β-catenin (A, E) Hematoxylin/eosin stain- ing, (B, F, H) immunostaining for β-catenin combined with Alcian blue (AB) staining, and (C-D, G, I) gene expression analysis by in situ hybridization with indicated probes for chondrogenic differentiation on consecutive transversal sections of the develop- ing nasal septum of a Col2a1-Cre;Apc15lox/15lox embryo and control littermate at E16.5. (F', G', H', I') High magnification pictures of the boxed regions in F, G, H, and I, respectively. Mesenchymal-like β-catenin-positive cells (arrow in F, H) were present between crumbled cartilage islands. Within these cartilage islands, although displaying chondrocytic morphology and an Alcian blue stained matrix, most of the β-catenin-positive cells did not express Sox9 (arrowheads in G') or Col2a1 (arrows in I'). Scale bars: 100 μm in A, E, F, H; 5 μm in F', H'. Page 10 of 14 (page number not for citation purposes) Page 10 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 Page 11 of 14 (page number not for citation purposes) Conclusion We show here for the first time that Apc, by negatively controlling the levels of β-catenin, is a critical regulator of the differentiation of skeletal progenitor cells. Condi- tional inactivation of the mouse Apc gene results in a het- erogeneous skeletal phenotype. Based on our results, we postulate that Apc-mediated control of the dosage of tran- scriptionally active β-catenin protein is directive for the differentiation program of skeletal precursor cells. In the vast majority of the skeletal precursors, loss of functional Apc leads to a strongly increased β-catenin level, resulting in the formation of an undifferentiated mesenchymal cell, which lacks differentiation potential for both osteogenic and chondrogenic lineages. When the inhibitory effect of a strongly increased β-catenin level in the skeletal precur- sors is reduced, highly active osteoblasts arise. Strong repression of β-catenin in these precursors is required for chondrogenesis. Support for our hypothesis on the importance of the dosage of Apc and β-catenin is provided by observations in Col2a1-Wnt14 transgenic mice [11]. Higher levels of Wnt14 expression resulting in a high level of β-catenin block differentiation of skeletal precursors into chondrocytes or osteoblasts, whereas lower levels of Wnt14 expression result in enhanced ossification. We pro- vide evidence that Apc plays a crucial role in modulating the β-catenin level during mouse skeletogenesis in a spa- tio-temporal regulated manner. In skeletal precursor cells, Apc is required for differentiation into both chondrocytes and osteoblasts. In addition, Apc is essential in chondro- cytes to maintain their phenotype and enable their matu- ration. Despite the evidence of functional osteoclasts, the intensely ossified proximal ribs show a strongly dimin- ished bone marrow cavity, rendering it likely that the increased bone formation is due to osteosclerosis. These observations are in agreement with other data, showing that enhanced canonical Wnt signaling can increase bone mass through stimulation of osteoblast activity rather than inhibition of osteoclast formation and activity [39- 41]. Such an osteopetrotic phenotype has only been seen in mice with conditional loss of functional Apc or consti- tutively active β-catenin in already differentiated osteob- lasts, resulting in dramatically increased bone deposition [17,42]. http://www.biomedcentral.com/1471-213X/9/26 http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 http://www.biomedcentral.com/1471-213X/9/26 bined precursor cells counteracting the noxious effect of β-catenin. bined precursor cells counteracting the noxious effect of β-catenin. bined precursor cells counteracting the noxious effect of β-catenin. bined precursor cells counteracting the noxious effect of β-catenin. Functional Apc is required to maintain the chondrocyte phenotype We have found clear evidence for the occurrence of chondrocyte dedifferentiation due to β-catenin accumula- tion in the nasal septum. Morphologically characterized chondrocytes, which were nuclear β-catenin-positive, lacked expression of typical chondrocyte markers. Fur- thermore, they were imbedded in an ECM containing sig- nificantly less proteoglycans. Given the noxious effect of increased β-catenin levels on chondrocyte formation (our data and [12,15]), these cells most likely have undergone Cre-mediated loss of func- tional Apc after completion of the initial stages of chondrocyte differentiation. Mouse models with an increased level of β-catenin in Col2a1-expressing cells show accelerated chondrocyte maturation [11,12]. We have found no indication for this phenomenon, implying that the high level of β-catenin due to loss of Apc does not result in chondrocyte maturation but in chondrocyte ded- ifferentiation. Our data suggest that accumulated β-cat- enin triggers this dedifferentiation program not only through inhibition of chondrogenic marker expression but also by enhancing the loss of ECM presumably through stimulation of matrix-degrading enzymes. It has been demonstrated that β-catenin increases expression and activity of a number of enzymes involved in matrix degradation [43-45]. β-Catenin stabilization has been associated with dedifferentiation of articular chondro- cytes in vitro upon serial monolayer culture, or treatment with retinoic acid or IL1β [46]. Dedifferentiated chondro- cytes have also been observed at other sites of endochon- dral bone formation in the Col2a1-Cre;Apc15lox/15lox embryos, however, the presence of these cells was most pronounced in the nasal septum. Altogether, our data indicate that Apc is required to suppress β-catenin for maintenance of the chondrocytic phenotype. Page 12 of 14 (page number not for citation purposes) Apc is crucial for both chondrogenic and osteogenic differentiation of skeletal precursors Wnt/β-catenin signaling represents a mechanism in mes- enchymal precursor cells for selecting between chondro- Page 11 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/9/26 References Whole mount β-galactosidase staining was performed as described [49], from E16.5 on after removal of the skin. For histology, immunohistochemistry, and in situ hybrid- ization, specimens were fixed in phosphate-buffered for- malin, embedded in paraffin, and sectioned at 6 μm. Hematoxylin/eosin, Nuclear red, Toluidine blue, and von Kossa stainings were performed according to standard procedures. For immunohistochemistry, sections were treated with 1% H2O2 in 40% methanol/60% TBS for 30 minutes to reduce endogenous peroxidase activity. For antigen retrieval the sections were boiled in Tris-EDTA pH 9.0 for 20 minutes. Blocking was performed with 5% blocking buffer for 30 minutes at 37°C (Boehringer Ingel- heim). Sections were incubated with the primary mouse monoclonal antibody against β-catenin (1:100; BD Trans- duction Laboratories) overnight at 4°C, followed by incu- bation with the second antibody biotin-conjugated rabbit anti-mouse IgG (1:300; Amersham Biosciences) for 45 minutes at 37°C. The biotinylated proteins were detected by incubation with horseradish peroxidase-conjugated streptavidin (1:200; Amersham Biosciences) for 30 min- utes at 37°C and visualized with DAB (Sigma). After counterstaining with Alcian blue for 15 minutes and hematoxylin for 1 minute, sections were dehydrated and embedded in Histomount (BDH). For in situ hybridiza- tion, digoxigenin-labeled single-stranded RNA probes were prepared using a DIG RNA labeling kit (Boehringer) following the manufacturers' instructions. All probes are available upon request. In situ hybridization was carried out as described [15,50]. Images were taken with a DXM- 1200 digital camera (Nikon). 1. Karsenty G: Genetics of skeletogenesis. Dev Genet 1998, 22:301-313. 1. Karsenty G: Genetics of skeletogenesis. Dev Genet 1998, 22:301-313. 2. Karsenty G, Wagner EF: Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2002, 2:389-406. 2. Karsenty G, Wagner EF: Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2002, 2:389-406. 3. Tuan RS: Biology of developmental and regenerative skele- togenesis. Clin Orthop Relat Res 2004:S105-S117. g p 4. Olsen BR, Reginato AM, Wang W: Bone development. Annu Rev Cell Dev Biol 2000, 16:191-220. g p 4. Olsen BR, Reginato AM, Wang W: Bone development. Annu Rev Cell Dev Biol 2000, 16:191-220. 5. Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B: The transcription factor Sox9 has essential roles in succes- sive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. 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J Cell Physiol 2006, 208:77-86. http://www.biomedcentral.com/1471-213X/9/26 http://www.biomedcentral.com/1471-213X/9/26 http://www.biomedcentral.com/1471-213X/9/26 BMC Developmental Biology 2009, 9:26 ditional Apc mouse model is currently in preparation (Robanus-Maandag et al., in preparation). Col2a1-Cre mice [27] were mated with Apc15lox/15lox mice. Of the off- spring, Col2a1-Cre+/-;Apc15lox/+ mice were mated with Apc15lox/15lox mice to obtain Col2a1-Cre+/-;Apc15lox/15lox mice. LacZ reporter mice were obtained from Dr. Xiao- hong Mao [28]. Routine mouse genotyping was per- formed on tail DNAs by PCR (Robanus-Maandag et al., in preparation). ditional Apc mouse model is currently in preparation (Robanus-Maandag et al., in preparation). Col2a1-Cre mice [27] were mated with Apc15lox/15lox mice. Of the off- spring, Col2a1-Cre+/-;Apc15lox/+ mice were mated with Apc15lox/15lox mice to obtain Col2a1-Cre+/-;Apc15lox/15lox mice. LacZ reporter mice were obtained from Dr. Xiao- hong Mao [28]. Routine mouse genotyping was per- formed on tail DNAs by PCR (Robanus-Maandag et al., in preparation). Cre mice were provided by TK and HMK; mutant mice were generated and genotyped by CAJB and ECR-M; embryo experimental work and analysis were performed by RLM; μCT analysis was performed by GR; data interpre- tation was carried out by RLM assisted by MK, GH, MAV, PA, CWGML, RF, JMW, and ECR-M; the manuscript was written by RLM with the assistance of all co-authors. All authors read and approved the final manuscript. Skeletal analysis We thank Christine Hartmann (IMP, Vienna, Austria) for the mouse Runx2, Sox9, Osc, and Ihh probes, Eero Vuorio (University of Turku, Finland) for the mouse Col1a1 and Col2a1 probes, and Willy Hofstetter (University of Bern, Switzerland) for the mouse Col10a1 probe. Skeletons of mouse embryos were stained with Alcian blue and Alizarin red for cartilaginous and mineralized tissues, respectively, according to standard procedures [47]. For micro-computed tomography (μCT) analysis, femora were recovered from 12-week-old mice after death and processed as described [48]. This work was financially supported by a short-term research fellowship from the European Society for Pediatric Endocrinology (RLM), an unre- stricted educational grant from IPSEN FARMACEUTICA BV (RLM), a research grant from The Human Growth Foundation (MK), and a research grant from the Association for International Cancer Research (CAJB). Transgenic mice All animal studies were approved by the ethical commit- tee of the Leiden University Medical Centre and complied with national laws relating to the conduct of animal experiments. The Apc15lox/+ mouse was generated by gene targeting in IB10 embryonic stem cells, using a 22.5 kb targeting vector containing loxP sites flanking the last exon of Apc, i.e. exon 15. LoxP sites were inserted in the BglII site of intron 14 and in the ApaI site approximately 350 bp downstream of the Apc polyadenylation signal. Exon 15 of the Apc gene encodes for codons 660 to 2842 of the Apc protein and harbours all the functional domains of Apc involved in β-catenin regulation as well as the C-terminal domains binding to microtubules, DLG, and EB1. Therefore, following Cre-mediated deletion of exon 15, functionality of the remaining protein will be fully impaired with respect to the main function of Apc, i.e. β-catenin regulating. Moreover, as deletion of exon 15 also removes the polyadenylation signal, no stable mRNA is produced and as a consequence no stable truncated pro- tein will be generated. A full description of this new con- Page 12 of 14 (page number not for citation purposes) Page 13 of 14 (page number not for citation purposes) Authors' contributions p J y 15. Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C: Canon- ical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell 2005, 8:727-738. 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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 14 of 14 (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 Authors' contributions Proc Natl Acad Sci USA 2005, 102:17406-17411. g g g 26. Fodde R, Smits R: Cancer biology. A matter of dosage. Science 2002, 298:761-763. 27. Schipani E, Ryan HE, Didrickson S, Kobayashi T, Knight M, Johnson RS: Hypoxia in cartilage: HIF-1alpha is essential for chondro- cyte growth arrest and survival. Genes Dev 2001, 15:2865-2876. 49. Dannenberg JH, Schuijff L, Dekker M, van der Valk M, te Riele H: Tis- sue-specific tumor suppressor activity of retinoblastoma gene homologs p107 and p130. 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https://openalex.org/W3127619881	https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0008596&type=printable	English	null	Echis carinatus snake venom metalloprotease-induced toxicities in mice: Therapeutic intervention by a repurposed drug, Tetraethyl thiuram disulfide (Disulfiram)	PLoS neglected tropical diseases	2,021	cc-by	13,755	Echis carinatus snake venom metalloprotease- induced toxicities in mice: Therapeutic intervention by a repurposed drug, Tetraethyl thiuram disulfide (Disulfiram) Gotravalli V. RudreshaID1, Amog P. UrsID2, Vaddarahally N. ManjuprasannaID1, Mallanayakanakatte D. Milan Gowda1, Krishnegowda Jayachandra1, Rajesh RajaiahID3, Bannikuppe S. VishwanathID1,3 1 Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore, Karnataka, India, 2 The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, United States of America, 3 Department of Studies in Molecular Biology, University of Mysore, Manasagangotri, Mysore, Karnataka, India a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * rajesh.r@biochemistry.uni-mysore.ac.in (RR); vishmy@biochemistry.uni-mysore.ac.in (BSV) Abstract Echis carinatus (EC) is known as saw-scaled viper and it is endemic to the Indian subconti- nent. Envenoming by EC represents a major cause of snakebite mortality and morbidity in the Indian subcontinent. Zinc (Zn++) dependent snake venom metalloproteases (SVMPs) present in Echis carinatus venom (ECV) is well known to cause systemic hemorrhage and coagulopathy in experimental animals. An earlier report has shown that ECV activates neu- trophils and releases neutrophil extracellular traps (NETs) that blocks blood vessels leading to severe tissue necrosis. However, the direct involvement of SVMPs in the release of NETs is not clear. Here, we investigated the direct involvement of EC SVMPs in observed patho- logical symptoms in a preclinical setup using specific Zn++ metal chelator, Tetraethyl thiuram disulfide (TTD)/disulfiram. TTD potently antagonizes the activity of SVMPs-mediated ECM protein degradation in vitro and skin hemorrhage in mice. In addition, TTD protected mice from ECV-induced footpad tissue necrosis by reduced expression of citrullinated H3 (citH3) and myeloperoxidase (MPO) in footpad tissue. TTD also neutralized ECV-induced systemic hemorrhage and conferred protection against lethality in mice. Moreover, TTD inhibited ECV-induced NETosis in human neutrophils and decreased the expression of peptidyl argi- nine deiminase (PAD) 4, citH3, MPO, and p-ERK. Further, we demonstrated that ECV- induced NETosis and tissue necrosis are mediated via PAR-1-ERK axis. Overall, our results provide an insight into SVMPs-induced toxicities and the promising protective effi- cacy of TTD can be extrapolated to treat severe tissue necrosis complementing anti-snake venom (ASV). Author summary India has highest incidence of deaths due to snakebite in the world. Echis carinatus (EC) is known as saw-scaled viper and its bite causes major mortality and morbidity in the Indian subcontinent. The abundant presence of zinc (Zn++) metalloproteases in Echis carinatus venom (ECV) is responsible for local tissue necrosis. An earlier report has shown that ECV activates neutrophils and leads to NETosis that blocks blood vessels leading to tissue necrosis. However, the toxin in ECV responsible for NETosis has not addressed. Here we investigated the Echis carinatus venom metalloproteases (ECVMPs) are responsible for NETosis and its associated tissue necrosis using Zn++ specific chelator, Tetraethyl thiuram disulfide (TTD). TTD inhibited ECVMPs-induced skin hemorrhage and footpad tissue necrosis by reduced expression of citrullinated H3 (citH3) and myeloperoxidase (MPO) in mice footpad tissue. TTD also neutralized ECV-induced systemic hemorrhage and con- ferred protection against lethality in mice. Moreover, TTD inhibited ECV-induced NETo- sis in human neutrophils and decreased the expression of p-ERK and NETosis markers. Further, we demonstrated that ECV-induced NETosis and tissue necrosis is mediated via PAR-1-ERK axis. Overall, our results provide an insight into SVMPs-induced toxicities and the promising protective efficacy of TTD can be extrapolated to treat severe tissue necrosis complementing anti-snake venom (ASV). Funding: The study was funded by the University Grants Commission of India, a statutory body set up by the Government of India under Ministry of Education (MRP-MAJOR-BIOC-2013-12157 to BSV) and Science and Engineering Research Board, a statutory body under the Department of Science and Technology, Government of India (EEQ/2017/000737 to RR). Funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. OPEN ACCESS Citation: Rudresha GV, Urs AP, Manjuprasanna VN, Milan Gowda MD, Jayachandra K, Rajaiah R, et al. (2021) Echis carinatus snake venom metalloprotease-induced toxicities in mice: Therapeutic intervention by a repurposed drug, Tetraethyl thiuram disulfide (Disulfiram). PLoS Negl Trop Dis 15(2): e0008596. https://doi.org/ 10.1371/journal.pntd.0008596 Editor: Stuart Robert Ainsworth, Liverpool School of Tropical Medicine, UNITED KINGDOM Received: July 13, 2020 Accepted: January 3, 2021 Published: February 2, 2021 Editor: Stuart Robert Ainsworth, Liverpool School of Tropical Medicine, UNITED KINGDOM Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pntd.0008596 Copyright: © 2021 Rudresha 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. 1 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities AL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities Data Availability Statement: All relevant data are within the manuscript and its Supporting information files. Venom Lyophilized powder of Echis carinatus venom (ECV) was purchased from Irula Snake- Catchers Co-operative Society Ltd., (Chennai, India). The required amount of venom was re- dissolved in PBS and centrifuged at 9000 g for 10 min to remove debris. The protein content of venom was determined according to the method of Lowry et al. [28]. Ethics statement Adult Swiss albino mice (6 to 8-week-old female) weighing 20–25 g were obtained from the Central Animal House Facility, Department of Studies in Zoology, University of Mysore, Mysuru, India. The animal experiments were approved by the Institutional Animal Ethical Committee, University of Mysore, Mysuru, India (Approval number: UOM/IAEC/20/2016). During all experiments, animal care and handling were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Human blood was drawn from the antecubital veins of healthy adult volunteers who were provided with written informed consent. All the experiments were approved by the Institu- tional Human Ethical Committee, University of Mysore, Mysuru, India (Approval number: IHEC-UOM No. 120 Ph.D/2015-16), and conducted in accordance with the ethical guidelines. Introduction According to the World Health Organization, snakebite is a global public health problem and a neglected tropical disease [1,2]. Snakebites are often associated with severe local manifesta- tions including inflammation, hemorrhage, blistering, skin damage, coagulopathy and pro- gressive tissue necrosis at the bitten site [3,4]. These local manifestations are nevertheless pathological condition, caused by a mixture of toxins rather than a single toxin present in the venom [4]. Unfortunately, treatment of snake envenomation-induced progressive tissue necrosis persists even after anti-snake venom (ASV) administration [5,6]. Hence, treating pro- gressive tissue necrosis is still a challenging issue for the existing strategies of snakebite man- agement. In addition, studies on progressive tissue necrosis induced by viper bite have clearly revealed the direct involvement of metzincin family matrix-degrading snake venom metallo- proteases (SVMPs) [4,7,8] and hyaluronidases (SVHYs) [9,10]. E. carinatus (EC) also known as a saw-scaled viper is the only Echis species found in India [11]. Echis bite is responsible for 10–20% mortality and causes severe local manifestations, due to rich in zinc (Zn++) dependent SVMPs [12–14]. In addition, ECV activates neutrophils and releases their nuclear and granular contents to the extracellular space leading to neutrophil extracellular traps (NETs); this process is described as NETosis [15]. A previous study showed that NETs released from neutrophils entrap the venom toxins and results in blockage of blood vessels leading to tissue necrosis [15]. However, the venom toxin/s responsible for NETs for- mation and cellular mechanism involved is unclear. Several studies have demonstrated that SVMPs of ECV are known to contribute to the pathophysiology by causing degradation of extracellular matrix (ECM) proteins resulting in hemorrhage at the site of injection [4,8,16]. Multiple scientific reports have demonstrated the direct involvement of SVMPs in disrupting the tissue architecture by degrading ECM proteins [7,17,18]. Hemorrhagic SVMPs act at the basement membrane and disrupt the capillary wall that results in extravasation [7,17,19]. Introduction Further experimental evidence suggests that the onset of micro-vessel damage is mediated by the degradation of type IV collagen by the action of 2 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities PLOS NEGLECTED TROPICAL DISEASES PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities PICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxici Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities SVMPs [7,19]. SVMPs have resemblance in catalytic site architecture and structural domains with metzincin family proteases such as MMPs and ADAMs [20]. A few reports showed the activation of MAPKs by MMPs via protease-activated receptor (PAR)-1 [21]. Since SVMPs are catalytically related to MMPs, we hypothesized that EC SVMPs-induce NETosis and intracel- lular signaling cascade via PAR-1. Here, we have demonstrated that EC SVMPs-induced NETosis is mediated via PAR-1-ERK signaling axis, responsible for severe tissue necrosis. Previously, we have shown the neutralizing abilities of Zn++ specific chelators against the snake venom-induced progressive tissue damage [22]. Very recently, Albulescu et al. demon- strated the therapeutic intervention of repurposed drug, 2, 3-dimercapto-1-propanesulfonic acid for hemotoxic snakebite [23]. Chelating agents are essential in restoring the physiological levels of MMPs, as their dysregulated activity reflects in debilitating conditions such as cancer and arthritis [24]. Many pharmacologically approved chelating agents have been extensively studied for inhibition of SVMPs [25,26]. Those molecules are failed to reach the clinical trial, because of their non-specific chelation property [27]. Consequently, a high affinity membrane permeable specific Zn++ chelator, Antabuse drug, Tetraethyl thiuram disulfide (TTD)/disulfi- ram repurposed as therapeutic for ECV-induced toxicities in preclinical setup and compared with PLA2 and hyaluronidase inhibitors, aristolochic acid (AA) and silymarin (SLN), respectively. PLA2 activity ECV PLA2 activity was performed according to the method of Patriarca et al. with some modi- fications [29]. E. coli was labeled with 14C-oleate, autoclaved and used to measure PLA2 activ- ity. ECV (0–50 μg) was added into a total reaction volume of 350 μl containing 5 mM CaCl2, 100 mM Tris-HCl buffer (pH 7.4) and 14C-oleate labeled E. coli cells (3.18×109) (corresponds to 10,000 cpm or 60 nmol lipid phosphorus) and incubated at 37˚C for 60 min. The reaction was terminated by adding 100 μl of 2N HCl and 100 μl of fatty acid free BSA (100 mg/ml). The tubes were vortexed, centrifuged at 20,000 g for 10 min and aliquot (140 μl) of supernatant was mixed with scintillation cocktail. The enzyme activity was determined by quantifying the free 14C-oleate released using Packard scintillation analyzer and expressed as nmols of free fatty acid released/min/mg of protein at 37˚C. For inhibition studies, similar reactions were carried out after pre-incubating 50 μg ECV with various concentrations of AA, SLN and TTD for 5 min at 37˚C. Inhibition was expressed as a percentage. Chemicals and reagents Tetraethyl thiuram (TTD), aristolochic acid (AA), silymarin (SLN), phorbol 12-myristate 13-acetate (PMA), porcine skin gelatin, collagen-I/IV, laminin, fibronectin, Hoechst stain, DNase 1, ficoll-paque, dextran and phosphatase inhibitor cocktail were obtained from Sigma- Aldrich (Bangalore, India). BSA, ethanol, dimethyl sulfoxide (DMSO; HPLC grade), Tween-20 and Hank’s balanced salt solution (HBSS) were purchased from HiMediaLaboratories, Pvt. Ltd. (Mumbai, India). SCH79797 (PAR-1 antagonist) and GB-83 (PAR-2 antagonist) were purchased from Cayman Chemicals (Michigan, USA). U0126 (MEK 1/2 inhibitor), antibodies 3 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities PLOS NEGLECTED TROPICAL DISEASES PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities against p-ERK, β-actin and cell lysis buffer were purchased from Cell Signaling Technology (Massachusetts, USA). HRP tagged anti-rabbit IgG and anti-mouse IgG were procured from Jackson ImmunoResearch (Philadelphia, USA). The rabbit polyclonal anti-citH3, rabbit poly- clonal anti-H3, mouse monoclonal anti-myeloperoxidase (anti-MPO) and anti-PAD4 were obtained from Abcam (Cambridge, UK). All other chemicals and reagents used in this study are analytical grade. Hyaluronidase activity Hyaluronidase activity of ECV was assayed according to the method of Reissig et al. with some modifications [30]. The reaction mixture (350 μl in 0.1 M sodium acetate buffer pH 5.5 with 0.15 M NaCl) containing ECV (0–100 μg) incubated separately with HA (50 μg) at 37˚C for 2½ h. After incubation, the reaction mixture was heated in a water bath for 5 min to stop the reaction and cooled to room temperature. Sodium tetraborate (50 μl; 0.8 M; pH 9.2) buffer was added followed by heating in a boiling water bath for 3 min. After cooling to room tem- perature, 1.5 ml of coloring reagent p-DMAB (1% in 9,1 ratio of glacial acetic acid and HCl) was added and incubated for 20 min at 37˚C and centrifuged at 1500 g for 10 min to remove turbidity, the absorbance of clear supernatant was measured at 585 nm. Activity was expressed as μmol of NAG released/min/mg protein. For inhibition studies, hyaluronidase activity was determined after pre-incubating 100 μg ECV with various concentrations of AA, SLN and TTD for 5 min at 37˚C. Inhibition was expressed as a percentage. ECV-induced skin hemorrhage in mice Hemorrhagic activity was performed as described by Kondo et al. with suitable modifications [34]. Mice were injected (n = 3; i.d.) with 5 μg of ECV and control mice received saline. After 2½ h, mice were sacrificed using pentobarbitone (30 mg/kg; i.p.) and the inner dorsal surface of the skin was surgically removed and photographed, and the hemorrhagic spot was quanti- tated using a graph sheet. For neutralization studies, 5 μg of ECV was pre-incubated with vari- ous doses of TTD (5, 10 and 20 mM) for 5 min at 37˚C and administered to the mice skin (i. d.). For challenge studies, various doses of TTD (13.25, 26.5 and 53 μg), AA (20 and 40 μg) and SLN (15 and 30 μg) were injected 15 min post ECV injection. Neutralization of hemorrhagic activity was measured in terms of decreased area of the hemorrhagic spot in comparison to ECV-induced hemorrhagic area. Gelatinolytic activity The gelatinolytic activity was performed by substrate gel assay as described by Heussen and Dowdle, with some modifications [32]. ECV, 5 μg was loaded onto a 10% SDS polyacrylamide gel (SDS-PAGE) impregnated with 0.08% of gelatinand electrophoresis was carried out under non-reducing condition at a 100 V for 2 h. After electrophoresis, SDS was removed by incubat- ing gel with 2.5% Triton X-100 for 1 h, followed by an extensive wash with distilled water. The gel was incubated overnight at 37˚C in incubation buffer, 50 mM Tris-HCl, pH 7.6 containing 0.9% NaCl, 10 mM CaCl2, 10 mM ZnCl2. The gel was stained with Coomassie brilliant blue- G250 (CBB-G250) and a clear zone indicates the gelatinolytic activity of ECV. For inhibition studies ECV was pre-incubated with different concentrations of TTD (1, 5, 10 and 20 mM), AA (10 and 20 mM) and SLN (10 and 20 mM) for 5 min at 37˚C and assay was performed as described above. ECM protein hydrolyzing activity ECM protein hydrolyzing activity was performed according to the method of Baramova et al. with slight modifications [33]. ECM proteins, collagen type-I/IV, laminin and fibronectin (50 μg each) were incubated with 5 μg of ECV, separately in a total reaction volume of 40 μl with Tris-HCl buffer (10 mM; pH 7.6) at 37˚C for 3 h. The reaction was terminated by adding 20 μl of reducing sample buffer (4% SDS, 6% β-mercaptoethanol and 1 M urea) and boiled for 5 min. An aliquot of 40 μl of this sample was loaded onto 7.5% SDS-PAGE and electrophoresis was carried out at 100 V for 2 h. After electrophoresis the cleavage pattern of ECM proteins was visualized by staining with CBB-G250. For inhibition studies, similar experiments were carried out by pre-incubating ECV with different concentrations of TTD (1, 5, 10 and 20 mM), AA (10 and 20 mM) and SLN (10 and 20 mM) for 5 min at 37˚C and electrophoresed as described above. Caseinolytic activity Proteolytic activity of ECV was assayed according to the method of Murata et al. with suitable modifications [31]. Fat free casein 0.4 ml (2%; 0.2 M Tris-HCl buffer; pH 8.5) was incubated with ECV (0–25 μg) and final volume make up to 1 ml with 0.2 M Tris-HCl (pH 8.5), incu- bated at 37˚C for 2½ h. The reaction was stopped by adding 1.5 ml of 0.44 M TCA and allowed to stand for 30 min. The mixture was centrifuged at 1,500 g for 15 min and 1.0 ml supernatant was mixed with 2.5 ml of 0.4 M sodium carbonate and 0.5 ml of 1:2 diluted Folin-Ciocalteu reagents. The colour developed was read at 660 nm. One unit of enzyme activity was defined as the amount of enzyme required to increase an absorbance of 0.01 at 660 nm/h at 37˚C. For inhibition studies, similar reactions were performed after pre-incubating 25 μg of venom with various concentrations of AA, SLN and TTD for 5 min at 37˚C. The proteolytic activity of ECV in the absence of inhibitors was considered as 100%. Inhibition was expressed as a percentage. 4 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities AL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities Isolation of neutrophils from human blood Human neutrophils were isolated from healthy volunteers blood, according to the method of Halverson et al. with slight modifications [39]. The blood was collected and mixed with acid citrate dextrose in a 5:1 volumetric ratio, followed by dextran sedimentation and hypotonic lysis to remove red blood cells. The cell pellet was suspended in 2 ml of PBS and subjected to density gradient centrifugation at 210 g using ficoll-paque for 30 min at 4˚C. The neutrophils settled at the bottom as a cell pellet were washed twice with PBS and centrifuged at 210 g and re-suspended in HBSS. The cells were counted using the Neubauer hemocytometer and the required cell density was adjusted using HBSS. Histopathological studies Mice were sacrificed using pentobarbitone (30 mg/kg; i.p.) and footpad tissues were excised and fixed for 24 h in Bouin’s fixative (picric acid: formaldehyde: glacial acetic acid, 30:10:2 v/ v), dehydrated with increasing concentrations of ethanol and embedded in paraffin. The tis- sues were sectioned into 4 μm thick using a microtome (R. Jung AG, Germany). Tissue sec- tions were processed and stained with hematoxylin and eosin as described by Svensson et al. with suitable modifications [36] and photographed using Axio Imager A2 microscope with LED—Zeiss (Oberkochen, Germany). ECV-induced mortality and systemic hemorrhage in mice Lethal toxicity of ECV was determined according to the method of Meier and Theakston, with some modifications [37]. To determine the anti-venom potential of TTD in preventive regi- men we followed our previous lethality study [38]. Briefly, ECV (1½ LD50; 3.31 mg/kg; n = 5) was pre-incubated with TTD (2.15 mg/kg) or effective dose ASV (ED ASV), separately for 5 min at 37˚C and injected intra peritoneally (i.p.) to mice. For challenge studies, TTD (2.15 mg/ kg) or ED ASV (mg anti-venom per mg venom) was injected 30 min post of venom adminis- tration (i.p.). The signs of toxicities were observed up to 24 h and survival time was recorded. For systemic hemorrhage and bleeding studies, mice received ECV (LD50; 2.21 mg/kg; n = 5; i. p.) and challenged with TTD (2.15 mg/kg; i.p.) or ED ASV, 30 min post ECV injection. After 3 h mice were sacrificed using pentobarbitone (30 mg/kg; i.p.) and peritoneal cavity was observed for symptoms and photographed. ECV-induced mice footpad tissue necrosis ECV-induced mice footpad tissue necrosis was performed as described by Rudresha et al. with suitable modifications [35]. Mice were anesthetized using pentobarbitone (30 mg/kg; i.p.) and ECV (LD50; 2.21 mg/kg body weight) was administered to the mice footpad (n = 5; Intraplan- tar injection). The time for onset of footpad injuries was recorded for each mouse. The severity of the injury was visually judged and scored based on a 5-point scale; 0-no injury, 1-edema with mild hemorrhage, 2-edema with severe hemorrhage causing discoloration of the footpad, 3-edema with severe hemorrhage and necrosis, 4-severe hemorrhage and necrotized footpad, 5-necrotized little toe detached from limb. The footpad injury observations were recorded 5 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities PLOS NEGLECTED TROPICAL DISEASES PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities PICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxici Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities every day for 8 days after venom injection. To evaluate the protection of ECV-induced tissue necrosis, TTD (2.15 mg/kg) was pre-incubated with ECV (2.21 mg/kg) for 5 min at 37˚C and administered to the mice footpad. For challenge studies, TTD (2.15 mg/kg) was administered to the venom injection site 30 min post venom injection. Previously, Katkar et al. showed that DNase 1 administration accelerates the healing of ECV-induced chronic wounds by degrading the deposited NETs. Hence, DNase 1 (100 U) was used as a positive control [15]. To assess the effect of SCH79797 (PAR-1 antagonist) on ECV-induced footpad tissue necrosis, SCH79797 (1.5 mg/kg) was administered to the mice footpad 15 min before to the ECV (½LD50; 1.10 mg/kg; n = 3) injection. The onset of footpad injuries was recorded as described above. Analysis of NET formation and its markers in human neutrophils NETs formation was analyzed using Hoechst stain according to the method of Katkar et al. with suitable modifications [15]. Human neutrophils (2×105/ml) were seeded on 13 mm round coverslips placed in 12 well plates in 500 μl of HBSS-1 and allowed cells to adhere to the 6 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities PICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxici Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities coverslips for 30 min at 37˚C in the presence of 5% CO2. Then, the cells were stimulated with different concentrations of ECV (5, 10, 25 and 50 μg/ml) for 2½ h. After incubation, cells were fixed with 4% paraformaldehyde for 30 min and stained with Hoechst stain (1:10,000) for 15 min. Cells were analyzed for NETs formation and images were acquired on a BA410 fluores- cence microscope (Motic) attached to a DS-Qi2 monochrome CMOS sensor camera. The NETs percentage was calculated in 5 non-overlapping fields per coverslip. For inhibition stud- ies, similar experiments were carried out by pre-incubating ECV (25 μg) with different con- centrations of TTD (1, 5, 10 and 20 mM), AA (10 and 20 mM) and SLN (10 and 20 mM) for 5 min at 37˚C and NETs percentage was calculated as described above. For the analysis of ECV-induced citH3, PAD4 and MPO expression, neutrophils were treated with ECV (25 μg) that was pre-treated with various concentrations of TTD (1, 5, 10 and 20 mM), AA (10 and 20 mM) and SLN (10 and 20 mM) at 37˚C for 5 min. After 2½ h, neutrophils were washed and lysed with cell lysis buffer containing PMSF and phosphatase inhibitor cocktail and stored at -20˚C overnight. Lysates were centrifuged at 9000 g for 10 min and tested for the expression of protein using Western blotting. To test the effect of pharmacological inhibitors on ECV-induced NETs and its markers, neutrophils were pre-sensitized without or with U0126 (MEK 1/2 inhibitor), SCH79797 (PAR-1antagonist) and GB-83 (PAR-2 antagonist) for 15 min. Then, neutrophils were stimu- lated with 25 μg of ECV for 2½ h at 37˚C, and NETs were quantitated using Hoechst stain. Cell lysates were used to test the expression of citH3, PAD4, MPO and ERK activation using Western blotting. Western blotting Mice were sacrificed using anesthesia pentobarbitone (30 mg/kg; i.p.) and ECV injected mice footpads were excised from respective days 1 to 8 and stored at -20˚C. The excised tissue sam- ples were sonicated for 30 seconds on ice bath (five passes of 10 seconds) with a 3.0 mm probe sonicator in cold cell lysis buffer containing (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glyceropho- sphate, 1 mM Na3VO4, 1 μg/ml leupeptin) (Cell Signaling Technology) and supplemented with PMSF and phosphatase inhibitor cocktail (Sigma Aldrich). Tissue homogenates were cen- trifuged at 9000 g for 10 min and the supernatant was used for protein quantification. Western blotting was performed as described by Rajaiah et al. with suitable modifications [40]. Briefly, an equal amount of proteins (25 μg) with Laemmli buffer was loaded on to SDS-PAGE and electrophoresis were carried out at 100 V for 2 h. After electrophoresis proteins were electro- blotted onto polyvinylidene difluoride (PVDF) membranes at 4˚C for 90 min. After blocking for 2 h in TBST (20 mM Tris, pH 7.5 with 150 mM NaCl and 0.05% Tween-20) containing 5% BSA, membranes were probed against anti-citH3 (1:1000) or anti-PAD4 (1:2000) or anti-MPO (1:1000) or anti-p-ERK (1:1000) antibodies overnight at 4˚C according to the manufacturer’s instructions (Abcam, UK and Massachusetts, USA). Membranes were washed extensively (4x) using TBST and incubated with HRP-conjugated anti-rabbit/mouse IgG (1:10,000) for 1 h at room temperature. The blots were developed with an enhanced chemiluminescence substrate for visualization (Alliance 2.7, Uvitec) and bands were quantitated using Image J software. H3 and β-actin were used as the loading control. TTD inhibits ECV-induced proteolytic activity, ECM protein degradation and hemorrhage We have tested the inhibitory efficacy of an Antabuse drug, TTD with chelating property on ECV-induced proteolytic activity, ECM protein degradation and hemorrhage in mice and compared with pharmacological inhibitors of PLA2 (AA) and hyaluronidase (SLN). To begin with, the effect of TTD on ECV-induced proteolytic activity was demonstrated by casein and gelatin as substrate. TTD inhibited ECV-induced caseinolytic and gelatinolytic activity in a concentration-dependent manner and the IC50 concentration of caseinolytic activity was found to be 3 mM (Fig 1A and 1B and S1C and S3A Figs). In addition, TTD also inhibited ECV-induced degradation of the ECM proteins, collagen, laminin and fibronectin in a Fig 1. Effect of TTD on ECV-induced ECM protein degradation in vitro and hemorrhage in mice. ECV was pre-incubated without or with various concentrations of TTD at 37˚C for 5 min and subjected for gelatinolytic, collagenolytic, laminin and fibronectin hydrolyzing activity. The gelatinolytic activity was performed by gelatin zymogram using 10% gel impregnated with 0.08% gelatin. Clear zones in the gel indicate the hydrolysis of gelatin by ECV (A). The area of gelatinolytic activity was quantitated using a graph sheet represented as area (mm2) (B). The data represented as mean ± SEM. p < 0.05, when compared ECV versus ECV + TTD. For collagen, laminin and fibronectin degradation analysis, the pre-incubated reaction mixture of ECV and TTD was incubated with 50 μg of collagen (Col) type I (C), type IV (D), laminin (Lam) (E) and fibronectin (Fib) (F) for 3 h at 37˚C. The hydrolyzing pattern was analyzed using 7.5% SDS-PAGE and visualized by staining with CBB-G250. Data are representative of two independent experiments. For skin hemorrhage, mice were injected (n = 3; i.d.) with 5 μg of ECV followed by the injection of different concentrations of TTD post 30 min at the site of ECV injection. After 180 min, dorsal patches of mice skin were photographed (G) and the hemorrhagic area was measured using graph sheets represented as area (mm2) (H). Data are representative of two independent experiments. Fig 1. Effect of TTD on ECV-induced ECM protein degradation in vitro and hemorrhage in mice. ECV was pre-incubated without or with various concentrations of TTD at 37˚C for 5 min and subjected for gelatinolytic, collagenolytic, laminin and fibronectin hydrolyzing activity. The gelatinolytic activity was performed by gelatin zymogram using 10% gel impregnated with 0.08% gelatin. Statistics Data represented as the mean ± SEM. One-way ANOVA followed by Bonferroni post hoc test was used to analyze more than two groups using Graph Pad Prism version 5.03 (La Jolla, USA). The comparison between the groups was considered significant if p< 0.05. 7 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxici PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities TTD inhibits ECV-induced proteolytic activity, ECM protein degradation and hemorrhage Clear zones in the gel indicate the hydrolysis of gelatin by ECV (A). The area of gelatinolytic activity was quantitated using a graph sheet represented as area (mm2) (B). The data represented as mean ± SEM. p < 0.05, when compared ECV versus ECV + TTD. For collagen, laminin and fibronectin degradation analysis, the pre-incubated reaction mixture of ECV and TTD was incubated with 50 μg of collagen (Col) type I (C), type IV (D), laminin (Lam) (E) and fibronectin (Fib) (F) for 3 h at 37˚C. The hydrolyzing pattern was analyzed using 7.5% SDS-PAGE and visualized by staining with CBB-G250. Data are representative of two independent experiments. For skin hemorrhage, mice were injected (n = 3; i.d.) with 5 μg of ECV followed by the injection of different concentrations of TTD post 30 min at the site of ECV injection. After 180 min, dorsal patches of mice skin were photographed (G) and the hemorrhagic area was measured using graph sheets represented as area (mm2) (H). Data are representative of two independent experiments. https://doi.org/10.1371/journal.pntd.0008596.g001 8 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities PICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxici Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities concentration-dependent manner (Fig 1C–1F). Furthermore, TTD was tested for its action on ECV-induced hemorrhage in mice skin in both pre-incubation and challenging studies. TTD efficiently neutralized ECV-induced hemorrhagic activity in pre-incubation and 30 min post ECV injection (Fig 1G and 1H and S3B and S3C Fig). PLA2 and hyaluronidase inhibitors, AA and SLN inhibited ECV-induced PLA2 and hyaluronidase activities, respectively (S1A and S1B Fig). On the other hand, both AA and SLN failed to inhibit ECV-induced ECM protein degra- dation (S2A–S2D Fig) and hemorrhagic activity in mice (S2E and S2F Fig). TTD protects ECV-induced mice footpad tissue necrosis with decreased expression of citrullinated H3 (citH3)/myeloperoxidase (MPO) and histopathological changes With promising results of in vitro inhibition of ECV-induced ECM proteins degradation and murine skin hemorrhage, TTD was tested for the neutralization of ECV-induced tissue necro- sis using mice footpad model. ECV injection to mice footpad resulted in progressive tissue necrosis that leads to the detachment of little toe from limb between 6–8 days. TTD adminis- tration neutralizes ECV-induced tissue necrosis and prevented the loss of little toe and, was able to restore the normal footpad morphology both in pre-incubation and challenging studies (Fig 2A and 2B and S3D and S3E Fig). Moreover, recently Katkar et al. reported that infiltrated neutrophils to the site of venom injection release chromatin content to the extracellular space as NETs that is responsible for local tissue necrosis [15]. Furthermore, Katkar et al. and Rudre- sha et al. demonstrated that the intervention of DNase 1 and plant DNase at a right time pro- tected ECV-induced tissue necrosis [15,41]. In addition, the excessive production of MPO and citH3 by the action of PAD4 has shown to be crucial for ECV-induced local tissue damage [15]. Similar to the previous study, ECV induced the expression of MPO and citH3, and it was efficiently inhibited by TTD (Fig 2C–2E). The inhibitory action of TTD on ECV-induced mice footpad necrosis and the expression of MPO and citH3 are more efficient and comparable with DNase 1 (Fig 2). In addition, the protective efficacy of TTD on ECV-induced footpad tis- sue necrosis was confirmed by histopathological studies using hematoxylin and eosin staining. Mice that received ECV alone showed extensive tissue damage. TTD treatment protects the ECV-induced histopathological changes (S4 Fig). TTD protects mice from ECV-induced lethality and neutralizes systemic hemorrhage In addition to the induction of progressive tissue necrosis, ECV is lethal when injected at 3.31 mg/kg body weight (1½LD50), and the average survival time is approximately 8 ± 2 h. Since TTD efficiently neutralized ECV-induced tissue necrosis and hemorrhage, its effect on ECV- induced mortality in mice was tested. TTD neutralized ECV-induced lethality and protected mice in both pre-incubation (100% survival—two independent experiments with 5 animals in each group) and challenge then treat (30 min post venom injection) (four of five animals sur- vived—two independent experiments with 5 animals in each group) (Fig 3A and 3B). The pro- tective effect of TTD was comparable to ED ASV (mg anti-venom per mg venom) both in pre- incubation and therapeutic regimens (Fig 3A and 3B). ECV is well-known for hemotoxic effect and its envenomation makes blood in-coagulable that leads to the systemic bleeding with dis- seminated intravascular coagulation [42]. In fact, ECV injection to mouse peritoneum caused severe bleeding and extravasation throughout the peritoneum (Fig 3C). As TTD protected mice from ECV-induced lethality, it neutralized ECV-induced bleeding in peritoneum even after 30 min post ECV injection and it was comparable with ED ASV as shown in Fig 3C. This indicates that TTD is a potential drug candidate that complements ASV during EC bite. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 9 / 24 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities Fig 2. Neutralization of ECV-induced mice footpad tissue necrosis by TTD. Mice footpads were injected with ECV (LD50; 2.21 mg/kg; n = 5). After 30 min, mice received either TTD or DNase 1 at the site of venom injection and footpads were photographed from day 1 to day 8 (A). Red arrow indicates edema and black arrow indicates tissue necrosis. ECV-induced footpad injury was measured manually on a scale of 1 to 5 (B). The level of ECV-induced citH3 and MPO in mouse footpad tissue in the absence or presence of either TTD or DNase 1 was analyzed by Western blotting (C) and quantitated using H3 and β-actin as a loading control for citH3 (D) and MPO (E), respectively. The data represented as mean ± SEM. p < 0.05, when compared ECV versus ECV + TTD and ECV versus ECV + DNase 1. TTD protects mice from ECV-induced lethality and neutralizes systemic hemorrhage htt //d i /10 1371/j l td 0008596 002 Fig 2. Neutralization of ECV-induced mice footpad tissue necrosis by TTD. Mice footpads were injected with ECV (LD50; 2.21 mg/kg; n = 5). After 30 min, mice received either TTD or DNase 1 at the site of venom injection and footpads were photographed from day 1 to day 8 (A). Red arrow indicates edema and black arrow indicates tissue necrosis. ECV-induced footpad injury was measured manually on a scale of 1 to 5 (B). The level of ECV-induced citH3 and MPO in mouse footpad tissue in the absence or presence of either TTD or DNase 1 was analyzed by Western blotting (C) and quantitated using H3 and β-actin as a loading control for citH3 (D) and MPO (E), respectively. The data represented as mean ± SEM. p < 0.05, when compared ECV versus ECV + TTD and ECV versus ECV + DNase 1. TTD inhibits ECV-induced NETs formation and activation of intracellular signaling in human neutrophils The time taken for mice mortality was recorded for 24 h and graph plotted as percent survival against the time of death (A). In the treatment model, mice received either TTD (2.15 mg/kg) or ED ASV, 30 min post ECV injection (i.p.) and the survival time was recorded for 24 h (B). For the neutralization of systemic hemorrhage, mice received (n = 5; i.p.) various concentrations of either TTD or ED ASV, 30 min post ECV (LD50; 2.21 mg/kg; i.p.) injection. Mice were sacrificed after 2 h and peritonea were photographed (C). Red arrow indicates the hemorrhage in the peritoneum cavity and black arrow indicated reduced hemorrhage in the peritoneum. Data are representative of two independent experiments. https://doi.org/10.1371/journal.pntd.0008596.g003 ECV-induced NETosis (S6A and S6B Fig). Moreover, ECV treated neutrophils showed increased expression of PAD4, citH3, and MPO and activation of ERK (Fig 4B). The impor- tance of PAD4 in DNA de-condensation by citH3 and DNA expulsion in both mouse and human neutrophils is well documented [47]. Furthermore, TTD significantly reduced ECV- induced NETosis and decreased the expression of PAD4, citH3 and MPO as well as activation of ERK in neutrophils (Fig 4A and 4B). TTD is a chelating agent that is known to inhibit SVMPs; therefore, these data clearly suggest that SVMPs are directly involved in the activation of ERK and NETs formation. TTD inhibits ECV-induced NETs formation and activation of intracellular signaling in human neutrophils Neutrophils are the first line innate immune cells recruited to sites of acute inflammation in response to chemotactic signals produced by injured tissue and tissue-resident macrophages [43,44]. During infection, neutrophils undergo degranulation and ultimately release chroma- tin as NETs that contribute to the killing of extracellular pathogens [45]. Previously, Setubal et al. demonstrated Bothrops bilineatus venom in the activation of neutrophils and the release of NETs [46]. Recently, Katkar et al. reported the discharged chromatin (NETs) upon ECV treatment is responsible for ECV-induced local tissue necrosis [15]. Similar to the previous reports, we observed ECV-induced chromatin discharge from human neutrophils in a concen- tration-dependent manner and it was effectively inhibited by TTD (Fig 4A and S5A Fig). On the other hand, the PLA2 and hyaluronidase inhibitors, AA and SLN are failed to inhibit the PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 10 / 24 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities Fig 3. Protection of mice against ECV-induced mortality and systemic hemorrhage by TTD. A lethal dose of ECV (1½LD50; 3.31 mg/kg) was pre- treated with TTD (2.15 mg/kg) or an effective dose of anti-snake venom (ED ASV) for 5 min at 37˚C and injected (n = 5; i.p.) to mice. The time taken for mice mortality was recorded for 24 h and graph plotted as percent survival against the time of death (A). In the treatment model, mice received either TTD (2.15 mg/kg) or ED ASV, 30 min post ECV injection (i.p.) and the survival time was recorded for 24 h (B). For the neutralization of systemic hemorrhage, mice received (n = 5; i.p.) various concentrations of either TTD or ED ASV, 30 min post ECV (LD50; 2.21 mg/kg; i.p.) injection. Mice were sacrificed after 2 h and peritonea were photographed (C). Red arrow indicates the hemorrhage in the peritoneum cavity and black arrow indicated reduced hemorrhage in the peritoneum. Data are representative of two independent experiments. https://doi.org/10.1371/journal.pntd.0008596.g003 Fig 3. Protection of mice against ECV-induced mortality and systemic hemorrhage by TTD. A lethal dose of ECV (1½LD50; 3.31 mg/kg) was pre- treated with TTD (2.15 mg/kg) or an effective dose of anti-snake venom (ED ASV) for 5 min at 37˚C and injected (n = 5; i.p.) to mice. ECV-induced NETs formation and tissue necrosis via PAR-1-ERK mediated axis It is well known that MMPs cleave PAR-1 at non-canonical sites, results in the activation of intracellular signaling cascade via MAPKs that leads to a diverse array of physiological func- tions [21,48]. Since MMPs and SVMPs are having structural homology in their catalytic site, we speculated that EC SVMPs activates ERK and NETs formation through PAR-1. To confirm whether ECV induces NETs formation via the PARs, we used PAR-1 and PAR-2 specific antagonists, SCH79797 and GB-83, respectively. SCH79797 is a selective antagonist of PAR-1 and it does not have any role in the inhibition of venom-induced toxicities by directly acting on ECV unlike TTD. SVMPs present in ECV instantaneously activate PAR-1 in the absence of 11 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities Fig 4. Inhibition of ECV-induced NETs formation by TTD. Human neutrophils were stimulated with ECV (25 μg) pre-incubated (5 min) without or with different concentrations of TTD for 180 min and NETs formation was observed and quantitated (A). ECV-induced citH3, PAD4 and MPO in neutrophil cell lysates were analyzed using Western blotting (B). Bands were quantitated using H3 as loading control for citH3 and β-actin as a loading control for MPO and PAD4 (C). The data represented as mean ± SEM. p < 0.05, when compared ECV versus ECV + TTD. https://doi.org/10.1371/journal.pntd.0008596.g004 Fig 4. Inhibition of ECV-induced NETs formation by TTD. Human neutrophils were stimulated with ECV (25 μg) pre-incubated (5 min) without or with different concentrations of TTD for 180 min and NETs formation was observed and quantitated (A). ECV-induced citH3, PAD4 and MPO in neutrophil cell lysates were analyzed using Western blotting (B). Bands were quantitated using H3 as loading control for citH3 and β-actin as a loading control for MPO and PAD4 (C). The data represented as mean ± SEM. p < 0.05, when compared ECV versus ECV + TTD. https://doi.org/10.1371/journal.pntd.0008596.g004 Fig 4 Inhibition of ECV induced NETs formation b TTD H man ne trophils ere stim lated ith ECV (25 μg) pre inc bated (5 min) itho t or Fig 4. Inhibition of ECV-induced NETs formation by TTD. ECV-induced NETs formation and tissue necrosis via PAR-1-ERK mediated axis Human neutrophils were stimulated with ECV (25 μg) pre-incubated (5 min) without or with different concentrations of TTD for 180 min and NETs formation was observed and quantitated (A). ECV-induced citH3, PAD4 and MPO in neutrophil cell lysates were analyzed using Western blotting (B). Bands were quantitated using H3 as loading control for citH3 and β-actin as a loading control for MPO and PAD4 (C). The data represented as mean ± SEM. p < 0.05, when compared ECV versus ECV + TTD. https://doi.org/10.1371/journal.pntd.0008596.g004 TTD and it is hard to control SVMPs activated PAR-1 signaling once it is activated. Hence, we have reduced the venom dose and injected SCH79797 before ECV injection. With this, we made an effort to establish the mechanism of SVMPs in the activation of PAR-1 that may have direct/indirect role in ECV-induced toxicities. In fact, ECV activated NETs formation was inhibited in the presence of SCH79797 and not by GB-83, suggesting that ECV-induced NETosis is mediated via PAR-1 in human neutrophils (Fig 5A and 5B). Further, ECV-induced expression of PAD4, CitH3 and activation of ERK was inhibited by SCH79797 (Fig 5C). On the other hand, MEK inhibitor, U0126 showed a partial effect on ECV-induced NETs formation and the expression of PAD4 and citH3 (Fig 5C). In support of in vitro results in human neutrophils, PAR-1 antagonist neutralized ECV-induced mice foot- pad tissue necrosis (Fig 6A and 6B). Overall, these data confirmed the involvement of EC SVMPs-induced tissue necrosis by inducing NETosis and activation of intracellular signaling via PAR-1 (Fig 7). 12 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities Fig 5. Effect of selective antagonists of ERK and PARs on ECV-induced NETosis and tissue necrosis. Neutrophils were pre-sensitized with selective antagonists of ERK (U0126), PAR-1 (SCH79797) and PAR-2 (GB-83) for 15 min, separately. Pre-sensitized neutrophils were stimulated with 25 μg of ECV for 180 min and cells were stained with Hoechst stain. Neutrophils were photographed under a microscope (A) and, NETs were quantitated and represented as percent NETosis (B). The data represented as mean ± SEM. p < 0.05, when compared ECV versus ECV + antagonists. ECV-induced NETs formation and tissue necrosis via PAR-1-ERK mediated axis The whole cell lysates were analyzed for the phosphorylated ERK, expression of PAD4 and citH3 using Western blotting (C). The p-ERK and PAD4 bands were quantitated using β-actin as a loading control. The citH3 bands were quantitated using H3 as a loading control. Data are representative of two independent experiments. htt //d i /10 1371/j l td 0008596 005 Fig 5. Effect of selective antagonists of ERK and PARs on ECV-induced NETosis and tissue necrosis. Neutrophils were pre-sensitized with selective antagonists of ERK (U0126), PAR-1 (SCH79797) and PAR-2 (GB-83) for 15 min, separately. Pre-sensitized neutrophils were stimulated with 25 μg of ECV for 180 min and cells were stained with Hoechst stain. Neutrophils were photographed under a microscope (A) and, NETs were quantitated and represented as percent NETosis (B). The data represented as mean ± SEM. p < 0.05, when compared ECV versus ECV + antagonists. The whole cell lysates were analyzed for the phosphorylated ERK, expression of PAD4 and citH3 using Western blotting (C). The p-ERK and PAD4 bands were quantitated using β-actin as a loading control. The citH3 bands were quantitated using H3 as a loading control. Data are representative of two independent experiments. https://doi.org/10.1371/journal.pntd.0008596.g005 Discussion Viper bites can induce progressive tissue necrosis that can result in permanent disability in the affected limb or digit [49]. Case reports on snakebite victims suggested that envenomation by hemotoxic venoms including Echis carinatus (EC) induces hematological complications, local pain, bleeding and edema at the bite site. Untreated Echis envenomation may involve multiple organs and the patient may suffer from, hematuria, renal failure, hemorrhage, anemia, hypo- tension and disseminated intravascular coagulation with capillary leak syndrome [50,51]. The LD50 of EC envenomation is 6.65 mg/kg and an average bite may yield about 40 mg of venom [52–54]. Generally, envenomation by EC is associated with a mortality rate of 10–20% if there PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 13 / 24 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n ECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake ve PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities Fig 6. Effect of the selective antagonist of PAR-1 on ECV-induced tissue necrosis. Mice footpads (n = 5) were pre-treated without or with PAR-1 antagonist (SCH79797) for 15 min and followed by the injection of ECV (½LD50; 1.10 mg/kg). Mice footpads were photographed from day 1 to day 8 (A) and tissue injury was measured manually on a scale of 1 to 5 (B). Red arrow indicates edema and black arrow indicates tissue necrosis. Data are representative of two independent experiments. https://doi.org/10.1371/journal.pntd.0008596.g006 Fig 6. Effect of the selective antagonist of PAR-1 on ECV-induced tissue necrosis. Mice footpads (n = 5) were pre-treated without or with PAR-1 antagonist (SCH79797) for 15 min and followed by the injection of ECV (½LD50; 1.10 mg/kg). Mice footpads were photographed from day 1 to day 8 (A) and tissue injury was measured manually on a scale of 1 to 5 (B). Red arrow indicates edema and black arrow indicates tissue necrosis. Data are representative of two independent experiments. Fig 6. Effect of the selective antagonist of PAR-1 on ECV-induced tissue necrosis. Mice footpads (n = 5) were pre-treated without or with PAR-1 antagonist (SCH79797) for 15 min and followed by the injection of ECV (½LD50; 1.10 mg/kg). Mice footpads were photographed from day 1 to day 8 (A) and tissue injury was measured manually on a scale of 1 to 5 (B). Red arrow indicates edema and black arrow indicates tissue necrosis. Discussion Data are representative of two independent experiments. https://doi.org/10.1371/journal.pntd.0008596.g006 is no initiation of effective treatment early. The major cause of mortality is due to increased bleeding after envenomation including venom-induced consumption coagulopathy or dissem- inated intravascular coagulation due to the prothrombin/thrombin-like enzymes present in the snake venom [14]. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 14 / 24 PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities Fig 7. Schematic representation of TTD and pharmacological inhibitors, site of action on ECV-induced toxicities. ECV-induced PAR-1-mediated ERK activation might be responsible for increased expression of PAD4, histone citrullination and MPO expressions that are accountable for severe tissue necrosis. TTD and pharmacological inhibitors interfere in ECV-induced signaling/tissue necrosis by inhibiting NETosis and chromatin release. https://doi.org/10.1371/journal.pntd.0008596.g007 EGLECTED TROPICAL DISEASES Re purposed drug, tetraethylthiuram disulfide neutralizes snake venom induced toxicities Fig 7. Schematic representation of TTD and pharmacological inhibitors, site of action on ECV-induced toxicities. ECV-induced PAR-1-mediated ERK activation might be responsible for increased expression of PAD4, histone citrullination and MPO expressions that are accountable for severe tissue necrosis. TTD and pharmacological inhibitors interfere in ECV-induced signaling/tissue necrosis by inhibiting NETosis and chromatin release. htt //d i /10 1371/j l td 0008596 007 https://doi.org/10.1371/journal.pntd.0008596.g007 https://doi.org/10.1371/journal.pntd.0008596.g007 SVMPs are one of the major toxins in most of the viper venoms including ECV and they primarily act on ECM components and are responsible for hemorrhagic activity [7,8,18,19,22]. The progressive tissue necrosis induced by viper bites mainly attributed to SVMPs, particularly P-III class metalloproteases [8]. In addition, SVMPs are hemotoxic in nature and interfere 15 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities ICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxici Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities with the hemostatic system in snakebite victims [55]. SVMPs are closely related to a disintegrin and metalloproteinases (ADAMs), thus they are also referred to as snake venom ADAMs [56,57]. SVMPs contains disintegrin-like (D), cysteine-rich (C), metalloproteinase and (M) domain, that harbors putative Zn++ binding sequence and bearing the typical structural fea- tures of the metzincin family of MMPs [20,57–59]. Discussion Alike MMPs, Zn++ on the M-domain of SVMPs plays a crucial role in the catalytic functions [20,57]. Hence, chelation of Zn++ metal ion by specific Zn++chelators rather than non-specific chelator is more effective in the manage- ment of local tissue necrosis induced by viper venoms [22]. Since SVMPs are directly responsi- ble for ECV-induced toxicities, the inhibition of SVMPs by TTD would be beneficial to manage ECV-induced toxicities. TTD was the first drug to treat chronic alcoholism and was approved by the FDA 1951 [60]. Since then, many studies have shown repurposing of TTD to treat diverse types of human malignant tumors including breast cancer, glioblastoma and pancreatic carcinoma [61,62]. TTD has also shown therapeutic potential in treating AIDS and it is found to be beneficial in treating Lyme disease in patients [63,64]. Very recently, the intervention of TTD in normalizing body weight in obese mice has been reported [65]. Besides, TTD has been shown to inhibit MMP-2 and MMP-9 activity by directly interacting with them via a Zn++ chelating mechanism [66]. Several scientific reports suggested that, many small inhibitors or chelators of SVMPs such as batimastat, marimastat, N,N,N0,N0-tetrakis (2-pyridylmethyl) ethane-1,2-diamine and dimer- caprol which targets the different classes of SVMPs-induced toxicities [67–71]. Some of these inhibitors were less effective as therapeutic regimen where the time lapse between venom and administration of inhibitors is prolonged [72]. Moreover, many small molecules and chelators were focused on interference in Viperinae snake venom-induced coagulopathy and local toxici- ties [69]. However, our findings highlight the efficacy of TTD in ECV-induced both local and systemic toxicities and, would be better repurposing to complement snakebite management. It has been demonstrated that 80–95% of an oral dose of TTD is absorbed from the gastrointesti- nal tract and it requires 1 to 2 h to peak serum concentration. TTD is rapidly distributed in adi- pose tissue, liver, spleen, adrenal gland and the brain. Prolonged administration of TTD is not known to induce tolerance and it is metabolized to diethyldithiocarbamate and mixed disulfides that are excreted via urine. The unabsorbed content of TTD is excreted in the feces [73,74]. TTD is known to cause hepatitis in 1 in 30,000 patients, which is sometimes fatal. There are rare reports of psychosis and confusional states and peripheral neuropathy and optic neuritis and, these effects were dose dependent. Moreover, TTD interacts with compounds that utilize the cytochrome P450 enzyme system [74,75]. Discussion Although the maximum recommended daily dose of TTD is 500 mg orally as an Antabuse [76], the long-term side effects of its use and dos- age requirements are still unknown that requires extensive in vivo research before they can be fully supported as a complementary therapy for snakebite management. Recently, Albulescu et al. showed that 2, 3-dimercapto-1-propanesulfonic acid, a derivative of dimercaprol effectively antagonizes the activity of Zn++ dependent SVMPs in vitro and neu- tralized ECV in mice [23]. Previously, we have reported the inhibitory potential of Zn++ specific chelating agents; N,N,N’,N’-tetrakis (2-pyridylmethyl) ethane-1,2-diamine, diethylene triami- nepenta acetic acid, TTD on ECV-induced toxicities [22]. In sight of these, we demonstrated that Zn++ chelating agent, TTD an Antabuse drug can be likely repurposed as a therapeutic can- didate in treating ECV-induced toxicities that are mediated by SVMPs. The proficient hydroly- sis of the basement membrane by SVMPs surrounding the blood vessels leads to immediate events of hemorrhage at the site of venom injection [18,77]. The progression of hemorrhage resulting in localized myonecrosis is due to extensive degradation of structural proteins and severe inflammation [46,78]. Initially, TTD successfully inhibited ECV-induced degradation of ECM proteins in a concentration-dependent manner and also neutralizes the hemorrhage PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 16 / 24 PLOS NEGLECTED TROPICAL DISEASES PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities PICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxici Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities induced by ECV upon challenging studies (Fig 1). On the other hand, AA and SLN inhibitors failed to inhibit the action of ECV-induced ECM protein degradation and hemorrhage. In sup- port of the neutralization of hemorrhage, TTD treatment could efficiently protect mice footpad from ECV-induced necrosis (Fig 2). ECV-induced footpad necrosis is evident after day 4 of injection and necrotized little toe detached from limb between 6–8 days of ECV injection. This prompted us to carry out mice footpad necrosis experiments till 8 days after ECV injection. The successful neutralization of ECV-induced ECM proteins degradation and hemorrhage by TTD indicates that SVMPs are the main toxins responsible for ECV-induced toxicities. Further, EC SVMPs are also hemotoxic and interfere in hemostasis by hydrolyzing clotting factors that lead to persistent coagulopathy and death [79]. Most SVMPs are α and β fibrinogenases that act on fibrinogen and making them truncated, and non-functional [79]. Discussion A few scientific reports have shown that inhibitors of SVMPs effectively protect mice from viperid snake venom-induced lethality [22,23]. Similarly, TTD was effective in protecting mice from ECV-induced lethality and systemic hemorrhage (Fig 3). These data clearly indicate that TTD has a beneficial effect on neutralizing ECV-induced toxicities in mice. Neutrophils are the first-line defense immune cells and efficiently arrest pathogens by NETosis at the site of infection [45,80]. Porto et al. demonstrated the infiltration of neutrophils at the site of viper venom injection [81]. However, the importance of NETosis in ECV-induced toxicities was not clear until Katkar et al. reported the critical role of NETosis in ECV-induced local tissue damage [15]. NETosis results in the blockage of blood vessels preventing venom from entering into the circulation. The accumulated venom-NETs complexes at the site of venom injection lead to the progressive tissue necrosis [15]. In addition, NETosis in non-heal- ing wounds is noticeable by increased expression of PAD4, citH3 and MPO level [15,82]. However, the previous study did not explain in the context of the toxin that is responsible for ECV-induced NETosis and toxicities [15]. The inhibition of ECV-induced NETosis and reduced levels of PAD4, citH3 and MPO expression by TTD confirms the direct involvement of EC SVMPs in the induction of NETosis. Nonetheless, the neutralized ECV-induced tissue necrosis and systemic hemorrhage by TTD correlated with the decreased ECV-induced NETosis. However, the mechanism of how ECV/SVMPs induce NETosis and toxicities is largely unknown. There are multiple scientific reports suggesting that the MMPs exert their effects by cleaving PARs and play an important role in vascular functions [21,48]. Moreover, MMPs bind and cleave the extracellular N-termi- nus of PAR-1 to release a tethered ligand and activate the intracellular G proteins across the membrane and initiate intracellular signaling cascade [21,83]. The inhibition of MMP-1 induced PAR-1 cleavage restricts the activation of MAPKs [84]. SVMPs belong to metzincin super-family and they are known to activate MAPKs signaling pathways in immune cells which results in elevated levels of pro-inflammatory mediators such as TNF-α, IL-1β and IL-6 leading to chronic inflammation [85]. Similarly, EC SVMPs mediates the phosphorylation of ERK in human neutrophils and it was completely inhibited by TTD (Fig 4). Similar to MMP- 1, EC SVMPs might cleave PAR-1 at the non-canonical site and activate downstream MAPKs signaling. Discussion Finally, ECV-induced NETosis and tissue necrosis in experimental animals are effectively neutralized by PAR-1 antagonists (Figs 5 and 6). Overall, current findings indicate that direct involvement of PAR-1 and downstream MAPKs signaling cascade in EC SVMPs- induced toxicities in mice (Fig 7). Supporting information S1 Fig. Inhibition of ECV-induced enzymatic activities by specific inhibitors. ECV was pre- incubated without or with various concentrations of AA/TTD/SLN at 37˚C for 5 min and sub- jected for PLA2 (A), hyaluronidase (B) and protease (C) activity. The inhibition was repre- sented as % inhibition and venom alone considered as 100% activity. p < 0.05, when compared ECV versus ECV + AA, ECV + SLN and ECV + TTD. (TIF) S2 Fig. Effect of AA and SLN on ECV-induced ECM protein degradation and hemorrhage in mice. ECV was pre-incubated without or with different concentrations of either AA (A) or SLN (C) at 37˚C for 5 min and subjected to gelatin zymogram as described in methods section. Clear zones in the gel indicate the hydrolysis of gelatin by ECV. Area of gelatinolytic activity was measured using graph sheet represented as area (mm2) (A and C). For collagen I (Col I), degradation, ECV was pre-incubated without or with increased concentrations of either AA (B) or SLN (D). Pre-incubated reaction mixture of ECV and inhibitors were further incubated with 50 μg of collagen I for 3 h at 37˚C and cleavage pattern was analyzed using 7.5% SDS-PAGE and visualized by staining with CBB-G250. For skin hemorrhage, mice were injected (n = 3; i.d.) with 5 μg of ECV followed by two different concentrations of AA and SLN after 30 min venom injection. After 180 min, dorsal patches of mice skin were photographed (E and F). Data are representative of two independent experiments. (TIF) S3 Fig. Inhibition of ECV-induced protease activity and local toxicities by TTD. Reaction mixture (1 ml) contained 0.4 ml of casein (2%) in 0.2 M Tris-HCl buffer pH 8.5 was incubated for 150 min at 37˚C with 25 μg of ECV and various concentrations of TTD (0–20 mM). The inhibition was represented as % inhibition and IC50 (median inhibitory concentration) of the TTD was calculated (A). For inhibition of skin hemorrhage, mice were injected (i.d.) with 5 μg of ECV that was pre-incubated with different concentrations of TTD (0–20 mM) at 37˚C for 5 min. After 180 min, dorsal patches of mice skin were photographed and IC50 (median inhibitory concentration) of the TTD was calculated (B and C). Conclusion There is an urgent need for effective snakebite treatments that can be administered in the remote areas where medical access is limited and also that can complement ASV. The current 17 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities AL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities findings suggested that TTD is a pharmacologically approved an Antabuse drug and that inhibits ECVMPs-induced NETosis in human neutrophils and footpad tissue necrosis in mice. Moreover, TTD also neutralized ECV-induced systemic hemorrhage and conferred protection against lethality in mice. Furthermore, we demonstrated that ECVMPs-induced NETosis and tissue necrosis is mediated via PAR-1-ERK axis. Overall, our results provide an insight into SVMPs-induced toxicities and the promising neutralizing potency of TTD can be exploited as first aid therapy, complementing ASV to treat snakebite-induced toxicities. Author Contributions Conceptualization: Gotravalli V. Rudresha, Rajesh Rajaiah, Bannikuppe S. Vishwanath. Formal analysis: Gotravalli V. Rudresha, Rajesh Rajaiah, Bannikuppe S. Vishwanath. Formal analysis: Gotravalli V. Rudresha, Rajesh Rajaiah, Bannikuppe S. Vishwanath. Funding acquisition: Rajesh Rajaiah, Bannikuppe S. Vishwanath. Funding acquisition: Rajesh Rajaiah, Bannikuppe S. Vishwanath. Investigation: Gotravalli V. Rudresha, Amog P. Urs, Vaddarahally N. Manjuprasanna, Malla- nayakanakatte D. Milan Gowda, Krishnegowda Jayachandra. Investigation: Gotravalli V. Rudresha, Amog P. Urs, Vaddarahally N. Manjuprasanna, Malla- nayakanakatte D. Milan Gowda, Krishnegowda Jayachandra. Project administration: Rajesh Rajaiah, Bannikuppe S. Vishwanath. Project administration: Rajesh Rajaiah, Bannikuppe S. Vishwanath. Supervision: Rajesh Rajaiah, Bannikuppe S. Vishwanath. Validation: Rajesh Rajaiah, Bannikuppe S. Vishwanath. Writing – original draft: Gotravalli V. Rudresha, Rajesh Rajaiah, Bannikuppe S. Vishwanath. Writing – original draft: Gotravalli V. Rudresha, Rajesh Rajaiah, Bannikuppe S. Vishwanath. Writing – review & editing: Gotravalli V. Rudresha, Amog P. Urs, Rajesh Rajaiah, Banni- kuppe S. Vishwanath. S5 Fig. ECV-induced NETosis and activation of intracellular signaling in neutrophils. S5 Fig. ECV-induced NETosis and activation of intracellular signaling in neutrophils. Human neutrophils were stimulated with ECV for 180 min and NETs formation was observed and quantitated (A). The whole cell lysates were analyzed for the phosphorylated ERK and NETosis markers using Western blotting. The p-ERK, MPO and PAD4 were quantitated using β-actin as a loading control and H3 as loading control for citH3. Data are representative of two independent experiments. (TIF) S5 Fig. ECV-induced NETosis and activation of intracellular signaling in neutrophils. Human neutrophils were stimulated with ECV for 180 min and NETs formation was observed and quantitated (A). The whole cell lysates were analyzed for the phosphorylated ERK and NETosis markers using Western blotting. The p-ERK, MPO and PAD4 were quantitated using β-actin as a loading control and H3 as loading control for citH3. Data are representative of two independent experiments. (TIF) S6 Fig. Effect of AA and SLN on ECV-induced NETs formation. Human neutrophils were stimulated (180 min) with ECV pre-incubated with different concentrations of either AA or SLN for 5 min at 37˚C and NETs formation was photographed under microscope (A) and quantitated as percent NETosis (B). The data represent the mean ± SD of three independent experiments. p < 0.05, when compared ECV versus ECV + AA and ECV + SLN. (TIF) Acknowledgments Authors thank Central Animal Facility, University of Mysore, for providing animals. Authors thank Prof. Manjunath Kini R, Dr. Nanjaraj UrsA N, Dr. Vikram Joshi and Dr. Suvilesh K N for providing valuable suggestions during the project. Authors also thank -Dr. Sumanth M S and Dr. Abhilasha K V for their help during experiments and neutrophils isolation. Supporting information For inhibition of tissue necrosis, mice foot- pads were injected with ECV (LD50; 2.21 mg/kg) pre-incubated with TTD (20 mM) at 37˚C for 5 min and footpads were photographed from day 1 to day 8 (D). Red arrow indicates edema and black arrow indicates tissue necrosis. ECV-induced footpad injury was measured manually on a scale of 1 to 5 (E). Data are representative of two independent experiments. (TIF) S4 Fig. Histochemical staining of ECV-induced tissue necrosis in mice footpad and its inhibition by TTD. Mice footpad was injected with ECV followed by TTD or DNase 1 injec- tion (30 min post venom injection). Mice were euthanized and footpad tissues were processed for histological sections and analyzed for tissue damage by H & E staining. PBS injected mouse footpad serves as control. (TIF) 18 / 24 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0008596 February 2, 2021 PLOS NEGLECTED TROPICAL DISEASES PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide n PLOS NEGLECTED TROPICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities ICAL DISEASES Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxici Re-purposed drug, tetraethylthiuram disulfide neutralizes snake venom-induced toxicities References 1. Gutierrez JM, Theakston RD, Warrell DA. 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https://openalex.org/W2022671112	https://diposit.ub.edu/dspace/bitstream/2445/166498/1/651744.pdf	English	null	A new reversal mode in exchange coupled antiferromagnetic/ferromagnetic disks: distorted viscous vortex	Nanoscale	2,015	cc-by	6,307	A new reversal mode in exchange coupled antiferromagnetic/ferromagnetic disks: distorted viscous vortex† Dustin A. Gilbert,a Li Ye,a Aïda Varea,b Sebastià Agramunt-Puig,c Nuria del Valle,c Carles Navau,c José Francisco López-Barbera,c,d Kristen S. Buchanan,e Axel Hoﬀmann,f Alvar Sánchez,c Jordi Sort,c,g Kai Liua and Josep Noguésc,d,g Open Access Article. Published on 28 April 2015. Downloaded on 5/29/2019 1:23:53 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Magnetic vortices have generated intense interest in recent years due to their unique reversal mecha- nisms, fascinating topological properties, and exciting potential applications. In addition, the exchange coupling of magnetic vortices to antiferromagnets has also been shown to lead to a range of novel phenomena and functionalities. Here we report a new magnetization reversal mode of magnetic vortices in exchange coupled Ir20Mn80/Fe20Ni80 microdots: distorted viscous vortex reversal. In contrast to the previously known or proposed reversal modes, the vortex is distorted close to the interface and viscously dragged due to the uncompensated spins of a thin antiferromagnet, which leads to unexpected asymme- tries in the annihilation and nucleation ﬁelds. These results provide a deeper understanding of the physics of exchange coupled vortices and may also have important implications for applications involving exchange coupled nanostructures. Magnetic vortices have long been studied and remain a topic of current interest due to their fascinating fundamental pro- perties and topological characteristics.1–4 This magnetization state, which arises from the competition between magneto- static, exchange and anisotropy energies in nanostructured ferromagnet (FM) materials, is characterized by a (counter-) clockwise in-plane curl of the magnetization (chirality) around an up or down out-of-plane central core (polarity).5,6 Recent demonstrations of chirality and polarity control7–11 have triggered renewed interests in these entities for spintronic applications,12–14 artificial Skyrmion lattices,15 and even bio- medical applications.16 has received ever increasing interest across many emerging frontiers of condensed matter physics, e.g., multiferroics,17,18 chiral ordering and exchange bias induced by Dzyaloshinskii– Moriya interactions,19,20 control of quantum magnets,21 AF spintronics,22,23 and triplet pairing in superconducting exchange biased heterostructures.24 When a magnetic vortex is coupled to an AF (exchange bias) novel eﬀects emerge, e.g., biased vortex reversal hysteresis loops, reversible non-zero remnant magnetization states, tunable angular dependent reversal modes, chirality-control, adjustable magnetization dynamics, or suppressed stochastic eﬀects.7,25–33 These eﬀects, which occur due to the imprinting of diﬀerent magnetic states in the AF,34,35 can lead to additional functionalities in vortex structures. Nanoscale A new reversal mode in exchange coupled antiferromagnetic/ferromagnetic disks: distorted viscous vortex† Dustin A. Gilbert,a Li Ye,a Aïda Varea,b Sebastià Agramunt-Puig,c Nuria del Valle,c Carles Navau,c José Francisco López-Barbera,c,d Kristen S. Buchanan,e Axel Hoﬀmann,f Alvar Sánchez,c Jordi Sort,c,g Kai Liua and Josep Noguésc,d,g This journal is © The Royal Society of Chemistry 2015 †Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5nr01856k aPhysics Department, University of California, Davis, CA, USA. E-mail: kailiu@ucdavis.edu bMinD-in2UB, Electronics Department, Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain cDepartament de Física, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain dICN2 – Institut Catala de Nanociencia i Nanotecnologia, Campus UAB, 08193 Bellaterra (Barcelona), Spain eDepartment of Physics, Colorado State University, Fort Collins, CO, USA fMaterials Science Division, Argonne National Laboratory, Argonne, IL, USA gInstitució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. E-mail: Josep.Nogues@uab.cat A new reversal mode in exchange coupled antiferromagnetic/ferromagnetic disks: distorted viscous vortex† Dustin A. Gilbert,a Li Ye,a Aïda Varea,b Sebastià Agramunt-Puig,c Nuria del Valle,c Carles Navau,c José Francisco López-Barbera,c,d Kristen S. Buchanan,e Axel Hoﬀmann,f Alvar Sánchez,c Jordi Sort,c,g Kai Liua and Josep Noguésc,d,g Furthermore, it has been predicted that in exchange coupled structures the vortex cores may be tilted along their thickness (in contrast to conventional vortices where the cores are straight) due to the pinning eﬀects of the AF.36,37 Such a tilted structure leads to additional asymmetries in the hysteresis loops, which are correlated with structural and magnetic parameters (e.g., dot geometry or AF/FM exchange strength). In fact, vortices exchange coupled to AFs have been prominently featured in key technologies such as magnetic random access memory and sensors.38 On the other hand, exchange bias [i.e., nominally the exchange coupling between a FM and an antiferromagnet (AF)] †Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5nr01856k aPhysics Department, University of California, Davis, CA, USA. E-mail: kailiu@ucdavis.edu bMinD-in2UB, Electronics Department, Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain cDepartament de Física, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain dICN2 – Institut Catala de Nanociencia i Nanotecnologia, Campus UAB, 08193 Bellaterra (Barcelona), Spain eDepartment of Physics, Colorado State University, Fort Collins, CO, USA fMaterials Science Division, Argonne National Laboratory, Argonne, IL, USA gInstitució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. E-mail: Josep.Nogues@uab.cat In this article we report a viscous vortex reversal mode in AF/FM exchange biased dots with a thick FM layer and varying AF thicknesses. By changing the AF thickness, tAF, its an- isotropy energy is systematically tuned, thus changing the rigidity of the AF spin structure from weak (“draggable” by the 9878 \| Nanoscale, 2015, 7, 9878–9885 View Article Online Nanoscale Paper FM layer) to rigid. This leads to a viscous vortex reversal mechanism in the dots, which deviates from the standard, biased and tilted vortex reversals. to enhanced HC, but no bias. For the thicker AF the anisotropy is suﬃciently large so that the spins remain rigidly oriented after the field cooling process.40,41 Major hysteresis loops for dots with 1 μm and 1.5 μm dia- meter are shown in Fig. 1(c–f). The symmetric pinched shape of the loops without AF, Fig. 1(c), is characteristic of a vortex state reversal. For dots with a thin IrMn layer (tAF = 3 nm), the major loops, Fig. 1(d), exhibit a much larger coercivity and a pronounced asymmetry. However, these dots do not show appreciable exchange bias, indicating that the AF has weak an- isotropy and is dragged during the FM reversal. A new reversal mode in exchange coupled antiferromagnetic/ferromagnetic disks: distorted viscous vortex† Dustin A. Gilbert,a Li Ye,a Aïda Varea,b Sebastià Agramunt-Puig,c Nuria del Valle,c Carles Navau,c José Francisco López-Barbera,c,d Kristen S. Buchanan,e Axel Hoﬀmann,f Alvar Sánchez,c Jordi Sort,c,g Kai Liua and Josep Noguésc,d,g For dots with thicker (tAF ≥5 nm) IrMn layers, Fig. 1(e and f), the exchange bias is clearly established, and the HC is less than that of the tAF = 3 nm samples. Close inspection of the loops reveals asym- metries in their shape, particularly for tAF = 5 nm, suggesting the presence of locally pinned spins. Interestingly, the major loop shape for dots with an AF is quite complex and not indicative of any traditional magnetization reversal modes. Major hysteresis loops Measured dependence of (b) ΔHA and (c) ΔHN on tAF for d = 1.0 and 1.5 µm circular dots. Error bars [smaller than symbol size in (b)] are determined by the radius of the curvature of the measured data at the nucleation (annihilation) corner. shed on 28 April 2015. Downloaded on 5/29/2019 1:23:53 PM. licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 28 April 2015. Downloaded on 5/29/2019 1:23:53 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 Schematic hysteresis loops are shown in (a) for unbiased, biased, tilted, and viscous vortex reversals. The unbiased vortex reversal is shown in dotted grey for reference. The viscous vortex reversal shows little to no exchange ﬁeld, but asymmetries in both the nucleation and annihilation. Symbols HN + (HN −) and HA + (HA −) represent nucleation ﬁelds from positive (negative) saturation and annihilation to positive (negative) saturation, respectively. Measured dependence of (b) ΔHA and (c) ΔHN on tAF for d = 1.0 and 1.5 µm circular dots. Error bars [smaller than symbol size in (b)] are determined by the radius of the curvature of the measured data at the nucleation (annihilation) corner. While the analytical theory for exchange bias induced vortex tilting by Guslienko and Hoﬀmann can qualitatively explain several of the experimentally observed eﬀects,36,42 some of the main experimental observations cannot be accounted for. For example, (i) the experimental ΔHA is not proportional to the macroscopic HE as assumed in the model; (ii) on decreasing the dot diameter, ΔHA decreases rather than increases as is expected from the calculations; and (iii) there is a HN asymmetry, which the theory assumes to be absent. These discrepancies suggest that the microscopic magnetic structure is far more complex than the one assumed in the theory. For instance, the model is mainly based on the depth dependence of the eﬀective exchange bias field, but it neglects the non-uniform spin structure at the interface. In particular, it is well accepted that the exchange bias eﬀect can be related to pinned and unpinned uncompensated spins in the AF/FM interface,43–48 giving rise to loop shifts and coercivity enhancements, respectively. In the biased vortex case, we can naively assume that the pinned uncompensated spins will be parallel to the cooling field, while the unpinned ones will form a curl mimicking the FM vortex. Major hysteresis loops Open Access Article. Published on 28 April 2015. Downloaded on 5/29/2019 1:23:53 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Major hysteresis loops of Fe20Ni80(30 nm)/Ir20Mn80(tAF) (FeNi/ IrMn) films are shown in Fig. 1(a) and the HC and HE trends are shown in Fig. 1(b). These plots show that the tAF = 0 film has a very small coercivity (HC = 0.4 Oe) and no bias (HE = 0), while the tAF = 3 nm film has a significantly increased HC (28 Oe) and a small HE (3.8 Oe). For tAF > 3 nm HC decreases (2.9–4.3 Oe), but HE is established (56–81 Oe). This behavior has been previously attributed to the anisotropy of the AF. Specifically, for a thin AF the anisotropy is exceedingly weak and is viscously dragged by the FM while it reverses,39 leading y gg y , g y g Fig. 1 (a) Hysteresis loops for continuous ﬁlms of FeNi/IrMn with diﬀerent tAF. (b) Dependence of \|HE\| and HC on tAF for the ﬁlms and 1.0 and 1.5 µm circular dots. Major hysteresis loops for the 1.0 and 1.5 µm circular dots with tAF of (c) 0 nm, (d) 3 nm, (e) 5 nm, and (f) 7 nm. Fig. 1 (a) Hysteresis loops for continuous ﬁlms of FeNi/IrMn with diﬀerent tAF. (b) Dependence of \|HE\| and HC on tAF for the ﬁlms and 1.0 and 1.5 µm Fig. 1 (a) Hysteresis loops for continuous ﬁlms of FeNi/IrMn with diﬀerent tAF. (b) Dependence of \|HE\| and HC on tAF for the ﬁlms and 1.0 and 1.5 µm circular dots. Major hysteresis loops for the 1.0 and 1.5 µm circular dots with tAF of (c) 0 nm, (d) 3 nm, (e) 5 nm, and (f) 7 nm. This journal is © The Royal Society of Chemistry 2015 Nanoscale, 2015, 7, 9878–9885 \| 9879 View Article Online Paper Paper Nanoscale Fig. 2 Schematic hysteresis loops are shown in (a) for unbiased, biased, tilted, and viscous vortex reversals. The unbiased vortex reversal is shown in dotted grey for reference. The viscous vortex reversal shows little to no exchange ﬁeld, but asymmetries in both the nucleation and annihilation. Symbols HN + (HN −) and HA + (HA −) represent nucleation ﬁelds from positive (negative) saturation and annihilation to positive (negative) saturation, respectively. This journal is © The Royal Society of Chemistry 2015 Nanoscale Paper Micromagnetic simulations results. The increasing and decreasing field branches of the loop are shown to have diﬀerent HA and HN [see Fig. 3(b)], with ΔHA and ΔHN of about 8 Oe, reproducing the asymmetries observed experimentally. To elucidate the origin of this asym- metry we examine the spin maps of each layer. In Fig. 3(c) we plot the orientation of the spin moments (at H ∼HC) at the AF/ FM interface and the top FM surface of the dot [labeled layers To highlight the origin of the novel reversal mode micromag- netic simulations were conducted. The simulated loop, shown in Fig. 3(a), exhibits a pinched loop shape, typical of vortex reversal, shifted along the field axis (with HE = 128 Oe and HC = 54 Oe), in good qualitative agreement with the experimental Open Access Article. Published on 28 April 2015. Downloaded on 5/29/2019 1:23:53 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. mulated hysteresis loop of a FM disk with 504 nm diameter and 30 nm thickness pinned to an AF layer. (b) Composition of the top half (i.e., M > 0) (black symbols) and the inverted bottom part (M < 0), corrected for the loop shift (red symbols). (c) Spin maps of the top ottom (green) FM layers at H ∼HC for decreasing (left) and increasing (right) ﬁelds. The background intensity corresponds to the diﬀer- my5) as red (positive) and blue (negative), as discussed in the text. The bottom images are enlarged views of the highlighted areas. The (HFC) and applied ﬁeld (HAppl) directions are shown by arrows. (d) Side- and (e) top-view schematic illustration of the magnetic spins, (blue) top, (yellow) middle, and (red) bottom, in a distorted vortex structure based on the simulations. © The Royal Society of Chemistry 2015 Nanoscale 2015 7 9878 9885 \| 9881 Fig. 3 (a) Simulated hysteresis loop of a FM disk with 504 nm diameter and 30 nm thickness pinned to an AF layer. (b) Composition of the top half of the loops (i.e., M > 0) (black symbols) and the inverted bottom part (M < 0), corrected for the loop shift (red symbols). (c) Spin maps of the top (black) and bottom (green) FM layers at H ∼HC for decreasing (left) and increasing (right) ﬁelds. The background intensity corresponds to the diﬀer- ence (my1 −my5) as red (positive) and blue (negative), as discussed in the text. Major hysteresis loops Hence, while the vortex near the FM/AF interface experiences a complex energy landscape, away from this interface it behaves more like a conventional vortex. These additional interface eﬀects, which should be enhanced for smaller sizes, could give rise to the discrepancies between the theory and experiments. A possible reversal mechanism proposed previously in exchange biased dots is the “tilted-vortex” model.36,37 In this model the interfacial moments in the FM are pinned by the exchange coupling to the AF and the vortex core position at this interface is displaced from the center, while further away from the interface the vortex is more centered, hence the core is tilted. As shown schematically in Fig. 2(a), the reversal mecha- nism is reflected in the major loops by the asymmetry of the positive and negative annihilation fields, HA + and HA −respecti- vely, after oﬀsetting the HE. That is, in an unbiased vortex HA + = −HA −, while in a biased vortex HA + −HE = −(HA −−HE), and in a tilted vortex HA + −HE ≠−(HA −−HE). Furthermore, the nucleation field, HN, should always be equally biased; for normal, biased and tilted vortex reversal HN + −HE = −(HN −− HE). We can thus define variables ΔHA = HA + + HA −−2HE and ΔHN = HN + + HN −−2HE. By identifying ΔHA and ΔHN these three reversal behaviors can be uniquely identified: ΔHA = ΔHN = HE = 0 for unbiased vortices, ΔHA = ΔHN = 0 and HE ≠0 for biased vortices, and ΔHA ≠0, ΔHN = 0 and HE ≠0 for tilted vortices. The trends for ΔHA and ΔHN are shown in Fig. 2(b and c), where the nucleation/annihilation fields are deter- mined from intercepts of the linear extrapolations of magneti- zation before and after nucleation/annihilation. It can be seen that indeed there is an asymmetry in HA, suggesting a tilted- vortex reversal. However, there is also an asymmetry in the nucleation field, which is not expected for any of the reversal behaviors discussed above. This journal is © The Royal Society of Chemistry 2015 9880 \| Nanoscale, 2015, 7, 9878–9885 View Article Online This journal is © The Royal Society of Chemistry 2015 First order reversal curve (FORC) analysis To gain a detailed understanding of the dot magnetization reversal, we have performed FORC studies on the microdot arrays [see ESI†].49–53 The FORC diagrams for the unbiased dots [Fig. 4(a and b)] show “butterfly”-like features of a stan- dard vortex reversal with three main peaks, identified in Fig. 4 (a) and discussed in the ESI (Fig. S1†): peak i corresponds to the initial vortex nucleation from positive saturation and sub- sequent annihilation approaching positive saturation;51 peak ii corresponds to re-nucleation from negative saturation, and is accompanied by a negative region that reflects the slope change along successive FORCs;52 peak iii identifies sub- sequent annihilation to positive saturation, manifesting asym- metries in the dot shape.50 For tAF = 0, features i and ii are of similar intensity [Fig. 4(a and b)] since the nucleation events are symmetric under field inversion. Fig. 4 FORC distributions for (left) 1.0 µm and (right) 1.5 µm diameter exchange biased dots with tAF of (a, b) 0 nm, (c, d) 3 nm, (e, f) 5 nm, and (g, h) 7 nm. fly”-like feature set. In addition, the nucleation/annihilation features are of comparable intensity. Recalling that ΔHA is nearly zero for these samples [Fig. 2(a)], this indicates that the reversal involves simply biased vortices, not tilted vortices. For tAF = 3 nm, intensities of the FORC features i and ii become asymmetric [Fig. 4(c and d)], indicating a deviation from the conventional vortex reversal and asymmetric magneti- zation reversal processes. The asymmetry is even more pro- nounced for tAF = 5 nm, where feature i has largely vanished. The suppression of feature i indicates a much-reduced irrever- sibility associated with the vortex nucleation/annihilation near the positive saturation, while the enhanced FORC peak ii shows that the primary irreversibility is due to the vortex nucleation/annihilation near the negative saturation. This vortex reversal asymmetry is consistent with a depth-depen- dent magnetization configuration in the dots,51,54 since the pinning induced by the AF is stronger at the FM/AF interface than at the FM free surface, as suggested by the simulations. In both tAF = 3 nm and 5 nm peak ii shifts to a larger local coercivity (HC* – see the ESI†), consistent with the proposed viscous drag reversal. For tAF = 5 nm the entire FORC distri- bution is shifted towards negative HB as the exchange bias is established [Fig. 4(e and f)]. This journal is © The Royal Society of Chemistry 2015 Micromagnetic simulations The bottom images are enlarged views of the highlighted areas. The cooling ﬁeld (HFC) and applied ﬁeld (HAppl) directions are shown by arrows. (d) Side- and (e) top-view schematic illustration of the magnetic spins, identiﬁed as (blue) top, (yellow) middle, and (red) bottom, in a distorted vortex structure based on the simulations. Nanoscale, 2015, 7, 9878–9885 \| 9881 This journal is © The Royal Society of Chemistry 2015 View Article Online Paper Fig. 4 FORC distributions for (left) 1.0 µm and (right) 1.5 µm diameter exchange biased dots with tAF of (a, b) 0 nm, (c, d) 3 nm, (e, f) 5 nm, and (g, h) 7 nm. Fig. 4 FORC distributions for (left) 1.0 µm and (right) 1.5 µm diameter exchange biased dots with tAF of (a, b) 0 nm, (c, d) 3 nm, (e, f) 5 nm, and 5 and 1 in Fig. 3(d)] in green and black, respectively. The color contrast in Fig. 3(c) identifies the magnetization diﬀerence in the two layers (my1 −my5) as red (positive) and blue (negative). The first remarkable result is that the core of the first and fifth layers seems to be at the same position (within one micromag- netic cell, 6 nm), indicating no vortex tilt, in contrast with theoretical predictions.36,37 However, as shown in Fig. 3(c) the vortices exhibit a clear distortion. While layer 1 (green) has a near perfect vortex structure, the interfacial spins in layer 5 tend to tilt towards the FC direction [see Fig. 3(d and e)]. The distortion is more pronounced along the ascending-field branch [Fig. 3(c) right panels] compared to the descending- field branch [left panels], as illustrated by the more intense background color. The origin of the major loop asymmetries seems to be related to diﬀerent degrees of distortion of the vortex structure close to the AF. This variation in the interfacial coupling is manifested diﬀerently in HA and HN depending on the previous saturation states and the field cooling direction. Discussions The reversal mechanism observed for tAF = 3 and 5 nm deviates from the three established behaviors discussed earlier (vortex, shifted vortex and tilted vortex), none of which predicts an asymmetry in HN. Originating from the drag of the AF and accompanied by a distortion of the vortex structure rather than a tilt (as shown by simulations), this magnetization reversal mechanism may be viewed as distorted viscous vortex reversal. Remarkably, the dependence of HE and HC, ΔHA and ΔHN and the evolution of the FORC features on tAF seem to indicate that the new reversal mode is dominated by the unpinned uncom- pensated spins, which explains its diﬀerences from the pro- posed tilted-vortex mode. Nevertheless, the asymmetries related to this new mechanism should be enhanced for thicker Finally, for tAF = 7 nm, shown in Fig. 4(g) and (h) (and 9 nm, not shown), the FORC distribution returns to a “butter- This journal is © The Royal Society of Chemistry 2015 9882 \| Nanoscale, 2015, 7, 9878–9885 View Article Online Nanoscale Paper FM layers (where the vortex distortion should increase) and moderately thin AFs (where the AF has weaker anisotropy and the drag should be larger). Even in nanostructures with thick AFs, distorted viscous vortex reversal may still emerge if the temperature is suﬃciently increased so that the AF anisotropy is concomitantly weakened.55 Interestingly, although the thick- ness of the FM layers in AF/FM dots is typically on the 10 nm scale, some hints of reversal asymmetries probably linked to this new reversal mode can be found in the literature.4,30,55,56 Note that the viscous drag of the magnetization due to the AF also occurs in thin films. However, in contrast to what is observed in nanostructures, in thin films the net eﬀect of this viscous drag is merely an increase in coercivity without any changes in the magnetization reversal modes.40,57 Importantly, the dragging of the AF layer is not only a general feature of exchange biased dots, but may also be relevant for virtually any exchange coupled system,58 e.g., in magnetically hard/soft exchange coupled nanostructures59–61 where the harder layer has insuﬃcient anisotropy to pin the softer layer. by a mask. The AF orientation was set by heating the sample to 520 K (above the blocking temperature of IrMn, TB = 420 K) then cooling to room temperature in an in-plane magnetic field, HFC = 2 kOe. Conclusions In summary, we have found a new distorted viscous vortex reversal mode in exchange biased FeNi/IrMn dots with varying AF thicknesses. Unbiased dots reverse via a vortex state, while dots with an AF layer undergo a much more complex reversal process: dots with thin AF layers reverse via a distorted viscous vortex state with an enhanced coercivity; once the AF layer is thick enough to have suﬃcient anisotropy energy, the magneti- zation reverses via a biased vortex state, and the coercivity enhancement is suppressed. This viscous vortex reversal mode and the asymmetries in the annihilation and nucleation fields are beyond the current understanding of exchange coupled vortices, and oﬀer interesting implications for device applications. Acknowledgements This work was supported by the US NSF (DMR-1008791 and ECCS-1232275), the 2014-SGR-1015 project of the Generalitat de Catalunya, and MAT2010-20616-C02, CSD2007-00041 and MAT2012-35370 projects of the Spanish Ministerio de Econo- mía y Competitividad (MinECO). Work at Argonne was sup- ported by the U. S. Department of Energy, Oﬃce of Science, Materials Science and Engineering Division. Fabrication was performed at the Center for Nanoscale Materials, which is sup- ported by DOE, Oﬃce of Science, Basic Energy Science under Contract No. DE-AC02-06CH11357. KL acknowledges support from the NSFC (11328402). AS acknowledges a grant from the ICREA Academia, funded by the Generalitat de Catalunya. ICN2 acknowledges support from the Severo Ochoa Program (MinECO, Grant SEV-2013-0295). Open Access Article. Published on 28 April 2015. Downloaded on 5/29/2019 1:23:53 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Note that the viscous drag of the magnetization due to the AF also occurs in thin films. However, in contrast to what is observed in nanostructures, in thin films the net eﬀect of this viscous drag is merely an increase in coercivity without any changes in the magnetization reversal modes.40,57 Importantly, the dragging of the AF layer is not only a general feature of exchange biased dots, but may also be relevant for virtually any exchange coupled system,58 e.g., in magnetically hard/soft exchange coupled nanostructures59–61 where the harder layer has insuﬃcient anisotropy to pin the softer layer. Simulations were conducted using the same geometric con- structions as the experimental system by iteratively solving Brown’s static equations64 using a 6 nm cubic mesh (consist- ent with the exchange length of FeNi65), making the simulated FM 5 cells thick. The polycrystalline FeNi was simulated using an exchange stiﬀness A = 1.3 × 10−11 J m−1, a saturation mag- netization MS = 8 × 105 A m−1, and magnetocrystalline an- isotropy KU = 0. The IrMn was modeled as 84% non-magnetic material and 16% (900 cells) randomly distributed magneti- cally contributing cells, representing uncompensated spins. The contributing cells are further divided into pinned and rotatable cells43–48 in a ratio of 4 : 5, giving a moderate loop shift, HE, and coercivity, HC. The pinned cells have their mag- netization (MS = 8 × 105 A m−1) fixed along the field-cool (FC) direction, while the unpinned ones have a large uniaxial an- isotropy (KU = 5 × 105 J m−1) in the FC direction. These uncom- pensated spins interact via exchange (assuming JAF–FM = JFM– FM) and magnetostatic interactions with the FM spins but only magnetostatically among themselves (since they represent the equivalent of isolated uncompensated spins in experimental systems). Since the results depend on the spatial distribution of the pinned and unpinned spins, the presented results are the average of 8 diﬀerent simulated configurations. The asymmetries inherent to the distorted viscous vortex reversal may have practical implications in the performance of magnetic devices based on exchange coupling. Thus, the poss- ible eﬀects of the distorted viscous vortex reversal should be taken into account in the design of such devices (e.g., tuning the thickness of the AF or FM layers or operating temperature to avoid this eﬀect). Methods Arrays of circular nanodots with diameters of 0.5, 1.0 and 1.5 μm and vertical structures of Ta(5 nm)/Fe20Ni80(30 nm)/ Ir20Mn80(tAF)/Pt(2 nm) [tAF = 0–9 nm] were fabricated on a naturally oxidized Si(001) substrate by electron-beam litho- graphy and DC magnetron sputtering from composite targets in 1.5 mTorr Ar. The dot sizes are optimal to achieve vortex structure. The FM layer was kept deliberately thick to promote tilted vortex reversal.36,37 Arrays with a common AF thickness were fabricated in a single run, with the other arrays shadowed Discussions Hysteresis loops and first-order reversal curve (FORC) measurements were recorded at room tempera- ture using a longitudinal magneto-optical Kerr eﬀect (MOKE) setup, following prior procedures,49,50,62,63 with loops measured along the cooling field axis, iteratively averaged at a rate of 7 Hz for ∼1000 cycles. FM layers (where the vortex distortion should increase) and moderately thin AFs (where the AF has weaker anisotropy and the drag should be larger). 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https://openalex.org/W4384156112	https://www.frontiersin.org/articles/10.3389/fimmu.2023.1205449/pdf	English	null	Recombinant BCG expressing the LTAK63 adjuvant increased memory T cells and induced long-lasting protection against Mycobacterium tuberculosis challenge in mice	Frontiers in immunology	2,023	cc-by	8,312	OPEN ACCESS OPEN ACCESS EDITED BY Jianping Xie, Southwest University, China REVIEWED BY Laurence Don Wai Luu, Univesity of Technology Sydney, Australia Suraj P. Parihar, Univesity of Cape Town, South Africa CORRESPONDENCE Luciana Cezar de Cerqueira Leite luciana.leite@butantan.gov.br RECEIVED 13 April 2023 ACCEPTED 29 June 2023 PUBLISHED 13 July 2023 CITATION Marques-Neto LM, Trentini MM, Kanno AI, Rodriguez D and Leite LCdC (2023) Recombinant BCG expressing the LTAK63 adjuvant increased memory T cells and induced long-lasting protection against Mycobacterium tuberculosis challenge in mice. Front. Immunol. 14:1205449. OPEN ACCESS EDITED BY Jianping Xie, Southwest University, China REVIEWED BY Laurence Don Wai Luu, Univesity of Technology Sydney, Australia Suraj P. Parihar, Univesity of Cape Town, South Africa CORRESPONDENCE Luciana Cezar de Cerqueira Leite luciana.leite@butantan.gov.br RECEIVED 13 April 2023 ACCEPTED 29 June 2023 Sout est U e s ty, C a REVIEWED BY Laurence Don Wai Luu, Univesity of Technology Sydney, Australia Suraj P. Parihar, Univesity of Cape Town, South Africa CORRESPONDENCE Luciana Cezar de Cerqueira Leite La´zaro Moreira Marques-Neto, Monalisa Martins Trentini, Alex Issamu Kanno, Dunia Rodriguez and Luciana Cezar de Cerqueira Leite La´zaro Moreira Marques-Neto, Monalisa Martins Trentini, Alex Issamu Kanno, Dunia Rodriguez and Luciana Cezar de Cerqueira Leite* La´zaro Moreira Marques-Neto, Monalisa Martins Trentini, Alex Issamu Kanno, Dunia Rodriguez and Luciana Cezar de Cerqueira Leite* Marques-Neto LM, Trentini MM, Kanno AI, Rodriguez D and Leite LCdC (2023) Recombinant BCG expressing the LTAK63 adjuvant increased memory T cells and induced long-lasting protection against Mycobacterium tuberculosis challenge in mice. Laborato´ rio de Desenvolvimento de Vacinas, Instituto Butantan, São Paulo, Brazil Vaccine-induced protection against Mycobacterium tuberculosis (Mtb) is usually ascribed to the induction of Th1, Th17, and CD8+ T cells. However, protective immune responses should also involve other immune cell subsets, such as memory T cells. We have previously shown improved protection against Mtb challenge using the rBCG-LTAK63 vaccine (a recombinant BCG strain expressing the LTAK63 adjuvant, a genetically detoxiﬁed derivative of the A subunit from E. coli heat-labile toxin). Here we show that mice immunized with rBCG-LTAK63 exhibit a long-term (at least until 6 months) polyfunctional Th1/Th17 response in the draining lymph nodes and in the lungs. This response was accompanied by the increased presence of a diverse set of memory T cells, including central memory, effector memory and tissue-resident memory T cells. TYPE Original Research PUBLISHED 13 July 2023 DOI 10.3389/fimmu.2023.1205449 TYPE Original Research PUBLISHED 13 July 2023 DOI 10.3389/fimmu.2023.1205449 tuberculosis, recombinant BCG, long-term protection, adjuvant, vaccine 2.1 Animals and immunization Speciﬁc-pathogen-free female BALB/c mice (4–8 weeks old), from Instituto Butantan – Central Animal Facility, were maintained in ABSL-2 racks ﬁtted with a HEPA-ﬁltered air intake and exhaust system. They were kept at the animal care facility of the Laboratório de Desenvolvimento de Vacinas, with water and food provided ad libitum. The temperature was maintained from 20–24°C, relative humidity of 40–70%, and a 12 h light/dark cycle. This study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the Committee of SBCAL (Sociedade Brasileira de Ciência em Animais de Laboratório) recommendations and was approved by the Animal Research Ethical Committee of Instituto Butantan (number: 3435250619). Classically, the Th1 cells (specially IFN-g+ or polyfunctional cells producing IFN-g, IL-2 and/or TNF-a) have been considered the most important correlates of protection for TB vaccines. As vaccine development progressed in the ﬁeld, Th17 and CD8+ T cells were also considered important cell populations to induce protective responses (9). In mice, immunization with BCG preferentially induces effector T cells and effector memory T cells (TEM - CD4+CD44+CD62-) and not central memory T cells (TCM - CD4+CD44+CD62+). The effector T cells have an immediate effect but are believed to be vulnerable to exhaustion from chronic infection and continuous exposure to mycobacteria, contrary to TCMs. Another recombinant BCG vaccine, VPM1002 (BCGDureC::hly) which is in phase III clinical trials, demonstrates that part of its protection against TB is related to an enhancement of the TCM population (10). The rBCG-LTAK63 strain used in this work was previously described (20). BCG or rBCG-LTAK63 were grown in Middlebrook 7H9 (Difco, Detroit, MI, USA) supplemented with 10% of OADC (oleic acid-albumin-dextrose-catalase; BBL, Cockeysville, MD, USA), 0.5% glycerol and 0.05% Tween 80 (7H9-OADC) or plated on Middlebrook 7H10 agar supplemented with 0.5% glycerol and OADC (7H10-OADC). Beyond TCM and TEM, tissue-resident memory T cells (TRM - KLRG1-PD-1+) have also been described as cell subsets involved in protection against TB. KLRG1 and PD-1 are considered important prognosis biomarkers (8, 11). TRM is a memory T cell subset that has a long lifespan in non-lymphoid tissues; they have low body recirculation capacity, but rapidly migrate through the resident organ parenchyma and differentiate into effector cells upon stimulation. In tuberculosis, the pulmonary TRMs were shown to quickly migrate into the lung after adoptive transfer and protect against Mtb infection (12–15). OPEN ACCESS After the challenge, the T cell phenotype in the lymph nodes and lungs were characterized by a decrease in central memory T cells, and an increase in effector memory T cells and effector T cells. More importantly, when challenged 6 months after the immunization, this group demonstrated increased protection in comparison to BCG. In conclusion, this work provides experimental evidence in mice that the rBCG-LTAK63 vaccine induces a persistent increase in memory and effector T cell numbers until at least 6 months after immunization, which correlates with increased protection against Mtb. This improved immune response may contribute to enhance the long- term protection. Front. Immunol. 14:1205449. doi: 10.3389/fimmu.2023.1205449 © 2023 Marques-Neto, Trentini, Kanno, Rodriguez and Leite. 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. 01 Frontiers in Immunology frontiersin.org 10.3389/ﬁmmu.2023.1205449 10.3389/ﬁmmu.2023.1205449 Marques-Neto et al. 1 Introduction (named rBCG-LTAK63). Immunization of mice with rBCG- LTAK63 increased innate and adaptive immune responses and improved the protection against Mtb challenge in comparison to BCG (19, 20). Here, we show that the immunization of mice with rBCG-LTAK63 enhances the generation of polyfunctional T cells, TCM, and TEM cells. Six months after immunization, these cells are still in higher numbers. At this time point, mice immunized with rBCG-LTAK63 when challenged with Mtb, displayed increased protection as compared with BCG. Tuberculosis (TB) is one of the deadliest infectious diseases in the world, responsible for more than 1.3 million deaths in 2021 (1). BCG is the only licensed vaccine against TB, providing protection against severe forms of TB, especially in children. However, as protection wanes, young individuals and adults exhibit variable protection and are more susceptible to pulmonary tuberculosis (2). Given BCG’s excellent safety record, adjuvant properties (heterologous protection), and effectiveness in newborns, several vaccines in development against TB seek to improve BCG’s protection (3–6). In this sense, the vaccine should confer durable protection and induce a prompt and robust immune response against the bacteria in the lungs (the primary site of infection). Therefore, the generation of memory subsets is one of the main goals sought to improve TB vaccines (7, 8). 2.1 Animals and immunization The development of TRM, however, was only achieved when BCG was used through the mucosal route, with intradermal/subcutaneous immunization failing to induce this cell population (8, 10). Finally, the level of T cell differentiation (reduced expression of KLRG1 marker, as well as the presence of the inhibitor marker PD-1) can indicate increased IL-2 producer cells that help to maintain effector T cell populations, as well as being less sensitive to exhaustion and apoptosis in chronic infection (12, 16, 17). To evaluate long-term immune response and protection, groups of mice (n=5) were immunized with BCG or rBCG-LTAK63 (1x106 CFU/100 mL) resuspended in phosphate-buffered saline (PBS- 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4) and administered subcutaneously in the back of the animals. Frontiers in Immunology 2.2 Intranasal infection with Mtb The intranasal infection was performed as described by Logan et al. (2008) (21). A frozen vial of Mycobacterium tuberculosis H37Rv (kept at -80°C) was thawed, and the inoculum was adjusted to 1.25x104 CFU/mL with PBS. Ninety and 180 days after immunization, the groups of mice were intranasally challenged with the Mtb suspension (500 CFU/40 µL in one nostril). To conﬁrm the bacterial load used, a single mouse from each group was euthanized at day 1 post-inoculation, and the lung homogenates were plated on 7H11-OADC agar. To determine protection, thirty days after infection, animals were euthanized, and the anterior and mediastinal right-lung lobes were collected, homogenized, and plated on 7H11-OADC agar. The bacterial load The LTK63 is a genetically detoxiﬁed E. coli heat-labile enterotoxin mutant that exhibits a potent mucosal adjuvanticity. It has been shown that LTK63 can activate several components of the immune response, including the recruitment and activation of neutrophils, NK cells, macrophages, dendritic cells, and B and T cells (18). We have previously developed a recombinant BCG (rBCG) strain expressing the subunit A of LTK63 as an adjuvant Frontiers in Immunology 02 frontiersin.org 10.3389/ﬁmmu.2023.1205449 Marques-Neto et al. lungs for each group in both time points (Figure 2B). Supplementary Figure 3 displays an example of lymph nodes analysis, based on FMO of a single functional T cell, producing IFN-g (Supplementary Figure 3A), TNF-a (Supplementary Figure 3B), or IL-17 (Supplementary Figure 3C). Cytokine events were background corrected based on this FMO. was determined by counting the CFU numbers after 14-21 days of incubation at 37°C. 3 Results Cells were plated in 96-well plates (CellWells™) and stimulated with 10 µg of BCG CFP (“culture ﬁltrate protein”, a proteinaceous supernatant of a BCG grown in Sauton medium for 14 days and concentrated through a 5,000 MWCO ﬁlter), ConA (positive control) or left unstimulated and incubated at 37°C and 5% CO2 for 4 h. Then, monensin (3 µM; eBioscience) was added and cultures were further incubated for another 4 h. Cells were then treated with 0.1% sodium azide (Sigma-Aldrich) in PBS for 30 min at room temperature and centrifuged at 400 x g for 15 min. The cellular phenotype was determined by permeabilization with Perm Fix/Perm Wash (BD Pharmingen) and incubation for 30 min with the following conjugated antibodies: TNF-a-FITC (clone MP6- XT22), IFN-g-PE (clone XMG1.2), CD4-PerCP (clone RM4-5), CD44-APCcy7 (clone IM7), IL-17-BV421 (clone TC11-18H10), CD62L-FITC (clone MEL-14), PD-1-PE (clone J43), KLRG1-APC (clone 2F1). 2.3 Cellular immune responses in draining lymph nodes and lungs The number of cells in each organ was quantiﬁed by multiplying the percentage of cells in each gate by the number of live cells counted in the Neubauer chamber. Flow cytometry analysis for speciﬁc effector T cell and memory T cell were performed as described in previous protocols (22, 23). Brieﬂy, 90 and 180 days after the immunization, axillar draining lymph nodes and lung lobes were collected. Draining lymph nodes were prepared as single-cell suspensions using 70-µm cell strainers (BD Biosciences), and the cells were resuspended in RPMI-1640 medium supplemented with 10% fetal calf serum, 0.15% sodium bicarbonate, 1% L-glutamine and 1% nonessential amino acids. 2.4 Statistical analysis Results were tabulated using the software GraphPad Prism 9 (GraphPad, La Jolla, CA, USA). The violin plot was plotted in Origin (Pro), Version Number (2022b – OriginLab Corporation, Northampton, MA, USA). The differences between groups were assessed using one-way ANOVA. Differences in p values < 0.05 were considered statistically signiﬁcant. All biological experiments were performed at least twice, repeating the immunization and assessments of immune response and protection”. Lung lobes were digested with DNAse IV (30 µg/mL) and collagenase III (0.7 mg/mL) for 30 min at 37°C. The digested lungs were prepared as single-cell suspensions using 70-µm cell strainers and erythrocytes lysed using an RBC lysing solution (0.15 M NH4Cl, 10 mM KHCO3). For both organs, viable cells were counted in a Neubauer chamber using Tripan Blue (0.2%), and cell concentration was adjusted to 1×106 cells/mL. All reagents were purchased from Sigma-Aldrich®, Merck KGaA, St. Louis, MO, USA. Frontiers in Immunology 3.1 rBCG-LTAK63 improves Th1, Th17, memory T cells, and protection, 90 days after immunization Protection against TB is correlated with an increased Th1/Th17 cytokine response observed at the time of challenge (Figure 1A). In agreement, here we show that mice immunized with rBCG-LTAK63 displayed a general increase in the Th1 and Th17 cell populations. At 90 days after immunization, there was an increase of a diverse milieu of CD4+ T cells expressing TNF-a, IFN-g and IL-17 either alone or in combination (double and triple polyfunctional cells) in draining lymph nodes and lungs (Figures 1B, C). In the lymph nodes, rBCG-LTAK63 immunization induced an increased percentage of CD4+ single TNF-a and IL-17-producing cells, and combinations of double TNF-a, IFN-g, and IL-17- producing cells at 90 days. The most signiﬁcant differences in terms of percentage and in the difference as compared to BCG were in CD4+TNF-a+ single positive, CD4+IFN-g+IL-17+ (double positive), and the triple polyfunctional T cells (Figure 1B). In the lungs as the target organ, the single CD4+ T cells producing IFN-g and TNF-a, and the double polyfunctional T cells were also increased as compared to BCG. In this case, the largest differences were seen with the CD4+IFN-g+ and the triple positive CD4+TNF- a+IFN-g+IL-17+ T cells (Figure 1C). Cell acquisition of 70,000 (draining lymph nodes) and 200,000 (lungs) total events per sample was performed using a BD FACS Canto II ﬂow cytometer and data analyzed using FlowJo™v10 Software (BD Life Sciences). The CD4+ effector T cell population was characterized as to expression of IFN-g, TNF-a and/or IL-17, either as single, double, or triple-positive cells. Memory T cell population was characterized as: naïve (CD4+CD44-CD62L+), central memory (TCM- CD4+CD44+CD62L+), effector memory (TEM-CD4+CD44+CD62L-), and tissue resident memory (TRM-CD4+PD-1+KLRG1-) cells. Regarding the numbers of polyfunctional T cells in the lymph node, the triple positive CD4+TNF-a+IFN-g+IL-17+ T cells showed the largest difference compared to BCG (Figure 1C). In the lungs, the double positive CD4+TNF-a+IL-17+ T cells were in larger numbers in the rBCG-LTAK63-immunized animals as compared to the BCG group, with a corresponding decrease in the numbers of CD4+IFN-g+IL-17+ T cells (Figure 1D). The gating strategy for all memory T cell subsets is shown in Supplementary Materials. Supplementary Figure 1 depicts gating for naïve/TEM/TCM (Supplementary Figure 1A) and an example of analysis in the lymph node for each group in both time points (Figure 1B). 3.1 rBCG-LTAK63 improves Th1, Th17, memory T cells, and protection, 90 days after immunization Supplementary Figure 2 shows gating for TRM (Supplementary Figure 2A) and an example of analysis in the Frontiers in Immunology 03 frontiersin.org 10.3389/ﬁmmu.2023.1205449 Marques-Neto et al. B C D A FIGURE 1 Increased induction of Th1, Th17, and polyfunctional cells in the draining lymph nodes and lungs of rBCG-LTAK63-immunized mice, 90 days after immunization. (A) Experimental design of the long-term immune response and protection performed. Created with BioRender.com. Twenty animals were immunized on day 0 with wild-type BCG or rBCG-LTAK63 or mock saline (n=20 per group). Immune responses were evaluated 90 and 180 days after immunization (n=5 per group). Challenges were performed 90 and 180 days after immunization and the protection was evaluated 30 days later (n=5 per group). In the last challenge evaluation (210 days after immunization) the immune response was also measured. Groups of BALB/c mice (n=5/group) were subcutaneously immunized with wild-type BCG or rBCG-LTAK63, and control groups received saline. Axillary lymph nodes (B) and lungs (C) were collected at 90 days after immunization and cellular suspensions were re-stimulated with CFP (culture ﬁltrate proteins) to evaluate the presence of CD4+ single and polyfunctional effector T cell subsets. Violin plots with box whiskers represent the data distribution, ( ) A A B C D B B C D C C FIGURE 1 Increased induction of Th1, Th17, and polyfunctional cells in the draining lymph nodes and lungs of rBCG-LTAK63-immunized mice, 90 days after immunization. (A) Experimental design of the long-term immune response and protection performed. Created with BioRender.com. Twenty animals were immunized on day 0 with wild-type BCG or rBCG-LTAK63 or mock saline (n=20 per group). Immune responses were evaluated 90 and 180 days after immunization (n=5 per group). Challenges were performed 90 and 180 days after immunization and the protection was evaluated 30 days later (n=5 per group). In the last challenge evaluation (210 days after immunization) the immune response was also measured. Groups of BALB/c mice (n=5/group) were subcutaneously immunized with wild-type BCG or rBCG-LTAK63, and control groups received saline. Axillary lymph nodes (B) and lungs (C) were collected at 90 days after immunization and cellular suspensions were re-stimulated with CFP (culture ﬁltrate proteins) to evaluate the presence of CD4+ single and polyfunctional effector T cell subsets. Violin plots with box whiskers represent the data distribution, median and outliers. (D) The pie charts depict the number of polyfunctional cells in evaluated organs. 3.2 The protective immune response induced by rBCG-LTAK63 immunization is maintained for up to 180 days after immunization To determine if the enhanced TEM and TCM cells at 90 days could increase the duration of protection, mice were immunized subcutaneously with 106 CFU (BCG or rBCG-LTAK63), and we assessed TEM and TCM generation, and protection against challenge 180 days later. At 180 days, the CD4+ T cells expressing TNF-a, IFN-g, or IL-17 remained at higher levels in rBCG- LTAK63-immunized animals in comparison to the BCG group in both organs (Figures 3A, B). In the lymph nodes, only CD4+TNF- a+IL-17+ double positive is present at a higher level (Figure 3A), while in the lungs, CD4+TNF-a+IL-17+, CD4+IFN-g+IL-17+ double positives are increased, together with the triple-positive T cells (Figure 3B). We had previously shown that rBCG-LTAK63-immunization induces protection against Mtb challenge in the intratracheal model of infection, 90 days after immunization (20). Hence, we here conﬁrmed protection against Mtb challenge using the intranasal model, 90 days after immunization with rBCG-LTAK63. Animals were administered 500 CFU of M. tuberculosis H37Rv intranasally and the bacterial load in the lungs was measured thirty days after the challenge. Also in the intranasal infection model, rBCG- LTAK63 immunization induces better protection than BCG, B C A FIGURE 2 Generation of memory T cells and protection of mice immunized with rBCG-LTAK63, 90 days after immunization. BALB/c mice (n=5/group) were immunized with either BCG or rBCG-LTAK63 (106 CFU); control groups received saline. Lymph node and lung cells were isolated after 90 days and were in vitro re-stimulated with CFP to evaluate memory T cell subsets. Memory T cells were characterized as naïve T cells (CD4+CD44-CD62L+), central memory T cells (TCM-CD4+CD44+CD62L+), effector memory T cells (TEM - CD4+CD44+CD62L-) present in the lymph nodes (A) and lungs (B) of immunized animals. Tissue-resident memory T cells were characterized as CD4+PD-1+KLRG-1- in the animal’s lungs (B). Violin plots with box whiskers represent the data distribution, median, and outliers. (C) Immunized and control animals were challenged intranasally with 500 CFU of M. tuberculosis H37Rv 90 days after immunization, and the lung bacillary load was assessed 30 days after infection. () Displays the statistical comparison between groups (p ≤0.05, p ≤0.01, p ≤0.001). Differences were considered statistically signiﬁcant when p ≤0.05 as compared to the saline or BCG group (one-way ANOVA). Bars represent mean ± S.D. The () above violin plots indicated comparisons with the saline control and the () bar showed all other group comparisons. 3.1 rBCG-LTAK63 improves Th1, Th17, memory T cells, and protection, 90 days after immunization () Represents the statistical comparison between groups (p ≤0.05, p ≤0.01, *p ≤0.001). Differences were considered statistically signiﬁcant when p ≤0.05 as compared to saline or BCG group (one-way ANOVA). The () above violin plots indicated comparison with the saline control and the () bar showed all other group comparisons. The ﬁgure shows a representative of two independent experiments. 04 Frontiers in Immunology Frontiers in Immunology frontiersin.org Marques-Neto et al. Marques-Neto et al. 10.3389/ﬁmmu.2023.1205449 Since an increased presence of effector CD4+ T cells was observed until 90 days after the immunization with rBCG- LTAK63, we assessed vaccine-induced memory T cells in the draining lymph nodes and lungs of immunized mice. In the lymph nodes, mice immunized with rBCG-LTAK63 displayed a tendency to decrease the naïve T cell population and signiﬁcantly increased TCM and TEM cells as compared to BCG (Figure 2A). In the lungs, the same tendency was observed; in this case, rBCG- LTAK63 immunization displayed signiﬁcantly larger percentages of the TCM and TEM cell populations. There was a trend to an increase in TRM in rBCG-LTAK63-immunized animals as compared to the saline group; however, this increase was not signiﬁcant (p value 0.17) (Figure 2B). reducing the bacillary load by more than two logs as compared to the non-immunized group and one log as compared to BCG (Figure 2C). 3.2 The protective immune response induced by rBCG-LTAK63 immunization is maintained for up to 180 days after immunization The ﬁgure shows a representative of two independent experiments. B A A C C FIGURE 2 Generation of memory T cells and protection of mice immunized with rBCG-LTAK63, 90 days after immunization. BALB/c mice (n=5/group) were immunized with either BCG or rBCG-LTAK63 (106 CFU); control groups received saline. Lymph node and lung cells were isolated after 90 days and were in vitro re-stimulated with CFP to evaluate memory T cell subsets. Memory T cells were characterized as naïve T cells (CD4+CD44-CD62L+), central memory T cells (TCM-CD4+CD44+CD62L+), effector memory T cells (TEM - CD4+CD44+CD62L-) present in the lymph nodes (A) and lungs (B) of immunized animals. Tissue-resident memory T cells were characterized as CD4+PD-1+KLRG-1- in the animal’s lungs (B). Violin plots with box whiskers represent the data distribution, median, and outliers. (C) Immunized and control animals were challenged intranasally with 500 CFU of M. tuberculosis H37Rv 90 days after immunization, and the lung bacillary load was assessed 30 days after infection. () Displays the statistical comparison between groups (p ≤0.05, p ≤0.01, *p ≤0.001). Differences were considered statistically signiﬁcant when p ≤0.05 as compared to the saline or BCG group (one-way ANOVA). Bars represent mean ± S.D. The () above violin plots indicated comparisons with the saline control and the () bar showed all other group comparisons. The ﬁgure shows a representative of two independent experiments. 05 Frontiers in Immunology frontiersin.org 10.3389/ﬁmmu.2023.1205449 Marques-Neto et al. B A B C A B C A FIGURE 3 Increased induction of Th1, Th17, and polyfunctional cells in the draining lymph nodes and lungs of rBCG-LTAK63-immunized mice, 180 days after immunization. Groups of BALB/c mice (n=5/group) were subcutaneously immunized with BCG or rBCG-LTAK63; the control group received saline. Axillary lymph nodes (A) and lungs (B) were collected at 180 days after immunization and cellular suspensions were re-stimulated with CFP (culture ﬁltrate proteins) to evaluate the presence of CD4+ effector T cell subsets. Violin plots with box whiskers represent the data distribution, median, and outliers. (C) The pie chart depicts the number of polyfunctional cells in evaluated organs. () Displays the statistical comparison between groups (p ≤0.05, p ≤0.01, *p ≤0.001). Differences were considered statistically signiﬁcant when p ≤0.05 as compared to saline or BCG group (one- way ANOVA). The () above violin plots indicated comparison with the saline control and the () bar showed all other group comparisons. 3.2 The protective immune response induced by rBCG-LTAK63 immunization is maintained for up to 180 days after immunization The ﬁgure shows a representative of two independent experiments. B B FIGURE 3 Increased induction of Th1, Th17, and polyfunctional cells in the draining lymph nodes and lungs of rBCG-LTAK63-immunized mice, 180 days after immunization. Groups of BALB/c mice (n=5/group) were subcutaneously immunized with BCG or rBCG-LTAK63; the control group received saline. Axillary lymph nodes (A) and lungs (B) were collected at 180 days after immunization and cellular suspensions were re-stimulated with CFP (culture ﬁltrate proteins) to evaluate the presence of CD4+ effector T cell subsets. Violin plots with box whiskers represent the data distribution, median, and outliers. (C) The pie chart depicts the number of polyfunctional cells in evaluated organs. () Displays the statistical comparison between groups (p ≤0.05, p ≤0.01, *p ≤0.001). Differences were considered statistically signiﬁcant when p ≤0.05 as compared to saline or BCG group (one- way ANOVA). The () above violin plots indicated comparison with the saline control and the () bar showed all other group comparisons. The ﬁgure shows a representative of two independent experiments. challenge with Mtb, reducing the bacillary load in the animals’ lungs by nearly two logs (Figure 4C). Regarding the number of polyfunctional T cells in the lymph node, the double positive CD4+IFN-g+IL-17+ T cells displayed the largest difference compared to BCG (Figure 3C). In the lungs, the double positive CD4+TNF-a+IFN-g+ T cells were in larger numbers in both groups, but the CD4+IFN-g+IL-17+ T cells were higher in the rBCG-LTAK63 group (Figure 3C). Frontiers in Immunology 4 Discussion TCM population showed no signiﬁcant difference between groups in both organs as compared to BCG. In this study, we show that immunization with rBCG-LTAK63 produces a broader range of effector cells than BCG. It also stimulates the production of more memory cells, primarily TCM. This leads to superior and longer-lasting protection against Mycobacterium tuberculosis. To obtain protection against TB, several CD4+ T cell subsets should be induced by immunization. Initially, Th1 and Th17 are the main effector cells associated with protection (24). Together, pre-existent TCM, after antigen re- exposure or infection, differentiates into TEM and then into Th1 or Th17 cells that migrate and exert their effector functions in infected tissues. A proportion of these T cells subsequently remain in the lung as TRM and constitute an efﬁcient frontline defense in the organ. These also can turn into Th1/Th17 effector cells, or rapidly recruit new effector cells after infection. In a chronic infection like Mtb, the longevity of the immune response and its resistance to continuous antigen exposure without exhaustion, is of equal importance. Hence, cells with lower expression of KLRG1 play a central role, because their proliferative potential can maintain the T cells in the tissue as the infection lasts (8, 25). The increase in the TEM population in the infected animal’s lungs indicates a possible differentiation from TCM into TEM and further into effector cells. Therefore, we also evaluated the Th1 and Th17 responses. The infection with Mtb increases CD4+TNF-a+, CD4+IFN-g+, and CD4+IL-17+ in the lymph nodes of rBCG- LTAK63 immunized animals (Figure 5C), while in the lungs, there was a drastic difference in CD4+IFN-g+, and CD4+IL-17+, when compared with BCG (Figure 5D). Finally, we compared the cell population dynamics across all time points during a longer period of immunization and infection. Regardless of the vaccine used, we can see that after a long period of immunization (180 dpi - before challenge), there is a tendency to decrease in all populations studied, most notably in the lungs of animals (Figure 6). Following infection, there is a decrease in the population of TCM cells in both organs and a considerable increase in the TEM cells in the lungs of the animals immunized with rBCG- LTAK63 (Figures 6G–J). 3.3 Challenge with Mtb induces TEM differentiation and Th1/Th17 recall in animals immunized with rBCG-LTAK63 An increase in TEM cell populations occurs in both organs at 180 days after rBCG-LTAK63 immunization, while there is no alteration of TEM in BCG groups (Figures 4A, B). Only rBCG- LTAK63 showed a higher percentage of TCM in the draining lymph node, as compared to BCG (Figure 4A). There is also an increase in TRM cells in the lungs of rBCG-LTAK63-immunized animals (Figure 4B). Mice were immunized with BCG or rBCG-LTAK63, challenged with Mtb 180 days later, and the memory cells (naive, TCM, and TEM) were examined using ﬂow cytometry 30 days later. TEM response in infected animal lymph nodes was higher in animals immunized with rBCG-LTAK63 than in those only infected (Figure 5A). In the lungs, TEM was higher in rBCG-LTAK63 group than in both the BCG and infected groups (Figure 5B). In terms of protection, even after 180 days, rBCG-LTAK63 immunization sustained higher protection against intranasal Frontiers in Immunology frontiersin.org 06 10.3389/ﬁmmu.2023.1205449 Marques-Neto et al. B C A FIGURE 4 Generation of memory T cells and protection of mice immunized with rBCG-LTAK63, 180 days after immunization. BALB/c mice (n=5/group) were immunized with either BCG or rBCG-LTAK63 (106 CFU); the control group received saline. Lymph node and lung cells were isolated after 180 days and in vitro re-stimulated with CFP to evaluate memory T cell subsets. (A) Memory T cells were characterized as naïve T cells (CD4+CD44-CD62L+), central memory T cells (TCM-CD4+CD44+CD62L+), effector memory T cells (TEM-CD4+CD44+CD62L-) present in the lymph nodes (A) and lungs (B) of immunized animals. Tissue-resident memory T cells were characterized as CD4+PD-1+KLRG-1- in the animal’s lungs (B). Violin plots with box whiskers represent the data distribution, median and outliers. (C) Animals were challenged intranasally with 500 CFU of Mycobacterium tuberculosis H37Rv 180 days after immunization, and the lung bacillary load was assessed 30 days after infection. () Displays the statistical comparison between groups (p ≤0.05, p ≤0.01, *p ≤0.001). Differences were considered statistically signiﬁcant when p ≤0.05 as compared to saline or BCG group (one-way ANOVA). Bars represent mean ± S.D. The () above violin plots indicated comparison with the saline control and the () bar showed all other group comparisons. The ﬁgure shows a representative of two independent experiments. B A C C GU Generation of memory T cells and protection of mice immunized with rBCG-LTAK63, 180 days after immunization. 3.3 Challenge with Mtb induces TEM differentiation and Th1/Th17 recall in animals immunized with rBCG-LTAK63 BALB/c mice (n=5/group) were immunized with either BCG or rBCG-LTAK63 (106 CFU); the control group received saline. Lymph node and lung cells were isolated after 180 days and in vitro re-stimulated with CFP to evaluate memory T cell subsets. (A) Memory T cells were characterized as naïve T cells (CD4+CD44-CD62L+), central memory T cells (TCM-CD4+CD44+CD62L+), effector memory T cells (TEM-CD4+CD44+CD62L-) present in the lymph nodes (A) and lungs (B) of immunized animals. Tissue-resident memory T cells were characterized as CD4+PD-1+KLRG-1- in the animal’s lungs (B). Violin plots with box whiskers represent the data distribution, median and outliers. (C) Animals were challenged intranasally with 500 CFU of Mycobacterium tuberculosis H37Rv 180 days after immunization, and the lung bacillary load was assessed 30 days after infection. () Displays the statistical comparison between groups (p ≤0.05, p ≤0.01, *p ≤0.001). Differences were considered statistically signiﬁcant when p ≤0.05 as compared to saline or BCG group (one-way ANOVA). Bars represent mean ± S.D. The () above violin plots indicated comparison with the saline control and the () bar showed all other group comparisons. The ﬁgure shows a representative of two independent experiments. 4 Discussion At the same time, there is an increase in the TNF-a (6B) and IL-17 (6F) producing CD4+ T cells in the lungs of the rBCG-LTAK63 group, but they remain stable in the BCG group. Frontiers in Immunology frontiersin.org 07 10.3389/ﬁmmu.2023.1205449 Marques-Neto et al. B C D A FIGURE 5 rBCG-LTAK63 induces higher effector and effector memory T cell after infection. BALB/c mice (n=5/group) were immunized with either BCG or rBCG-LTAK63 (106 CFU); the control group received saline. Animals were challenged intranasally with 500 CFU of Mycobacterium tuberculosis H37Rv 180 days after immunization; lymph nodes and lung cells were isolated 30 days after infection. Memory T cells were characterized as naïve T cells (CD4+CD44-CD62L+), central memory T cells (TCM-CD4+CD44+CD62L+), effector memory T cells (TEM - CD4+CD44+CD62L-) present in the lymph nodes (A) and lungs (B) of immunized animals. The lymph nodes (C) and lung (D) cells were isolated 30 days after infection to evaluate the presence of CD4+ effector T cell subsets. Violin plots with box whiskers represent the data distribution, median, and outliers. () Displays the statistical comparison between groups (p ≤0.05, *p ≤0.001). Differences were considered statistically signiﬁcant when p ≤0.05 as compared to infection or BCG (one-way ANOVA). The () above violin plots indicated comparison with the saline control and the () bar showed all other group comparisons. The ﬁgure shows a representative of two independent experiments. B A B C D D FIGURE 5 rBCG-LTAK63 induces higher effector and effector memory T cell after infection. BALB/c mice (n=5/group) were immunized with either BCG or rBCG-LTAK63 (106 CFU); the control group received saline. Animals were challenged intranasally with 500 CFU of Mycobacterium tuberculosis H37Rv 180 days after immunization; lymph nodes and lung cells were isolated 30 days after infection. Memory T cells were characterized as naïve T cells (CD4+CD44-CD62L+), central memory T cells (TCM-CD4+CD44+CD62L+), effector memory T cells (TEM - CD4+CD44+CD62L-) present in the lymph nodes (A) and lungs (B) of immunized animals. The lymph nodes (C) and lung (D) cells were isolated 30 days after infection to evaluate the presence of CD4+ effector T cell subsets. Violin plots with box whiskers represent the data distribution, median, and outliers. () Displays the statistical comparison between groups (p ≤0.05, *p ≤0.001). Differences were considered statistically signiﬁcant when p ≤0.05 as compared to infection or BCG (one-way ANOVA). 4 Discussion The () above violin plots indicated comparison with the saline control and the () bar showed all other group comparisons. The ﬁgure shows a representative of two independent experiments. The protective mechanism(s) of polyfunctional CD4+ T cells induced by vaccines or natural infection are still unknown. However, it has been considered that cells that express multiple effector functions may be more effective at controlling Mtb infection than cells that produce a single cytokine. We had previously shown that rBCG-LTAK63 elicited an increased protective response (as compared with BCG) when immunized mice were challenged with H37Rv or a highly virulent Beijing strain (intratracheally) at 90 days after immunization (20). Here we conﬁrmed the previous results in an intranasal challenge model and show that when immunized mice were challenged after 180 days, this improved protection is maintained (Figures 2, 4). Immunization with rBCG-LTAK63 increases Th1 and Th17 single and polyfunctional responses in the lymph node and lungs, for up to 180 days, in contrast to BCG. This long-term protective response is directly associated with the production of Th1 responses, which activate macrophages, stimulate phagocytosis, phagosome maturation, nitrogen reactive production, and improve antigen presentation (26). At the same time, Th17 cells mediate antibacterial and pro-inﬂammatory responses, contributing to the generation of protective immune responses and memory cells, and support Th1 cell reactivity by down-regulating IL-10 and up-regulating IL-12 production. These responses can protect against tuberculosis infection in the absence of a Th1 response (27, 28). We have previously demonstrated that intraperitoneal inoculation of rBCG-LTAK63 induced increased recruitment of CD4+ lymphocytes (19). Moreover, in vitro studies with human macrophages demonstrated that rBCG-LTAK63 upregulated interferon-inducible, antimicrobial, and inﬂammatory cytokines, and induced tissue repair genes when compared to BCG. Speciﬁcally, rBCG-LTAK63-infected macrophages produced higher levels of inﬂammatory cytokines including IL-12(p70), Frontiers in Immunology frontiersin.org 08 Marques-Neto et al. 10.3389/ﬁmmu.2023.1205449 B C D E F G H I J A URE 6 namics of the T cell population show increased TEM and effector T cells after Mtb challenge in the lungs of rBCG-LTAK63 immunized animals. 4 Discussion lution of the cell populations of immunized animals at 90 days (90dpi) and 180 days after immunization (180 dpi), and 30 days after challenge dpc): CD4+TNF-a+ T cells in lymph nodes (A) and lungs (B); CD4+IFN-g+ T cells in lymph nodes (C) and lungs (D); CD4+IL-17+ T cells in lymph es (E) and lungs (F); TCM (CD4+CD44+CD62L+) cell populations in lymph nodes (G) and lungs (H); TEM (CD4+CD44+CD62L-) in the lymph es (I) and lungs (J). Bars represent ± S.D. Statistical difference (p ≤0.05) as compared to the prior timepoint in two-way ANOVA test. B A A C D D C E E F G H G G H J J I I I J J FIGURE 6 Dynamics of the T cell population show increased TEM and effector T cells after Mtb challenge in the lungs of rBCG-LTAK63 immunized animals. Evolution of the cell populations of immunized animals at 90 days (90dpi) and 180 days after immunization (180 dpi), and 30 days after challenge (30dpc): CD4+TNF-a+ T cells in lymph nodes (A) and lungs (B); CD4+IFN-g+ T cells in lymph nodes (C) and lungs (D); CD4+IL-17+ T cells in lymph nodes (E) and lungs (F); TCM (CD4+CD44+CD62L+) cell populations in lymph nodes (G) and lungs (H); TEM (CD4+CD44+CD62L-) in the lymph nodes (I) and lungs (J). Bars represent ± S.D. *Statistical difference (p ≤0.05) as compared to the prior timepoint in two-way ANOVA test. TNF-a, and IL-15 (29). Our work demonstrates that immunization with rBCG-LTAK63 induces TCM cells in the lymphoid organ (Figure 2), as well as TRM cells in the lungs (Figure 4). IL-15 (together with IL-7 and IL-2) plays a crucial function in memory T cell development and homeostasis and may explain the TRM and TEM generation. However, the TCM generation seems to be IL-15 independent, and the mechanism by which rBCG-LTAK63 induces TCM is still unknown (30–32). In the TCM and TEM cell population study, it was demonstrated that rBCG-LTAK63 enhances the TCM response and, as expected, this response is maintained in the lymphoid organ while also increased in the animal’s lungs. This improvement is one of the most auspicious characteristics of rBCG-LTAK63 described here. In adoptive transfer studies, TCM generated by VPM1002 immunization was demonstrated to be partly responsible for its increased protection (10). Frontiers in Immunology 09 frontiersin.org Marques-Neto et al. 10.3389/ﬁmmu.2023.1205449 Marques-Neto et al. 4 Discussion After the infection, it is expected that the TCM cells differentiate into TEM cells, which migrate from the lymphoid organ to the lungs (3). TCM are not different between the recombinant vaccine and wild- type BCG, while TEM cells are increased in the lungs after infection (Figures 5, 6). This can indicate a possible differentiation of TCM into TEM. Differentiation of TEM will induce an increase in effector T cells (Th1/Th17), and we can see this enhancement in lymph node CD4+TNF-a+/CD4+IFN-g+/CD4+IL-17+, and in the lungs CD4+IFN- g+/CD4+IL-17+ (Figure 6). Our previous work showed that rBCG- LTAK63 reduces NF-kB, IL-12, IFN-g, TNF-a, and IL-17 after challenge while increasing TGF-b (20). Our results differ from the previous one, most likely due to the method used. In that case, cytokine production was evaluated using RNA transcription, which measures the total cytokine expressed in the tissue. The reduction in total inﬂammatory cytokine production correlates with the decrease in CFU and in the inﬂammation area. Here we show the increase in speciﬁc T-cell response, which agrees with the later paper that showed an increase in CD4+TNF-a+ cells in animals immunized with rBCG- LTAK63, ﬁfteen days after H37Rv infection (19). correlates with the longer-lasting protection observed against challenge. These ﬁndings suggest that rBCG-LTAK63 can induce a more durable and stable immune response and protection, which could address some of the current BCG vaccine issues. Funding We acknowledge the support from FAPESP (Projects 2017/ 24832-6, 2019/06454-0 and 2019/02305-0) and Fundação Butantan. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Conﬂict of interest LL has a patent application on the use of rBCG-LTAK63 as a vaccine against Mtb. The remaining 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. The T CD8 cell populations did not reveal any signiﬁcant differences between BCG and rBCG-LTAK63 (data not shown). The genetically detoxiﬁed LTKA63 protein does not display the same toxicity as LTA, which is an adenylyl cyclase activator; however, LTKA63 maintains part of the adjuvanticity of the original protein. Since neither LTA nor LTAK63 produce cross- presentation, phagosome scape, or any other CD8-inducing function, it was not expected that rBCG-LTAK63 would have this effect. It is important to note that here we do not explore the inﬂuence of rBCG-LTAK63 on crucial cell populations involved in tuberculosis protection and protective immunity development (i.e., dendritic cells, monocytes, macrophages), and we use a single gender and mouse strain (34, 35). The next stages should address these limitations using mice strains with diverse tuberculosis susceptibility (e.g., CBA, C3HeB/FeJ, DBA/2, and 129SvJ), different animal genders, and evaluating other possible processes associated with the rBCG-LTAK63 protective effect. Author contributions LM-N, MT, DR, AK, and LL conceived and designed the experiments; MT and LM-N performed the experiments and collected data; LM-N, MT, DR, AK, and LL processed and analyzed the data; LM-N, MT, AK, and LL wrote the manuscript, and all authors critically revised the manuscript. The long-term protection induced against tuberculosis can be associated with other memory T cells such as the TRM cells; KLRG- 1/PD-1 marked T cells are one of the most prominent subsets (16, 17). TRM cells are non-lymphoid tissue memory cells that were shown to be induced in BCG only when the vaccine is intranasally delivered (15, 33). They are considered to be highly protective against tuberculosis (14, 33). Here, the immunization with BCG or rBCG-LTAK63 was performed subcutaneously. Surprisingly, rBCG-LTAK63 improved the generation of TRM (Figure 4), which reaches statistical signiﬁcance at 180 days after immunization. Again, this can be associated to IL-15 production, which also plays an important role in TRM generation and maintenance (30). It is important to observe that a limitation to this study subset is in the characterization of the TRM population. While the expression of PD-1+ KLRG1- has been used as a marker for TRM, these cells can also be found in the vasculature, BAL, and parenchyma. Therefore, in order to conﬁrm that these are actually lung tissue resident cells, we could include CXCR3 as a marker in vitro or perform in vivo CD45 labeling. Ethics statement The animal study was reviewed and approved by 3435250619. Frontiers in Immunology Marques-Neto et al. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu.2023.1205449/ full#supplementary-material Overall, our ﬁndings show that rBCG-LTAK63 immunization increased the levels of several memory T cell subsets, which Frontiers in Immunology frontiersin.org 10 10.3389/ﬁmmu.2023.1205449 Marques-Neto et al. References Neto LMM, Zufelato N, de Sousa-Júnior AA, Trentini MM, da Costa AC, Bakuzis AF, et al. Speciﬁc T cell induction using iron oxide based nanoparticles as subunit vaccine adjuvant. Hum Vaccin Immunother (2018) 14:2786–801. doi: 10.1080/ 21645515.2018.1489192 6. Roth A, Jensen H, Garly ML, Djana Q, Martins CL, Sodemann M, et al. Low birth weight infants and Calmette-Guérin bacillus vaccination at birth: community study from Guinea- Bissau. Pediatr Infect Dis J (2004) 23:544–50. doi: 10.1097/01.inf.0000129693.81082.a0 24. Cooper AM. Cell-mediated immune responses in tuberculosis. 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Nascimento IP, Rodriguez D, Santos CC, Amaral EP, Rofatto HK, Junqueira- Kipnis AP, et al. Recombinant BCG expressing LTAK63 adjuvant induces superior protection against Mycobacterium tuberculosis. Sci Rep (2017) 7:2109. doi: 10.1038/ s41598-017-02003-9 3. Angelidou A, Diray-Arce J, Conti MG, Smolen KK, van Haren SD, Dowling DJ, et al. BCG As a case study for precision vaccine development: lessons from vaccine heterogeneity, trained immunity, and immune ontogeny. Front Microbiol (2020) 11:332. doi: 10.3389/fmicb.2020.00332 3. Angelidou A, Diray-Arce J, Conti MG, Smolen KK, van Haren SD, Dowling DJ, et al. BCG As a case study for precision vaccine development: lessons from vaccine heterogeneity, trained immunity, and immune ontogeny. Front Microbiol (2020) 11:332. doi: 10.3389/fmicb.2020.00332 21. Logan KE, Gavier-Widen D, Hewinson RG, Hogarth PJ. Development of a Mycobacterium bovis intranasal challenge model in mice. Tuberculosis (2008) 88:437– 43. doi: 10.1016/j.tube.2008.05.005 4. Lalor MK, Floyd S, Gorak-Stolinska P, Ben-Smith A, Weir RE, Smith SG, et al. BCG Vaccination induces different cytokine proﬁles following infant BCG vaccination in the UK and Malawi. J Infect Dis (2011) 204:1075–85. doi: 10.1093/infdis/jir515 4. Lalor MK, Floyd S, Gorak-Stolinska P, Ben-Smith A, Weir RE, Smith SG, et al. BCG Vaccination induces different cytokine proﬁles following infant BCG vaccination in the UK and Malawi. J Infect Dis (2011) 204:1075–85. doi: 10.1093/infdis/jir515 22. Junqueira-Kipnis AP, de Oliveira FM, Trentini MM, Tiwari S, Chen B, Resende DP, et al. Prime-boost with Mycobacterium smegmatis recombinant vaccine improves protection in mice infected with Mycobacterium tuberculosis. PloS One (2013) 8: e78639. doi: 10.1371/journal.pone.0078639 5. Garly ML, Martins CL, Balé C, Baldé MA, Hedegaard KL, Gustafson P, et al. BCG Scar and positive tuberculin reaction associated with reduced child mortality in West Africa. a non-speciﬁc beneﬁcial effect of BCG? Vaccine (2003) 21:2782–90. doi: 10.1016/s0264-410x(03)00181-6 23. References The acquired immune response to the mucosal adjuvant LTK63 imprints the mouse lung with a protective signature. J Immunol (2007) 179:5346–57. doi: 10.4049/jimmunol.179.8.5346 35. Kurtz SL, Rossi AP, Beamer GL, Gatti DM, Kramnik I, Elkins KL. The diversity outbred mouse population is an improved animal model of vaccination against tuberculosis that reﬂects heterogeneity of protection. mSphere (2020) 5:10.1128. doi: 10.1128/msphere.00097-20 19. Carvalho Dos Santos C, Rodriguez D, Kanno Issamu A, Cezar De Cerqueira Leite L, Pereira Nascimento I. Recombinant BCG expressing the LTAK63 adjuvant 11 Frontiers in Immunology Frontiers in Immunology frontiersin.org
https://openalex.org/W2220948796	https://europepmc.org/articles/pmc4696659?pdf=render	English	null	Assessment of Oral Fluid HIV Test Performance in an HIV Pre-Exposure Prophylaxis Trial in Bangkok, Thailand	PloS one	2,015	public-domain	4,614	RESEARCH ARTICLE Background Rapid easy-to-use HIV tests offer opportunities to increase HIV testing among populations at risk of infection. We used the OraQuick Rapid HIV-1/2 antibody test (OraQuick) in the Bangkok Tenofovir Study, an HIV pre-exposure prophylaxis trial among people who inject drugs. Editor: Dimitrios Paraskevis, University of Athens, Medical School, GREECE Received: October 9, 2015 Accepted: December 9, 2015 Published: December 30, 2015 Editor: Dimitrios Paraskevis, University of Athens, Medical School, GREECE Received: October 9, 2015 Accepted: December 9, 2015 Published: December 30, 2015 Editor: Dimitrios Paraskevis, University of Athens, Medical School, GREECE Received: October 9, 2015 Accepted: December 9, 2015 Published: December 30, 2015 Assessment of Oral Fluid HIV Test Performance in an HIV Pre-Exposure Prophylaxis Trial in Bangkok, Thailand Pravan Suntharasamai1, Michael Martin2,3, Kachit Choopanya1, Suphak Vanichseni1, Udomsak Sangkum1, Pairote Tararut2, Wanna Leelawiwat2, Rapeepan Anekvorapong4, Philip A. Mock2, Thitima Cherdtrakulkiat2, Manoj Leethochawalit4, Sithisat Chiamwongpaet4, Roman J. Gvetadze3, Janet M. McNicholl3, Lynn A. Paxton3, Somyot Kittimunkong5, Marcel E. Curlin2,3 1 Bangkok Tenofovir Study Group, Bangkok, Thailand, 2 Thailand MOPH – U.S. CDC Collaboration, Nonthaburi, Thailand, 3 Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 4 Bangkok Metropolitan Administration, Bangkok, Thailand, 5 Thailand Ministry of Public Health, Nonthaburi, Thailand 1 Bangkok Tenofovir Study Group, Bangkok, Thailand, 2 Thailand MOPH – U.S. CDC Collaboration, Nonthaburi, Thailand, 3 Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 4 Bangkok Metropolitan Administration, Bangkok, Thailand, 5 Thailand Ministry of Public Health, Nonthaburi, Thailand a1111 Znd9@cdc.gov * Znd9@cdc.gov Methods The Bangkok Tenofovir Study was a randomized, double-blind, placebo-controlled trial. We tested participants’ oral fluid for HIV using OraQuick monthly and blood using a nucleic-acid amplification test (NAAT) every 3 months. We used Kaplan-Meier methods to estimate the duration from a positive HIV NAAT until the mid-point between the last non-reactive and first reactive oral fluid test and proportional hazards to examine factors associated with the time until the test was reactive. Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. OPEN ACCESS Citation: Suntharasamai P, Martin M, Choopanya K, Vanichseni S, Sangkum U, Tararut P, et al. (2015) Assessment of Oral Fluid HIV Test Performance in an HIV Pre-Exposure Prophylaxis Trial in Bangkok, Thailand. PLoS ONE 10(12): e0145859. doi:10.1371/ journal.pone.0145859 Methods The Bangkok Tenofovir Study was a randomized, double-blind, placebo-controlled trial con- ducted in 17 drug-treatment clinics that showed that daily oral tenofovir disoproxil fumarate (tenofovir) can reduce HIV transmission among people who inject drugs by 49%.[9] The study protocol, consent process, and trial materials were approved by the Bangkok Metropolitan Administration and Thailand Ministry of Public Health Ethical Review Committees and the U.S. Centers for Disease Control and Prevention (CDC) Institutional Review Board. An inde- pendent Data and Safety Monitoring Board conducted annual safety reviews and one interim efficacy review. Participants provided written informed consent to participant in the study. The trial was registered with ClinicalTrials.gov Identifier: NCT00119106. Trial Registration ClinicalTrials.gov NCT00119106. Assessment of Oral Fluid HIV Test Performance Introduction Globally, half of those infected with HIV do not know their status.[1] These individuals do not have access to life-saving antiretroviral therapy and may unknowingly transmit HIV to others. In addition, people often learn they are infected with HIV late in their illness.[2] In response, UNAIDS has urged countries to ensure that by 2020, 90% of people infected with HIV know their status,[3] and the World Health Organization has called for national HIV programs to identify people living with HIV as early as possible and link them to HIV services in a timely manner.[4] Rapid HIV tests have created opportunities to increase testing by providing results the same day, often within 30 minutes, and by extending testing beyond medical facilities.[5] In 2004, the U.S. Food and Drug Administration approved the OraQuick Advance Rapid HIV-1/2 Anti- body Test (OraSure Technologies, Inc., Bethlehem, Pennsylvania, USA) for use with oral fluid, and in 2012 it became the first over-the-counter HIV test approved for home use.[6] Oral fluid HIV tests have not been approved to diagnose HIV in Thailand.[7,8] We used an oral fluid HIV test in the Bangkok Tenofovir Study, an HIV pre-exposure pro- phylaxis trial among people who inject drugs.[9] We chose the test because it could be done in drug treatment clinics, provided a result in 20 minutes, did not require a blood draw, and had good reported sensitivity and specificity.[10] Here, we describe the performance of the test. Discussion Competing Interests: The authors have declared that no competing interests exist. The oral fluid HIV test performed well at screening, suggesting it can be used when rapid results and non-invasive tools are preferred. However, participants receiving tenofovir took longer to develop a reactive oral fluid test result than those receiving placebo. Thus, among people using pre-exposure prophylaxis, a blood-based HIV test may be an appropriate choice. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. We screened 3678 people for HIV using OraQuick. Among 447 with reactive results, 436 (97.5%) were confirmed HIV-infected, 10 (2.2%) HIV-uninfected, and one (0.2%) had inde- terminate results. Two participants with non-reactive OraQuick results were, in fact, HIV- infected at screening yielding 99.5% sensitivity, 99.7% specificity, a 97.8% positive predic- tive value, and a 99.9% negative predictive value. Participants receiving tenofovir took lon- ger to develop a reactive OraQuick (191.8 days) than participants receiving placebo (16.8 days) (p = 0.02) and participants infected with HIV CRF01_AE developed a reactive Ora- Quick earlier than participants infected with other subtypes (p = 0.04). Funding: The US Centers for Disease Control and Prevention (CDC) sponsored the trial in collaboration with the Bangkok Metropolitan Administration (BMA). CDC and BMA staff participated in study design, data collection, analysis, and interpretation of the results. The corresponding author had access to all the data and decided to submit the manuscript for publication. Study drug was donated by Gilead Sciences. PLOS ONE \| DOI:10.1371/journal.pone.0145859 December 30, 2015 1 / 9 Participants and procedures HIV-uninfected people who met eligibility criteria and provided written informed consent were randomly assigned to receive daily oral tenofovir 300 mg or placebo.[11] Participant screening and enrollment began in June 2005 and follow-up continued through October 2012. 2 / 9 PLOS ONE \| DOI:10.1371/journal.pone.0145859 December 30, 2015 Assessment of Oral Fluid HIV Test Performance At screening, enrollment, and each monthly visit, staff observed participants collect an oral fluid specimen and tested the specimen using OraQuick Rapid HIV-1/2 Antibody Test (Ora- Quick).[12] OraQuick is manufactured for distribution outside the United States and is identi- cal to the U.S. Food and Drug Administration approved OraQuick Advance Rapid HIV-1/2 Antibody test.[10] We confirmed reactive oral fluid HIV tests using enzyme-immunoassays (EIA) (Genetic Systems HIV-1/ HIV-2 plus O EIA, Redmond, WA, USA) and Western blot (Bio-Rad, Redmond, WA, USA). We collected blood specimens from participants at enrollment, months 1, 2, 3, and every 3 months thereafter, for safety assessments. Blood collected at 3-monthly visits from participants with non-reactive oral fluid HIV test results was tested for HIV using EIA. We tested the final blood specimens from participants with consistently non-reactive results for HIV using nucleic-acid amplification testing (NAAT) (Aptima HIV-1 RNA Qualitative Assay, GenProbe Inc, San Diego, CA, USA). Samples with positive NAAT results were confirmed and viral load determined using real-time polymerase chain reaction (PCR) (COBAS TaqMan v 1.0, Roche Molecular Systems, Branchburg, NJ, USA). We determined the last NAAT negative and first NAAT positive specimen using real-time PCR. The lower limit of detection was 47 copies/mL. We used TruGene (Siemens HealthCare Diagnostic, Tarrytown, NY, USA) to determine HIV subtype. OraSure personnel trained CDC staff on the biological principles of the OraQuick test, kit storage, specimen collection, testing and interpretation, and quality control.[10] CDC staff trained clinic-based staff who performed participant testing. After training, performance was assessed using proficiency panels from the Model Performance Evaluation Program and the Thailand National Institute of Health. CDC staff visited each clinic monthly to review HIV testing records and observe staff performing tests. Statistical analysis If the HIV NAAT was positive before the oral fluid test, we estimated the time until the oral fluid test was reactive using the date of the first positive NAAT and the mid-point date between the last non-reactive and first reactive oral fluid test. We used the Kaplan-Meier method to esti- mate the time from a positive HIV NAAT until a reactive oral fluid test result, using the mid- point date, and a Cox proportional hazards model, including study drug group, to determine factors associated with the time until the test was reactive using the likelihood ratio chi-square test.[13] We compared HIV RNA concentrations when the HIV NAAT was positive by study drug group using a non-parametric Wilcoxon test; to assess RNA concentration by other fac- tors, we adjusted for study drug group using van Elteren’s test.[13,14] We excluded data from participants lost to follow-up for more than 3 months who had a positive NAAT when they returned, and censored data of participants who did not have a reactive oral fluid HIV test the date of their final test. We used SAS version 9.3 (SAS Institute, Cary, North Carolina, USA) to analyze data. Assessment of Oral Fluid HIV Test Performance Fig 1. HIV test results of participants in the Bangkok Tenofovir Study, 2005–2012. OraQuick, OraQuick Rapid HIV-1/2 Antibody Test; EIA, enzyme immune assay; NAAT, nucleic-acid amplification test. aTwo participants with reactive OraQuick tests during follow-up were later found to have been HIV- infected before enrollment. bPlasma collected at 3-monthly visits from participants with a non-reactive OraQuick test result was tested for HIV using EIA. d i 10 1371/j l 0145859 001 Fig 1. HIV test results of participants in the Bangkok Tenofovir Study, 2005–2012. OraQuick, OraQuick Rapid HIV-1/2 Antibody Test; EIA, enzyme immune assay; NAAT, nucleic-acid amplification test. aTwo participants with reactive OraQuick tests during follow-up were later found to have been HIV- infected before enrollment. bPlasma collected at 3-monthly visits from participants with a non-reactive OraQuick test result was tested for HIV using EIA. doi:10.1371/journal.pone.0145859.g001 doi:10.1371/journal.pone.0145859.g001 a positive predictive value of 97.8% (95% CI: 88.8% to 100%), and a negative predictive value of 99.9% (95% CI: 96.5% to 100%). Among those screened, 2413 enrolled. Their median age was 31 years (range, 20–59), and 1924 (79.7%) were male. Enrolled participants had 115,241 oral fluid HIV tests done from June 2005 through June 2012, ranging from 1 to 88 tests per participant, and from 4079 to 10,973 tests per clinic. Among those enrolled, 62 participants had a reactive oral fluid HIV test result during fol- low-up; 46 (74.2%) participants were confirmed HIV-infected and 16 (25.8%) had falsely reac- tive results. Two of the confirmed HIV-infected participants were later found to have been infected before enrollment (described above) and excluded from analysis (Fig 1). The 16 falsely reactive results were reported from nine clinics and occurred between June 2006 and February 2012 during study visits ranging from the first monthly visit to the month 81 visit. Five partici- pants were diagnosed with HIV infection using EIA and Western Blot testing and did not have a reactive oral fluid HIV test result reported. Final blood specimens collected from two partici- pants tested positive for HIV using NAAT; one was confirmed, while the other could not be confirmed because there was insufficient blood and the participant could not be reached for repeat testing (Fig 1). Among the 50 participants with confirmed incident HIV infection, 10 were lost to follow- up for more than 3 months and returned HIV-infected. We excluded these participants from further analysis. Results From June 2005 through July 2010, 3678 people were screened using the oral fluid OraQuick HIV test. Among those screened, 447 participants had a reactive result: 436 (97.5%) were con- firmed HIV-infected, 10 (2.2%) had a falsely reactive result, and one (0.2%) had an indetermi- nate Western blot result and was excluded from analysis (Fig 1). Two participants with non- reactive oral fluid HIV test results were later found to have been HIV-infected before enroll- ment, based on blood EIA and Western blot testing. These results yielded a sensitivity of 99.5% (95% confidence interval [CI]: 90.4% to 100%), a specificity of 99.7% (95% CI: 96.3% to 100%), 3 / 9 PLOS ONE \| DOI:10.1371/journal.pone.0145859 December 30, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0145859 December 30, 2015 Of the 40 remaining participants, 14 (35.0%) had a reactive oral fluid HIV test the same day the NAAT was reactive, and, including these 14, 27 (67.5%) participants had reac- tive oral fluid HIV test results within 3 months (i.e., 84 days) of the positive NAAT. Staff 4 / 9 PLOS ONE \| DOI:10.1371/journal.pone.0145859 December 30, 2015 Assessment of Oral Fluid HIV Test Performance reported reactive test results for 8 (20.0%) participants more than 3 months after the positive NAAT. The eight participants came from six clinics, and the first positive NAAT results occurred between March 2006 and June 2010 and on study visits ranging from month 9 to month 54. In addition, 5 (11.9%) participants with non-reactive OraQuick results were diag- nosed with HIV infection using EIA and Western Blot testing. Censoring these five participants the day of their final OraQuick test, the median time from the NAAT to the mid-point between the last non-reactive and first reactive oral fluid HIV test for the 40 participants evaluated was estimated to be 17.4 days (i.e., 62% of a 28 day month; interquartile range [IQR], 2.8–131.3 days). The HIV RNA level was higher among participants receiving placebo (median = 227,863 copies/ml) at the time the NAAT was reactive than among participants receiving tenofovir (median = 5532 copies/ml; p = 0.02). We successfully amplified and sequenced HIV from 37 of the 40 HIV-infected participants evaluated; 32 (86.5%) were infected with subtype CRF01_AE, and the median plasma HIV RNA concentration was 161,878 copies/ml (IQR, 6683–541,521 copies/ml; geometric mean = 53,077 copies/ml) at the time the NAAT was positive. This was higher, but not statistically different (p = 0.15, controlling for study drug group), than the median (30,056 copies/ml; IQR, 3935–70,898 copies/ml; geometric mean = 14,840 copies/ml) of the eight participants infected with other subtypes (subtype B’ = 4, B’/CRF01_AE recombi- nant = 1, unable to type = 3). The median time from the first positive NAAT to the mid-point between the last non-reac- tive and first reactive oral fluid HIV test for participants randomized to tenofovir was 191.8 days and for participants randomized to placebo was 16.8 days (p = 0.02). The median time for participants infected with subtype CRF01_AE was 16.8 days and for participants infected with other subtypes was 191.8 days (controlling for study drug group, p = 0.04) (Fig 2A–2B and S1 Dataset). The difference between study drug groups remained when we controlled for subtype (p = 0.05). The median time from the first positive NAAT to the oral fluid HIV test mid-point did not differ by sex (p = 0.20), age (p = 0.73), or time until the oral fluid HIV test kit expiration date (p = 0.59). PLOS ONE \| DOI:10.1371/journal.pone.0145859 December 30, 2015 Discussion We successfully implemented oral fluid HIV testing in 17 drug-treatment clinics in Bangkok. The test sensitivity and specificity were consistent with performance measures reported by the manufacturer[10] and in other studies.[15,16] The positive predictive value of 97.8% in this population of people who inject drugs shows that the test is a useful screening tool, but positive results need to be confirmed with additional HIV testing. The negative predictive value was 99.9% suggesting that when used to screen for HIV infection, care providers and people who inject drugs can have confidence that a negative oral fluid HIV test result, in the absence of a recent HIV exposure, is accurate. The oral fluid HIV test was reactive within 3 months (i.e., 84 days) of the first positive NAAT in 27 (67.5%) participants with incident HIV infection. Falsely reactive oral fluid HIV test results did not cluster by clinic, time on study, or calendar time. Consistent with a previous study showing that antiretroviral therapy can decrease the sensitivity of the oral fluid HIV test, [17] we found that participants receiving tenofovir took longer to develop a reactive test result than participants receiving placebo. We also found that participants infected with subtype CRF01_AE developed a reactive oral fluid HIV test faster than participants infected with other subtypes. Participants with CRF01_AE infection had higher, but not statistically different, plasma HIV RNA concentrations the day the NAAT was positive than participants with other subtypes. Another larger study found that those infected with CRF01_AE had significantly 5 / 9 PLOS ONE \| DOI:10.1371/journal.pone.0145859 December 30, 2015 Assessment of Oral Fluid HIV Test Performance ig 2. Kaplan-Meier estimates of time (28 day months) from date HIV was detected using nucleic acid amplification until the etween the last non-reactive and first reactive oral fluid HIV test. (A) By study drug group: tenofovir or placebo. (B) Controlling fo IV subtype: CRF01_AE or other subtypes. oi:10.1371/journal.pone.0145859.g002 Fig 2. Kaplan-Meier estimates of time (28 day months) from date HIV was detected using nucleic acid amplification until the mid-point date between the last non-reactive and first reactive oral fluid HIV test. (A) By study drug group: tenofovir or placebo. (B) Controlling for study drug group, by HIV subtype: CRF01_AE or other subtypes. doi:10.1371/journal.pone.0145859.g002 Fig 2. Discussion Kaplan-Meier estimates of time (28 day months) from date HIV was detected using nucleic acid amplification until the mid-point date between the last non-reactive and first reactive oral fluid HIV test. (A) By study drug group: tenofovir or placebo. (B) Controlling for study drug group, by HIV subtype: CRF01_AE or other subtypes. Fig 2. Kaplan-Meier estimates of time (28 day months) from date HIV was detected using nucleic acid amplification until the mid-point date between the last non-reactive and first reactive oral fluid HIV test. (A) By study drug group: tenofovir or placebo. (B) Controlling for study drug group, by HIV subtype: CRF01_AE or other subtypes. doi:10.1371/journal.pone.0145859.g002 doi:10.1371/journal.pone.0145859.g002 PLOS ONE \| DOI:10.1371/journal.pone.0145859 December 30, 2015 6 / 9 Assessment of Oral Fluid HIV Test Performance higher plasma RNA concentrations during the first 3 months of infection than those infected with other subtypes.[18] Higher HIV RNA levels among participants with CRF01_AE infec- tion may speed or increase the antibody response, decreasing the time to a reactive oral fluid HIV test result. This study has several limitations. Oral fluid specimens must be evaluated within 40 min- utes and cannot be preserved; therefore, we were unable to re-test specimens to confirm the results. Oral fluid HIV tests were done monthly but blood was collected every 3 months. Thus, when NAAT testing of blood showed that HIV infection occurred before a reactive oral fluid HIV test, we had to estimate the time from HIV infection until the oral fluid test was reactive. Also, only 8 participants had positive HIV NAAT results more than 3 months before a reactive oral fluid test, limiting analysis of falsely non-reactive results. Thailand’s Ministry of Public Health has recognized the importance of increasing HIV test- ing, and has issued guidance for HIV testing with same day results.[19] The OraQuick test is easy to use, does not require a blood draw, and provides a result in 20 to 40 minutes, attributes that make it a useful tool for HIV screening in settings where laboratory facilities are not avail- able, and a rapid result and non-invasive tools are preferred. However, oral fluid-based tests are less sensitive than blood-based assays in people with early HIV infection.[16,20,21,22] Dur- ing the Bangkok Tenofovir Study, staff reported non-reactive monthly oral fluid HIV test results for 8 participants for more than 3 months (84 days) after HIV infection. Acknowledgments Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the U.S. Centers for Disease Control and Prevention. Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the U.S. Centers for Disease Control and Prevention. We thank the study participants, clinic staff, and the members of the Bangkok Tenofovir Study Group. necessarily represent the views of the U.S. Centers for Disease Control and Prevention. We thank the study participants, clinic staff, and the members of the Bangkok Tenofovir Study Group. Supporting Information S1 Dataset. Data used for Kaplan-Meier estimates and proportional hazards analysis. If the first positive HIV NAAT occurred the same day as the first reactive oral fluid HIV test, we esti- mated the time from positive NAAT to reactive oral fluid HIV test was 0 days. If the HIV NAAT was positive before the oral fluid test, we estimated the time until the oral fluid test was reactive as Time (the proportion of 28 days) from positive HIV NAAT until the mid-point date between the last non-reactive and first reactive oral fluid HIV test; Expiry 1 = more than 7 months until oral fluid HIV test kit expired, 2 = 7 months or less until expired; CRF01_AE 1 = yes, 2 = no; Sex 1 = male, 2 = female; Group 1 = tenofovir, 2 = placebo; Age 1 = 30 years or older, 2 = less than 30 years old. (XLS) Discussion Thus, in a set- ting where repeat HIV testing will be done among people at high risk of HIV infection, for example among people receiving pre-exposure prophylaxis, a blood-based test or fourth gener- ation test that simultaneously detects HIV-1 p24 antigen and antibody, may be a more appro- priate choice.[5,23] Bangkok Tenofovir Study Group Principal Investigator: Kachit Choopanya; Advisory Group: Sompob Snidvongs Na Ayudhya, Sithisat Chiamwongpaet, Kraichack Kaewnil, Praphan Kitisin, Malinee Kukavejworakit, Manoj Leethochawalit, Pitinan Natrujirote, Saengchai Simakajorn, Wonchat Subhachaturas; Study Clinic Coordination Team Lead: Suphak Vanichseni; Study Clinic Coordination Team 7 / 9 PLOS ONE \| DOI:10.1371/journal.pone.0145859 December 30, 2015 Assessment of Oral Fluid HIV Test Performance Members: Boonrawd Prasittipol, Udomsak Sangkum, Pravan Suntharasamai; Bangkok Metro- politan Administration: Rapeepan Anekvorapong, Chanchai Khoomphong, Surin Koochar- oenprasit, Parnrudee Manomaipiboon, Siriwat Manotham, Pirapong Saicheua, Piyathida Smutraprapoot, Sravudthi Sonthikaew, La-Ong Srisuwanvilai, Samart Tanariyakul, Montira Thongsari, Wantanee Wattana, Kovit Yongvanitjit; Thailand Ministry of Public Health: Sumet Angwandee, Somyot Kittimunkong; Thailand Ministry of Public Health—U.S. Centers for Dis- ease Control and Prevention Collaboration: Wichuda Aueaksorn, Benjamaporn Chaipung, Nartlada Chantharojwong, Thanyanan Chaowanachan, Thitima Cherdtrakulkiat, Wannee Chonwattana, Rutt Chuachoowong, Marcel Curlin, Pitthaya Disprayoon, Kanjana Kamkong, Chonticha Kittinunvorakoon, Wanna Leelawiwat, Robert Linkins, Michael Martin (Lead author, znd9@cdc.gov), Janet McNicholl, Philip Mock, Supawadee Na-Pompet, Tanarak Pli- pat, Anchala Sa-nguansat, Panurassamee Sittidech, Pairote Tararut, Rungtiva Thongtew, Dar- arat Worrajittanon, Chariya Utenpitak, Anchalee Warapornmongkholkul, Punneeporn Wasinrapee; U.S. Centers for Disease Control and Prevention: Jennifer Brannon, Monique Brown, Roman Gvetadze, Lisa Harper, Lynn Paxton, Charles Rose; Johns Hopkins University: Craig Hendrix, Mark Marzinke. Author Contributions Conceived and designed the experiments: PS MM KC SV US PT WL RA PAM JMM LAP MEC SK. Performed the experiments: MM PT WL RA PAM MEC. Analyzed the data: MM PAM TC RJG. Wrote the paper: PS MM PAM ML SC JMM LAP MEC SK. References 1. World Health Organization. Consolidated Guidelines on the use of antiretroviral drugs for treating and preventing HIV infection. Geneva 2013. Available: http://www.who.int/hiv/pub/guidelines/arv2013/en/. Accessed 27 May 2015 1. World Health Organization. Consolidated Guidelines on the use of antiretroviral drugs for treating and preventing HIV infection. Geneva 2013. Available: http://www.who.int/hiv/pub/guidelines/arv2013/en/. Accessed 27 May 2015 2. CDC. Advancing HIV prevention: new strategies for a changing epidemic—United States, 2003. MMWR Morb Mortal Wkly Rep. 2003; 52: 329–332. PMID: 12733863 3. UNAIDS. 90-90-90: An ambitious treatment target to help end the AIDS epidemic. Geneva 2014. Avail- able http://www.unaids.org/en/resources/documents/2014/90-90-90. Accessed 27 May 2015 4. World Health Organization. Service delivery approaches to HIV testing and counselling (HCT): a strate- gic HTC programme framework. Geneva 2012. Available: http://apps.who.int/iris/bitstream/10665/ 75206/1/9789241593877_eng.pdf. Accessed 27 May 2015. 5. Branson BM. State of the art for diagnosis of HIV infection. Clin Infect Dis. 2007; 45 Suppl 4: S221–225. doi: 10.1086/522541 PMID: 18190290 6. Food and Drug Administration. OraQuick1 In-Home HIV Test Summary of Safety and Effectiveness. 2012. Available: http://www.fda.gov/downloads/BiologicsBloodVaccines/BloodBloodProducts/ ApprovedProducts/PremarketApprovalsPMAs/UCM312534.pdf. Accessed 27 May 2015. 7. Thailand Food and Drug Administration. Medical Device Control in Thailand. 1990. Available: http:// www.fda.moph.go.th/fda-net/html/product/mdcd/eng/attach01_1.asp. Accessed 27 May 2015. 8. Thailand Ministry of Public Health, Department of Medical Sciences. Diagnositic HIV tests available in Thailand. 2014. Available: http://ttp.dmsc.moph.go.th/ttp/th/file/file2download/2557/kit_evaluation/ TEST%20KIT%20update%20(2014%20May%2012).pdf. Accessed 27 May 2015. 9. Choopanya K, Martin M, Suntharasamai P, Sangkum U, Mock PA, Leethochawalit M, et al. Antiretrovi- ral prophylaxis for HIV infection in injecting drug users in Bangkok, Thailand (the Bangkok Tenofovir Study): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet. 2013; 381: 2083–2090. doi: 10.1016/S0140-6736(13)61127-7 PMID: 23769234 10. OraSure Technologies. OraQuick Advance Rapid HIV-1/2 Antibody Test [package insert]. 2004. Avail- able: http://www.fda.gov/downloads/BiologicsBloodVaccines/ucm091917.pdf. Accessed 27 May 2015. 11. Martin M, Vanichseni S, Suntharasamai P, Sangkum U, Chuachoowong R, Mock PA, et al. 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https://openalex.org/W2337169759	https://pure.mpg.de/pubman/item/item_2354449_2/component/file_2354448/J_Infect_Dis_2016_214_379.pdf	English	null	Lower Viral Loads and Slower CD4<sup>+</sup>T-Cell Count Decline in MRKAd5 HIV-1 Vaccinees Expressing Disease-Susceptible HLA-B*58:02	The journal of infectious diseases (Online. University of Chicago Press)/The Journal of infectious diseases	2,016	cc-by	9,605	Received 9 November 2015; accepted 2 March 2016; published online 6 March 2016. Presented in part: 8th IAS Conference on HIV Pathogenesis, Treatment, and Prevention, Van- couver, Canada, 19–22 July 2015. Oral poster discussion WEPDA0102. aPresent affiliations: Institute of Cancer Research, Royal Cancer Hospital, London (J. H.), and Department of Infectious Diseases and Microbiology, Oxford University Hospitals NGS Trust, John Radcliffe Hospital, Oxford, United Kingdom (P. C. M.). Correspondence: E. M. Leitman, Department of Paediatrics, University of Oxford, the Peter Medawar Building for Pathogen Research, South Parks Road, Oxford OX1 3SY, United Kingdom (ellen.leitman@st-hughs.ox.ac.uk). Lower Viral Loads and Slower CD4+ T-Cell Count Decline in MRKAd5 HIV-1 Vaccinees Expressing Disease- Susceptible HLA-B58:02 g Keywords. HLA class I; HIV-1 vaccine; Phambili trial; Gag-speciﬁc CD8+ T cells. responses against HIV-1. Gag-speciﬁc CD8+ T-cell responses are associated with lower viremia levels and are dominant in people with protective HLA alleles, such as HLA-B57:03 [1, 5]. In contrast, disease-susceptible HLA alleles, such as HLA- B58:02, typically present relatively ineffective non-Gag re- sponses [6] associated with high viral set points [5]. This prompts the hypothesis that a successful vaccine might induce Gag-speciﬁc responses in subjects who would not target this protein in natural infection. HLA class I alleles are an important predictor of disease course in human immunodeﬁciency virus type 1 (HIV-1) infection [1]. In sub-Saharan Africa, HLA-B57, HLA-B58:01, and HLA- B81:01 are protective against disease progression, while HLA-B18:01 and HLA-B58:02 are associated with rapid pro- gression [2–4]. One contributory factor is the role of HLA mol- ecules in presenting particular epitopes to induce CD8+ T-cell Two recent randomized efﬁcacy trials of the MRKAd5 sub- type B HIV-1 Gag/Pol/Nef T-cell vaccine provide a unique op- portunity to address this hypothesis [7, 8]. The ﬁrst study, Step, was conducted in the Americas, Australia, and the Caribbean, where HIV-1 subtype B predominates [7].Both interim and fol- low-up analyses reported vaccine-enhanced HIV-1 acquisition [7, 9]. The second trial, Phambili, tested the efﬁcacy of the same vaccine in South Africa, where HIV-1 subtype C is en- demic [8,10].Following Step discontinuation, Phambili was ter- minated 9 months into the study, having enrolled 801 subjects (27%) of the originally scheduled 3000 [10]. As in Step, the Received 9 November 2015; accepted 2 March 2016; published online 6 March 2016. Correspondence: E. M. Leitman, Department of Paediatrics, University of Oxford, the Peter Medawar Building for Pathogen Research, South Parks Road, Oxford OX1 3SY, United Kingdom (ellen.leitman@st-hughs.ox.ac.uk). Lower Viral Loads and Slower CD4+ T-Cell Count Decline in MRKAd5 HIV-1 Vaccinees Expressing Disease- Susceptible HLA-B58:02 at Deutsches RheumaforschungsZentrum und http://jid.oxfordjournals.org/ Downloaded from Ellen M. Leitman,1 Jacob Hurst,2,a Masahiko Mori,1 James Kublin,3 Thumbi Ndung’u,6,7,8,11 Bruce D. Walker,6,7 Jonathan Carlson,5 Glenda E. Gray,9,10 Philippa C. Matthews,2,a Nicole Frahm,3,4 and Philip J.R. Goulder1,7 at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from 1Department of Paediatrics, and 2Nuffield Department of Medicine, University of Oxford, United Kingdom; 3HIV-1 Vaccine Trials Network, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 4Department of Global Health, University of Washington, Seattle, and 5eScience Group, Microsoft Research, Redmond, Washington; 6Ragon Institute of MGH, MIT, and Harvard, Cambridge, Massachusetts; 7HIV-1 Pathogenesis Programme, Doris Duke Medical Research Institute, University of KwaZulu-Natal, 8KwaZulu-Natal Research Institute for Tuberculosis and HIV-1, University of KwaZulu-Natal, Durban, 9South African Medical Research Council, Cape Town, and 10Perinatal HIV-1 Research Unit, University of the Witwatersrand, Johannesburg, South Africa; and 11Max Planck Institute for Infection Biology, Berlin, Germany Background. HLA strongly inﬂuences human immunodeﬁciency virus type 1 (HIV-1) disease progression. A major contribu- tory mechanism is via the particular HLA-presented HIV-1 epitopes that are recognized by CD8+ T-cells. Different populations vary considerably in the HLA alleles expressed. We investigated the HLA-speciﬁc impact of the MRKAd5 HIV-1 Gag/Pol/Nef vaccine in a subset of the infected Phambili cohort in whom the disease-susceptible HLA-B58:02 is highly prevalent. p g y p Methods. Viral loads, CD4+ T-cell counts, and enzyme-linked immunospot assay–determined anti-HIV-1 CD8+ T-cell respons- es for a subset of infected antiretroviral-naive Phambili participants, selected according to sample availability, were analyzed. Results. Among those expressing disease-susceptible HLA-B58:02, vaccinees had a lower chronic viral set point than placebo recipients (median, 7240 vs 122 500 copies/mL; P = .01), a 0.76 log10 lower longitudinal viremia level (P = .01), and slower progres- sion to a CD4+ T-cell count of <350 cells/mm3 (P = .02). These differences were accompanied by a higher Gag-speciﬁc breadth (4.5 vs 1 responses; P = .04) and magnitude (2300 vs 70 spot-forming cells/106 peripheral blood mononuclear cells; P = .06) in vaccinees versus placebo recipients. Conclusions. In addition to the known enhancement of HIV-1 acquisition resulting from the MRKAd5 HIV-1 vaccine, these ﬁndings in a nonrandomized subset of enrollees show an HLA-speciﬁc vaccine effect on the time to CD4+ T-cell count decline and viremia level after infection and the potential for vaccines to differentially alter disease outcome according to population HLA composition. Clinical Trials Registration. NCT00413725, DOH-27-0207-1539. at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from Neither protective nor disease-susceptible vaccine increased the risk of HIV-1 acquisition and did not re- duce viremia levels in early infection [8, 10]. subsequently became HIV-1 infected. HLA-B18:01, also strongly associated with rapid progression [2,3],restricts a dom- inant CD8+ T-cell epitope in Nef [5]. Although Nef-speciﬁc CD8+ T-cell responses have also been associated with poor con- trol of HIV-1 [5, 15–17], the immunodominance of the HLA- B18:01–restricted CD8+ T-cell response might be unaffected by the MRKAd5 HIV-1 Gag/Pol/Nef vaccine. Post hoc Step studies, however, demonstrated lower viremia levels in vaccinees with a greater breadth of Gag-speciﬁc CD8+ T-cell activity [11], consistent with the notion of Gag-speciﬁc ef- ﬁcacy, but also suggested the possibility of lower viral loads in vaccinees expressing protective HLA alleles [12, 13]. To examine this question in a population substantially dissimilar in the HLA alleles expressed from that studied in Step, we here tested in the South African Phambili cohort the hypothesis that the MRKAd5 HIV-1 vaccine might indeed have an HLA-speciﬁc effect. We set out, therefore, to investigate the impact of the MRKAd5 HIV-1 vaccine in the Phambili trial, particularly on the HLA- B58:02–expressing individuals, to determine whether immune control of HIV-1 was improved in vaccinees who subsequently became infected. This study has limitations that we wish to high- light, including postrandomization bias, sample selection bias, and low subject numbers (see Discussion), but it provides poten- tially important insights into HLA-dependent vaccine effects on the postinfection disease course and immune response to HIV-1. Speciﬁcally, we hypothesized that subjects expressing HLA- B58:02, the most prevalent HLA-B allele in South Africans [2, 5], might beneﬁt from the MRKAd5 HIV-1 vaccine. The ra- tionale was that the HLA-B58:02–restricted CD8+ T-cell re- sponse is solely Env speciﬁc and, therefore, associated with poor immune control [5, 14]; HLA-B58:02 indeed is strongly associated with rapid HIV-1 disease progression in southern Africa [2, 3, 6]. Hence, vaccine-mediated induction of Gag- speciﬁc responses in subjects expressing HLA-B58:02 would, hypothetically, improve disease course in vaccinees who 380 • JID 2016:214 (1 August) • Leitman et al The Journal of Infectious Diseases® © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/infdis/jiw093 HLA-B58:02–Speciﬁc Vaccine Effect • JID 2016:214 (1 August) • 379 Table 1. Baseline Characteristics of the 60 Infected Phambili Participants Studied in This Report Characteristic Vaccine Placebo Total Subjects 37 (62) 23 (38) 60 (100) Age at enrollment, y 24 (21.5–28.5) 24 (22–32) 24 (22–29) Sex Female 21 (57) 18 (78) 39 (65) Male 16 (43) 5 (22) 21 (35) Study site Soweto, Johannesburg 15 (41) 7 (30) 22 (37) Emavundleni, Cape Town 8 (22) 6 (26) 14 (23) CAPRISA, Klerksdorp 5 (14) 4 (17) 9 (15) eThekwini, Durban 7 (19) 3 (13) 10 (17) Medunsa, Polokwane 2 (5) 3 (13) 5 (8) Time since diagnosis, mo. Time point 1 2 (1–2.5) 2 (1–3) 2 (1–3) Time point 2 12 (11–12) 11 (11–12) 12 (11–12) Duration of follow-up data, time since diagnosis, mo. 17 (16–18) 17 (16–19) 17 (16–18) Started ART 5 (14) 6 (26) 11 (18) HLA typea Protective B57:03 12 (32) 3 (13) 15 (25) B58:01 2 (5) 0 (0) 2 (3) B81:01 7 (19) 3 (13) 10 (17) Disease susceptible 4 (11) 0 (0) 4 (7) B18:01 0 (0) 1 (4) 1 (2) B58:02 7 (19) 7 (30) 14 (23) Neither protective nor disease-susceptible 18 (49) 13 (57) 31 (52) Early viral set point,b copies/mL 41 850 (3 675–234 075) 88 500 (11 485–238 500) 71 700 (6 475–238 500) Data are no. (%) of subjects or median value (interquartile range). There were no significant differences between vaccine and placebo groups for any of the characteristics. Abbreviation: ART, antiretroviral therapy. a HLA types were grouped by their association with disease progression, as specified in “Methods” section. b Defined as the median of human immunodeficiency virus type 1 load measurements 2–3 months after diagnosis, as specified in “Methods” section. Table 1. Baseline Characteristics of the 60 Infected Phambili Participants Studied in This Report Study Design The Phambili Ancillary Study included 100 HIV-1–infected Phambili participants (Supplementary Figure 1). Sixty of them, 380 • JID 2016:214 (1 August) • Leitman et al for whom HLA types, peripheral blood mononuclear cell (PBMC), viral load, and CD4+ T-cell count data were available, compose the focus of this report (Table 1). For the remaining 40 individuals, no PBMC were available to assess CD8+ T-cell re- sponses, and thus only selected viral load and CD4+ T-cell count data were included in additional analyses; material for HLA typ- ing was available for 25 participants. Subjects were categorized as having protective HLA-B expression (HLA-B57:02/57:03/58:01/ 81:01); disease-susceptible HLA-B expression (HLA-B18:01/ 58:02), and neither protective nor disease-susceptible HLA-B ex- pression (eg, HLA-B42:01/44:03) [2, 3]. For the most prevalent HLA-B allele, HLA-B58:02 (phenotypic frequency, 23%), there were sufﬁcient subject numbers to compare vaccinees (n = 7) to placebo recipients (n = 7). All participants provided written informed consent; ethics committee at each clinical site and the University of Oxford approved the study. Fisher exact test (Prism v5.0c). Only pre-ART viral loads were used. Early viral set point was deﬁned as the geometric mean of HIV-1 load measurements 2–3 months after diagnosis [7, 10]. To determine the viral set point more accurately, we also de- ﬁned the long-term chronic set point as the median of all pre-ART viral loads available as (1) including the diagnostic (acute) measurement, (2) excluding the diagnostic measure- ment, (3) including all measurements from >2 weeks after diag- nosis, (4) including all measurements from >1 month after diagnosis, or (5) including all measurements from >3 months after diagnosis. Chronic set points were very similar irrespective of the deﬁnition, and results using deﬁnition 4 are reported here because this may most accurately reﬂect the beginning of the chronic phase [21, 22]. Set-point differences between vaccinees and placebo recipients were analyzed using the Mann–Whitney U test (Prism v5.0c). at Deutsches RheumaforschungsZentrum und Max http://jid.oxfordjournals.org/ Downloaded from at Deutsches RheumaforschungsZentrum http://jid.oxfordjournals.org/ Downloaded from at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from To identify associations between HLA expression and recog- nition of HIV-1–speciﬁc peptides, we used enzyme-linked im- munospot (ELISPOT) data from 1031 untreated subjects with chronic natural HIV-1 infection from the previously described Sinikithemba (enrolled in 2003–2008) and Gateway (enrolled in 2012–2013) cohorts in Durban, South Africa [5, 18]. HLA-Independent Vaccine Effects We ﬁrst compared the baseline characteristics between vaccine and placebo recipients in the selected subgroup of 60 subjects for whom PBMCs were available to test the impact of the MRKAd5 HIV-1 vaccine on postinfection CD8+ T-cell activity. These showed no signiﬁcant differences in age, sex, HLA-B ge- notype, or early viral set point (Table 1). As previously reported [7, 10, 27], median early viral set point did not differ signiﬁ- cantly between vaccine and placebo recipients among these 60 patients (41 850 copies/mL [interquartile range {IQR}, 3657–234 075 copies/mL] vs 88 500 copies/mL [IQR, 11 485– 238 500 copies/mL], P = .34; Table 1). We observed a trend Study Design The medi- an viral load in these subjects was 25 700 copies/mL (range, 3690–119 000 copies/mL), and the median CD4+ T-cell count was 404 cells/mm3 (range, 273–549 cells/mm3). PBMCs from each individual were screened with a panel of peptides spanning the entire C-clade 2001 consensus sequence [5] and analyzed (Supplementary Materials). To compare the CD8+ T-cell re- sponses in the HLA-B58:02–positive South African Phambili subjects to those in HLA-B58:02–positive South African sub- jects naturally infected with HIV-1, we used ELISPOT data from 66 HLA-B58:02–positive subjects from the Sinikithemba cohort because individuals in both groups would have had a similar geographical location, genetic background, timing of in- fection, and infection with similar/related viral species (Supple- mentary Table 3). Linear mixed-effects models were constructed to investigate the effects of vaccination, sex, age, anti–adenovirus serotype 5 (Ad5) antibody levels, and herpes simplex virus type 2 (HSV- 2) serostatus on longitudinal viremia levels. Random intercept models were constructed with the grouping by participant iden- tiﬁer in R, using the lme4 library [23];P values were obtained by comparing models with and those without vaccination as a ﬁxed effect (analysis of variance [ANOVA]) [24]. The log-rank test (Prism v5.0c) and univariate Cox regression models (SPSS v22.0) were used to compare time to disease pro- gression between vaccine and placebo groups, deﬁned by a CD4+ T-cell count of <350 cells/mm3, the World Health Organization’s CD4+ T-cell count criterion for ART initiation [25]. Only pre- ART CD4+ T-cell counts were included; the impact of sex, age, Ad5 antibody levels, and HSV-2 serostatus was checked. Differences in ELISPOT responses between vaccinees and placebo recipients were analyzed in Prism v5.0c by the Mann– Whitney U test (2-group analyses) or the Kruskal–Wallis test (3-group analyses). To identify associations between expression of HLA alleles and recognition of HIV-1–speciﬁc peptides, we used a decision tree based on the Fisher exact test (Supplementary Materials) [26]. Interferon γ (IFN-γ) ELISPOT Assays Blinded to the vaccine/placebo treatment assignments, we screened PBMCs from the 60 subjects with available cells to quantify IFN-γ ELISPOT responses to pools (n = 36) of overlap- ping peptides spanning the entire C-clade HIV-1 proteome, based on the 2001 C-clade consensus [5]. For each subject, cells obtained 2 and 12 months after diagnosis (Table 1) were tested [19, 20]. Responses were divided into 4 groups: speciﬁc to Gag, speciﬁc to Pol, speciﬁc to Nef, or speciﬁc to other (Env, Tat, Rev, Vpu, Vpr, and Vif) proteins. Breadth was de- ﬁned as the number of pools testing positive (>50 spot-forming cells [SFCs]/106 PBMCs, after background subtraction). Statistical Analysis Baseline characteristics (Table 1) between vaccine and placebo recipients were analyzed using the Mann–Whitney U test or the HLA-B58:02–Speciﬁc Vaccine Effect • JID 2016:214 (1 August) • 381 Figure 1. Longitudinal viremia levels, CD4+ T-cell counts, and CD8+ T-cell responses in the 60 infected vaccinees and placebo recipients, irrespective of HLA. A, Chronic viral set point (defined for each individual as the median of all measurements from >3 months after diagnosis). Each patient’s set point is shown as a circle (vaccine recipients) or triangle (placebo recipients), and horizontal lines and numbers denote median values. Statistical analysis was performed by the Mann–Whitney U test. B, Examination of the effect of vaccination on longitudinal viremia levels before antiretroviral therapy initiation. The slopes (thick lines) generated from a linear mixed-effects model are plotted on top of data points and viral load trajectories for individual patients (thin lines). Statistical analysis was performed by analysis of variance. C, Kaplan–Meier curves showing time to reach a CD4+ T-cell count of <350 cells/mm3 in vaccinees and placebo recipients. Statistical analysis was performed by the log-rank test. D, Breadth and magnitude of CD8+ T-cell responses at the second time point in vaccinees and placebo recipients. In Tukey box plots, horizontal lines indicate median values, boxes show interquartile ranges, and whiskers show minimum and maximum values. Statistical analysis was performed by the Mann–Whitney U test. The column termed “Total” shows the combined values of responses to all proteins; the column termed “Other” includes responses to Env, Rev, Tat, Vpu, Vif, and Vpr. Abbreviations: IFN-γ, interferon γ; PBMC, peripheral blood mononuclear cell; SFC, spot- forming cells. at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from Figure 1. Longitudinal viremia levels, CD4+ T-cell counts, and CD8+ T-cell responses in the 60 infected vaccinees and placebo recipients, irrespective of HLA. A, Chronic viral set point (defined for each individual as the median of all measurements from >3 months after diagnosis). Each patient’s set point is shown as a circle (vaccine recipients) or triangle (placebo recipients), and horizontal lines and numbers denote median values. Statistical analysis was performed by the Mann–Whitney U test. B, Examination of the effect of vaccination on longitudinal viremia levels before antiretroviral therapy initiation. The slopes (thick lines) generated from a linear mixed-effects model are plotted on top of data points and viral load trajectories for individual patients (thin lines). Statistical Analysis Statistical analysis was performed by analysis of variance. C, Kaplan–Meier curves showing time to reach a CD4+ T-cell count of <350 cells/mm3 in vaccinees and placebo recipients. Statistical analysis was performed by the log-rank test. D, Breadth and magnitude of CD8+ T-cell responses at the second time point in vaccinees and placebo recipients. In Tukey box plots, horizontal lines indicate median values, boxes show interquartile ranges, and whiskers show minimum and maximum values. Statistical analysis was performed by the Mann–Whitney U test. The column termed “Total” shows the combined values of responses to all proteins; the column termed “Other” includes responses to Env, Rev, Tat, Vpu, Vif, and Vpr. Abbreviations: IFN-γ, interferon γ; PBMC, peripheral blood mononuclear cell; SFC, spot- forming cells. toward a lower median chronic viral set point in vaccinees in the 60 subjects (12 055 copies/mL [IQR, 3805–137 500 copies/mL] vs 103 600 copies/mL [IQR, 9190–141 800 copies/mL], P = .055; Figure 1A). However, this difference did not reach statistical sig- niﬁcance, even when all of the viral load data were analyzed using a linear mixed-effects model (P = .058; Figure 1B). It is important to take note of the fact that 12 vaccinees expressed protective alleles (HLA-B57/58:01/81:01), compared with 3 placebo recipients, although this also did not reach statistical signiﬁcance (P = .13). Additionally, there was no signiﬁcant dif- ference in time to disease progression (ie, time to a CD4+ T-cell count of <350cells/mm3) between vaccine and placebo groups (P = .25; Figure 1C). included in the vaccine (Env, Vif, Vpr, Vpu, Tat, and Rev). However, there was no signiﬁcant difference in the Pol- or Nef-speciﬁc responses between vaccine and placebo recipients either, despite pol/nef inclusion in the vaccine. 382 • JID 2016:214 (1 August) • Leitman et al age, Ad5 antibody levels, and HSV-2 status (Supplementary Table 1). age, Ad5 antibody levels, and HSV-2 status (Supplementary Table 1). only signiﬁcant contributor to this difference was placebo /vac- cine treatment assignment, and the difference between vaccine and placebo recipients remained signiﬁcant with addition of the 3 HLA-B58:02–positive subjects with determined HLA types but unavailable PBMCs (P = .03, by the log-rank test; HR, 0.29 [95% CI, .06–.99; P = .04, by a Cox regression model; Supplementary Table 2]). Finally, among the HLA-B58:02– positive subjects studied, one individual also expressed protective HLA-B58:01, and exclusion of this subject did not materially alter the ﬁndings described (viral set point difference, P = .01; time to a CD4+ T-cell count of <350 cells/mm3, P = .04). Similarly, no signiﬁcant difference was observed with respect to HLA-C alleles expressed among the HLA-B58:02–positive sub- jects studied; in each case, HLA-C06:02 was expressed in linkage disequilibrium with HLA-B58:02. only signiﬁcant contributor to this difference was placebo /vac- cine treatment assignment, and the difference between vaccine and placebo recipients remained signiﬁcant with addition of the 3 HLA-B58:02–positive subjects with determined HLA types but unavailable PBMCs (P = .03, by the log-rank test; HR, 0.29 [95% CI, .06–.99; P = .04, by a Cox regression model; Supplementary Table 2]). Finally, among the HLA-B58:02– positive subjects studied, one individual also expressed protective HLA-B58:01, and exclusion of this subject did not materially alter the ﬁndings described (viral set point difference, P = .01; time to a CD4+ T-cell count of <350 cells/mm3, P = .04). Similarly, no signiﬁcant difference was observed with respect to HLA-C alleles expressed among the HLA-B58:02–positive sub- jects studied; in each case, HLA-C06:02 was expressed in linkage disequilibrium with HLA-B58:02. HLA-Speciﬁc Vaccine Effects on Viral Load and CD4+ T-Cell Counts: Disease-Susceptible HLA Alleles With respect to the disease-susceptible HLA alleles, HLA- B18:01 and HLA-B58:02, none of the vaccinees with available PBMCs and HLA data expressed HLA-B18:01, and analyses were limited to the HLA-B58:02–positive subjects (7 vaccinees and 7 placebo recipients; Table 2). Median chronic viral set point was signiﬁcantly lower in HLA-B58:02–positive vaccinees (7240 copies/mL [IQR, 4090–63 000 copies/mL] vs 122 500 copies/mL [IQR, 103 600–677 500 copies/mL]; P = .01; Figure 2C). This difference was especially clear when all pre-ART longitudinal data points were interrogated using a mixed-effects model (P = .01; Figure 2C). These data show that vaccination in the HLA-B58:02–positive subjects on aver- age resulted in a 0.76 log10 reduction in chronic viral set point and that no other factors (sex, age, Ad5 antibody levels, and HSV-2 serostatus) had a signiﬁcant inﬂuence. The viral load differences between vaccinees and placebo recipients remained signiﬁcant when the 3 additional HLA-B58:02–positive sub- jects were added from the Phambili participants with deter- mined HLA types but unavailable PBMCs (P = .03, by the Mann–Whitney U test; P = .03, by an ANOVA mixed-effects model). HLA-Speciﬁc Vaccine Effects on Viral Load and CD4+ T-Cell Counts: Protective HLA Alleles To investigate whether these vaccine-mediated effects on the CD8+ T-cell responses were associated with HLA-speciﬁc im- mune control, as observed for protective HLA alleles in the Step trial [12], we ﬁrst compared chronic viral set points and time to a CD4+ T-cell count of <350 cells/mm3 in the subset of infected Phambili subjects expressing any of the protective alleles HLA-B57/58:01/81:01 (Table 2). Although subject numbers provided limited power (3 placebo recipients vs 12 vaccinees expressed HLA-B57/58:01/81:01), there was evi- dence of vaccine-mediated immune protection through these HLA alleles (Figure 2A and 2B), with a signiﬁcantly more- rapid time to disease progression (ie, a CD4+ T-cell count of <350 cells/mm3) in placebo recipients (P = .009). Placebo/ vaccine treatment assignment was the only signiﬁcant contrib- utor to this difference in a univariate analysis that included sex, Examining the impact of the MRKAd5 HIV-1 vaccine on CD8+ T-cell IFN-γ responses after infection in Phambili sub- jects with available PBMCs, we observed that vaccinees had sig- niﬁcantly higher Gag-speciﬁc breadth and magnitude than placebo recipients (P = .03 and P = .02, respectively, 2 months after diagnosis; and P = .05 and P = .02, respectively, 12 months after diagnosis; Figure 1D). These assays were undertaken blind- ed to the subjects’ vaccination assignments. There was no differ- ence between the 2 groups in the responses toward proteins not 382 • JID 2016:214 (1 August) • Leitman et al Table 2. Demographic Characteristics Stratified by HLA Group Table 2. Demographic Characteristics Stratified by HLA Group HLA-Speciﬁc Vaccine Effects on Viral Load and CD4+ T-Cell Counts: Neither Protective nor Disease-Susceptible HLA Alleles Analysis of the subjects expressing neither protective nor disease-susceptible alleles showed no differences between vaccine and placebo groups in chronic viral set points or time to a CD4+ T-cell count of <350 cells/mm3 (Figure 3A and 3B). Representative observations are illustrated by the HLA-B44:03–expressing subjects (6 in each group; Figure 3C and 3D). Progression to a CD4+ T-cell count of <350 cells/mm3 was also signiﬁcantly slower in HLA-B58:02–positive vaccinees as compared to placebo recipients (P = .02, by the log-rank test [Figure 2D]; hazard ratio [HR], 0.22 [95% CI, .05–.91; P = .04, by a Cox regression model; Supplementary Table 2]). Again, the Table 2. Demographic Characteristics Stratified by HLA Group Characteristic Protective HLA Neither Protective nor Disease- Susceptible HLA Disease-Susceptible HLA-B58:02 Vaccine (n = 12) Placebo (n = 3) Vaccine (n = 18) Placebo (n = 13) Vaccine (n = 7) Placebo (n = 7) Age at enrollment, y 25.5 (22.5–30.5) 32 (22–32) 22.5 (21.5–30.5) 23 (22–26) 22 (21–27) 26 (21–34) Sex Female 7 (58) 3 (100) 9 (50) 10 (77) 5 (71) 5 (71) Male 5 (42) 0 (0) 9 (50) 3 (23) 2 (29) 2 (29) Circumcision statusa Circumcised 2 (40) . . . 5 (56) 1 (33) 0 (0) 0 (0) Uncircumcised 3 (60) . . . 4 (44) 2 (67) 2 (100) 2 (100) Adenovirus 5 statusb Seropositive 9 (75) 2 (67) 17 (94) 12 (92) 6 (86) 4 (57) Seronegative 3 (25) 1 (33) 1 (6) 1 (8) 1 (14) 3 (43) HSV-2 status at enrollment Positive 9 (75) 2 (67) 10 (56) 5 (38) 2 (29) 5 (71) Negative 3 (25) 1 (33) 8 (44) 8 (62) 5 (71) 2 (29) Data are no. (%) of subjects or median value (interquartile range). There were no significant differences between vaccine and placebo groups within each HLA subset for any characteristic. Abbreviation: HSV-2, herpes simplex virus type 2. a Data are for men circumcised at baseline or during study but before infection and those uncircumcised throughout study or before infection. b Seropositivity was defined as a titer of >18, and seronegativity was defined as a titer of ≤18. at Deutsches RheumaforschungsZentrum und http://jid.oxfordjournals.org/ Downloaded from at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from a Data are for men circumcised at baseline or during study but before infection and those uncircumcised throughout study or before infection. b Seropositivity was defined as a titer of >18, and seronegativity was defined as a titer of ≤18. HLA-Speciﬁc Vaccine Effects on Anti–HIV-1 CD8+ T-Cell Responses HLA Speciﬁc Vaccine Effects on Anti HIV 1 CD8 T Cell Responses To investigate whether the apparent effects on reduced viral load and slower time to a CD4+ T-cell count of <350 cells/ mm3 observed might be related to HLA-speciﬁc induction of altered CD8+ T-cell responses in the vaccinees versus placebo HLA-B58:02–Speciﬁc Vaccine Effect • JID 2016:214 (1 August) • 383 Figure 2. Chronic viral set point and CD4+ T-cell counts in infected Phambili subjects expressing protective HLA-B57/58:01/81:01 or disease-susceptible HLA-B58:02 alleles. A–C, Data for the participants expressing protective alleles (12 vaccinees and 3 placebo recipients). D–F, Data for the participants expressing HLA-B58:02 (7 vaccinees and 7 placebo recipients). A and C, Chronic viral set point (defined for each individual as the median of all measurements from >3 months after diagnosis) is shown at left. Each patient’s set point is shown as circles (vaccine recipients) or triangles (placebo recipients). Horizontal lines and numbers denote median values for vaccine and placebo groups. Statistical analysis was performed by the Mann–Whitney U test. Examination of the effect of vaccination on longitudinal viral load values before antiretroviral therapy initiation is shown at right. The slopes (thick lines) generated from a linear mixed-effects model are plotted on top of data points and viral load trajectories (thin lines) for individual patients. Statistical analysis was performed by analysis of variance. B and D, Kaplan–Meier curves showing time to reach a CD4+ T-cell count of <350 cells/mm3 in vaccinees and placebo recipients. Statistical analysis was performed by the log-rank test. at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from Figure 2. Chronic viral set point and CD4+ T-cell counts in infected Phambili subjects expressing protective HLA-B57/58:01/81:01 or disease-susceptible HLA-B58:02 alleles. A–C, Data for the participants expressing protective alleles (12 vaccinees and 3 placebo recipients). D–F, Data for the participants expressing HLA-B58:02 (7 vaccinees and 7 placebo recipients). A and C, Chronic viral set point (defined for each individual as the median of all measurements from >3 months after diagnosis) is shown at left. Each patient’s set point is shown as circles (vaccine recipients) or triangles (placebo recipients). Horizontal lines and numbers denote median values for vaccine and placebo groups. Statistical analysis was performed by the Mann–Whitney U test. Examination of the effect of vaccination on longitudinal viral load values before antiretroviral therapy initiation is shown at right. HLA-Speciﬁc Vaccine Effects on Anti–HIV-1 CD8+ T-Cell Responses A and B, Data for the participants expressing neither protective nor disease-susceptible alleles overall (18 vaccinees and 13 placebo recipients). C and D, Example of a subset of the subjects from panels A and B who express HLA-B44:03 and for whom there were sufficient subject numbers to compare vaccine (n = 6) and placebo (n = 6) groups. A and C, Chronic viral set point (defined for each individual as the median of all measurements from >3 months after diagnosis) is shown at left. Each patient’s set point is shown as a circle (vaccine recipients) or triangle (placebo recipients), and horizontal lines and numbers denote median values for vaccine and placebo groups. Statistical analysis was performed by the Mann–Whitney U test. Examination of the effect of vaccination on longitudinal viral load values before antiretroviral therapy initiation is shown at right. The slopes (thick lines) generated from a linear mixed-effects model are plotted on top of the data points and viral load trajectories (thin lines) for individual patients. Statistical analysis was performed by analysis of variance. B and D, Kaplan–Meier curves showing the time to reach a CD4+ T-cell count of <350 cells/mm3 in vaccinees and placebo recipients. Statistical analysis was performed by the log-rank test. Abbreviation: NS, not significant. at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from Figure 3. Chronic viral set point and CD4+ T-cell counts in the infected Phambili subjects expressing neither protective nor disease-susceptible HLA alleles. A and B, Data for the participants expressing neither protective nor disease-susceptible alleles overall (18 vaccinees and 13 placebo recipients). C and D, Example of a subset of the subjects from l A d B h HLA B44 03 d f h th ffi i t bj t b t i ( 6) d l b ( 6) A d C Ch i i l t Figure 3. Chronic viral set point and CD4+ T-cell counts in the infected Phambili subjects expressing neither protective nor disease-susceptible HLA alleles. A and B, Data for the participants expressing neither protective nor disease-susceptible alleles overall (18 vaccinees and 13 placebo recipients). C and D, Example of a subset of the subjects from panels A and B who express HLA-B44:03 and for whom there were sufficient subject numbers to compare vaccine (n = 6) and placebo (n = 6) groups. HLA-Speciﬁc Vaccine Effects on Anti–HIV-1 CD8+ T-Cell Responses The slopes (thick lines) generated from a linear mixed-effects model are plotted on top of data points and viral load trajectories (thin lines) for individual patients. Statistical analysis was performed by analysis of variance. B and D, Kaplan–Meier curves showing time to reach a CD4+ T-cell count of <350 cells/mm3 in vaccinees and placebo recipients. Statistical analysis was performed by the log-rank test. recipients, we ﬁrst analyzed these in HLA-B58:02–positive study subjects. Median Gag-speciﬁc breadth was 4.5-fold higher in vaccinees (4.5 responses [IQR, 2–5 responses] vs 1 response [IQR, 0–3 responses]; P = .04; Figure 4A), and the median Gag- speciﬁc magnitude was 32-fold higher (2300 SFCs/106 PBMCs [median, 603–6105 SFCs/106 PBMCs] vs 70 SFCs/106 PBMCs [0–1520 SFCs/106 PBMCs]; P = .06; Figure 4A). Additionally, we compared the CD8+ T-cell responses in the Phambili sub- jects to those of HLA-B58:02–positive unvaccinated individu- als with natural chronic HIV-1 C-clade infection (n = 66; Figure 4A), most of whom (79%) were females recruited in South Africa (Supplementary Table 3). Here also we observed that the Phambili HLA-B58:02–positive vaccinees had a signiﬁcantly higher Gag-speciﬁc breadth as compared to non-Phambili in- fected subjects (P = .04), while the difference between HLA- B58:02–positive placebo recipients and non-Phambili subjects was not signiﬁcant. The median Gag-speciﬁc magnitude was >2-fold higher in HLA-B58:02–positive vaccinees as compared to non-Phambili HLA-B58:02–positive subjects (1100 SFCs/106 PBMCs [IQR, 255–2050 SFCs/106 PBMCs]; P = .2). No signiﬁ- cant associations were found between Gag-speciﬁc breadth/ magnitude and viral loads or CD4+ T-cell count in HLA- B58:02–positive vaccinees (Supplementary Figure 2). In contrast, in subjects expressing neither protective nor dis- ease-susceptible alleles, the difference between the groups in median Gag-speciﬁc breadth and magnitude was more modest (2 responses [IQR, 1.5–3 responses] vs 1 response [IQR, 0–2 re- sponses; P = .09]; and 529 SFCs/106 PBMCs [IQR, 166–2163 SFCs/106 PBMCs] vs 180 SFCs/106 PBMCs [IQR, 0–377 SFCs/ 106 PBMCs; P = .06]; Figure 4B). No signiﬁcant differences were observed in Gag breadth and magnitude in the subjects express- ing protective HLA (Figure 4C). Although the MRKAd5 HIV-1 Gag/Pol/Nef vaccine ap- peared to boost Gag-speciﬁc responses overall in vaccinees 384 • JID 2016:214 (1 August) • Leitman et al Figure 3. Chronic viral set point and CD4+ T-cell counts in the infected Phambili subjects expressing neither protective nor disease-susceptible HLA alleles. HLA-Speciﬁc Vaccine Effects on Anti–HIV-1 CD8+ T-Cell Responses A and C, Chronic viral set point (defined for each individual as the median of all measurements from >3 months after diagnosis) is shown at left. Each patient’s set point is shown as a circle (vaccine recipients) or triangle (placebo recipients), and horizontal lines and numbers denote median values for vaccine and placebo groups. Statistical analysis was performed by the Mann–Whitney U test. Examination of the effect of vaccination on longitudinal viral load values before antiretroviral therapy initiation is shown at right. The slopes (thick lines) generated from a linear mixed-effects model are plotted on top of the data points and viral load trajectories (thin lines) for individual patients. Statistical analysis was performed by analysis of variance. B and D, Kaplan–Meier curves showing the time to reach a CD4+ T-cell count of <350 cells/mm3 in vaccinees and placebo recipients. Statistical analysis was performed by the log-rank test. Abbreviation: NS, not significant. HLA-B58:02–Speciﬁc Vaccine Effect • JID 2016:214 (1 August) • 385 DISCUSSION within the 60 subjects studied here (Figure 1D), there were no signiﬁcant differences for the CD8+ T-cell responses toward Pol, Nef, or Env, Vif, Vpr, Vpu, Tat, or Rev in the 3 HLA groups (Figure 4). However, of note, in addition to having a signiﬁ- cantly broader Gag-speciﬁc response, HLA-B58:02–positive vaccinees tended to have a narrower Nef-speciﬁc response (P = .06; Figure 4A). The potential relevance of this is discussed below. This study set out to investigate whether the HLA-speciﬁc vac- cine effects suggested in the Step trial [12, 13, 28] could be dem- onstrated in the ethnically dissimilar Phambili cohort. Half of the Step trial participants who subsequently became infected were white, whereas none of the Phambili cohort was white. Ex- pression of HLA class I molecules, such as HLA-B42:01, HLA- B58:02, and HLA-B81:01, for example, is observed in 0% of white individuals [29] and in 20%, 23%, and 10%, respectively, of Black South Africans [2, 5]. The principal ﬁnding in the current study is an HLA-speciﬁc vaccine effect in subjects ex- pressing the disease-susceptible HLA-B58:02. HLA-B58:02– positive vaccinees had a signiﬁcantly lower viral set point and progressed more slowly to a CD4+ T-cell count of <350 cells/ mm3, compared with HLA-matched placebo recipients. This Together, our ﬁndings support the original hypothesis that the MRKAd5 HIV-1 vaccine could improve disease outcome by boosting beneﬁcial Gag-speciﬁc responses [5] in HLA- B58:02–positive individuals who, during naturally acquired in- fection, do not generate HLA-B58:02–restricted Gag responses but produce predominantly Env-speciﬁc ineffective responses (Figure 5). HLA-B58:02–Speciﬁc Vaccine Effect • JID 2016:214 (1 August) • 385 Figure 4. CD8+ T-cell responses in the infected Phambili subjects expressing disease-susceptible HLA-B58:02 or those expressing neither protective nor disease-susceptible alleles or those expressing protective HLA alleles. A, Breadth and magnitude of CD8+ T-cell responses at the second time point in HLA-B58:02–positive vaccinees, placebo recipients, and C clade chronically infected unvaccinated B58:02-positive individuals. B, Breadth and magnitude of CD8+ T-cell responses at the second time point in vaccinees and placebo recipients expressing neither protective nor disease-susceptible HLA-B alleles. C, Breadth and magnitude of CD8+ T-cell responses at the second time point in vaccinees and placebo recipients expressing protective HLA-B alleles. In Tukey box plots, horizontal lines indicate median values, boxes show interquartile ranges, and whiskers show minimum and maximum values. Statistical analysis was performed by the Kruskal–Wallis and Mann–Whitney U tests. DISCUSSION The column termed “Total” shows the combined values of responses to all proteins; the column termed “Other” includes responses to Env, Rev, Tat, Vpu, Vif, and Vpr. Abbreviations: IFN-γ, interferon γ; NS, not significant; PBMC, peripheral blood mononuclear cell; SFC, spot-forming cells. at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb o http://jid.oxfordjournals.org/ rom Figure 4. CD8+ T-cell responses in the infected Phambili subjects expressing disease-susceptible HLA-B58:02 or those expressing neither protective nor disease-susceptible alleles or those expressing protective HLA alleles. A, Breadth and magnitude of CD8+ T-cell responses at the second time point in HLA-B58:02–positive vaccinees, placebo recipients, and C clade chronically infected unvaccinated B58:02-positive individuals. B, Breadth and magnitude of CD8+ T-cell responses at the second time point in vaccinees and placebo recipients expressing neither protective nor disease-susceptible HLA-B alleles. C, Breadth and magnitude of CD8+ T-cell responses at the second time point in vaccinees and placebo recipients expressing protective HLA-B alleles. In Tukey box plots, horizontal lines indicate median values, boxes show interquartile ranges, and whiskers show minimum and maximum values. Statistical analysis was performed by the Kruskal–Wallis and Mann–Whitney U tests. The column termed “Total” shows the combined values of responses to all proteins; the column termed “Other” includes responses to Env, Rev, Tat, Vpu, Vif, and Vpr. Abbreviations: IFN-γ, interferon γ; NS, not significant; PBMC, peripheral blood mononuclear cell; SFC, spot-forming cells. log10 copies/mL [6, 30], similar to that in the HLA-B58:02– positive placebo recipients here (5.1 log10 copies/mL), corre- sponding to rapid progression and a high risk of further transmissions [2–4, 31]. The 17-fold reduction in viral set point in HLA-B58:02–positive vaccinees observed here equates to a >5-year AIDS-free period without ART and a >2-fold reduction in onward transmission risk [31].Second, HLA-B58:02 is highly prevalent in sub-Saharan Africa (20%–25% of South Africans) [2, 5]. Thus, a vaccine effect on postinfection disease progression in subjects expressing protective HLA-B57/58:01/81:01 (24% of South Africans [2, 5]) and in those expressing HLA-B58:02 would favorably affect disease course in approximately half of the population. result was associated with an increased breadth and magnitude of Gag-speciﬁc CD8+ T-cell responses in vaccinees expressing HLA-B58:02. In contrast, in Phambili subjects expressing nei- ther protective nor disease-susceptible alleles, these vaccine ef- fects were not observed. DISCUSSION HLA-dependent differential immunodominance hierarchy of CD8+ T-cell responses in chronically C clade–infected, antiretroviral-naive, South African adults. CD8+ T-cell immunodominance hierarchy in subjects in the 3 HLA groups (allele frequency ≥5%), expressing protective HLA, neither protective nor disease-susceptible HLA, and disease-susceptible HLA. Targeting frequency denotes the percentage of subjects making detectable responses in interferon γ enzyme-linked immunospot assays to the named human immunodeficiency virus type 1 peptide. Numbers on the x-axis refer to the particular overlapping peptide (of 410 spanning the C clade proteome) to which a response was detected. The x in each column denotes the median viral load of responders to that peptide. with higher viral loads and the same observation is made here (Supplementary Figures 2 and 3). Thus, the ﬁnding of a lower viral loads in B58:02-positive subjects with both higher Gag- speciﬁc and lower Nef-speciﬁc responses may be the result of a combination of these effects. vaccine might therefore be expected to improve outcome by boosting beneﬁcial Gag responses [5,11]. A 70-fold higher mag- nitude and a 4–5-fold greater breadth of Gag-speciﬁc responses were observed in HLA-B58:02–positive vaccine recipients, but the necessary samples were unavailable to determine whether these were restricted by HLA-B58:02 or by other HLA alleles expressed by these subjects. The limitations of this study include small subject numbers, the potential for postrandomization bias [35], and the caveats that apply to any post hoc analysis. To address the possibility that the differences in postinfection outcomes were caused by the differences in characteristics associated with infection risk, rather than by vaccination, we investigated the impact of HLA on HIV-1 acquisition among 329 (of 801) Phambili subjects with available HLA data. There was no such inﬂuence in either vaccine or placebo recipients. This suggests that HLA does not confound the association of vaccination status with disease pro- gression [36] and is consistent with ample data showing the im- pact of HLA on disease progression but not on HIV-1 acquisition [1, 37]. Skewing could have been introduced by unintended sam- ple selection bias: PBMCs were only available for 60 of the 100 HIV-1–infected subjects, and the 40 subjects with unavailable samples and thus unknown CD8+ T-cell responses showed a fast- er time to a CD4+ T-cell count of <350 cells/mm3. DISCUSSION The described HLA-speciﬁc vaccine effect was suggested in the Step study in relation to the protective HLA-B27:05/ B57:01/B58:01 [12, 13, 28]. Analysis here also showed a 1.3 log10 lower viral load and signiﬁcantly slower progression to CD4+ T-cell count of <350 cells/mm3 (P = .009) in vaccinees expressing protective HLA-B57:02/57:03/58:01/81:01 [2, 3], compared with HLA-matched placebo recipients. However, these analyses were limited by numbers (only 3 placebo recipients). The HLA-B58:02–speciﬁc effect observed supports the orig- inal hypothesis. HLA-B58:02–restricted CD8+ T-cell responses are almost exclusively directed against Env, which is associated with a high viral load [5] (Figure 5). The MRKAd5 HIV-1 Our ﬁnding of reduced viral load and slower time to a CD4+ T-cell count of <350 cells/mm3 in HLA-B58:02–positive vacci- nees is signiﬁcant. First, viral load in natural infection is 4.9–5.2 386 • JID 2016:214 (1 August) • Leitman et al Figure 5. HLA-dependent differential immunodominance hierarchy of CD8+ T-cell responses in chronically C clade–infected, antiretroviral-naive, South African adults. CD8+ T-cell immunodominance hierarchy in subjects in the 3 HLA groups (allele frequency ≥5%), expressing protective HLA, neither protective nor disease-susceptible HLA, and disease-susceptible HLA. Targeting frequency denotes the percentage of subjects making detectable responses in interferon γ enzyme-linked immunospot assays to the named human immunodeficiency virus type 1 peptide. Numbers on the x-axis refer to the particular overlapping peptide (of 410 spanning the C clade proteome) to which a response was detected. The x in each column denotes the median viral load of responders to that peptide. at Deutsches RheumaforschungsZentrum und Max http://jid.oxfordjournals.org/ Downloaded from at Deutsches RheumaforschungsZentrum und http://jid.oxfordjournals.org/ Downloaded from at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from Figure 5. HLA-dependent differential immunodominance hierarchy of CD8+ T-cell responses in chronically C clade–infected, antiretroviral-naive, South African adults. CD8+ T-cell immunodominance hierarchy in subjects in the 3 HLA groups (allele frequency ≥5%), expressing protective HLA, neither protective nor disease-susceptible HLA, and disease-susceptible HLA. Targeting frequency denotes the percentage of subjects making detectable responses in interferon γ enzyme-linked immunospot assays to the named human immunodeficiency virus type 1 peptide. Numbers on the x-axis refer to the particular overlapping peptide (of 410 spanning the C clade proteome) to which a response was detected. The x in each column denotes the median viral load of responders to that peptide. Figure 5. HLA-B58:02–Speciﬁc Vaccine Effect • JID 2016:214 (1 August) • 387 Notes Acknowledgments. We thank the National Institute of Allergy and In- fectious Diseases (NIAID), National Institutes of Health (NIH), and the NIAID-funded HIV Vaccine Trials Network (HVTN), for providing clinical data from the HVTN503/Phambili trial; members of the HVTN503/Pham- bili team, for their contributions and discussions during our study; and the Phambili trial subjects and the staff at the trial clinical sites, for their participation. 17. Novitsky V, Gilbert P, Peter T, et al. Association between virus-speciﬁc T-cell re- sponses and plasma viral load in human immunodeﬁciency virus type 1 subtype C infection. J Virol 2003; 77:882–90. 18. Payne R, Muenchhoff M, Mann J, et al. Impact of HLA-driven HIV adaptation on virulence in populations of high HIV seroprevalence. Proc Natl Acad Sci U S A 2014; 111:E5393–400. 19. Altfeld MA, Trocha A, Eldridge RL, et al. Identiﬁcation of dominant optimal HLA-B60- and HLA-B61-restricted cytotoxic T-lymphocyte (CTL) epitopes: rapid characterization of CTL responses by enzyme-linked immunospot assay. J Virol 2000; 74:8541–9. E. M. L., P. C. M., and P. J. R. G. were responsible for study design. E. M. L. performed laboratory-based assays. E. M. L., J. H., J. C., P. C. M., and P. J. R. G. analyzed and interpreted the data. N. F. assisted with longi- tudinal data access. E. M. L. wrote the ﬁrst draft of the manuscript. All co- authors provided comments to E. M. L. on the manuscript. E. M. L. and P. J. R. G. were responsible for the ﬁnal version of the manuscript. 20. Addo MM, Yu XG, Rathod A, et al. Comprehensive epitope analysis of human immunodeﬁciency virus type 1 (HIV-1)-speciﬁc T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J Virol 2003; 77:2081–92. Financial support. This work was supported by the NIH (grant RO1AI46995 to P. J. R. G.), the Wellcome Trust (grant WT104748MA to P. J. R. G.), the NIAID (grant UM1AI068618 to N. F. and grants 5U01 AI068614, 5U01 AI068618, 5U01 AI068635, 5U01 AI069453, 5U01 AI069519, and 5U01 AI069469 to the HIV Vaccine Trials Network), the National Institute of Health Research (NIHR to P. C. M.), the South African Research Chairs Initiative, the Victor Daitz Foundation, and the Howard Hughes Medical Institute an (Inter- national Early Career Scientist Award to T. N.). 21. Wright JK, Novitsky V, Brockman MA, et al. References 1. Goulder PJ, Walker BD. HIV and HLA class I: an evolving relationship. Immunity 2012; 37:426–40. 2. Kiepiela P, Leslie AJ, Honeyborne I, et al. Dominant inﬂuence of HLA-B in me- diating the potential co-evolution of HIV and HLA. Nature 2004; 432:769–75. g p 3. Leslie A, Matthews PC, Listgarten J, et al. Additive contribution of HLA class I 3. Leslie A, Matthews PC, Listgarten J, et al. Additive contribution of HLA class alleles in the immune control of HIV-1 infection. J Virol 2010; 84:9879–88. 3. Leslie A, Matthews PC, Listgarten J, et al. Additive contribution of HLA class alleles in the immune control of HIV-1 infection. J Virol 2010; 84:9879–88. 4. Carlson JM, Brumme CJ, Martin E, et al. Correlates of protective cellular immu- nity revealed by analysis of population-level immune escape pathways in HIV-1. J Virol 2012; 86:13202–16. 5. Kiepiela P, Ngumbela K, Thobakgale C, et al. CD8+ T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med 2007; 13:46–53. at Deutsches RheumaforschungsZentrum und http://jid.oxfordjournals.org/ Downloaded from at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from 6. Ngumbela KC, Day CL, Mncube Z, et al. Targeting of a CD8T cell env epitope presented by HLA-B5802 is associated with markers of HIV disease progression and lack of selection pressure. AIDS Res Hum Retroviruses 2008; 24:72–82. In conclusion, although the MRKAd5 HIV-1 Gag/Pol/Nef vaccine increased the infection risk among vaccine recipients in the Step and Phambili trials [7, 8, 10], these data suggest its potential role as a therapeutic vaccine operating in an HLA- speciﬁc manner, with the capacity to beneﬁt individuals expressing disease-susceptible alleles. Even in ART recipients, CD8+ T cells may contribute to suppression of viremia [41], and so-called shock and kill cure strategies [42] may likely re- quire effective anti–HIV-1 CD8+ T-cell activity to achieve success- ful eradication of viral reservoirs [43]. Any theoretical increased risk of HIV-1 infection resulting from adenoviral-based vaccines in subjects already infected with HIV-1 would be low as compared to the potential therapeutic beneﬁt of inducing an effective anti– HIV-1 T-cell response. These studies indicate that the dramatic differences in HLA composition between distinct populations may contribute to variation in vaccine efﬁcacy. 7. Buchbinder SP, Mehrotra DV, Duerr A, et al. Efﬁcacy assessment of a cell- mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial. References Lancet 2008; 372:1881–93. 8. Gray GE, Moodie Z, Metch B, et al. Recombinant adenovirus type 5 HIV gag/pol/ nef vaccine in South Africa: unblinded, long-term follow-up of the phase 2b HVTN 503/Phambili study. Lancet Infect Dis 2014; 14:388–96. 9. Duerr A, Huang Y, Buchbinder S, et al. Extended follow-up conﬁrms early vaccine-enhanced risk of HIV acquisition and demonstrates waning effect over time among participants in a randomized trial of recombinant adenovirus HIV vaccine (Step Study). J Infect Dis 2012; 206:258–66. 10. Gray GE, Allen M, Moodie Z, et al. Safety and efﬁcacy of the HVTN 503/Phambili study of a clade-B-based HIV-1 vaccine in South Africa: a double-blind, rando- mised, placebo-controlled test-of-concept phase 2b study. Lancet Infect Dis 2011; 11:507–15. 11. Janes H, Frahm N, DeCamp A, et al. MRKAd5 HIV-1 Gag/Pol/Nef vaccine- induced T-cell responses inadequately predict distance of breakthrough HIV-1 se- quences to the vaccine or viral load. PLoS One 2012; 7:e43396. 12. Fitzgerald DW, Janes H, Robertson M, et al. An Ad5-vectored HIV-1 vaccine elic- its cell-mediated immunity but does not affect disease progression in HIV-1- infected male subjects: results from a randomized placebo-controlled trial (the Step study). J Infect Dis 2011; 203:765–72. 13. Altfeld M, Goulder PJ. The STEP study provides a hint that vaccine induction of the right CD8+ T cell responses can facilitate immune control of HIV. J Infect Dis 2011; 203:753–5. Supplementary Data 14. Troyer RM, McNevin J, Liu Y, et al. Variable ﬁtness impact of HIV-1 escape mu- tations to cytotoxic T lymphocyte (CTL) response. PLoS Pathog 2009; 5:e1000365. Supplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to beneﬁt the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. 15. Masemola A, Mashishi T, Khoury G, et al. Hierarchical targeting of subtype C human immunodeﬁciency virus type 1 proteins by CD8+ T cells: correlation with viral load. J Virol 2004; 78:3233–43. 16. Betts MR, Ambrozak DR, Douek DC, et al. Analysis of total human immunode- ﬁciency virus (HIV)-speciﬁc CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection. J Virol 2001; 75:11983–91. DISCUSSION However, the absence of data on the CD8+ T-cell response in this group of 40 more-rapidly progressing subjects does not alter the validity of the ﬁnding in relation to the 60 we were able to study. Although previous studies have shown a correlation between increasing Gag breadth and decreasing viral load [5, 15, 32, 33], including in vaccinees in the Step study [28], this was not observed here (Supplementary Figures 2 and 3). In part this is due to our small study numbers. In addition, Gag-speciﬁc responses are not equally potent. For example, p24-speciﬁc re- sponses are associated with better viremic control than p17- or p15-speciﬁc responses [34]. However, in this study the design of the Gag peptide pools made these subanalyses not possible. Furthermore, sample nonavailability prevented the HLA restric- tion of the CD8+ T-cell responses in the vaccinees to be deter- mined. In large cohort studies, on average, Gag-speciﬁc breadth is associated with a lower viral load, but in small studies the var- iation between the different Gag and non-Gag responses will strongly inﬂuence these analyses. Furthermore, the vaccine- mediated increase in Gag breadth may be associated with changes in the non–Gag-speciﬁc responses. On average Nef- speciﬁc and Env-speciﬁc responses are associated with higher viral loads [5, 15]. In our study, Nef-speciﬁc breadth in infected HLA-B58:02–positive vaccinees was 4-fold lower than in HLA- B58:02–positive placeborecipients (P = .06).Nef-speciﬁc breath has been consistently shown in previous studies to be associated Finally, although low CD4+ T-cell count and high viral load are, separately, strongly correlated with an increased risk of opportunistic infections and mortality [38, 39], these measures do not necessarily equate with disease [40]. However, the HLA-B58:02–Speciﬁc Vaccine Effect • JID 2016:214 (1 August) • 387 observation of progression to a CD4+ T-cell count of <350 cells/ mm3 within 6 months of infection in >80% of the HLA- B58:02–positive placebo recipients, together with a viral set point of 5.1 log10 copies/mL in these subjects, is consistent with rapid progression observed in natural infection in HLA- B58:02–positive individuals [6, 30]. The 1.3 log10 lower viral set point in HLA-B58:02–positive vaccinees and the inability of >50% to meet the CD4+ T-cell count criteria for ART initi- ation by 600 days after infection are consistent with vaccine- mediated protection against rapid progression. Notes Inﬂuence of Gag-protease-mediated replication capacity on disease progression in individuals recently infected with HIV-1 subtype C. J Virol 2011; 85:3996–4006. 22. Gounder K, Padayachi N, Mann JK, et al. High frequency of transmitted HIV-1 gag HLA class I-Driven immune escape variants but minimal immune selection over the ﬁrst year of clade C infection. PLoS One 2015; 10:e0119886. over the ﬁrst year of clade C infection. PLoS One 2015; 10:e0119886 23. Bates D, Maechler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. J Stat Softw 2014; 67:1–48. 24. Long JD. Longitudinal data analysis for the behavioral sciences using R. SAGE Publications, Inc., 2012. Potential conﬂicts of interest. All authors: No reported conﬂicts. All authors have submitted the ICMJE Form for Disclosure of Potential Con- ﬂicts of Interest. Conﬂicts that the editors consider relevant to the content of the manuscript have been disclosed. 25. WHO. HIV/AIDS Programme. Antiretroviral therapy for HIV infection in adults and adolescents: recommendations for a public health approach. 2006 revision. http://www.who.int/hiv/pub/guidelines/en/. Accessed 18 March 2015. 388 • JID 2016:214 (1 August) • Leitman et al 34. Honeyborne I, Prendergast A, Pereyra F, et al. Control of human immunodeﬁcien- cy virus type 1 is associated with HLA-B13 and targeting of multiple gag-speciﬁc CD8+ T-cell epitopes. J Virol 2007; 81:3667–72. 26. Kloverpris HN, Harndahl M, Leslie AJ, et al. HIV control through a single nucle- otide on the HLA-B locus. J Virol 2012; 86:11493–500. 27. Gray G, Buchbinder S, Duerr A. Overview of STEP and Phambili trial results: two phase IIb test-of-concept studies investigating the efﬁcacy of MRK adenovirus type 5 gag/pol/nef subtype B HIV vaccine. Curr Opin HIV AIDS 2010; 5:357–61. 35. Mehrotra DV, Li X, Gilbert PB. A comparison of eight methods for the dual-end- point evaluation of efﬁcacy in a proof-of-concept HIV vaccine trial. Biometrics 2006; 62:893–900. 28. Janes H, Friedrich DP, Krambrink A, et al. Vaccine-induced gag-speciﬁc T cells are associated with reduced viremia after HIV-1 infection. J Infect Dis 2013; 208:1231–9. 36. Shepherd BE, Gilbert PB, Jemiai Y, Rotnitzky A. Sensitivity analyses comparing outcomes only existing in a subset selected post-randomization, conditional on co- 36. Shepherd BE, Gilbert PB, Jemiai Y, Rotnitzky A. Sensitivity analyses comparing outcomes only existing in a subset selected post-randomization, conditional on co- variates, with application to HIV vaccine trials. Biometrics 2006; 62:332–42. 29. Cao K, Hollenbach J, Shi X, Shi W, Chopek M, Fernandez-Vina MA. HLA-B58:02–Speciﬁc Vaccine Effect • JID 2016:214 (1 August) • 389 Notes Analysis of the frequencies of HLA-A, B, and C alleles and haplotypes in the ﬁve major ethnic groups of the United States reveals high levels of diversity in these loci and con- trasting distribution patterns in these populations. Hum Immunol 2001; 62:1009–30. 37. Vince N, Bashirova AA, Lied A, et al. HLA class I and KIR genes do not protect against HIV type 1 infection in highly exposed uninfected individuals with hemo- philia A. J Infect Dis 2014; 210:1047–51. at Deutsches RheumaforschungsZentrum und http://jid.oxfordjournals.org/ Downloaded from 30. Lazaryan A, Lobashevsky E, Mulenga J, et al. Human leukocyte antigen B58 super- type and human immunodeﬁciency virus type 1 infection in native Africans. J Virol 2006; 80:6056–60. 38. Seage GR III, Losina E, Goldie SJ, Paltiel AD, Kimmel AD, Freedberg KA. The relationship of preventable opportunistic infections, HIV-1 RNA, and CD4 Cell counts to chronic mortality. J Acquir Immune Deﬁc Syndr 2002; 30:421–8. at Deutsches RheumaforschungsZentrum und Max Planck Institut fuer Infgektionsb on October 27, 2016 http://jid.oxfordjournals.org/ Downloaded from 31. Fraser C, Hollingsworth TD, Chapman R, de Wolf F, Hanage WP. Variation in HIV-1 set-point viral load: epidemiological analysis and an evolutionary hypoth- esis. Proc Natl Acad Sci USA 2007; 104:17441–6. 39. Mellors JW, Rinaldo CR Jr, Gupta P, White RM, Todd JA, Kingsley LA. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 1996; 272:1167–70. 32. Geldmacher C, Currier JR, Herrmann E, et al. CD8 T-cell recognition of multiple epitopes within speciﬁc Gag regions is associated with maintenance of a low steady-state viremia in human immunodeﬁciency virus type 1-seropositive pa- tients. J Virol 2007; 81:2440–8. 40. Group I-ES, Committee SS, Abrams D, et al. Interleukin-2 therapy in patients with HIV infection. N Engl J Med 2009; 361:1548–59. 41. Silvestri S. Animal models of HIV prevention and cure. Seattle, WA: CROI, 2015. 41. Silvestri S. Animal models of HIV prevention and cure. Seatt 33. Radebe M, Gounder K, Mokgoro M, et al. Broad and persistent Gag-speciﬁc CD8+ T-cell responses are associated with viral control but rarely drive viral escape dur- ing primary HIV-1 infection. AIDS 2015; 29:23–33. 42. Deeks SG. HIV: shock and kill. Nature 2012; 487:439–40. 43. Deng K, Pertea M, Rongvaux A, et al. Broad CTL response is required to clear la- tent HIV-1 due to dominance of escape mutations. Nature 2015; 517:381–5.
https://openalex.org/W2605712521	https://periodicos.ufsc.br/index.php/revistacfh/article/download/2178-4582.2016v50n2p265/33924	Portuguese	null	Intensidades reflexivas sob a égide do caos	Revista de Ciências Humanas	2,016	cc-by	1,230	Revista de Ciências HUMANAS, Florianópolis, v. 50, n. 2, p. 265-267, jul-dez 2016 Revista de Ciências HUMANAS, Florianópolis, v. 50, n. 2, p. 265-267, jul-dez 2016 Editorial Intensidades reflexivas sob a égide do caos Reflective intensities under the aegis of chaos http://dx.doi.org/10.5007/2178-4582.2016v50n2p265 O ano de 2016 foi marcado por fortes golpes as instituições nacionais, em um franco processo de desconstrução de valores e sentidos pautados em direitos humanos e sociais. Vimos explodir afirmações pautadas em emoções negativas, objetivadas em ódio, acompanhadas por uma racionalidade precon- ceituosa e excludente, as quais são capazes de fornecer a inteligibilidade de múltiplas formas de violência. Sabemos que nenhuma emoção caminha sem uma racionalidade que a sustente, ampare e lhe dê a inteligibilidade que necessita para formar o argu- mento voltado a uma determinada prática e, sendo assim, percebemos que o ódio tão presente nos discursos nas redes sociais no Brasil se alimenta de uma pensabilidade pautada na ideia de um universo naturalizado. Neste universo está presente quase que uma concepção fascista, obviamente caótica, mas tra- vestida de democracia. Para resistir e construir múltiplas estratégias de luta é preciso outras emo- ções e também outras pensabilidades que possam nos servir de argumento e inteligibilidade para um caminho outro de relações entre os humanos, assim como entre os humanos e o universo. É sob esta perspectiva que apresento o volume 50, número 2, com artigos que podem nos servir na construção de novas argumentações. Abrimos o número com o artigo de Francisco Canella intitulado “O movi- mento dos sem-teto em Florianópolis: mudanças no perfil dos atores e práticas (1990 – 2014)”, no qual o autor analisa as recentes ocupações urbanas da região da Grande Florianópolis (SC), tendo por foco a Ocupação Contestado, que demarcou a retomada de lutas organizadas pelo acesso à terra urbana. Em seguida, o artigo “De vizinhos a piqueteiros: movimentos de trabalhado- res desempregados e grupos subalternos na Argentina recente”, de Renake B. David das Neves, analisa as transformações na ação e no pensamento político de setores populares na Argentina a partir do surgimento dos Movimentos de Trabalhadores Desempregados (piqueteiros), em meados dos anos 1990, expressando facetas da reconfiguração da relação entre capital e trabalho no capitalismo contemporâneo (pós-1970). Lívia M. C. dos Santos e Bader B. Sawaia, analisam, a partir de Vigotski, as afetações que manifestações artís- 265 MAHEIRIE, Kátia. Editorial: Intensidades reflexivas sob a égide do caos ticas tradicionais promovem em um bairro no artigo “Um mergulho no ‘mor- ro do querosene’ e o encontro com os artistas do invisível: reflexões sobre arte, comun(idade), afeto e práxis psicossocial”. Editorial As autoras evidenciam que atividades artísticas, quando realizadas na rua, promovem encontros que fa- vorecem configurações comunitárias, ao mesmo tempo em que explicitam as contradições e relações de poder que permeiam o cotidiano. Suzana M. Gotardo, Hervacy Brito, Maria Carolina de A. Freitas, Maria Elizabeth B. de Barros e Vânia C. de Araújo, no artigo “O processo de forma- ção de comissões de saúde do trabalhador da educação: experiência e políti- ca”, problematizam a prática de formação empreendida em Projeto Piloto de Comissões de Saúde do Trabalhador da Educação (COSATEs) no município de Serra/ES, articulando com conceitos oriundos de obras de Hannah Arendt, Walter Benjamin e Giorgio Agamben. O artigo “Luhmann, Lotman e o pro- blema da fronteira: uma reflexão teórico-crítica em torno das categorias de inclusão e exclusão”, de Jan Steffens e Suene de S. Dantas, apresentam o atual debate sobre inclusão e exclusão na Alemanha, problematizado o conceito de fronteira, compreendida como uma dimensão espaço-temporal onde o diálogo entre sistemas psíquicos e entre sistemas sociais se efetua. O artigo “O desamparo dos adolescentes pobres na cidade de Maringá”, de autoria de Angela M. P. Caniato e Monica S. Capelasso, a partir da Psico- política de Freud e Theodor Adorno, mostram como diferentes expressões da violência fragilizam a identidade subjetiva dos adolescentes de classes socio- econômicas desfavorecidas. Lucienne Martins-Borges e Allyne Fernandes O. Barros, no artigo “Homicídios conjugais: notícias publicadas em jornais do sudeste do Brasil”, fazem um mapeamento inicial dos homicídios conjugais por meio de uma pesquisa documental, consultando as matérias publicadas nos jornais O Estado de São Paulo e Jornal da Tarde entre 2000 e 2010. “A psi- cose ordinária e seus índices: uma investigação à luz da clínica borromeana”, de autoria de Marconi M. da C. Guedes e Márcia M. R.V. Luchina, aborda a noção de psicose ordinária e suas manifestações na clínica psicanalítica atual, a partir das formulações da clínica borromeana de Jacques Lacan, interrogan- do, quais são os seus índices e fundamentos conceituais. “Capacitação da pessoa com deficiência intelectual ao mercado de traba- lho numa APAE do sul de Santa Catarina”, de Laysa K. Cardoso e Cristina A. R. Kern, é o artigo que trata de um estudo de caso em uma APAE do sul de Santa Catarina, para conhecer as medidas adotadas pela instituição na capa- citação para a inclusão da pessoa com deficiência intelectual no mercado de trabalho. Natanael Ítalo A. Editorial da C. Marreiros no artigo “Do direito à educação à perspectiva ressocializadora: análise de uma escola pública em uma peniten- ciária”, analisa se as propostas de ensino de uma instituição dentro de uma pe- 266 Revista de Ciências HUMANAS, Florianópolis, v. 50, n. 2, p. 265-267, jul-dez 2016 nitenciária estavam de acordo com as Diretrizes Curriculares Nacionais para oferta de educação de jovens e adultos em situação de privação de liberdade nos estabelecimentos penais. Deivis Perez e Carla Messias, no artigo intitulado “As aplicações da au- toconfrontação no exame do trabalho docente”, discute o uso do dispositivo autoconfrontação em investigações sobre o trabalho docente, realizadas por pesquisadores de um grupo de pesquisa da área da Linguística Aplicada. O artigo “Uma revisão de literatura sobre técnicas de pesquisa utilizadas nos estudos da relação criança com os espaços abertos”, de Patrícia M. S. Peres, Luana dos S. Raymundo e Ariane Kuhnen, analisa as publicações de periódi- cos em Psicologia Ambiental dos anos de 2002 até 2012, encontrando, através dos descritores “children and outdoors” 18 estudos, e identifica o predomínio de técnicas tradicionais combinadas a técnicas como desenho, trajetos de ca- minhada e autofotografia. O artigo “O risco de desastre e as cidades: uma análise discursiva sobre práticas em defesa civil”, de autoria de Juliana C. B. da Silva e Jaileila M. de A. Menezes, discute o modelo de risco de desastre que fundamenta algumas práticas em defesa civil no Brasil, por meio da análise de discurso realizada em documentos oficiais brasileiros. Daniel R. Santiago da Silva e Úrsula A. Matthias, no artigo “Autonomia apesar da dependência: a construção de uma antropologia dimensional no diálogo entre Frankl e Hartmann”, trata de um estudo teórico onde são apresentados alguns pontos da Ontologia Dimensio- nal de Nicolai Hartmann e a relação destes com a Antropologia Ontológico- Dimensional de Viktor Frankl. “Design em paralaxe: uma discussão sobre a coexistência de diferentes abordagens do design na contemporaneidade”, arti- go de autoria de Patrícia Wielewicki e Rui M. F. Roda, estabelece um debate entre teóricos do campo do design e de outras áreas do saber, discutindo temas como identidade, valor e função no design, propondo o conceito ‘design em paralaxe’, como capaz de contribuir para a reflexão sobre as recorrentes carac- terizações estereotipadas do design. Intensas reflexões a nos brindar para novas lutas e resistências! Desejamos a todas e todos, boa leitura! Editorial Kátia Maheirie Editora Geral Kátia Maheirie 267
W1908654922.txt	https://www.scielo.br/j/rsp/a/95YnWLRjRLQG6ZbHQ8tZySs/?lang=en&format=pdf	en		Economic crisis and counter-reform of universal health care systems: Spanish case	Revista de saúde pública/Revista de Saúde Pública	2,015	cc-by	1,827	Rev Saúde Pública 2015;49:34 Brief Communication Paulo Antônio de Carvalho FortesI Economic crisis and counter-reform of universal health care systems: Spanish case Regina Ribeiro Parizi CarvalhoII Marília Cristina Prado LouvisonIII DOI:10.1590/S0034-8910.2015049005469 Crise econômica e contrarreforma dos sistemas universais de saúde: caso espanhol ABSTRACT The economic crisis that has been affecting Europe in the 21st century has modified social protection systems in the countries that adopted, in the 20th century, universal health care system models, such as Spain. This communication presents some recent transformations, which were caused by changes in Spanish law. Those changes relate to the access to health care services, mainly in regards to the provision of care to foreigners, to financial contribution from users for health care services, and to pharmaceutical assistance. In crisis situations, reforms are observed to follow a trend which restricts rights and deepens social inequalities. DESCRIPTORS: Health Systems, economics. Spain. Health Care Reform. European Union, economics. Equity in Health. Health Policy. RESUMO I Departamento de Prática de Saúde. Faculdade de Saúde Pública. Universidade de São Paulo. São Paulo, SP, Brasil II Centro de Desenvolvimento e Pesquisa. Instituto de Assistência Médica ao Servidor Público Estadual. São Paulo, SP, Brasil III Departamento de Prática de Saúde. Faculdade de Saúde Pública. Universidade de São Paulo. São Paulo, SP, Brasil Correspondence: Regina Ribeiro Parizi Carvalho Rua Dr. Diogo de Faria, 1311 Apto 51 Vila Clementino 04037-005 São Paulo, SP, Brasil E-mail: pariziregina@gmail.com Received: 3/25/2014 Approved: 9/24/2014 Article available from: www.scielo.br/rsp A crise econômica que afeta a Europa neste século XXI tem modificado os sistemas de proteção social dos países que adotaram, no século XX, o modelo de sistema de saúde universalista, como a Espanha. Esta comunicação apresenta algumas transformações recentes, causadas por mudanças na legislação espanhola. Estas se deram no acesso às prestações de saúde, principalmente com relação ao atendimento a estrangeiros, no aporte financeiro dos usuários às prestações sanitárias e na assistência farmacêutica. Verifica-se que em situações de crise há tendências de reforma no sentido de restringir direitos, aprofundando desigualdades sociais. DESCRITORES: Sistemas de Saúde, economia. Espanha. Reforma dos Serviços de Saúde. União Europeia, economia. Equidade em Saúde. Política de Saúde. 2 The economic crisis that has been affecting Europe in the 21st century has modified social protection systems in the countries that adopted, in the 20th century, universal health care system models, such as Portugal, Italy, Greece, the United Kingdom, and Spain.2 This communication, as part of a study conducted on economic crisis trends on health care policies and the national health care system in Spain, presents some recent transformations that were caused by changes in the legislation. These processes relate to the access to health care services, to financial contribution from users for health care services, and to pharmaceutical assistance. Having many points that may be closely related to Brazil’s public health care system, despite the historical differences and the distinct political, social, and economic contexts of those countries, it seems to us that it is important to understand what has been happening with the Spanish health care system during this time of economic crisis. In Spain, from 1978 to 1986, the health care system consisted of mandatory insurance, in the form of social security, and it was available to insured workers and their beneficiaries. As of Law 14/1986, which was called Ley General de la Sanidad (General Health Care Law), principles and guidelines were established, and allowed health care to become universal and accessible to all Spanish, regardless of their employment relationship statuses.3 The system started to be financed by general taxes, rather than by mandatory contributions from workers and companies. A national health care system was implemented, along the lines of the Brazilian Unified Health System (SUS), joining old and fragmented regional and corporate service networks, regardless of their operation conditions. A National Health Care System was created, considering the set of health care services from the central government and from the Autonomous Communities (CCAA), in a way that was similar to Brazilian state governments, but with increased autonomy, which were then responsible for planning and managing health care center and specific service networks. The Interterritorial Council of the Spanish National Health Service was organized as a coordination instrument between the national scope and the CCAA, in an effort to reach consensus among involved parties, a task that, in Spain, is partly performed by the Tripartite Intermanagement Committee. Similar to SUS, it gathers representatives from federal, state, and municipal governments. However, in SUS social participation spaces have been organized in the decision-making agencies, such as the Conselho Nacional de Saúde and other state and municipal councils. Economic crisis and health care systems Fortes PAC et al Based on constitutional regulations and on the Autonomy Statutes, all CCAA have progressively taken over responsibilities regarding public health care, in a decentralization process that differs from the one existing in Brazil, as two government levels exist in Spain – the national and the regional one – which do not establish local competences, rights, or duties such as SUS.4 The Law from 1986, in article 1, aimed to protect the health of all Spanish people, including non-residents, and of all foreign citizens who resided in the national territory. The coverage of the universal system went from 86.0% of insured people, in 1982, to 97.0% of the population in 1987.1 The legal grounds, up to 2012, enabled the system to provide coverage of health care services to the whole Spanish population and to all foreigners, even the illegal ones. That situation has been changing after the Real Decreto-ley 16/2012, from April 20, 2012, was enacted. It deals with urgent measures in order to ensure the sustainability of the National Health Care System. That regulation was created during a crucial period when the country was hit by the economic crisis. It contained measures that were considered urgent, on the need for economic and social adjustments, on the alleged claims that they would ensure sustainability in the National Health Care System, contain expenditures, and target improvements in quality and safety for the provision of services. Some changes stand out in the Spanish health care system, as of 2012, when the legislation changed the health care model, which shifted from a health care system that was conceived under a model of universal rights, back to a social security model, with insured individuals and beneficiaries. Out of the changes included in that law, article 3 of the Real Decreto stands out – “regarding the condition of the insured party” – where it is established that the Spanish health assistance, as provided by the publicly-funded National Health Care System, must be assured to individuals who fit the status of insured persons; that is, workers who are affiliated to the Social Security Program, pensioners in the system, and beneficiaries of periodic payments such as unemployment insurance. Another change is the increased access restrictions to the system. Thus, the persons with Spanish nationality or who are citizens from other Member States of the European Union, from the European Economic Area, or from Switzerland were excluded, as they have never made contributions to the funding of the social security system. The same is true for individuals whose income exceeds one hundred thousand (100,000) euros a year. 3 Rev Saúde Pública 2015;49:34 Illegal foreigners, who are neither registered nor authorized as residents in the country, lose the right to health care, and it may be granted only in emergency situations – due to serious diseases or accidents, regardless of their causes – and during pregnancy, labor, and postpartum situations. However, all people under 18 years old should be granted health care in the same conditions as the Spanish people. Other measures that extended restrictions were the increase in financial contributions at the time services are provided and the division of those services in three categories, named basic, supplementary and accessory. In supplementary and accessory modalities, insured people must make copayments when receiving care. The common basic portfolio of health care services comprises all health care activities regarding prevention, diagnosis, treatment, and rehabilitation, which are performed in health centers or in social health centers, as well as emergency transportation, whose full cover is made possible by public funding. The common supplementary portfolio includes all services whose provision is conducted in outpatient facilities, and cover the provision of pharmaceutical assistance, prosthetics, diet products, and non-emergency medical transportation. The portfolio of accessory services includes activities such as occupational therapy, services, and techniques that are not deemed essential, or are adjuvant in supporting the treatment of chronic pathologies. In regards to outpatient pharmaceutical assistance, the Real Decreto outlined specific regulations and guidelines that restrict the free distribution of medications. The access to those products makes users subject to significant copayments, which are proportional to their income levels. These values are updated annually. Each CCAA may incorporate techniques, technologies, and procedures in their portfolios which are not covered in the national regulations, with their own additional funds. Those listed measures are inserted in the core of the discussion, and in political and judicial disputes, not only in Spain, but also in the whole European community. Thus, important protests are being conducted, involving the population, health care professionals, the Spanish judicial system, and some of the organizations that develop European agreements. Those protests criticize the change in the universal health care model.1 Some of those debates question whether the adopted measures will be efficient and effective, with a significant financial impact, or if they will increase injustice and iniquity to individuals, thus hindering public health, and therefore, the country’s economy. That scenario, it is important to remember, was the one which consolidated the creation of the first universal health care system in the United Kingdom, in 1948. During crisis situations in countries whose social protection systems are greatly important, reforms that restrict rights may cause damage to the most underprivileged people, deepening inequalities and social exclusion. In the European economic crisis, which has included several restrictive measures in social wellness policies in the region, the health care area has been submitted to the most expressive alterations, with questionable effects. Those measures show that the neoliberal project, despite being the economic model that is responsible for the crisis, is still the formula used to solve budget problems, above all when health care is not considered an essential responsibility of the State. REFERENCES 1. Abellán Perpiñan JM, Sánchez Martinez FI, Martinez Pérez JE, Méndez Martinez I. El sistema sanitario público en España y sus comunidades autónomas: sostenibilidad y reformas. Bilbao: Fundación BBVA; 2013. 3. García-Armesto S, Abadía-Taira MB, Durán A, Hernández-Quevedo C, Bernal-Delgado E. Spain: health system review. Health Syst Transit. 2010;12(4):1-295, xix-xx. 2. Conill EM. Sistemas comparados de saúde. In: Campos GWS, Minayo MCS, Akerman M, Drumond Júnior M, Carvalho YM. Tratado de saúde coletiva. 2.ed. Rio de Janeiro: Hucitec; Fiocruz; 2009. p.563-613. 4. Sacardo DP, Fortes PAC, Tanaka OY. Novas perspectivas na gestão do sistema de saúde da Espanha. Saude Soc. 2010;19(1):170-9. DOI:10.1590/S0104-12902010000100014 The authors declare no conflict of interest
https://openalex.org/W2883733646	https://europepmc.org/articles/pmc6068476?pdf=render	English	null	Effects of Applying Different Resonance Amplitude on the Performance of the Impedance-Based Health Monitoring Technique Subjected to Damage	Sensors	2,018	cc-by	11,198	Received: 15 June 2018; Accepted: 11 July 2018; Published: 13 July 2018 Abstract: Smart materials such as piezoelectric transducers can be used for monitoring the health of building structures. In this study, a structural health monitoring technique known as the electromechanical impedance (EMI) method is investigated. Although the EMI method has the advantage of using a single piezoelectric patch that acts both as the actuator and as the sensor, there are still many issues to be addressed. To further understand the problem, the performance of the EMI method on a structure subjected to progressive damage at different resonance frequency ranges and peak amplitudes was investigated using three different statistical metrics: root-mean-square deviation (RMSD), mean absolute percentage deviation (MAPD) and correlation coefﬁcient deviation (CCD). Metal plates were used throughout the study. The results acquired could be used to further understand the damage identiﬁcation performance of the EMI method. Keywords: electromechanical impedance; piezoelectric transducer; damage detection; structural health monitoring; nondestructive testing Received: 15 June 2018; Accepted: 11 July 2018; Published: 13 July 2018 sensors sensors sensors Sensors 2018, 18, 2267; doi:10.3390/s18072267 Article Effects of Applying Different Resonance Amplitude on the Performance of the Impedance-Based Health Monitoring Technique Subjected to Damage Wongi S. Na , Dong-Woo Seo, Byeong-Cheol Kim and Ki-Tae Park Sustainable Infrastructure Research Center, Korea Institute of Civil Engineering & Building Technology (KICT), Gyeonggi-Do 10223, Korea; dwseo@kict.re.kr (D.-W.S.); bckim@kict.re.kr (B.-C.K.); ktpark@kict.re.kr (K.-T.P.) Correspondence: wongi84@naver.com; Tel.: +82-31-9100-155 Wongi S. Na , Dong-Woo Seo, Byeong-Cheol Kim and Ki-Tae Park Sustainable Infrastructure Research Center, Korea Institute of Civil Engineering & Building Technology (KICT), Gyeonggi-Do 10223, Korea; dwseo@kict.re.kr (D.-W.S.); bckim@kict.re.kr (B.-C.K.); ktpark@kict.re.kr (K.-T.P.) Correspondence: wongi84@naver.com; Tel.: +82-31-9100-155 Wongi S. Na , Dong-Woo Seo, Byeong-Cheol Kim and Ki-Tae Park Sustainable Infrastructure Research Center, Korea Institute of Civil Engineering & Building Technology (KICT), Gyeonggi-Do 10223, Korea; dwseo@kict.re.kr (D.-W.S.); bckim@kict.re.kr (B.-C.K.); ktpark@kict.re.kr (K.-T.P.) Correspondence: wongi84@naver.com; Tel.: +82-31-9100-155 Wongi S. Na , Dong-Woo Seo, Byeong-Cheol Kim and Ki-Tae Park Sustainable Infrastructure Research Center, Korea Institute of Civil Engineering & Building Technology (KICT), Gyeonggi-Do 10223, Korea; dwseo@kict.re.kr (D.-W.S.); bckim@kict.re.kr (B.-C.K.); ktpark@kict.re.kr (K.-T.P.) Correspondence: wongi84@naver.com; Tel.: +82-31-9100-155 1. Introduction As civil infrastructures age, factors in the surrounding environment eventually cause their components to deteriorate over time, weakening the integrity of the structure. Most of the buildings around us are made from concrete, metals and composite materials, and as such, maintenance has always been a vital issue. There are many non-destructive ways of checking the status of a structure, including acoustic emission, radiographic, ultrasonic, liquid penetrant, eddy-current testing, magnetic-particle, and more. As the number of structures requiring management is increasing and in order to minimize the need for scheduled maintenance, an effective structural health monitoring system is the preferred choice compared to conventional non-destructive methods, The electromechanical impedance (EMI) method is a structural health monitoring technique that uses a single piezoelectric (PZT) transducer to act as both sensor and actuator [1]. The EMI method uses high frequency structural excitation, usually higher than 20 kHz, through a bonded PZT transducer to detect changes in structural mechanical impedance. The application of high frequency ranges means this method can detect local damage up to a few meters depending on the properties of the host structure. When applying the EMI method, a suitable frequency range needs to be manually determined through trial and error. To accomplish this, various frequency ranges are swept in order to search for a range with multiple peaks, and this range is selected to perform the EMI method. This is a vital step, as the absence of any peaks can result in a failure to identify damage as virtually no variations in impedance signatures will be observed, such as in composite structures [2]. Since the existence of these peaks is usually difﬁcult to predict, the effect of different amplitudes on the performance of the EMI method should be investigated to achieve better understanding of the method. www.mdpi.com/journal/sensors Sensors 2018, 18, 2267; doi:10.3390/s18072267 www.mdpi.com/journal/sensors 2 of 13 Sensors 2018, 18, 2267 To date, many investigations have been performed regarding the EMI method. In [2], the author showed that it is possible to distinguish between crack damage and debonding damage using a metal disc, which is similar to the concept shown later in this study. The main idea of the study was to create a ﬁxed resonance frequency range and use an averaging technique to distinguish between the two different types of damage. 1. Introduction In [3–6], environmental factors such as temperature variation effect, corrosive solution exposure, and the durability of PZT transducers were investigated and showed that the EMI method has several issues that needed to be resolved. The idea of using steel wire to minimize the temperature effect was proposed in [3]. Here, a PZT transducer was attached at one end of the wire while the other end is attached to a pipe with temperatures increasing up to 300 ◦C. The results showed that the PZT transducer did not exceed 40 ◦C. Na et al. investigated the performance of the EMI method on adhesive joints of glass-epoxy composite plates subjected to corrosive solution [4]. The results showed that damage to the adhesive can be monitored with the EMI method. Wandowski et al. [5] conducted experiments to detect delamination of CFRP panels whereby the authors applied a technique to compensate the effects of signature variation due to change in temperature. Yang et al. [6] also investigated the EMI technique subjected to temperature variations. In addition, the durability of the PZT transducers was also tested by measuring the impedance signature for up to 15 months. The results showed that a silicone rubber layer over the PZT transducer reduced the impedance signature changes during this time period. In Baptista and Filho [7], Bhalla et al. [8], Panigrahi et al. [9] and Wandowski et al. [10], the studies focused on minimizing the cost of performing the EMI method, as an HP4294 impedance analyzer can cost up to US$40,000. This was achieved by using devices such as a function generator, oscilloscope, FFT analyzer and an AD5933 evaluation board. The experimental results have shown that such systems can also measure impedance signatures with promising repeatability and reliability. Wandowski et al. [10] compared the performance of the AD5933 evaluation board with the impedance analyzer, HIOKI IM3570 and the results indicated that the evaluation board showed promising results. Although there are many more studies that have been conducted related to the EMI method, no investigation has been carried out on the performance of the method when subjected to different amplitude sizes. Thus, it would be important to know how different statistical metrics perform when subjected to different peak amplitudes as the non-model-based EMI method relies heavily on the signature acquired after the attachment of the PZT transducer. 1. Introduction In this study, amplitude is decreased by attaching additional PZT transducers in series where various statistical metrics are used to analyze the acquired impedance signatures subjected to damage. 2. The EMI Method The PZT used for this study was the model PSI-5A4E manufactured by Piezo Systems Inc., Cambridge, MA, USA. The size of the PZT sheet was 72.4 mm × 72.4 mm with a thickness of 0.508 mm, and it was cut into the required sizes for the study. Notice that the wiring of the test specimen is taped onto the edge of the high table to allow it to be left in the air. Since this study involves creating damage to a metal plate, one needs to manually pick up the plate and replace it on the table after creating damage. Leaving the specimen in the air can eliminate any signature variations caused by changes in boundary conditions, as the impedance signatures are extremely sensitive to any changes. Figure 1. Electromechanical impedance experiment configuration. Figure 1. Electromechanical impedance experiment conﬁguration. Figure 1. Electromechanical impedance experiment configuration. Figure 1. Electromechanical impedance experiment conﬁguration. After measuring the electrical impedance of the PZT, the next step is to quantify the intensity of the damage using a statistical method. In this study, three different equations were used, the root- mean-square deviation (RMSD), mean absolute percentage deviation (MAPD) and the correlation coefficient deviation (CCD), which are shown as Equations (2)–(4), respectively. In the equations, ሺܼ௞ሻ௜ represents the reference impedance signature and ሺܼ௞ሻ௝ isthe corresponding signature. N is the number of impedance signatures with the symbols ܼ̅ signifying mean values and ߪ௓ signifying standard deviation. For the study, the real part of the impedance signature was used for data analysis, as it has been experimentally proven to be less sensitive to temperature variations than the imaginary part of the signature [11]. In general, the single value obtained from any of the three equations will be higher with an increase in damage, as the impedance signature will change more severely. The final step in the EMI method is to define a threshold value which is manually defined by experts in this field. Then, any values that exceeds this threshold value is considered to be damaged. ଶ ே ே ଵଶ ⁄ After measuring the electrical impedance of the PZT, the next step is to quantify the intensity of the damage using a statistical method. In this study, three different equations were used, the root-mean-square deviation (RMSD), mean absolute percentage deviation (MAPD) and the correlation coefﬁcient deviation (CCD), which are shown as Equations (2)–(4), respectively. 2. The EMI Method The one-dimensional equation proposed by Liang et al. (1994) shows how the EMI method works. In Equation (1), the electrical admittance Y(ω) is a combined function of Zs(ω) and Za(ω), which are the mechanical impedance of the host structure and the PZT transducer, respectively. Other variables in the equation, which are I, V, ω, a, εT 33, δ, d3x, Y E xx represent the PZT output current, PZT input voltage, input frequency, geometric constant, dielectric constant, loss tangent, piezoelectric constant and Young’s modulus, respectively. Y(ω) = iωa εT 33(1 −iδ) − Zs(ω) Zs(ω) + Za(ω)d2 3xY E xx (1) (1) To measure the electrical impedance of a PZT transducer, a commercialized AD5933 evaluation board is utilized throughout this study where all the experiments were conducted at a room temperature of 22 ± 0.2 ◦C. The board is manufactured by Analog Devices Co., Norwood, MA, USA, and retails for less than US$100. The experimental setup is shown in Figure 1. The AD5933 evaluation board is connected to a laptop, which can operate the device using the software provided within the package. The advantage of this device is its weight, as it is very light and its small size makes it portable. It can measure impedance up to 100 kHz with 511 data points and is fully powered by a USB cable 3 of 13 3 of 13 Sensors 2018, 18, 2267 Sensors 2018, 18, x FOR that is connected to the laptop. The PZT used for this study was the model PSI-5A4E manufactured by Piezo Systems Inc., Cambridge, MA, USA. The size of the PZT sheet was 72.4 mm × 72.4 mm with a thickness of 0.508 mm, and it was cut into the required sizes for the study. Notice that the wiring of the test specimen is taped onto the edge of the high table to allow it to be left in the air. Since this study involves creating damage to a metal plate, one needs to manually pick up the plate and replace it on the table after creating damage. Leaving the specimen in the air can eliminate any signature variations caused by changes in boundary conditions, as the impedance signatures are extremely sensitive to any changes. portable. It can measure impedance up to 100 kHz with 511 data points and is fully powered by a USB cable that is connected to the laptop. 3. Impedance Signature Peak Reduction Effect on EMI Performance p g To evaluate the performance of the EMI method when the impedanc To evaluate the performance of the EMI method when the impedance signature is reduced, three 15 mm square PZT transducers were prepared. Three test cases were created and tested, where the ﬁrst case “C_1” involved attaching one of the three PZT transducers to the center of the 100 mm square metal plate with a thickness 0.3 mm, as shown in Figure 2. This conﬁguration was then connected to the AD5933 evaluation board, and then progressive damage was created using a metal cutter along the dotted line up to 20 mm. During this process, impedance signatures were measured by exciting the PZT transducer at every 2 mm of damage in the frequency range of 25 kHz to 65 kHz in the 80 Hz interval. Here, the frequency range was chosen by sweeping various ranges and selecting the most appropriate range with multiple resonance peaks. Thus, including the reference signature measured before any damage was introduced, 11 impedance signatures were acquired. The second test case “C_2” was conducted by attaching an additional PZT transducer in series to the C_1 conﬁguration (also shown in Figure 2). This additional PZT is left freely in the air, as the attachment will affect the amplitude of the impedance signatures as the equivalent impedance of the two transducers is changed. In this case, since the capacitance of the PZT transducer is directly proportional to its size, the amplitude will decrease. For this second test (C_2), progressive damage is introduced in the same manner. Again, impedance signatures were measured for every 2 mm of damage up to 20 mm in the same frequency range as above, resulting in 11 impedance signatures. The third test case “C_3” involved attaching another PZT transducer in series onto the C_2 conﬁguration, which further increases the overall area of the PZT transducers. Again, progressive damage was introduced along the dotted line and the impedance signatures were measured in an identical manner to the previous two test cases. Through these three experiments (C_1, C_2 and C_3), 33 impedance signatures were acquired in total. p p g 15 mm square PZT transducers were prepared. Three test cases were created and tested, where the first case “C_1” involved attaching one of the three PZT transducers to the center of the 100 mm square metal plate with a thickness 0.3 mm, as shown in Figure 2. 3. Impedance Signature Peak Reduction Effect on EMI Performance p g To evaluate the performance of the EMI method when the impedanc This configuration was then connected to the AD5933 evaluation board, and then progressive damage was created using a metal cutter along the dotted line up to 20 mm. During this process, impedance signatures were measured by exciting the PZT transducer at every 2 mm of damage in the frequency range of 25 kHz to 65 kHz in the 80 Hz interval. Here, the frequency range was chosen by sweeping various ranges and selecting the most appropriate range with multiple resonance peaks. Thus, including the reference signature measured before any damage was introduced, 11 impedance signatures were acquired. The second test case “C_2” was conducted by attaching an additional PZT transducer in series to the C_1 configuration (also shown in Figure 2). This additional PZT is left freely in the air, as the attachment will affect the amplitude of the impedance signatures as the equivalent impedance of the two transducers is changed. In this case, since the capacitance of the PZT transducer is directly proportional to its size, the amplitude will decrease. For this second test (C_2), progressive damage is introduced in the same manner. Again, impedance signatures were measured for every 2 mm of damage up to 20 mm in the same frequency range as above, resulting in 11 impedance signatures. The third test case “C_3” involved attaching another PZT transducer in series onto the C_2 configuration, which further increases the overall area of the PZT transducers. Again, progressive damage was introduced along the dotted line and the impedance signatures were measured in an identical manner to the previous two test cases. Through these three experiments (C_1, C_2 and C_3), 33 impedance signatures were acquired in total. Figure 2. The setup for three test cases for evaluating the EMI performance subjected to amplitude reduction. Figure 2. The setup for three test cases for evaluating the EMI performance subjected to amplitude reduction. Figure 2. The setup for three test cases for evaluating the EMI performance subjected to amplitude reduction. Figure 2. The setup for three test cases for evaluating the EMI performance subjected to amplitude reduction. Figure 3 shows all the impedance signatures for the three cases where the difference in the height of the amplitudes can be clearly seen. The amplitudes, in general, significantly decrease from C_1 to C_3. The frequency ranges around 30 kHz, 44 kHz and 65 kHz are a good example of this. 2. The EMI Method In the equations, (Zk)i represents the reference impedance signature and (Zk)j is the corresponding signature. N is the number of impedance signatures with the symbols Z signifying mean values and σZ signifying standard deviation. For the study, the real part of the impedance signature was used for data analysis, as it has been experimentally proven to be less sensitive to temperature variations than the imaginary part of the signature [11]. In general, the single value obtained from any of the three equations will be higher with an increase in damage, as the impedance signature will change more severely. The ﬁnal step in the EMI method is to deﬁne a threshold value which is manually deﬁned by experts in this ﬁeld. Then, any values that exceeds this threshold value is considered to be damaged. RMSD = ൬෍ ൣܴ݁ሺܼ௞ሻ௝−ܴ݁ሺܼ௞ሻ௜൧ ෍ ሾܴ݁ሺܼ௞ሻ௜ሿଶ ௞ୀଵ ൘ ௞ୀଵ ൰ (2) MAPD = 1 ܰ෍หൣܴ݁ሺܼ௞ሻ௝−ܴ݁ሺܼ௞ሻ௜൧ܴ݁ሺܼ௞ሻ௜ ⁄ ห ே ௞ୀଵ (3) CD = 1 − 1 ܰߪߪ ෍ൣܴ݁ሺܼ௞ሻ௝−ܴ݁ሺܼ̅ሻ௝൧ ே ∙ሾܴ݁ሺܼ௞ሻ௜−ܴ݁ሺܼ̅ሻ௜ሿ (4) RMSD = N ∑ k=1 h Re(Zk)j −Re(Zk)i i2. N ∑ k=1 [Re(Zk)i]2 !1/2 (2) MAPD = 1 N N ∑ k=1 \|[Re(Zk)j −Re(Zk)i]/Re(Zk)i\| (3) MSD = ൬෍ ൣܴ݁ሺܼ௞ሻ௝−ܴ݁ሺܼ௞ሻ௜൧ ෍ ሾܴ݁ሺܼ௞ሻ௜ሿଶ ௞ୀଵ ൘ ௞ୀଵ ൰ (2) MAPD = 1 ܰ෍หൣܴ݁ሺܼ௞ሻ௝−ܴ݁ሺܼ௞ሻ௜൧ܴ݁ሺܼ௞ሻ௜ ⁄ ห ே (3) RMSD = N ∑ k=1 h Re(Zk)j −Re(Zk)i i2. N ∑ k=1 [Re(Zk)i]2 !1/2 (2) ൰ !1/2 (2) ௞ୀଵ = 1 − 1 ܰ ෍ൣܴ݁ሺܼ௞ሻ௝−ܴ݁ሺܼ̅ሻ௝൧ ே ∙ሾܴ݁ሺܼ௞ሻ௜−ܴ݁ሺܼ̅ሻ௜ሿ (4) MAPD = 1 N N ∑ k=1 \|[Re(Zk)j −Re(Zk)i]/Re(Zk)i\| (3) (3) 4 of 13 Sensors 2018, 18, 2267 CCD = 1 − 1 NσZjσZi N ∑ k=1 [Re(Zk)j −Re Z j]· Re(Zk)i −Re Z i (4) R PEER REVIEW 4 of 13 nature Peak Reduction Effect on EMI Performance (4) of 13 3. Impedance Signature Peak Reduction Effect on EMI Performance p g To evaluate the performance of the EMI method when the impeda 3. Impedance Signature Peak Reduction Effect on EMI Performance p g To evaluate the performance of the EMI method when the impedanc Sensors 2018, 18, x FOR PEER REVIEW 5 of 13 progressive damage for C_3. The peak that exists at 66 kHz is reduced and virtually disappears after 20 mm of progressive damage. Figure 3. Impedance signatures for C_1, C_2 and C_3 experiment. Figure 3. Impedance signatures for C_1, C_2 and C_3 experiment. Figure 3. Impedance signatures for C_1, C_2 and C_3 experiment. Figure 3. Impedance signatures for C_1, C_2 and C_3 experiment. Using the impedance signatures shown in Figure 3, three statistical metrics (RMSD, MAPD and CCD) were calculated and are displayed in Table 1. The first column represents the damage intensity from 0 mm to 20 mm, the second to fourth columns are the RMSD values (RC_1, RC_2, RC_3), the fifth to seventh columns are the MAPD values (MC_1, MC_2, MC_3) and the last three columns are the CCD values (CC_1, CC_2, CC_3). For the RMSD values, RC_1 starts at 7.38% and ends at 13.53%, RC_2 starts at 5.78% and ends at 7.27%, and RC_3 starts at 1.63% and ends at 4.18%. In addition, averaging the 10 RMSD values for each test cases results in 11.46%, 6.04% and 3.25% for RC_1, RC_2 and RC_3, respectively. These results prove that with smaller amplitudes, the RMSD values will be lower when subjected to the same level of damage. Furthermore, the averaged RMSD values of 6.04% and 3.25% are 53% (6.04/11.46) and 28% (3.25/11.46), respectively. Using the impedance signatures shown in Figure 3, three statistical metrics (RMSD, MAPD and CCD) were calculated and are displayed in Table 1. The ﬁrst column represents the damage intensity from 0 mm to 20 mm, the second to fourth columns are the RMSD values (RC_1, RC_2, RC_3), the ﬁfth to seventh columns are the MAPD values (MC_1, MC_2, MC_3) and the last three columns are the CCD values (CC_1, CC_2, CC_3). For the RMSD values, RC_1 starts at 7.38% and ends at 13.53%, RC_2 starts at 5.78% and ends at 7.27%, and RC_3 starts at 1.63% and ends at 4.18%. In addition, averaging the 10 RMSD values for each test cases results in 11.46%, 6.04% and 3.25% for RC_1, RC_2 and RC_3, respectively. These results prove that with smaller amplitudes, the RMSD values will be lower when subjected to the same level of damage. Furthermore, the averaged RMSD values of 6.04% and 3.25% are 53% (6.04/11.46) and 28% (3.25/11.46), respectively. Table 1. 3. Impedance Signature Peak Reduction Effect on EMI Performance p g To evaluate the performance of the EMI method when the impedanc First, examining the impedance signatures between 28 kHz and 32 kHz, the center of the resonance peaks seems to shift slightly rightward from C_1 to C_3 with the overall amplitudes decreasing, which possibly indicates that progressive damage is changing the resonance frequency of the host structure. For C_1, C_2 and C_3 the maximum peak-to-peak heights of the resonance are around 15 kΩ, 7 kΩ and 5 kΩ, respectively. Next, examining the impedance signatures between 43 kHz and 46 kHz shows a similar result where resonance is shifting rightward as damage progresses. However, the maximum peak-to-peak amplitudes for the three test cases are around 14 kΩ, 5 kΩ and 1.5 kΩ from C_1 to C_3, proving that an increase in PZT area does not have a linear relationship to the maximum height of Figure 3 shows all the impedance signatures for the three cases where the difference in the height of the amplitudes can be clearly seen. The amplitudes, in general, signiﬁcantly decrease from C_1 to C_3. The frequency ranges around 30 kHz, 44 kHz and 65 kHz are a good example of this. First, examining the impedance signatures between 28 kHz and 32 kHz, the center of the resonance peaks seems to shift slightly rightward from C_1 to C_3 with the overall amplitudes decreasing, which possibly indicates that progressive damage is changing the resonance frequency of the host structure. For C_1, C_2 and C_3 the maximum peak-to-peak heights of the resonance are around 15 kΩ, 7 kΩand 5 kΩ, respectively. Next, examining the impedance signatures between 43 kHz and 46 kHz shows a similar result where resonance is shifting rightward as damage progresses. However, the maximum peak-to-peak amplitudes for the three test cases are around 14 kΩ, 5 kΩand 1.5 kΩfrom 5 of 13 Sensors 2018, 18, 2267 C_1 to C_3, proving that an increase in PZT area does not have a linear relationship to the maximum height of the peak-to-peak amplitudes. Finally, at the frequency range between 63 kHz and 67 kHz, the resonance is again shifting in the rightward direction with damage. This can be clearly seen in the C_1 test. In addition, another observation made here is the decrease in the peak amplitudes with progressive damage for C_3. The peak that exists at 66 kHz is reduced and virtually disappears after 20 mm of progressive damage. 4. Creating Various Resonance Frequency Ranges attached on top of the metal disc with a 25 mm diame were created using two different metal discs with thi In the previous section, experiments were used to conﬁrm that a decrease in the amplitude of the impedance signature will also decrease the RMSD and MAPD values (but not CCD values). Thus, to further the investigation, the amplitude reduction effect on the EMI method subjected to damage in various frequency ranges should be examined. To achieve this, the conventional method of attaching a PZT transducer to the surface of the host structure is changed in order to create resonance frequency ranges in various regions. In Na et al. [4], the authors introduced a technique for creating resonance frequency ranges in certain regions regardless of the properties of the host structure. The core of this technique involves attaching a PZT transducer on to a metal disc, then attaching the metal disc onto the host structure. Here, the resonance frequency range can be either increased or decreased simply by changing the thickness of the metal disc. Thus, in this study, this idea is applied to create various frequency ranges where additional free PZT transducers were utilized to reduce peak amplitudes. This conﬁguration can be seen in Figure 4a, where a 15 mm square PZT transducer is attached on top of the metal disc with a 25 mm diameter and 3 mm thickness, then, two more devices were created using two different metal discs with thicknesses of 4 mm and 5 mm. These three devices were used to create 9 test cases referred to as “T3_x”, “T4_x” and “T5_x”, where the ﬁrst number represents the thickness of the metal in mm and variable x is the total number of PZT transducers connected. For example, T3_1 is the 3 mm metal disc with only the PZT transducer attached, T3_2 is the same with an additional free PZT transducer attached in series, and T3_3 has another free PZT transducer attached to T3_2 in series. g were used to create 9 test cases referred to as “T3_x”, “T4_x” and “T5_x”, where the first number represents the thickness of the metal in mm and variable x is the total number of PZT transducers connected. For example, T3_1 is the 3 mm metal disc with only the PZT transducer attached, T3_2 is the same with an additional free PZT transducer attached in series, and T3_3 has another free PZT transducer attached to T3_2 in series. 3. Impedance Signature Peak Reduction Effect on EMI Performance p g To evaluate the performance of the EMI method when the impedanc Creating Variou I th i 6 of 13 ude of of 27.55%. For the MAPD values of MC_2, the average value was signiﬁcantly decreased to 2.62% (from 18.42%) where the values start from 1.58% and end at 3.46%. This shows that the amplitude reduction of the impedance signature has a vital impact on the MAPD values. Next, the values for MC_3 have an average of 1.64%, starting from 0.71% and ending at 2.12%. the impedance signature will also decrease the RMSD and MAPD values (but not CCD values). Thus, to further the investigation, the amplitude reduction effect on the EMI method subjected to damage in various frequency ranges should be examined. To achieve this, the conventional method of attaching a PZT transducer to the surface of the host structure is changed in order to create resonance frequency ranges in various regions In Na et al [4] the authors introduced a technique for creating Compared to the RMSD and MAPD values, the CCD values, the results show a different pattern. The highest average value is observed for CC_2 with a value of 5.46%, followed by 3.94% for CC_3 and 1.92% for CC_1. As expected, the CCD index is insensitive to variations in amplitude as it is based on the correlation coefﬁcient. However, when comparing RC_3, MC_3 and CC_3 average values, CC_3 has the highest with 3.94%. This experimentally shows that when there are many peaks with only small amplitudes in the signature, CCD is the preferred choice of the three statistical metrics. frequency ranges in various regions. In Na et al. [4], the authors introduced a technique for creating resonance frequency ranges in certain regions regardless of the properties of the host structure. The core of this technique involves attaching a PZT transducer on to a metal disc, then attaching the metal disc onto the host structure. Here, the resonance frequency range can be either increased or decreased simply by changing the thickness of the metal disc. Thus, in this study, this idea is applied to create various frequency ranges where additional free PZT transducers were utilized to reduce peak amplitudes. This configuration can be seen in Figure 4a, where a 15 mm square PZT transducer is 3. Impedance Signature Peak Reduction Effect on EMI Performance p g To evaluate the performance of the EMI method when the impedanc Three statistical metric values for C_1, C_2 and C_3 experiment. Damage RMSD (%) MAPD (%) CCD (%) (mm) RC_1 RC_2 RC_3 MC_1 MC_2 MC_3 CC_1 CC_2 CC 2 7.38 5.78 1.63 11.51 1.58 0.71 0.90 4.66 1 4 10.61 3.92 2.29 16.10 1.70 1.03 1.63 2.37 1 6 10.06 5.50 2.09 15.07 2.20 1.08 1.49 4.51 1 8 10.95 4.72 3.25 15.77 2.33 1.68 1.73 3.37 3 10 11.68 5.94 4.19 17.38 2.80 2.05 1.94 5.20 6 12 14.24 7.03 3.41 19.58 3.16 1.75 2.77 7.06 4 14 13.32 6.43 3.72 18.36 2.96 1.91 2.46 6.07 4 16 11.59 6.92 3.84 21.12 2.95 1.97 1.91 6.75 5 18 11.22 6.93 3.86 21.79 3.07 2.05 1.81 6.94 5 20 13.53 7.27 4.18 27.55 3.46 2.12 2.54 7.64 5 Average 11.46 6.04 3.25 18.42 2.62 1.64 1.92 5.46 3 Numbers in different colors represent colors in picture. Table 1. Three statistical metric values for C_1, C_2 and C_3 experiment. Damage (mm) RMSD (%) MAPD (%) CCD (%) RC_1 RC_2 RC_3 MC_1 MC_2 MC_3 CC_1 CC_2 CC_3 2 7.38 5.78 1.63 11.51 1.58 0.71 0.90 4.66 1.06 4 10.61 3.92 2.29 16.10 1.70 1.03 1.63 2.37 1.90 6 10.06 5.50 2.09 15.07 2.20 1.08 1.49 4.51 1.62 8 10.95 4.72 3.25 15.77 2.33 1.68 1.73 3.37 3.68 10 11.68 5.94 4.19 17.38 2.80 2.05 1.94 5.20 6.05 12 14.24 7.03 3.41 19.58 3.16 1.75 2.77 7.06 4.05 14 13.32 6.43 3.72 18.36 2.96 1.91 2.46 6.07 4.81 16 11.59 6.92 3.84 21.12 2.95 1.97 1.91 6.75 5.10 18 11.22 6.93 3.86 21.79 3.07 2.05 1.81 6.94 5.13 20 13.53 7.27 4.18 27.55 3.46 2.12 2.54 7.64 5.97 Average 11.46 6.04 3.25 18.42 2.62 1.64 1.92 5.46 3.94 Numbers in different colors represent colors in picture. Table 1. Three statistical metric values for C_1, C_2 and C_3 experiment. RMSD (%) MAPD (%) C Table 1. Three statistical metric values for C_1, C_2 and C_3 experiment. For the MAPD values, MC_1 is generally larger than the values of RC_1 with an average of 18.42%. The first 2 mm results in a value of 11.51% and increases as the damage progresses, ending For the MAPD values, MC_1 is generally larger than the values of RC_1 with an average of 18.42%. The ﬁrst 2 mm results in a value of 11.51% and increases as the damage progresses, ending with a value Sensors 2018, 18, 2267 4. 4. Creating Various Resonance Frequency Ranges attached on top of the metal disc with a 25 mm diame were created using two different metal discs with thi In Figure 4b, the 9 impedance signature measurements are displayed using the three devices that were created. First, examining the three impedance signatures of T3_1, T3_2 and T3_3, there is a decrease in the amplitudes of the resonance peak located between 33 kHz and 38 kHz when additional PZT transducers are attached. Here, it can be said that the resonance is that of the metal disc. The height of the amplitude for T3_1, which was the difference between the highest and lowest points of the impedance signature for this study, is about 16 kΩ. For T3_2 and T3_3, the heights of the peak amplitudes are about 7 kΩ and 3.5 kΩ, respectively. Regarding the other 6 test cases, T4_1, T4_2, T4_3, T5_1, T5_2, and T5_3, the heights of the peak amplitudes are roughly 16 kΩ, 8 kΩ, 4.5 kΩ, 24 kΩ, 16 kΩ, and 10 kΩ, respectively. Overall, the resonance peaks are concentrated in 3 different regions, 33 kHz~38 kHz, 41 kHz~46 kHz and 50 kHz~55 kHz, thus it is possible to investigate the performance of the EMI method subjected to damage at various frequency ranges. Compared to the other cases, the amplitudes are relatively larger for the peaks at the 50 kHz~55 kHz frequency range. Thus, it can be assumed that the metal disc with a thickness of 5 mm might perform better when the EMI method is applied. The results of this test are discussed further later in this paper. In the previous section, experiments were used to conﬁrm that a decrease in the amplitude of the impedance signature will also decrease the RMSD and MAPD values (but not CCD values). Thus, to further the investigation, the amplitude reduction effect on the EMI method subjected to damage in various frequency ranges should be examined. To achieve this, the conventional method of attaching a PZT transducer to the surface of the host structure is changed in order to create resonance frequency ranges in various regions. In Na et al. [4], the authors introduced a technique for creating resonance frequency ranges in certain regions regardless of the properties of the host structure. g were used to create 9 test cases referred to as “T3_x”, “T4_x” and “T5_x”, where the first number represents the thickness of the metal in mm and variable x is the total number of PZT transducers connected. 5. Conducting the EMI Method with Various Signature Amplitudes 5. Conducting the EMI Method with Various Signature Amplitudes The damage intensity of each of the cases was assumed to be the same, as the distance from the PZT transducers was identical. For each test case, 11 impedance signatures were acquired in total (including a reference signature) in the frequency ranges between 25 kHz to 65 kHz. Figure 5b, c shows the test specimen for the remaining 6 tests (T4_1, T4_2, T4_3, T5_1, T5_2, and T5_3), where the tests were identical to Figure 5a with different metal disc thicknesses. Figure 5. Test setup for (a) T3_x; (b) T4_x; (c) T5_x. Figure 5. Test setup for (a) T3_x; (b) T4_x; (c) T5_x. Figure 5. Test setup for (a) T3_x; (b) T4_x; (c) T5_x. Figure 5. Test setup for (a) T3_x; (b) T4_x; (c) T5_x. Figure 6 shows the 33 impedance signatures acquired from the T3_1, T3_2 and T3_3 experiments, where resonance peaks are concentrated between 36 kHz and 44 kHz. Compared with the impedance signature before the PZT device was attached onto the metal plate as shown in Figure 4b, one can observe that the resonance frequency has shifted rightward, about 5 kHz. Also, additional resonance frequency ranges have appeared at around 31 kHz and 45 kHz. Since the largest resonance at 36 kHz~44 kHz is the resonance of the metal disc, one can assume that the resonance at 31 kHz and 45 kHz is the resonance of the metal plate. However, regardless of the resonance being either the metal plate or the disc, signature variations can be visually identified in the resonance frequency ranges. Finally, the impedance signature beyond 46 kHz has no resonance where the T3_1 signature shifts in the downward direction subjected to damage. For T3_2 and T3_3, the impedance signatures seemed to be unaffected by damage, showing virtually no sign of any shift movement. Figure 6 shows the 33 impedance signatures acquired from the T3_1, T3_2 and T3_3 experiments, where resonance peaks are concentrated between 36 kHz and 44 kHz. Compared with the impedance signature before the PZT device was attached onto the metal plate as shown in Figure 4b, one can observe that the resonance frequency has shifted rightward, about 5 kHz. Also, additional resonance frequency ranges have appeared at around 31 kHz and 45 kHz. 5. Conducting the EMI Method with Various Signature Amplitudes 5. Conducting the EMI Method with Various Signature Amplitudes Figure 5 shows the EMI method used to test 9 cases in order to evaluate the damage detection performance using 3 different resonance frequency ranges. Figure 5a shows the three test cases, which will be referred to as T3_1, T3_2 and T3_3 hereafter. Again, the ﬁrst number represents the thickness of the metal disc, with the second number representing the number of PZT transducers connected for the test. The PZT attached metal disc is adhered to the center of the metal plate using an epoxy glue and was left at room temperature for 24 h to ensure full curing. For the test, damage was created using the same method introduced in Section 2, where a metal cutter was used to cut the metal plate up to 20 mm with impedance signatures being measured at every 2 mm of damage. These three test cases were conducted on the same metal plate as shown in the ﬁgure. The damage intensity of each of the cases was assumed to be the same, as the distance from the PZT transducers was identical. For each test case, 11 impedance signatures were acquired in total (including a reference signature) in the frequency ranges between 25 kHz to 65 kHz. Figure 5b, c shows the test specimen for the remaining 6 tests (T4_1, T4_2, T4_3, T5_1, T5_2, and T5_3), where the tests were identical to Figure 5a with different metal disc thicknesses. Figure 5 shows the EMI method used to test 9 cases in order to evaluate the damage detection performance using 3 different resonance frequency ranges. Figure 5a shows the three test cases, which will be referred to as T3_1, T3_2 and T3_3 hereafter. Again, the first number represents the thickness of the metal disc, with the second number representing the number of PZT transducers connected for the test. The PZT attached metal disc is adhered to the center of the metal plate using an epoxy glue and was left at room temperature for 24 h to ensure full curing. For the test, damage was created using the same method introduced in Section 2, where a metal cutter was used to cut the metal plate up to 20 mm with impedance signatures being measured at every 2 mm of damage. These three test cases were conducted on the same metal plate as shown in the figure. 4. Creating Various Resonance Frequency Ranges attached on top of the metal disc with a 25 mm diame were created using two different metal discs with thi For example, T3_1 is the 3 mm metal disc with only the PZT transducer attached, T3_2 is the same with an additional free PZT transducer attached in series, and T3_3 has another free PZT transducer attached to T3_2 in series. In Figure 4b, the 9 impedance signature measurements are displayed using the three devices that were created. First, examining the three impedance signatures of T3_1, T3_2 and T3_3, there is a Figure 4. Different PZT attachment (a) configuration; (b) device impedance signatures. Figure 4. Different PZT attachment (a) conﬁguration; (b) device impedance signatures. Figure 4. Different PZT attachment (a) configuration; (b) device impedance signatures. Figure 4. Different PZT attachment (a) conﬁguration; (b) device impedance signatures. In Figure 4b, the 9 impedance signature measurements are displayed using the three devices that were created. First, examining the three impedance signatures of T3_1, T3_2 and T3_3, there is a decrease in the amplitudes of the resonance peak located between 33 kHz and 38 kHz when additional PZT transducers are attached. Here, it can be said that the resonance is that of the metal disc. The height 7 of 13 Sensors 2018, 18, 2267 of the amplitude for T3_1, which was the difference between the highest and lowest points of the impedance signature for this study, is about 16 kΩ. For T3_2 and T3_3, the heights of the peak amplitudes are about 7 kΩand 3.5 kΩ, respectively. Regarding the other 6 test cases, T4_1, T4_2, T4_3, T5_1, T5_2, and T5_3, the heights of the peak amplitudes are roughly 16 kΩ, 8 kΩ, 4.5 kΩ, 24 kΩ, 16 kΩ, and 10 kΩ, respectively. Overall, the resonance peaks are concentrated in 3 different regions, 33 kHz~38 kHz, 41 kHz~46 kHz and 50 kHz~55 kHz, thus it is possible to investigate the performance of the EMI method subjected to damage at various frequency ranges. Compared to the other cases, the amplitudes are relatively larger for the peaks at the 50 kHz~55 kHz frequency range. Thus, it can be assumed that the metal disc with a thickness of 5 mm might perform better when the EMI method is applied. The results of this test are discussed further later in this paper. Sensors 2018, 18, x FOR PEER REVIEW 7 of 13 5. Conducting the EMI Method with Various Signature Amplitudes 5. Conducting the EMI Method with Various Signature Amplitudes Since the largest resonance at 36 kHz~44 kHz is the resonance of the metal disc, one can assume that the resonance at 31 kHz and 45 kHz is the resonance of the metal plate. However, regardless of the resonance being either the metal plate or the disc, signature variations can be visually identiﬁed in the resonance frequency ranges. Finally, the impedance signature beyond 46 kHz has no resonance where the T3_1 signature shifts in the downward direction subjected to damage. For T3_2 and T3_3, the impedance signatures seemed to be unaffected by damage, showing virtually no sign of any shift movement. 8 of 13 med Sensors 2018, 18, 2267 the downward d t b ff t d Figure 6. Impedance signatures for the T3_1, T3_2 and T3_3 experiment. Figure 6. Impedance signatures for the T3_1, T3_2 and T3_3 experiment. ensors 2018, 18, x FOR PEER REVIEW 8 of 1 Figure 6. Impedance signatures for the T3_1, T3_2 and T3_3 experiment. Figure 6. Impedance signatures for the T3_1, T3_2 and T3_3 experiment. FOR PEER REVIEW Figure 7 shows the 33 impedance signatures acquired from the T4_1, T4_2 and T4_3 experiments where resonance peaks are concentrated between 43 kHz and 49 kHz. Speciﬁcally, , there are two resonance frequency regions which are at 43 kHz~45 kHz and 45 kHz~49 kHz where the ﬁrst is the resonance of the metal plate as shown in the previous ﬁgure. We know that the second resonance is the resonance of the metal disc, as it was experimentally proven that the resonance shifts rightward after the attachment of a metal plate. In addition, resonance is larger with more dynamic activities in the 43 kHz~45 kHz range compared to the previous ﬁgure, as the resonance of the metal disc has inﬂuenced the outcome. The small resonance located at 30 kHz~33 kHz is smaller when compared to the previous ﬁgure, and one possible cause of this is the thicker metal disc used for the experiment, as T4_x is 1 mm thicker than T3_x, thus making the PZT transducer 1 mm further away from the host structure. For the frequency range above 55 kHz where no resonance can be observed, the impedance signatures for T4_1 show a downward shift, with the T4_2 and T4_3 impedance signatures remaining virtually unchanged. 5. Conducting the EMI Method with Various Signature Amplitudes 5. Conducting the EMI Method with Various Signature Amplitudes Figure 7 shows the 33 impedance signatures acquired from the T4_1, T4_2 and T4_3 experiments where resonance peaks are concentrated between 43 kHz and 49 kHz. Specifically, , there are two resonance frequency regions which are at 43 kHz~45 kHz and 45 kHz~49 kHz where the first is the resonance of the metal plate as shown in the previous figure. We know that the second resonance is the resonance of the metal disc, as it was experimentally proven that the resonance shifts rightward after the attachment of a metal plate. In addition, resonance is larger with more dynamic activities in the 43 kHz~45 kHz range compared to the previous figure, as the resonance of the metal disc has influenced the outcome. The small resonance located at 30 kHz~33 kHz is smaller when compared to the previous figure, and one possible cause of this is the thicker metal disc used for the experiment, as T4_x is 1 mm thicker than T3_x, thus making the PZT transducer 1 mm further away from the host structure. For the frequency range above 55 kHz where no resonance can be observed, the impedance signatures for T4_1 show a downward shift, with the T4_2 and T4_3 impedance signatures remaining virtually unchanged. Figure 7. Impedance signatures for the T4_1, T4_2 and T4_3 experiment. Figure 7. Impedance signatures for the T4_1, T4_2 and T4_3 experiment. Figure 7. Impedance signatures for the T4_1, T4_2 and T4_3 experiment. Figure 7. Impedance signatures for the T4_1, T4_2 and T4_3 experiment. Figure 8 shows the 33 impedance signatures acquired from the T5_1, T5_2 and T5_3 experiments where resonance peaks are concentrated between 53 kHz and 59 kHz. At first glance, the impedance signatures have a relatively smaller number of peaks compared to the previous two figures. As such, one can assume that the values obtained from the statistical methods (RMSD, MAPD, CCD) would be smaller compared to the test cases, T3_x and T4_x. This will be further discussed in Section 6. For the small resonance around 32 kHz, the peaks seem to decrease and almost disappear with the T5_3 signatures. The resonance here is smaller than the resonance observed in the previous figure, as the PZT transducer is further away from the host structure due to the use of a thicker metal disc. 6.1. Analyzing the Data for T3_x, T4_x and T5_x Cases Table 2 shows the values calculated using the three statistical methods for Figure 6. When the values in Table 1 are compared with the C_1, C_2 and C_3 test cases, the averaged values for each column are generally lower. In addition, most of the values increase as the damage progresses. The RMSD values for RT3_1 range from 5.33% to 11.03% with an average value of 9.18%. The average values for RT3_2 and RT3_3 are 2.95% and 2.16%, respectively. This shows that it is best to have impedance signatures with a large amplitude for high RMSD values, as one of the ﬁnal steps in identifying damage using the EMI method is to deﬁne a threshold value. Thus, large RMSD values subjected to damage can allow the user to deﬁne a threshold value which can maximize the chance of differentiating a damaged structure from an intact one. The averaged values for MT3_1, MT3_2 and MT3_3 are 13.56%, 1.30% and 0.93%, respectively, which is a similar result to the data in Table 1. Again, a decrease in the amplitudes of the impedance signatures had a signiﬁcant impact on the MAPD values, as 1.3% is only approximately 1/10 of 13.56%. For the CCD values, the average values are 2.95%, 3.98% and 2.05% for CT3_1, CT3_2 and CT3_3, respectively. This result shows that the reduction in the amplitudes does not have as large an impact, as it did with the RMSD and MAPD values. Table 3 shows the values calculated using the three statistical methods for Figure 7. With the extra 1 mm thickness of the metal disc, one might expect that the values calculated using statistical methods would be lower compared to the T3_x cases. However, all the averaged values for each column are larger compared to those in Table 2. For the RMSD values, the averaged values for RT4_1, RT4_2 and RT4_3 are 9.52%, 5.61% and 3.28%, respectively. For the MAPD values, the averaged values are 19.11%, 2.33% and 1.31% for MT4_1, MT4_2 and MT4_3, respectively, experimentally proving once again that the height of the peaks of the impedance signatures are very important when using the MAPD method. For the CCD values, the averaged values are 6.15%, 10.00% and 3.58% for CT4_1, CT4_2 and CT4_3, respectively. One of the reasons why the calculated values are relatively larger in this case is the overlap of the resonance of the metal disc and the plate. 5. Conducting the EMI Method with Various Signature Amplitudes 5. Conducting the EMI Method with Various Signature Amplitudes Figure 8 shows the 33 impedance signatures acquired from the T5_1, T5_2 and T5_3 experiments where resonance peaks are concentrated between 53 kHz and 59 kHz. At ﬁrst glance, the impedance signatures have a relatively smaller number of peaks compared to the previous two ﬁgures. As such, one can assume that the values obtained from the statistical methods (RMSD, MAPD, CCD) would be smaller compared to the test cases, T3_x and T4_x. This will be further discussed in Section 6. For the small resonance around 32 kHz, the peaks seem to decrease and almost disappear with the T5_3 signatures. The resonance here is smaller than the resonance observed in the previous ﬁgure, as the PZT transducer is further away from the host structure due to the use of a thicker metal disc. 9 of 13 Sensors 2018, 18, 2267 signatures. T PZT t d Figure 8. Impedance signatures for the T5_1, T5_2 and T5_3 experiment. Figure 8. Impedance signatures for the T5_1, T5_2 and T5_3 experiment. Figure 8. Impedance signatures for the T5_1, T5_2 and T5_3 experiment. Figure 8. Impedance signatures for the T5_1, T5_2 and T5_3 experiment. 6. Results and Discussion 6.1. Analyzing the Data for T3_x, T4_x and T5_x Cases 6.1. Analyzing the Data for T3_x, T4_x and T5_x Cases As shown in Figure 7, this seems to amplify the height of the resonance peaks in the frequency range that corresponds to the resonance of the metal plate. Table 4 shows the values calculated using the three statistical methods for Figure 8. With the thicker metal disc, one can expect that most of the calculated values will be lower compared to the values in Tables 2 and 3. However, when we compare the values in Table 4 with those in Table 2, more than half of the averaged values are larger. These are the RMSD averaged values of 3.66%, 3.32%, all the MAPD averaged values of 35.95%, 3.09%, 1.29%, and the CCD value of 2.73%. This shows that while the 5 mm thick metal disc is roughly two times thicker than the 3 mm thick metal disc, this difference does not have too much of an effect on the statistical values. In addition, the averaged 10 of 13 Sensors 2018, 18, 2267 value of MT5_1 is 35.91%, approximately triple the averaged value of MT3_1 (13.56%). Next, comparing Table 4 with Table 3, only the three averaged values of RT5_3 (3.32%), MT5_1 (35.95%) and MT5_2 (3.09%) have higher values. For the RMSD and MAPD averaged values, the number decreases from 7.28% to 3.32% and from 35.95% to 1.29%, respectively. For the CCD values, the results are the opposite, as the value increases from 2.06% to 2.73%, regardless of the fact that the impedance signature amplitudes are decreasing. Table 2. Three statistical metric values for the T3_1, T3_2 and T3_3 experiment. Damage (mm) RMSD (%) MAPD (%) CCD (%) RT3_1 RT3_2 RT3_3 MT3_1 MT3_2 MT3_3 CT3_1 CT3_2 CT3_3 2 5.33 1.82 1.39 6.21 0.69 0.55 1.11 1.56 0.92 4 7.09 2.12 1.18 10.87 0.88 0.55 1.81 2.03 0.73 6 10.53 2.36 2.04 19.77 0.99 0.88 3.73 2.49 1.78 8 8.82 2.86 2.89 13.50 1.28 0.99 2.69 3.51 3.37 10 8.61 3.13 2.21 13.24 1.37 0.86 2.53 4.25 2.04 12 9.41 2.92 2.11 14.91 1.33 0.94 3.00 3.75 1.89 14 9.65 2.93 2.30 12.76 1.33 1.01 3.12 3.74 2.22 16 10.46 3.25 2.58 16.29 1.46 1.15 3.61 4.57 2.72 18 10.87 4.24 2.20 15.66 1.90 1.08 3.88 7.56 2.03 20 11.03 3.83 2.65 12.34 1.79 1.27 4.02 6.31 2.76 Average 9.18 2.95 2.16 13.56 1.30 0.93 2.95 3.98 2.05 Numbers in different colors represent colors in picture. Table 2. Numbers in different colors represent colors in picture. 6.2. Regression Analysis on Experimental Data 6.2. Regression Analysis on Experimental Data In this section, a linear regression analysis is performed using the results from the previous subsection to evaluate the performance of the EMI method subjected to different frequency ranges and statistical metrics (RMSD, MAPD and CCD). Figure 9a shows the scatter plot using T3_1 results from Table 2 (the 2nd, 5th and 8th columns). Three lines of best ﬁt are drawn where the coefﬁcient of determination (R2) is 0.67 for RMSD, 0.13 for MAPD and 0.66 for CCD. As damage increases, all three lines show an increasing trend where the slopes have virtually the same steepness. The R2 of 0.13 obtained from the MAPD values is considerably lower than the other two statistical metrics, and one of the reasons for this is the randomness of the MAPD values. This randomness can be caused by damage to either a node or an anti-node of the metal plate, which can have a signiﬁcant effect on the impedance signatures. In Figure 9b, the remainder of the results from the previous subsection are used to plot a bar graph of R2 values, where the three bars (RMSD, MAPD and CCD) at T3_1 is of Figure 9a. By observation, while the T3_x, T4_x and T5_x cases have different resonance frequency ranges, it seems that this difference does not have a signiﬁcant impact on the regression analysis results as it appears to be random. For example, among the T3_x cases, the T3_2 case seems to have highest R2 values. Among the T4_x cases, the T4_1 case has the highest R2 values, while of the T5_x cases, the T5_3 case has the highest R2 values. These results show the complexity of the EMI method data analysis. However, one common feature that can be noted is that the MAPD values are always higher compared to the RMSD and the CCD values, with the exception of the T3_1 case. In addition, the RMSD values are always higher than the CCD values except for the T5_2 case. Overall, the three statistical metrics have similar values for most of the cases, with the T5_3 case having the highest values out of all the values. Another observation that can be made here is that when the impedance signature amplitude decreases from T4_1 to T4_3, the R2 values also decrease, whereas the T5_x cases result in the exact opposite (R2 values increasing from T5_1 to T5_3). 6.2. Regression Analysis on Experimental Data This shows that the size of the amplitudes does not have that signiﬁcant an effect on the R2 values. Sensors 2018, 18, x FOR PEER REVIEW 11 of 13 determination (R2) is 0.67 for RMSD, 0.13 for MAPD and 0.66 for CCD. As damage increases, all three lines show an increasing trend where the slopes have virtually the same steepness. The R2 of 0.13 obtained from the MAPD values is considerably lower than the other two statistical metrics, and one of the reasons for this is the randomness of the MAPD values. This randomness can be caused by damage to either a node or an anti-node of the metal plate, which can have a significant effect on the impedance signatures. In Figure 9b, the remainder of the results from the previous subsection are used to plot a bar graph of R2 values, where the three bars (RMSD, MAPD and CCD) at T3_1 is of Figure 9a. By observation, while the T3_x, T4_x and T5_x cases have different resonance frequency ranges, it seems that this difference does not have a significant impact on the regression analysis results as it appears to be random. For example, among the T3_x cases, the T3_2 case seems to have highest R2 values. Among the T4_x cases, the T4_1 case has the highest R2 values, while of the T5_x cases, the T5_3 case has the highest R2 values. These results show the complexity of the EMI method data analysis. However, one common feature that can be noted is that the MAPD values are always higher compared to the RMSD and the CCD values, with the exception of the T3_1 case. In addition, the RMSD values are always higher than the CCD values except for the T5_2 case. Overall, the three statistical metrics have similar values for most of the cases, with the T5_3 case having the highest values out of all the values. Another observation that can be made here is that when the impedance signature amplitude decreases from T4_1 to T4_3, the R2 values also decrease, whereas the T5_x cases result in the exact opposite (R2 values increasing from T5_1 to T5_3). This shows that the size of the amplitudes does not have that significant an effect on the R2 values. Figure 9. Results for (a) T3_1 statistical metric values; (b) T3_x, T4_x and T5_x R2 values. Figure 9. 6.1. Analyzing the Data for T3_x, T4_x and T5_x Cases Three statistical metric values for the T3_1, T3_2 and T3_3 experiment. Table 3. Three statistical metric values for the T4_1, T4_2 and T4_3 experiment. p Damage (mm) RMSD (%) MAPD (%) CCD (%) RT4_1 RT4_2 RT4_3 MT4_1 MT4_2 MT4_3 CT4_1 CT4_2 CT4_3 2 5.61 2.86 2.19 8.85 1.13 0.81 2.12 2.46 1.66 4 7.39 2.92 3.57 14.82 1.21 1.27 3.56 2.55 4.14 6 9.38 5.66 3.05 13.76 2.15 1.25 5.69 9.48 2.98 8 8.85 5.67 3.13 15.61 2.34 1.18 5.06 9.14 3.14 10 9.25 6.36 3.27 15.05 2.57 1.37 5.41 11.47 3.43 12 7.67 5.97 3.14 17.54 2.27 1.30 3.81 10.20 3.18 14 9.06 5.49 3.05 18.54 2.23 1.25 5.21 8.68 3.01 16 10.94 5.20 3.30 24.93 2.34 1.37 7.62 7.93 3.52 18 13.28 7.63 3.48 29.70 3.44 1.37 11.12 17.55 3.90 20 13.77 8.30 4.66 32.27 3.60 1.89 11.90 20.56 6.88 Average 9.52 5.61 3.28 19.11 2.33 1.31 6.15 10.00 3.58 Numbers in different colors represent colors in picture. Table 4. Three statistical metric values for the T5_1, T5_2 and T5_3 experiment. Table 4. Three statistical metric values for the T5_1, T5_2 and T5_3 experiment. Damage (mm) RMSD (%) MAPD (%) CCD (%) RT5_1 RT5_2 RT5_3 MT5_1 MT5_2 MT5_3 CT5_1 CT5_2 CT5_3 2 5.67 1.66 2.38 22.36 0.89 0.80 1.29 0.61 1.42 4 4.97 3.17 2.24 25.02 1.39 0.85 1.04 1.71 1.29 6 7.09 3.32 2.81 35.83 1.88 1.01 1.92 1.85 1.91 8 8.07 3.49 3.21 43.96 2.15 1.17 2.43 2.02 2.43 10 8.44 3.39 3.25 42.95 2.48 1.26 2.64 1.92 2.50 12 8.39 3.37 3.26 29.65 2.29 1.38 2.62 1.91 2.52 14 6.35 4.35 3.56 26.86 3.43 1.37 1.58 3.06 2.98 16 8.18 4.91 3.52 37.23 5.75 1.45 2.50 3.83 2.91 18 7.44 4.23 4.24 50.54 4.90 1.69 2.09 2.90 4.16 20 8.23 4.68 4.72 45.11 5.73 1.88 2.53 3.50 5.13 Average 7.28 3.66 3.32 35.95 3.09 1.29 2.06 2.33 2.73 Numbers in different colors represent colors in picture. 11 of 13 Sensors 2018, 18, 2267 11 of 13 6.2. Regression Analysis on Experimental Data Results for (a) T3_1 statistical metric values; (b) T3_x, T4_x and T5_x R2 values. Figure 9. Results for (a) T3_1 statistical metric values; (b) T3_x, T4_x and T5_x R2 values. Figure 9. Results for (a) T3_1 statistical metric values; (b) T3_x, T4_x and T5_x R2 values. 7. Conclusions 7. Conclusions In this study, a structural health monitoring method known as the electromechanical impedance method was investigated, by subjecting a plate to progressive damage under different resonance frequency ranges and peak amplitude heights. The first part of the study consisted of using a square metal plate with a 15 mm square piezoelectric (PZT) transducer attached at the center plate. The test was to create 20 mm of progressive damage with impedance signatures being measured at every 2 mm step, with 11 impedance signatures being acquired in total. This data was then compared with the second test in which the damage created was identical to the first test, but this time with a free PZT transducer attached in series to reduce the amplitudes of the impedance signature peaks. The third test was conducted with damage created identically to the previous tests, with another free PZT transducer attached to the series circuit. From these three tests (C_1, C_2 and C_3), 33 impedance signatures were measured and these were analyzed using three different statistical metrics which In this study, a structural health monitoring method known as the electromechanical impedance method was investigated, by subjecting a plate to progressive damage under different resonance frequency ranges and peak amplitude heights. The ﬁrst part of the study consisted of using a square metal plate with a 15 mm square piezoelectric (PZT) transducer attached at the center plate. The test was to create 20 mm of progressive damage with impedance signatures being measured at every 2 mm step, with 11 impedance signatures being acquired in total. This data was then compared with the second test in which the damage created was identical to the ﬁrst test, but this time with a free PZT transducer attached in series to reduce the amplitudes of the impedance signature peaks. The third test was conducted with damage created identically to the previous tests, with another free PZT transducer attached to the series circuit. From these three tests (C_1, C_2 and C_3), 33 impedance 12 of 13 12 of 13 Sensors 2018, 18, 2267 signatures were measured and these were analyzed using three different statistical metrics, which were root-mean-square deviation (RMSD), mean absolute percentage deviation (MAPD) and correlation coefﬁcient deviation (CCD). The results of these tests show that with the decrease in amplitude with the attachment of additional PZT transducers, the RMSD and MAPD values were also decreased in general. 7. Conclusions 7. Conclusions However, for the CCD values, this effect was not seen, which experimentally showed that the height of the resonance amplitudes was not a signiﬁcant factor when analyzing the impedance signature data with a CCD statistical metric. Here, the results show that MAPD is the most appropriate method to use with large amplitudes as MC_1 results in 11.51% increasing up to 27.55% with 20 mm of damage. However, with small amplitudes, CCD performed the best with CC_3 starting from 1.06% increasing up to 5.97% with 20 mm damage. The second part of the study involved investigating the performance of the EMI method at different frequency ranges. To achieve this, the conventional method of attaching the PZT transducer was changed by using a metal disc, which was ﬁrst introduced in Na et al. [4]. Three metal discs (T3_1 to T5_3) with different thicknesses were used to create 9 test cases. For each test, progressive damage was introduced in the same manner as in the ﬁrst part of the study, and three tests were conducted for each metal plate. Afterwards, three statistical metrics were calculated, and showed a similar pattern to the ﬁrst part of the study. The RMSD and MAPD values generally decreased with a decrease in the resonance amplitudes while the CCD values had no speciﬁc pattern, experimentally proving once again that the resonance amplitudes had very little effect on the CCD values. Here, the MAPD method performed the best with largest amplitudes as MT3_1 resulted in 12.34%, MT4_1 with 32.27% and MT5_1 with 45.11% when the test specimen was damaged up to 20 mm. In addition, the CCD method performed the best at 20mm damage with small amplitudes as CT3_3 resulted in 2.76%, CT4_3 with 6.88% and CT5_3 with 5.13%. This demonstrates that it is better to acquire an impedance signature with large amplitudes. Finally, linear regression analysis on all the RMSD, MAPD and CCD values obtained from the above tests was conducted to evaluate the performance of the EMI method. By ﬁtting a line of best ﬁt on all cases, it was found that the coefﬁcient of determination (R2) value was highest for the MAPD values (8 out of 9 cases) compared to both the RMSD and CCD values. In addition, no signiﬁcant pattern was observed when conducting the EMI method at different frequency ranges, which demonstrates the complexity of analyzing impedance signature data. 7. Conclusions 7. Conclusions One of the reasons for this is the sensitivity of the EMI method as many factors can cause the impedance signatures to change. These include the bonding condition of the epoxy adhesive, small differences in the dimensions of the metal plates, small temperature differences, etc. For this reason, research to compensate unwanted signature changes is an important area which should be researched further. However, the knowledge gained from this study can be used to understand the EMI method even further, bringing us one step closer to using this method for practical applications. Author Contributions: W.S.N. is the principal author for the research involved in writing and planning the overall work, D.-W.S. and B.-C.K. conducted the experiments and K.-T.P. analyzed the results from the experiments. Funding: The research was supported by a grant from “Evaluation techniques for cable system/earth anchor by micro and macro measured data (20180009-001)” funded by the Korea Institute of Civil engineering and building Technology (KICT), South Korea. Conﬂicts of Interest: Authors declare no conﬂict of interest. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References 1. Liang, C.; Sun, F.P.; Rogers, C.A. Coupled electromechanical analysis of adaptive material system determination of the actuator power consumption and system energy transfer. J. Intell. Mater. Syst. Struct. 1994, 5, 12–20. [CrossRef] 1. Liang, C.; Sun, F.P.; Rogers, C.A. Coupled electromechanical analysis of adaptive material system determination of the actuator power consumption and system energy transfer. J. Intell. Mater. Syst. Struct. 1994, 5, 12–20. [CrossRef] 2. Na, W.S. Distinguishing crack damage from debonding damage of glass ﬁber reinforced polymer plate using a piezoelectric transducer based nondestructive testing method. Compos. Struct. 2017, 159, 517–527. [CrossRef] 13 of 13 Sensors 2018, 18, 2267 3. Na, W.S.; Lee, H. Experimental investigation for an isolation technique on conducting the electromechanical impedance method in high-temperature pipeline facilities. J. Sound Vib. 2016, 383, 210–220. [CrossRef] 4. Na, S.; Tawie, R.; Lee, H.K. Electromechanical impedance method of ﬁber-reinforced plastic adhesive joints in corrosive environment using a reusable piezoelectric device. J. Intell. Mater. Syst. Struct. 2012, 23, 737–747. [CrossRef] 5. Wandowski, T.; Malinowski, P.H.; Ostachowicz, W.M. Delamination detection in CFRP panels using EMI method with temperature compensation. Compos. Struct. 2016, 151, 99–107. [CrossRef] 6. Yang, Y.; Lim, Y.Y.; Soh, C.K. Practical issues related to the application of the electromechanical impedance technique in the structural health monitoring of civil structures: I. Experiment. Smart Mater. Struct. 2008, 17, 035008. [CrossRef] 7. Baptista, F.G.; Vieira Filho, J. A new impedance measurement system for PZT-based structural health monitoring. IEEE Trans. Instrum. Meas. 2009, 58, 3602–3608. [CrossRef] 8. Bhalla, S.; Gupta, A.; Bansal, S.; Garg, T. Ultra low-cost adaptations of electro-mechanical impedance technique for structural health monitoring. J. Intell. Mater. Syst. 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https://openalex.org/W2803312841	https://bmcpublichealth.biomedcentral.com/track/pdf/10.1186/s12889-018-5471-0	English	null	Protocol: a multi-level intervention program to reduce stress in 9-1-1 telecommunicators	BMC public health	2,018	cc-by	7,845	Protocol: a multi-level intervention program to reduce stress in 9-1-1 telecommunicators Hendrika Meischke1, Michelle Lilly2, Randal Beaton3, Rebecca Calhoun4, Ann Tu5, Scott Stangenes1, Ian Painter1, Debra Revere6 and Janet Baseman7 Abstract Suite 400, Seattle, WA 98105, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Correspondence: stangs@uw.edu 1Northwest Center for Public Health Practice, University of Washington, 1107 NE 45th St. Suite 400, Seattle, WA 98105, USA Full list of author information is available at the end of the article Meischke et al. BMC Public Health (2018) 18:570 https://doi.org/10.1186/s12889-018-5471-0 Meischke et al. BMC Public Health (2018) 18:570 https://doi.org/10.1186/s12889-018-5471-0 Abstract Background: Nationwide, emergency response systems depend on 9-1-1 telecommunicators to prioritize, triage, and dispatch assistance to those in distress. 9-1-1 call center telecommunicators (TCs) are challenged by acute and chronic workplace stressors: tense interactions with citizen callers in crisis; overtime; shift-work; ever-changing technologies; and negative work culture, including co-worker conflict. This workforce is also subject to routine exposures to secondary traumatization while handling calls involving emergency situations and while making time urgent, high stake decisions over the phone. Our study aims to test the effectiveness of a multi-part intervention to reduce stress in 9-1-1 TCs through an online mindfulness training and a toolkit containing workplace stressor reduction resources. Methods/design: The study employs a randomized controlled trial design with three data collection points. The multi-part intervention includes an individual-level online mindfulness training and a call center-level organizational stress reduction toolkit. 160 TCs will be recruited from 9-1-1 call centers, complete a baseline survey at enrollment, and are randomly assigned to an intervention or a control group. Intervention group participants will start a 7-week online mindfulness training developed in-house and tailored to 9-1-1 TCs and their call center environment; control participants will be “waitlisted” and start the training after the study period ends. Following the intervention group’s completion of the mindfulness training, all participants complete a second survey. Next, the online toolkit with call- center wide stress reduction resources is made available to managers of all participating call centers. After 3 months, a third survey will be completed by all participants. The primary outcome is 9-1-1 TCs’ self-reported symptoms of stress at three time points as measured by the C-SOSI (Calgary Symptoms of Stress Inventory). Secondary outcomes will include: perceptions of social work environment (measured by metrics of social support and network conflict); mindfulness; and perceptions of social work environment and mindfulness as mediators of stress reduction. Discussion: This study will evaluate the effectiveness of an online mindfulness training and call center-wide stress reduction toolkit in reducing self-reported stress in 9-1-1 TCs. The results of this study will add to the growing body of research on worksite stress reduction programs. Trial registration: ClinicalTrials.gov Registration Number: NCT02961621 Registered on November 7, 2016 (retrospectively registered). Keywords: 9-1-1 dispatcher, Emergency medical services, Mindfulness, Stress reduction * Correspondence: stangs@uw.edu 1Northwest Center for Public Health Practice, University of Washington, 1107 NE 45th St. * Correspondence: stangs@uw.edu 1Northwest Center for Public Health Practice, University of Washington, 1107 NE 45th St. Suite 400, Seattle, WA 98105, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background Rameshbabu et al. (2013) reported that rotating shifts, common in 9-1-1 TCs, and their accompanying inad- equate sleep were negatively associated with physical health outcomes in call center personnel [13]. Long hours of sedentary, high mental demand computer work, also ubiquitous in TCs, have been found to increase the risk for musculoskeletal symptoms [14] which can also be associated with psychological distress [15, 16]. Work- place environmental conditions such as lighting, ventila- tion, temperature, break/lunch room availability, and workstation ergonomics have also been found to exacer- bate work-related stress in TC workforces [11, 12]. In addition, frequent technology updates including incre- mental and major “upgrades” in technologies, also com- mon in the TC workplace, can cause “technostress” which has been shown in other worker groups to be associated with decreased job satisfaction and declines in productiv- ity, commitment to the organization, and intention to remain on the job. [17] g The effectiveness and efficiency of an emergency re- sponse system depends on 9-1-1 telecommunicators (TCs), the emergency call receivers and dispatchers who are the first first-responders to assist people in distress. 9-1-1 TCs prioritize and triage incoming calls delivered via myriad channels (cell and landline phone; TTY for deaf-and-hard-of-hearing citizens); collect and distill call information; dispatch police, firefighters, emergency medical technicians, and paramedics; and may issue medical and other instructions to callers while they await the assistance dispatched to the scene. Each 9-1-1 TC fields thousands of calls every year, ranging from trivial to life-threatening, while simultaneously monitor- ing and entering data into up to six different computer screen displays in real time during each call. Much of the research on stress in first responders has been focused on police and firefighters, and the relation- ship between their trauma exposures and post-traumatic stress disorder (PTSD) [1, 2]. Although it is known that vicarious or secondary exposure can create secondary traumatic stress (i.e., behaviors and emotions resulting from knowledge about a traumatizing event that was ex- perienced by another person and the desire to help that person) [3, 4] or compassion fatigue (i.e., cumulative stress) [4–7] in first responders, few studies have focused specifically on the occupational stressors and work- related stress symptoms of 9-1-1 TCs. Project aims The objective of this study is to develop and test the effectiveness of an evidence-based, multi-level interven- tion program designed to reduce stress in 9-1-1 TCs. The project’s primary aim is to: develop, implement, and test the effectiveness of an individual-level online mindfulness training to reduce stress in 9-1-1 telecommunicators. The secondary aims are to: The secondary aims are to: develop and test the effectiveness of an organizational-level online worksite stress reduction toolkit utilized by 9-1-1 TC managers that is designed to reduce or minimize sources of stress in the 9-1-1 call center environment. test if the multi-level intervention program will lead to increases in TCs’ positive perceptions of the call center social work environment. test if participation in the mindfulness training increases mindfulness. The adverse outcomes associated with exposure to acute and chronic occupational stressors are costly and include physical and mental diseases, and impaired work performance, which can threaten the attainment of an organization’s critical mission(s) [10]. In addition to trauma exposures, 9-1-1 TCs are subject to those stressors commonly encountered by non-emergency call center workforces, including: fast-paced and time-urgent decision-making; tense interactions with distressed and sometimes abusive callers; time pressure to meet call processing requirements; and a negative work culture, which can include co-worker conflict, and a lack of appreciation or recognition from supervisors [11, 12]. test if increases in TC’s positive perceptions of the call center social work environment and increases in mindfulness serve as mediators of stress reduction in 9-1-1 TCs. Background Troxell (2008), one of the first to study 9-1-1 TCs, reported high peri- traumatic distress (i.e., responses occurring at the time of a trauma and immediately after) and a positive rela- tionship between peritraumatic distress and compassion fatigue in 9-1-1 TCs [8]. Pierce and Lilly (2012) added to these findings by assessing traumatic call exposure, peri- traumatic distress, and PTSD symptoms in 9-1-1 TCs, reporting high levels of peritraumatic distress and a moderate, positive relationship between peritraumatic distress and PTSD symptom severity. This research sug- gests that direct, physical “incident scene” exposure(s) to traumata may not be necessary to cause or to increase risk for PTSD in 9-1-1 TCs [9]. Meischke et al. BMC Public Health (2018) 18:570 Page 2 of 10 Page 2 of 10 Conceptual framework Borrowing from the literature on occupational stress, in- dividual stress, and coping response we developed a con- ceptual framework that describes how this multilevel intervention is expected to effect stress in 9-1-1 tele- communicators (Fig. 1). At the individual level, mindful- ness training is hypothesized to increase an individual’s Meischke et al. BMC Public Health (2018) 18:570 Page 3 of 10 Fig. 1 Conceptual Framework Fig. 1 Conceptual Framework Mindfulness has also been associated with reduced stress in firefighters and paramedics [32] and MBIs have also been associated with fewer PTSD symptoms in combat veterans [33–38]. Specific to 9-1-1 TCs, a recent cross sec- tional/non-intervention study showed that increased mindfulness was inversely correlated with self-reported symptoms of stress [39]. And regarding delivery of mind- fulness training, online MBIs have been shown to be effective in a number of workplace settings [40–46]. Mindfulness has also been associated with reduced stress in firefighters and paramedics [32] and MBIs have also been associated with fewer PTSD symptoms in combat veterans [33–38]. Specific to 9-1-1 TCs, a recent cross sec- tional/non-intervention study showed that increased mindfulness was inversely correlated with self-reported symptoms of stress [39]. And regarding delivery of mind- fulness training, online MBIs have been shown to be effective in a number of workplace settings [40–46]. ability to regulate their emotional response to stressors, whereas the call center manager-directed organizational level intervention is hypothesized to reduce worksite stressors and increase perceived social support by imple- mentation of targeted strategies and worksite policies. Mindfulness and stress While there is no universally agreed upon definition, mindfulness can be defined as the individual’s ability to pay attention to and be aware of present moment ex- perience, closely observe and describe sensations, per- ceptions, thoughts, and feelings, and act with full awareness [18]. A growing literature base has established the salutary effects of mindfulness-based interventions (MBIs). MBIs model, teach and cultivate the inner atten- tional resources of a trainee with the goal of learning to recognize and accept stress responses. Through recogni- tion and acceptance, individuals learn to no longer rely on avoidance or suppression of emotional responses. Avoid- ance and suppression of emotional responses have been connected to greater stress levels and psychopathology following exposure to distressing events [19–23]. Mindful- ness has been reported to be positively correlated with compassion satisfaction [24, 25] and inversely correlated with compassion fatigue [26–28] and burnout [29–31]. Population and procedures The study population consists of 9-1-1 TCs working in multiple 9-1-1 call centers serving urban, suburban and rural areas in the United States. All participating call centers respond to 9-1-1 calls for fire, medical or police emergencies, with many centers responding to all three emergency call types. Worksite environment and stress In occupations such as the 9-1-1 TCs’, in which work demands are high and worker control is low, employee wellness and stress-mediated outcomes can impact attrition, turnover, burnout/exhaustion and absenteeism [47, 48]. Worksite characteristics affect employees’ prod- uctivity, satisfaction, and stress; however, an array of interventions may improve the quality of the work envir- onment, and worksite health promotion activities can support employees’ healthy behaviors. The role of managers in particular, as a resource and support to em- ployees has been highlighted as a significant component of organizational-level interventions that can reduce or mitigate stress [49–51]. In addition, interpersonal conflict between employees is a stressor amenable to intervention, Page 4 of 10 Meischke et al. BMC Public Health (2018) 18:570 Page 4 of 10 for example, by training supervisors to adopt strategies for conflict management [52] and assisting organizations in adoption of anti-bullying policies [53]. Other known strat- egies for promoting organization-level wellness include healthy nutrition and exercise programs [54] and the re- duction or elimination of environmental hazards in the workplace [11]. Thus, an array of interventions have been documented to improve the quality of the work environ- ment, reduce symptoms of stress, and improve worker health- but the vast majority of this research has been conducted with non-9-1-1 TC workers. a baseline stress survey and will then be randomized to ei- ther a mindfulness training intervention or wait-list con- trol group. Following the mindfulness training, both the intervention and control groups will complete a post- training survey. Next, managers from all participating call centers will be provided access to the online Toolkit. After three months of access, all 9-1-1 TCs (training and control groups) will be asked to complete a post-toolkit survey. Implementation protocols After completing the baseline survey, participants will be assigned a study ID number and are randomized to either start the online mindfulness training (the inter- vention group) or are assigned to the wait-list control group. Online stress reduction toolkit The mindfulness training is developed by clinicians and investigators who are trained mindfulness teachers and practitioners. The training is adapted from the widely-used Mindfulness-Based Stress Re- duction (MBSR) program, an evidence-based training program originally implemented as in-person training [55, 56]. For our project, the MBSR training content was modified, abbreviated, and revised to accommo- date an online format and tailored to meet the con- straints and needs of the 9-1-1 TC population. Following the intervention group participants’ comple- tion of the mindfulness training and completion of the second survey, managers of all participating call centers will be provided with a link to access the Toolkit and are asked to share the link with other administrative personnel at their call center who may be in a position to implement any of the suggested activities. Managers will have access to the Toolkit for 3 months and will re- ceive bi-monthly emails encouraging them to use the Toolkit content and directing them to specific modules of the Toolkit. After 3 months access to the Toolkit, all participants complete the third survey. Organizational-level stress reduction toolkit The online Toolkit is comprised of several modules providing evidence-based and expert-informed content and strategies for call center managers to consider and implement in an effort to reduce select stressors and increase network support in their worksites. The spe- cific topics were selected based upon consultations and guidance from our research program advisory board consisting of call center managers from throughout our catchment area. The on-line toolkit includes modules Description, Design & Development of Interventions Individual-level mindfulness training Two weeks following the end of the online mindful- ness training all participants will be asked to complete the second survey. Two weeks following the end of the call-center-level toolkit availability, all participants will be asked to complete the third survey. The online mindfulness training is composed of seven modules to be completed weekly, over a 7-week time period. Each weekly online module is expected to require participants between 20 to 30 min to complete. Each individual module consists of the following components: Methods/design Study design The study is a longitudinal multi-stage study design that consists of a randomized controlled trial to evaluate the effectiveness of an online mindfulness training in reducing symptoms of stress in 9-1-1 call-center TCs, followed by 3 months access to an online call center stress reduction activity toolkit for the call center managers. We will re- cruit TCs from multiple enrolled 9-1-1 call centers nation- wide. As shown in Fig. 2, at enrollment, TCs will complete Recruitment will be conducted in two stages. First, the study team will conduct an outreach campaign to 9-1-1 call centers, using industry publications and listserv an- nouncements, to enroll call centers into the study. Next, at enrolled call centers, 9-1-1 TCs will be recruited through staff announcements, recruitment flyers, email, Fig. 2 Study Design Fig. 2 Study Design Meischke et al. BMC Public Health (2018) 18:570 Page 5 of 10 Page 5 of 10 focused on the following content areas: Conflict man- agement; Bullying in the Workplace; and Reducing Technostress; with additional resources on Health and Wellness. Content is specifically tailored for managers and for their utility in 9-1-1 call centers. and word-of-mouth. After obtaining electronic informed consent, participants will be asked to provide demo- graphic (age range, gender) and other information (length of employment, etc.) and complete the baseline survey. All participant information is stored on computers. Online mindfulness training During the 7-week training period, the intervention group will be contacted each week, receiving two emails: one containing the link to that week’s training module and one providing additional suggestions for incorporat- ing that week’s stress reduction activities into daily liv- ing. Participants will be encouraged to complete the training on a designated weekday to allow approximately 7 days between each module. Participants will also be encouraged to practice the mindfulness skills introduced in the module throughout the week for approximately 10 min every day. A short video introducing the theme for the week A few paragraphs describing the theme and activities for the week An audio-guided meditative stress reduction exercise Suggestions for brief mindfulness activities that can be performed throughout the day A brief check-in form for participants to let study staff know how the training is going g g g A moderated discussion board on which participants can post questions/comments if they wish Procedures Respondents from each enrolled call center who are eli- gible, have provided their informed consent, and have completed the baseline survey, will be randomized into either the mindfulness training group or wait-list control group. Covariates We will collect the following data that may serve as co- variates or confounders with our main outcome. Demographics: Participants are asked to provide their age, gender, and years of experience as 9-1-1 TCs. Overcommitment (OC): OC refers to an individual’s exhaustive coping style which can adversely impact the health and well-being. [69, 70]. In our prior work, OC has been positively related to symptoms of stress and negatively related to mindfulness in a sample of 9-1-1 TCs [39]. OC items are drawn from the Effort-Reward Imbalance (ERI) scale used to Measurements Main outcome The study main outcome is the TC’s self-reported stress as measured by the Calgary Symptoms of Stress Inven- tory (C-SOSI), a validated 56-item scale designed to assess various subjective symptoms of stress with 8 factor derived subscales, each consisting of 6-9 items: Depression, Anger, Muscle Tension, Cardiopulmonary Page 6 of 10 Meischke et al. BMC Public Health (2018) 18:570 Arousal, Sympathetic Arousal, Neurological/GI, Cognitive Disorganization, and Upper Respiratory Symptoms [57]. Subjects are asked to indicate the frequency with which they have experienced a particular symptom during the prior one week timeframe. measure occupational efforts relative to rewards [69]. We will add one item to increase its relevance to 9-1-1 TC work. Overtime: 9-1-1 TCs will be asked if they have worked overtime, and if it was voluntary or mandatory. Sample size and power calculations We anticipate randomizing a total of 160 9-1-1 TCs into two groups (~ 80 per group), and obtaining outcome data on survey measures from a minimum of 65 per group after accounting for attrition (allowing for a loss to follow-up of about 20%). A rough estimate of a clinic- ally meaningful effect size was obtained after review of studies of mindfulness interventions that used the C- SOSI or SOSI measure as an outcome. We found seven studies [75–81] that reported sufficient information to estimate the effect size and within-participant standard deviations. These suggest that an effect size of 0.4 to 0.5 represents a clinically meaningful effect size. Based on our prior work measuring 9-1-1 TC stress utilizing the C-SOSI, this corresponds to a change in C-SOSI score of about 15 [39]. To give some context, in an unpublished analysis of stress in this population, a change in C-SOSI score of 15 was associated with a 30% increase in the use of sick days. Power calculations were performed using a two-sample t-test of the pre-post difference in C-SOSI scores, under the assumption of an effect size of 0.5, a type I error rate of 5%. To achieve 80% power re- quires data on 65 participants per group; allowing for a loss to follow up rate of 18% would require a total of 160 participants. Mediating variables Acute stressor or stress event: Call center managers will be asked if there was an unusual organization- wide acute stressful event during the study period. Data regarding the following variables hypothesized to serve as mediators to the main outcome will be collected. Job Demands - mental: Measured with the Mental Demands subscale (5 items) from the National Institutes for Occupational Safety and Health (NIOSH) Generic Job Stress Questionnaire. [71, 72] Social Support: Perceptions regarding social support (at work with coworkers and at home with family and friends), assessed by two separate visual analogue scales (VAS). Prior research has established the reliability and concurrent validity of these measures in another first responder sample [58]. Job Demands - physical: Measured with the Job Requirements subscale (10 items) from the NIOSH Generic Job Stress Questionnaire. [71, 73] Network Conflict: The degree of conflict experienced at home and at work, assessed by two separate VAS. Prior research has established the reliability and concurrent validity of these measures in another first responder sample [58]. Technostress: Technostress is measured using items drawn from the Techno-uncertainty and Techno- insecurity sub-scales of the Technostress creators’ Scale [74] and we will add one Techno-insecurity item specific to 9-1-1 TC work. p p Mindfulness: The Mindful Attention Awareness Scale (MAAS) will assess attentional sensitivity to psychological, somatic, and environmental cues [59]. Prior research has documented internal consistency reliability estimates were of good quality (α ranged = .89–.93) and test–retest reliability correlations between the repeated MAAS measures were all of medium-to-large magnitude and statistically significant [60]. The Five Facet Mindfulness Questionnaire (FFMQ) will assess five factors associated with mindfulness. Sound psychometric properties for the FFMQ have been consistently observed, including construct validity for the global FFMQ score, as well as adequate to strong internal consistency for the global score and subscales (α = .67-.93) [61–68]. The five factor hierarchical structure has been confirmed in samples of mediators and non-mediators [61, 68]. Statistical analyses The primary hypothesis for this intervention study is that TCs who are randomized to the online mindfulness training will report fewer symptoms of stress compared to TCs who are randomized to the wait-listed control group. Page 7 of 10 Meischke et al. BMC Public Health (2018) 18:570 Funding This project was supported by grant number ROH010536 funded by the National Institute for Occupational Safety and Health (NIOSH), the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention or the Department of Health and Human Services. The funder had no role in the design of the study, has no role in the collection, analysis, and interpretation of data, and had no role in writing the manuscript. Effect of training participation on stress: Training participation will be examined using two measures: number of trainings completed as measured by access logs for viewing training videos and self- report mean number of days per week on which mindfulness was practiced. The effects of level of Discussion Emergency response systems depend on 9-1-1 telecom- municators, the emergency call receivers and dispatchers who are the first first-responders to assist callers con- tacting 9-1-1 for aid in emergencies. Research shows that this workforce is exposed to acute and chronic oc- cupational stressors; both trauma exposures specific to emergency response, and those stressors commonly en- countered by non-emergency call center workforces. This study will evaluate if an online mindfulness training and call center-wide stress reduction resources can re- duce stress in 9-1-1 TCs. The results of this study will add to the growing body of research on worksite stress reduction programs, the use of online mindfulness train- ing in workforce stress reduction, and stress reduction in 9-1-1 telecommunicators. Secondary analyses The secondary outcomes examined will include mindful- ness and perceptions of social work environment. We anticipate conducting the following analyses: Effect of the intervention on mindfulness: We will examine the effect of the intervention on mindfulness score using the same model as the primary analysis with MAAS score as the outcome. Effect of intervention on perceptions of social work environment: We will examine the effect of the intervention on perceptions of social work environment (social support and network conflict) using the same model as the primary analysis with the 4 VAS. Abbreviations C SOSI C l S C-SOSI: Calgary Symptoms of Stress Inventory; ERI: Effort-Reward Imbalance; FFMQ: Five Facet Mindfulness Questionnaire; MAAS: Mindfulness Attention Awareness Scale; MBI: Mindfulness-Based Intervention; MBSR: Mindfulness- Based Stress Reduction; NIOSH: National Institute for Occupational Safety and Health; OC: Overcommitment; PTSD: Post-Traumatic Stress Disorder; SOSI: Symptoms of Stress Inventory; TC: Telecommunicator; VAS: Visual Analogue Scale Analysis of mindfulness and perceptions of social work environments as mediators of stress reduction: We will conduct analyses of the mediation effect of mindfulness (MAAS measures) and perceptions of social work environments (workplace social support and conflict VAS measures) on the intervention, estimating the direct and indirect causal effects using the approach of Imai [73]. Primary analyses participation will be examined using repeated measures mixed effects models, with the effect of participation level assessed by interaction terms between level of participation and follow-up time. The models will in- clude level of participation as a covariate, fixed effect for time (3 levels; baseline, post intervention and follow up), site, time–by-level-of-participation interaction and a random intercept for participant. To examine the primary hypothesis we will use repeated measures mixed effects models, with differences assessed by interaction terms between randomization group and follow-up time. The models will include fixed effects for group (2 levels) and time (3 levels; baseline, post interven- tion and follow up), site, time–by-treatment interaction, and an interaction term between the implementation of the center wide toolkit and the third time point, with a random intercept for participant. Differences between groups will be assessed using the time-by-treatment inter- action term. Effect of online Toolkit: A secondary aim of this study is to determine the extent to which an online Toolkit for reducing occupational stress in call centers improves call center climate four months post implementation of the Toolkit. We will use a multi-level model to examine the effects of imple- mentation of the toolkit (as measured through the manager survey) on mean call center stress levels and call center climate, as measured by workplace support and conflict measures. The primary outcome variable will be the C-SOSI symptoms of stress scale. Hypotheses will be conducted at the 0.05 level of significance without adjustment for multiple comparisons, with group assigned according to intent-to-treat. The effects of possible differential drop-out rates be- tween the two groups will be assessed using sensitivity analyses under the assumption that data is not missing at random. 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https://openalex.org/W3136289939	https://www.e3s-conferences.org/articles/e3sconf/pdf/2021/20/e3sconf_emmft2020_10040.pdf	English	null	Environmental aspects of the theme park development in the Russian Federation	E3S web of conferences	2,021	cc-by	2,961	1 Introduction The word ‘recreation’ derived from Latin words: re, meaning ‘again’; and creare, meaning ‘to create, ‘give existence to’. The term seems to stem from 14th-century English language when it was used in the context of ‘refreshment or curing of a sick person’ [1]. Modern medicine acknowledges the importance of this process for human physical, psychological, and cognitive health. The importance of recreation and leisure in the life of a modern human is hard to overstate. Stress is a common companion of a modern person because society puts a high value on productivity, which decreases spare time left after work and other daily routines. Modern media are becoming progressively more engaging, allowing for more enjoyable activities, and ‘offline leisure’ as it’s sometimes called, is struggling to compete. This issue is prevalent among youth especially, as TV and computer games can keep a young person engage for hours on end. These activities are not harmful by themselves, however, when exercised in abundance, they can lead to a number of health-related issues (both physical and psychological), most of which stem from the stationary and solitary nature of these activities. This underlines the importance of forming alternative recreational opportunities and contributed heavily to the emergence of recreational areas. Environmental aspects of the theme park development in the Russian Federation Mariya Tihonova1,, Tatyana Simankina1, and Aida Kormishova2 1Peter the Great St. Petersburg Polytechnic University, Polytechnicheskaya str., 29, St. Petersburg, 195251, Russian Federation 2 2State University of Management, Ryazansky Prospect, 99, Moscow, Russian Federation 2State University of Management, Ryazansky Prospect, 99, Moscow, Russian Federation Abstract. Recreation and leisure are of paramount importance in human life. This article makes a comparison of different recreational areas categories. The main focus of this research is the current situation of Russian amusement and theme parks market. A comparison of Russian and European theme parks, their attributes, qualities, popularity and revenue is given. The research uses data gathering, analysis and synthesis to illustrate the insufficiencies of theme park development in the Russian Federation. The completed research suggests that Russia severely lags behind other European countries in terms of theme park quantity and quality. Factors that contribute towards this insufficiency are revealed in the conclusion of this research. E3S Web of Conferences 244, 10040 (2021) EMMFT-2020 E3S Web of Conferences 244, 10040 (2021) EMMFT-2020 https://doi.org/10.1051/e3sconf/202124410040 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 2 Materials and methods This research uses information from open databases on the Internet was used. The sources are linked in text and listed in the “Reference” section of this paper. The subject of this research is recreational areas. For the sake of research, the subject was arbitrarily subdivided into two categories, as explained below. 1. Temporary recreational areas Temporary recreational areas form at the location of mass recreational events. As the name implies, these zones are temporary in nature: they cease to exist when the event ends. Temporary recreational areas form at the location of mass recreational events. As the name implies, these zones are temporary in nature: they cease to exist when the event ends. p p y y An example is Rio Carnival. The event is held yearly on the streets of Rio de Janeiro, the second most populous municipality in Brazil. In 2019 the event hosted a total of 1.62 million visitors, and amassed over 1.4 billion dollars in revenue [2]. An example is Rio Carnival. The event is held yearly on the streets of Rio de Janeiro, the second most populous municipality in Brazil. In 2019 the event hosted a total of 1.62 million visitors, and amassed over 1.4 billion dollars in revenue [2]. Another example is Oktoberfest, a yearly outdoor festival. This event happens yearly in Munich, Bavaria, Germany and is the most prominent local festival. In 2019 it hosted 6.3 million visitors and generated over 1.3 billion dollars [3 ,4]. Temporary recreational areas are able to provide a short-term boost to the recreational market of an area, but their temporary nature can make them unavailable for certain population groups. These events are more often than not strongly themed, which decreases the target audience even further. g 2. Permanent recreational areas g 2. Permanent recreational areas Permanent recreational areas are stationary in nature and are active for the significant part of the year (except for possible seasonal shutdowns). Examples of such areas are listed below. Permanent recreational areas are stationary in nature and are active for the significant part of the year (except for possible seasonal shutdowns). Examples of such areas are listed below. Sanya is China’s most southern tourist centre and a resort. Located in Hainan province, on an eponymous island, it is China’s only tropical resort. Corresponding author: marie.milson@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 244, 10040 (2021) EMMFT-2020 https://doi.org/10.1051/e3sconf/202124410040 The importance of this topic lies in the central place recreation takes in human’s life. The creation and development of recreational areas can be an answer to the ever-increasing demand for recreation. Historically leisure has been more available to the upper class, who are more financially capable and have to endure fewer working hours. The advent of technology has enabled long-range travel, which enhanced global tourism. As the average income improves, mode disposable income is being allocated towards tourism and leisure. Recreation is important for all categories of people, which means that the consumer base of the industry greatly varies in income, interests, lifestyle, and capability to travel. Below is the analysis of recreational areas that can accommodate for a variety of consumer categories. The biggest tourism market has historically formed in Europe. Most prominent in that regard are Southern and West Europe. Germany has been hosting traditional fares in Hanover, Frankfurt, Leipzig for decades. Since recreational resources are not readily available in every part of the world, more and more people engage in wellness and education travel. E3S Web of Conferences 244, 10040 (2021) EMMFT-2020 Amusement parks are a subcategory of permanent recreational areas. They combine the festive nature of temporary areas but are more accessible to the public due to their permanent nature. They can compete with more traditional tourist locations, like museums and sights, as they allow to target tourists of various age, status, and wealth. Gathering many forms of businesses in one place, they are able to target a wide range of audiences, which helps accommodate whole families in terms of entertainment. They are often huge spectacular facilities that can become a local landmark, Such sites become a go-to place for tourists and secure huge profits. Highly developed regions may have multiple such facilities, which and serve as badges of prestige for the local tourism industry. Disneyland in Paris, France is a prime example of an amusement park (Fig 1). Fi 2 Di l d P i F [21] Fig. 2. Disneyland, Paris, France [21]. Disneyland in Paris hosts 2 theme parks, 8 hotels, a golf course, a trading center, and more. The resort hosts a total of 7 roller coasters. Their speeds range from 45 km/h on the height of 3 meters (which accommodate for the youngest of guests) to more extreme ones – up to 92 km/h, 24 meters high [7]. Beyond entertainment, the resort features a massive outlet center, «Val d'Europe» which contains 190 shops and 30 restaurants. Spanning over 1 million square feet, it’s the most lucrative attraction for every shopper. q y pp Disneyland Paris had hosted more than 16 million attendants in 2012. At an average price of 50 Euros per ticket, that equates to 800 million Euros in profit from tickets alone. [8] In 2019 however, the number decreased to 9,745 million visitors [9]. [8] In 2019 however, the number decreased to 9,745 million visitors [9]. Another example of a successful theme park is PortAventura World, located in Salou, a city in Catalonia, Spain. Buit around the PortAventura theme park in 2002, it attracts more than 3,5 million visitors yearly The park contains 9 roller coasters, more than 10 flat and water rides, and 6 themed hotels [10]. In 2013 a 49.9 percent stake at PortAventura has been purchased by a U.S.-based private equity firm for 439 million Euros [11]. The estimated yearly income is around 215 million dollars with 4,85 million yearly visitors [12, 13]. 2 Materials and methods With local population of around 700 thousand people, the region accommodated more than 83 million abroad and domestic tourists in 2019. Total tourism revenue is estimated to be 15 U.S. billion dollars yearly [5]. Central Park is an urban park located in the city centre of New York City, U.S. With an area of more than 3,4 square kilometers, it is one of the most visited tourist sites worldwide [6]. Its 65 million dollar budget is being managed by a non-profit municipal organization, The Conservancy. The park is free to enter. Below is the comparison of amusement and theme parks from different parts of world. 2 https://doi.org/10.1051/e3sconf/202124410040 E3S Web of Conferences 244, 10040 (2021) EMMFT-2020 3 The Russian experience With a price of admission of 163 US dollars for a family of four, it’s about twice less expensive to enter than Disneyland Paris [15]. 3 The Russian experience 3 E3S Web of Conferences 244, 10040 (2021) EMMFT-2020 https://doi.org/10.1051/e3sconf/202124410040 In terms of recreational areas development, Russia is lagging behind other European countries. The sphere is not developed with only a handful of available examples. Many large and ambitious projects have been stalled by the financial crisis. In terms of recreational areas development, Russia is lagging behind other European countries. The sphere is not developed with only a handful of available examples. Many large and ambitious projects have been stalled by the financial crisis. An example of a finished such project is Sochi Park. It is built in Sochi, a southern city that is sometimes called the “Russian Riviera”. The city hosted the XXII Olympic Winter Games, and multiple matches of the 2018 World Cup, which has bolstered the city’s attractiveness among tourists. Considering the visa difficulties. However, the park has to rely on the local resident market. The park features a total of 23 rides, 1 themed hotel. The guests are greeted by famous Russian movie and cartoon heroes like Zmey Gorinych [14]. The Dream Island, another Russian theme park, opened its doors in 29 of February, 2020 [15], (Fig 2). Sometimes referred to as “Russian Disneyland”, its estimated construction costs are over 1,5 billion dollars. Featuring a 72 acres glass dome, it’s the biggest indoor theme park in Europe. Fig. 2. Dream Island, Moscow, Russia [21]. It features famous cartoon characters like Hello Kitty, Teenage Mutant Ninja Turtles, as well as characters of Soyuzmultfilm, a Russian movie studio. The park is operational all year round, which should help it secure a stable profit. With a price of admission of 163 US dollars for a family of four, it’s about twice less expensive to enter than Disneyland Paris [15]. Fig. 2. Dream Island, Moscow, Russia [21]. It features famous cartoon characters like Hello Kitty, Teenage Mutant Ninja Turtles, as well as characters of Soyuzmultfilm, a Russian movie studio. The park is operational all year round, which should help it secure a stable profit. With a price of admission of 163 US dollars for a family of four, it’s about twice less expensive to enter than Disneyland Paris [15]. It features famous cartoon characters like Hello Kitty, Teenage Mutant Ninja Turtles, as well as characters of Soyuzmultfilm, a Russian movie studio. The park is operational all year round, which should help it secure a stable profit. 4 Results and discussion The result of the study is a comparison of the selected recreational areas. The comparison is made in table 1. The comparison shows that even Sochi Park, one of the grandest theme parks in the Russian Federation, fails to capture the financial success and popularity of its counterparts around the world. Dream Island, a prime Russian theme park can not be found on the lists of most popular theme parks [24]. This fact can be attributed to a relatively high admission price and low value for the price. Comparison made in table 2 suggests that theme parks in Russian Federation are almost 9 times less prevalent when compared to the rest of Europe. These findings, combined with 4 4 E3S Web of Conferences 244, 10040 (2021) EMMFT-2020 https://doi.org/10.1051/e3sconf/202124410040 the decadent popularity figures, reveal the dire situation of theme parks development in the Russian Federation. Table 2 compares the rate of theme parks to the local population figures in Russian Federation as compared to the rest of European countries. Table 1. Comparison of the selected recreational areas. Name Area, km2 Seasonal availability Workplaces Estimated yearly visitors, million Estimated yearly income, million dollars Temporary Rio-de-Janeiro Carnival - 5 days a year, 1,3% - 1,62 1400 Oktoberfest - 15 days a year, 4 % - 6,30 1300 Permanent Disneyland Paris 19 Year round, 100% 17000 16 >800 Port Aventura World 1,20 190 days a year, 52% 2000 [18] 4,85 [16] 215 [17] Sanya, China 1900 Year round, 100% N/A 83 15000 New York City Central Park 3,41 Year round, 100% 350 38 Non-profit Sochi Park 0,25 240 days a year, 66% N/A 1 [18] 33,6[18] Table 1. Comparison of the selected recreational areas. Table 2. The rate of theme parks to the local population figures in Russian Federation. Table 2. The rate of theme parks to the local population figures in Russian Federation. Number of theme parks Population, million people Number of theme parks per population, parks/million people Europe (except Russia) 14 [22] 145,9 [23] 0,07 Russia 381 [22] 601,3 [23] 0,63 5 Conclusions The result of the analysis made in this paper could be interpreted as follows. Theme parks are a popular form of recreational areas. Theme parks are also capable of attracting massive amounts of tourists, including international tourists. Most successful amusement and theme parks show high revenue figures, surpassing the 1 billion dollar mark in some cases [17]. Unfortunately, Russia is severely lagging behind its European peers in terms of theme parks design and implementation. 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Number of employees of Port Aventura Entertainment SAU from 2010 to 201 [Digital source]. – Access link: https://www.statista.com/statistics/779396/annual-number-of- employees-port-aventura/ (Retrieved 14.11.2020) 19. Number of employees of Port Aventura Entertainment SAU from 2010 to 201 [Digital source]. – Access link: https://www.statista.com/statistics/779396/annual-number-of- employees-port-aventura/ (Retrieved 14.11.2020) 20. Disneyland Paris Top Tips [Digital source]. – Access link: https://joynerjournal.co.uk/2020/03/06/disneyland-paris-top-tips/] (Retrieved 19.11.2020) 21. Island of Dreams park in Moscow [Digital source]. – Access link: https://en.wikipedia.org/wiki/Dream_Island_(amusement_park)#/media/File:Island_of _Dreams2.jpg (Retrieved 17.11.2020) 22. S.B. Park, J. Kim, Y.K. Lee, C.M. Ok, Visualizing theme park visitors’ emotions using social media analytics and geospatial analytics, Tourism Management 80, 104127 (2020) 23. 2019 Revision of World Population Prospects [Digital source]. – Access link: https://population.un.org/wpp/ (Retrieved 25.11.2020) 24. Adult admission price to the most popular theme parks in Europe as of 2018 [Digital source] – Access link: https://www.statista.com/statistics/921444/most-popular-theme- parks-in-europe-by-entry-price/ (Retrieved 13.11.2020) 7
W2055846071.txt	https://zenodo.org/records/2403814/files/article.pdf	de		Über generalisierte multiple Epitheliome der Haut	Archives of dermatological research	1,912	public-domain	9,682	Ober generalisierte multiple Epitheliome der Haut. Von Privatdozent Dr. St, W e i d e n f e l d (Wien). Besondere hufmerksamkeit schenkt man in jfingster Zeit dem huftreten multipler Karzinome oder Epitheliome, weil man dutch dieses Vorkommnis~ einzelnen Fragen ihrer Pathogenese niiherzutreten glaubt. So wurden yon T s u n o d a multiple Karzinome der Magenschleimhaut, aus der K 1i n ik R i e h 1 in Wien und aus anderen Orten multiple Karzinome tier Rfickenhaut beschrieben. Bekanntlich stehen sich in der Anschauung fiber die Genese des Krebses zwei Meinungen gegenfiber. R i b b e r t glaubt, das Karzinom durch eine Epithe!absehniirung, die mechanisch durch das Bindegewebe veranlal3t wird, entstanden und zieht fiir diese Anschauung das unizentrische Wachstum des Karzinoms als Beweis heran. H a n s e m a n n dagegen sieht als primiire Ursache die Anaplasie der Zellen, w~ihrend er ifir das Wachstum derselben, iiuBere Schiidigungen, traumatische, bakteritische heranzieht, huch R i b b e r t mul3 fiir das Wachstum der Epithelzellen eine Emanzipation derselben yon dem umliegenden Gewebe annehmen, wodurch sich fast eine Ahnlichkeit mit der Ansicht H a n s e m a n n s ergibt, der yon einem h l t r u i s m u s der Zellen spricht, worunter er die gegenseitige Beeinflussung der Zellen versteht, soweit diese sich auf Wachstums- und Vermehrungsvorgi~nge bezieht. Nun aber kommen auch Karzinome und Epitheliome vor, die sicher keinen unizentrischen, sondern einen plurizentrischen Ausgang aufweisen, 468 Weidenfeld. was soviel heil~t, dab an einem zirkumskripten Herd mehrere Krebsfoki sich bilden. Diese Form des plurizentrischen Entstehens der Epitheliome fiihrt hiniiber zu den multiplen Epitheliomen iiberhaupt, indem man nur anzunehmen braucht, dal] die einzelnen Krebsfoki disseminiert fiber eine grSl~ere F]~che sich ausbreiten. Es darf nicht unerwiihnt bleiben, dal~ die yon den meisten pathologischen Anatomen zuriickgewiesene Anschauung eines parasit~iren Ursprungs des Karzinoms in einigen ausgezeichneten Forschern Vertreter finder (Levin, C z e rny), wenn auch zugegeben werden muB, dab ein Beweis bis jetzt nach gar keiner Richtung hin mit Sicherheit erbracht worden ist. Die multiplen Karzinome jedoch, die fiber eine grSBere Partie ausgebreitet erscheinen, kSnnten als Beweis fiir die letztere Theorie herangezogen werden, wenn nicht andere gewichtigere Momente dagegen sprechen wiirden. Ich will es nun hier nicht versuehen, auf das Pro und Kontra dieser Erw~gungen n~her einzugehen, da ich die Absicht habe, in einer demn~chst erscheinenden Arbeit, die sich mit der Frage befassen soll, darauf zuriickzukommen, und will im folgenden vor allem einen Fall publizieren, tier, wie mir scheint, ein sehr seltenes Vorkommnis darstellt. A n a m n e s e : Patientin, 38 Jahre alt, Professorsgattin. Mit 14 Jahren war Patientin sehr blutarm und mager und erhielt deshalb dreimal tiiglich einen El~lSffel Ronzegnowasser durch acht Monate. In den folgenden Jahren weiB sie nichts Genaueres fiber den weiteren Verbrauch von Arsenikw~issern anzugebem Vom 19.--20. Lebensjahre wiederum sehr starke Abmagerung, wogegen sie wieder Solutio arsenicalis Fowleri erhielt. Da sie zu g]eicher Zeit fiirchtete, an Tuberkulose zu erkranken, nahm sie auch Kreosotpillen, so dai~ sie durch mehrere ffahre mit eiazelnen Unterbrechungen Kreosot und Fowlertropfen nahm. Im Jahre 1900 ~soll sie sich eine Verletzung mit einem Kistennagel an der Ful]sohle zuo gezogen haben. Einige Monate nachher bildete sich genau an derselben Stelle eine schwielige Verdickung~ wie ein Hfihnerauge~ die sehr schmerzhaft war und vom Hiihneraugenoperateur immer entfernt werden muBte, wobei, wie sie sich ausdrfickte, alas rote Fleisch zutage trat. Einige Monate sparer wurde das Hfihnerauge so schmerzhaft, dab es yon ihrem Bruder, tier Arzt ist, weggekratzt werden mul~te; unter der weggekratztea Schwiele befand sich nach ihrer Aussage Eiter. Durch 9--3 Jahre muBte ihr Bruder immer wieder das Hfihnerauge wegkratzen, unter welchem sioh immer Eiter befand. Die Schmerzen dabei waren so groB, dab Patientin sich Umschl~ge mit essigsaurer Tonerde machen muBte. 1901 Heirat, 0ber generalisierte multiple Epitheliome der Haut. 469 1902 traten nach der Geburt, nach ihrer Angabe, schwarze Flecke an der Kleiderfurehe auf, die naeh 2--3 Jahren der Sitz fiir krustenartige Gebilde wurden. 1905 bemerkte der Hiihneraugenoperateur am linken Fuise ghnlich wie am reehten eine Schwiele, die nicht mehr zuheilte. Im Jahre 1907 bemerkte sie auch an der Auisenflgehe des linken 0berschenkels eine solehe krustSse Stelle. Da die Schwie]e am rechten Fuis ihr sehr web tat, konsultierte sie 1907 einen Operateur, der ihr sowoh] die Schwiele am FuI~ als am 0berschenkel mit rauchender Salpeters~iure weg~tzte. Am 0bersehenkel heilte die Stelle aus, rezidivierte abet, wenn auch nicht in der gleichen Ausdehnung, worauf auf Rat de8 Arztes graues Pilaster auf die Wunde aufgelegt wurde, jedoch ohne irgendwelchen Nutzem Sp~ter gtzte er die Stelle mit Kall causticum, worauf sich nach Abfall des Sehorfes wieder eine Schwiele bildete~ unter der Eiter war. 1908 wurde nun yon einem konsultierten Dermatologen graues Pilaster sowohl auf den Fuis als auf die roten Stellen am 0berschenkel gelegt, ,~p~ter Priizipitalsalbe auf die ulzerierte Stelle an der Sohle und innerlieh Sajodin, worauf jedoch unter tier Salbe heftige Schmerzen an der Fuissohle auftraten. (Ich will gleich bier hinzufiigen, dais die runde Form der Tumoren uud der Geschwiire: die Generalisierung des Prozesses fiber die ganze Haut, die Polymorphie der Effioreszenzen, das gleichzeitige l~ebeneinander yon schuppend roten Effloreszenzen und die Gesehwfire den Gedanken sehr nahe erscheinen lassen, dais es sich bier um Lues handela kSnnte, weshalb die yon vielen 8eiten angeordneten antiluetisehen Kuren ihre Erkliirung finden.) Im Friihjahr 1908 wurden die Schmerzen enorm und der behandelnde Arzt (Dr. J e r u s a l e m ) verschrieb ihr zuerst milde Salben, sp~ter Scharlaehsalbe. Im Januar 1908 wurde ihr von einem Internisten Atoxyl in sehr groisen Mengen verabreieht, wobei sehr heftige Magenkriimpfe auftraten. Sajodin wurde fortgesetzt. Im 8p~tsommer wurden ihr Jothion-Einreibungen gemaeht~ die yon sehr starker Abmageruag gefolgt waren. Im Friihjahr 1909 bekam Patientln wieder Arsazetin-Einspritzungen, w o r a u f a 11e S t e 11e n r a p i d w u e h s e n u n d s i e h v e r g r S i s e r t e n . Im Juni 1909 wurden, da die Stellen sehr grois wurden und wieder wueherten, Queeksilbereinreibungen verabfolgt, lokal Pr~zipitatsalbe, worauf die Stelle am Fuis ungeheuer sehmerzhaft wurde und bis zu Apfe]grSise heranwuchs~ so dab Patientin fast dauernd ans Bert gefesselt war. Zu gleieher Zeit wucherte aueh eine Stelle am Kopfe (reehtes Scheitelbein), die bis zur Hfhnereigr6ise heranwuehs und auch sehr schmerzte. Patientin ist die Tochter eines Arztes, eiue Schwester starb an Tuberkulose, ein Bruder lebt und ist gesun~. Sonst war Patientin nie krauk~ Periode und sonstige Funktionen normal~ Patientin g ebar einmal ein gesundes Kind, des lebt. Herr Dr. J e r u s a 1em stellte mir freundlichst folgende 1%tizen zur Verfiigung: Am 17. Mai 1908 befand sieh an der rechten Fu~sohle und an der linken Ferse je ein Klavus mit einer tiefen Rhagade, angeblich sehon seit Monaten bestehend; erfolglos mit Lapis behandelt. Tgglich mehrere Stunden Stauung mittels Gummibinde am Oberschenkel; kein wesentlieher 470 Weidenfeld. Erfolg bis 24. Juni. Am 21. September 1908 Befund unverfindert, hellergrol]e Ulzerationen am Kopfe, Yerruka in der rechten retro Maleolargegend. Exkochteation. Janaar 1909 An/isthesin, Scharlachrot, Lichtbehandlung ohne Erfolg. Sommer 1909 Quecksilberkur and innerlich Jod in Ischl. S t a t u s p r a e s e n s September 1909: 35j~hrige Patientin kr/iftig gebaut, Paniculus adiposus ziemlich stark entwickelt, ziemlich gut erhalten. An der rechten Fu/~sohle, an Stelle des Gro~zehenballens, finder sich eine hiihnereigro~e, pilzhutfSrmige, rotgranulierende Gesehwulst, die bei Berfihrung sehr schmerzhaft, leieht blutend ist und deren R/inder fiberh/ingend sind. An der Mittelzehe, plantarw~rts, finder sich ein hellergro~er, runder, seichter Substanzverlust, dessen R~nder nirgends eleviert sind, dessen Basts jedoeh rein granulierend und rot ist. Die g a n z e P l a n t a r h a ut der Ferse und des ~ul~eren Ful~randes sehr stark verdickt und beim Drfiberfahren mit der Hand sehr rauh sich aaffihlend (man hat das GefilM, als wenn kleine hornartige Spitzen vorstehen w/irden). Bei n~iherem Zusehen sieht man tats/iehlich innerhalb der verdickten Haut punktfSrmige bis linsengro~e, warzenartige, sehr harte Exkreszenzen in die Haut eingesprengt, aus welcher sie sich mit einiger Mfihe herauslSsen lassen, wobei Patientin heftige Schmerzen verspfirt und zum Tell das blntige Korium zutage tritt. Solche Effloreszenzen finden sich zu Dutzenden in die Haut eingesprengt. An der Ferse der linken Fu~sohle findet sich ein kronengrol]er Substanzverlust, nieht sehr tier, dessen R~nder nicht deutlich eleviert erscheinen, keine Farbenver~inderung aufweisen and dessen Basis gleiehfalls feinst rot granulierend erscheint. Beide H o h l h / ~ n d e in gleicher Weise wie die Fal~sohlen an verschiedenen Stellen schwielenartig verdiekt und an diesen Stellen mit sebr ~hnlichen, warzenartigen Exkreszenzen bedeekt wie die Ful]sohlen. Einzelne yon diesen sind erbsengro~, zum Teil aaeh mit Krusten bedeekt, die in der verrukSsen Oberfl/~che eingelagert erscheinen. Dabei 16st sich an der einen oder der anderea dieser Effloreszenzen, an einer kleineren oder grS]eren Zirkumferenz die Exkreszenz ab and man sieht eine scharf begrenzte, leicht rot granulierende Basis zutage treten. Auch an den Seitenr~ndern der Finger und am Dorsum der H~inde finden sich 5--6 soleher Exkreszenzen yon sehr derber Beschaffenheit oder kleine Substanzverluste nach Abfallen der warzenartigen Exkreszenzen. Die rechte and linke Hand zeigen keine wesentliehen Intensit/itsunterschiede. Am Rficken der Patientin finden sieh fiber und zwischen den Schulterbliittern 5--6 papillenartige, linsengrol]e oder flache, mitderberHornhaut bekleidete, scbarf begrenzte Effloreszenzen. Am Kreuzbein finden sieh gleichfalls einige 10--15 steeknadelkopfbis linsengrol]e, zum Tell mit derber Hornhaut bedeckte, sehrharte, zum Tell schwarzbraune oder rStliche, im Hautniveau liegende, oder etwas elevierte, scharf begrenzte Effloreszenzen. Vorne am Bauche in der Kleiderfurehe finden sich gleichfalls 5--6 disseminierte linsen- bis erbsengrol]e Effloreszenzen, yon denen eine sehr Uber generalisierte multiple Epitheliome der Haut. 471 stark Framboesieform hat (~ihnlieh einem grol3en Akrochordon) und zum Tell an ihrer Zirkumferenz sich yon der Unterlage losl6st, wobei ein rotes Korium zutage tritt. Uber der reehten Sehulter in der Halsgegend finder sich ein kreuzergrol3er~ roter aber seharfbegrenzter, zugleich sehuppender Herd. Ahnliche sehr kleine schuppende Herde mit scharfer Begrenzung finden sieh auch am Kreuz und in der Bauchgegend. Diese Herde erreichen mitunter die GrS~e einer Linse. Der Herd a n der Halsgegend ist etwas derb, leicht eleviert, an einer Stelle haften die Schuppen starker als an der anderen, wo sie abgestreift erscheinen. An der rechten Kopfseite (Seheitelbeingegend) finder sieh eine nnBgrol~e, rot granulierende Gesehwulst, mit pilzhutfSrmig fiberh~ngenden Riindern, die bei Beriihrung leicht b h t e t und sehr schmerzhaft ist. Nirgends finden sieh Andeutungen einer gesehwellten Driise, kein Jucken, Mundschleimhaut und Gesichtshaut frei. Patientin liegt im Bette, da sie dureh die grol~e Sehmerzhaftigkeit der Gesehwulst an ihrem FuBe am Gehen gehinder~ ist. Am 28. September 1909 Operation, Athernarkose, Exstirpation des Tumors saint Fascia plantae. An der linken Ful]sohle und am Kopfe werden die Ulzerationen nur mit dem scharfen LSffel exkoehleiert und fulguriert. Am Abdomen Exkochleation zweier gro~er warzenartiger Gebilde. Am Hals Exzision eines roten Fleckes zu Zweeken der mikroskopischen Untersuehung. Die fibrigen Stellen des K6rpers, wie H~inde, Handriieken, Kreuz, Abdomen werden mit RSntgenstrahlen behandelt. Im November rezidivierte die (~eschwulst an der reehten Ful3sohle. Es traten innerhalb der Narbe sowohl, als auch in den zentralen noeh nicht fiberh~uteten Anteilen des Operationsfeldes neue Geschwulstmassen hervor, weswegen eine neuerliche Exzision am 9. November und zwar jetzt welt ins Gesunde vorgenommen wurde. Der restierende Substanzverlust war handtellergrol] und wird naeh T i e r s c h gedeckt. Die Epidermislamellen stammen yon einer andern Person (Kinderfrgulein der betreffenden Dame). Im weiteren Verlaufe heilten einige L~ppchen an, das iibrige granulicrte. Im Laufe des J~inner 1910 war die ganze Ful~sohle geheilt, nur am Kopfe blieb ein kreuzergrol~es, mit kallSsen R~indern umsgumtes Gesehwfir, das durch Monate mit intermittierenden RSntgenstrahlen bebandelt wurde, sich oft rfiekbildet und immer wieder rezidivierte, weswegen am 27./I. 1910 dasselbe yon J e r u s a l e m neuerdings exkochleiert und mit dem Paquelin verschorft wurde. Im weiteren Verlaufe wurde auch das Kopfgesehwfir und die Narbe an der Fu~sohle einer neuerlichen intensiven R6ntgenbestrahlung zugefiihrt. Im Janner 1911 mul~te eine neuerliche Operation am Kopfe vorgenommen werden, da die Geschwulst sich bedeutend vergrSl~erte und wiederum pilzhutfSrmig zu wuchern begann. In Atherrausch mul]te die ganze Geschwulst saint Periost cntfernt werden~ wobei der Knochen sich als elfenbeinartig troeken und hart crwies. Bis April granulierte wohl das Geschwfir, ohne aber sich vollkommen iiberhauten zu wollen, so Arch. f. Dermat. u. Syph. Bd. CXI. 30 472 Weidenfeld. dab eine Sonnenlichtbehandlung unternommen wurde, die naeh Abstoiiung einer fiinfkronengroBen Knochenlamelle Eade April die tt~utung rasch herbeifiihrte. WAhrend des ganzen Verlaufes wurde niemals aufgehSrt, mit R6ntgenstrahlen die einzelnen warzea- und schwielenartigen Effloreszenzen am Stamme and den Extremitiiten za behandeln, wobei es oft zur Exsudation eines eitrigen Exsudates unter den Effloreszenzen kam, die im weiteren Verlaafe zur Exfoliatioa der Effloreszenzen and zur tteilung ffihrte. Es traten aber immer wieder Rezidiven an den ersten Lokalisationen und aeue Effloreszenzen auf, so dab die R6ntgenbehandlung eiae kontinuierliehe bleiben muBte. S t a t u s v o m E n d e Mai 1911. Am Dorsum der H~nde findet man einzclne warzenartige linsengroBe Effloreszenzen sowohl unter den Fingern als auch am Dorsum und Palma manus. Die tIaut fiber der Palma manas ist rauh verdickt (Folgezustande der zahlreichen RSntgenbehaadlungea). Am Kopfe die granulierende Wunde nach Exstirpation des Karzihorns. An den Sehultern u. zw. sowohl fiber der Spina scapulae als auch zwischeu den Schultern vereiazelte braune Fleeke als Resultate der RSntgenbehandlung bier gewesener Effloreszenzen. Zahlreiche stecknadelkopf- bis linsengroBe Effloreszenzen finden sich fiber dem Kreuzbeia and an den Seitenteilen der Lenden, bciderseits je 15--20 Effloreszenzen. Die Effloreszenzen sind zum Teile grau~ braun, ieiast verrak5s oder schuppig oder es finden sich aueh rStliche, rail feinen Schuppen bedeckte Effloreszenzen, wobei die Schuppe wegkratzbar ist und der PapillarkSrper blutet. Diese Arten sind sehr wenlg eleviert~ auBerdem aber finden sich Effloreszenzea~ die etwas mehr eleviert sind~ deren verrukSse Exkreszenzen mit einer Kruste lest verwaehsen sind. so dab sie nur schwer yon der Unterlage abhebbar sind. Sp~rliche Effloreszenzen finden sich an der AuBenseite der Ober- uad Uaterschenkel. Die FaBsohlen sind sehwielig verdickt; innerhalb dieser schwieligen Verdickangen finder man zum Teil mit weiBea, festhaftendea Epidermisschuppen bedeckte Effloreszenzen eingesprengt. Die Exstirpationsnarbe sieht tadellos aus ohue Rezidiv. Das Allgemeinbefinden tier Patientin ist tadellos, und hat eiuige Kilo an KSrpergewicht zugenommen. Mikroskopisoher Befund. Die Untersuchung betrifft drei Stellen: 1. die exzidierte Stelle an der FuBsohle, 2. die exchloeierten Stellen am Kopfe and an der Ferse, 3. eine exzidierte Stelle fiber der rechten Klavikula. Die letztere stellt sich, wie schon oben angefiihrt, als eine schuppige, rStliche Stelle dar. Bei schwacher VergrSBerung sieht man das Stratum corneum fiber der gesunden Haut wohl [Jber generalisierte multiple Epitheliome der Haut. 473 erhalten, einige Schichten yon normalen Hornhaudammellen zeigend, ohne hnzeichen einer Parakeratose. Das Epithel ist mehrfach gesehichtet, man kann deutlieh des Stratum granulosum und die Basalzellenschichte erkennen, die Papilleu jedoch sind nicht wahrnehmbar; nur hie und da finder sich eine arkadenfSrmige VorwSlbung, die aber zumeist dureh Einsenkung der Follikeln hervorgebracht sin& Je n~her man jedoeh zu der krankhaften Stelle kommt, um so deutlicher werden auch Papillen sichtbar. (Jber der krankhaften Stelle sieht man dann allm~hlich die Papillen breiter~ m~chtiger werden. Auch die Epidermisforts~tze werden sehr breit. Im zentralen Anteile des exstirpierteu Stiickes verlieren die Papillen das kolbige Aussehen, werden spitzer, indem die Epidermisforts~tze durch ihre Konfiguration ihnen ein solches Aussehen aufzwingem Das Stratum corneum bleibt nun fiber der ganzen Stelle zum grS~iteu Teile erhalten, sower man es bei sehwacher Vergr51]erung zu beurteilen vermag. Dagegen ~ndert sich, wie schon angefiihrt, das Rete Malpighii, indem die Epidermisforts~tze sehr massig heranwachsen und aus einem sehr dichtea Zellhaufen zu bestehen scheinen, wodureh das Aussehen wesentlich ver~ndert wird. Naeh den zentralen Partien jedoch verliert sich dieses Epithel vollst~ndig, man sieht nut ein sehr diinnes, aus fiaehgestellten Kerneu bestehendes Band fiber die weitere Stelle hiniiberziehen. Die K u t i s ist auch in den ~iufleren Partien etwas zellreicher, die Gefiil3e yon einem Zellmantel umgeben. Je mehr man jedoch gegen die zentralen Anteile des Pr~iparates kommt~ um so zahlreicher wird der Papillaranteil und an vielen Stellen hebt sich die Epidermis yon diesem zellreichen Anteil der Kutis ab~ so dal] zwischen beiden ein Raum besteht. Dieser Zellreichtum erstreekt sich, nur sp~rlich den Gefiifien folgend, in die unteren Partien der Kutis. so dab auch hier, wenn auch sehr sp~rliehe Infiltrationen um die GefiiBe herum sichtbar werden. Im zentralsten Teile finden sich netzfSrmig angeordnete, diinnste Zellstr~nge, zwischen denen ein sehr lockeres, stellenweise sehr zellarmes, nicht gut fiirbbares Gewebe sich befindet, das an der Peripherie gegen die uutere Kutislagen zu wieder yon einem etwas dichteren, zellreichen, mit spindelfSrmigen Kernen durchsetzten Gewebe ums~iumt wird. 30* 474 We[denfeld. B e i s t a r k e r V e r g r S l ] e r u n g erweist sich an derPeripherie die Epidermis mit etwas starker gefiigtem Stratum corneum bedeckt. Das Stratum granulosum ist gewShnlich einreihig und nicht an allen Stellen in deutlichster Ausbildung vorhanden. Das Rete weist 3w~ Zellagen auf, wobei jedoch die Zellkonturen nieht deutlich ausgepr~igt sind, die Zellkerne sehr stark gef~irbt erseheinen, hie und da yon einem perinuklearen Halo ums~umt. Die Basalzellen zeigen sehr gute Ausbildungen an ihrem der Kutis zugewendeten Teile, indem hier die Zone sehr breit erscheint, auch sind die Zellen hier zum Teile besser abgegrenzt als in den hSheren Teilen. Die Kutis erzeugt sehr geringe Papillenbildung, in den hSheren Lagen zeigt sic eia feinfaseriges Gewebe, yon nicht sehr zahlreichen Zellen durehsetzt. An der Grenze gegen die Kutis und innerhalb derselben finden sich starker ausgebildete Kapillaren, die B h t enthalten und die ~on einem schmalen Zellmantel ums~umt erscheinen. Die gegen die zentralen Anteile des Pr~parates liegenden, schon oben beschriebenen, st~irkeren Epidermisforts~tze erweisen sich bei der stiirkeren VergrSl]erung als bedeutend anders zusammengesetzt als die normale Epidermis. H i e r findet man das Stratum coraeum nieht bl~tterartig, wie peripherwiirts, sondern starker gefiigt nur als schmalen Saum erhalten, das Rete aus 4--5 platten Zellagen bestehend, in dem die Kerne flach gedriickt erscheinen. Die bei schwacher VergrSl]erung zu beobachtenden Epidermisforts~tze erweisen sieh gleichfalls aus Zellen mit grol]en Kernen zusammengesetzt, wobei die Kerne 5iters verschiedene GrSl]e haben~ dagegen das Protoplasma an ~ielen Stellen undeutlich gef~rbt erscheint, so dal~ man mitunter an einer oder der andern Stelle die Zellkonturen schwer erkennen kann, an anderen jedoeh deutlich das bekannte Aussehen des Stratum dentatum vor sich hat. An vielen Stellen sieht man aber Anlagen zur Gr~ppierung im Sinne yon Epithelnestern, wobei die zentralen Zellkonturen versehwimmen und yon bl~schenfSrmigen Kernen, zum Teile yon bizarren Formen durchsetzt sind, w~hrend die peripheren Zellen plattgedriickte Kerne haben und schalenartig die zentrale Stelle umgeben. Solche Nester finden sieh bier sehr zahlreich. Die naeh dem Zentrum zu gelegenen Epidermisforts~tze erscheinen aus noch 1Jber generalisierte multiple Epiiheliome der ttaut. 475 viel dichteren Epidermisanhiiufungen zu bestehen, so daI~ das Protoplasma verschwindet und man nut die sehr stark gefttrbten Ninglichen oder ovalen Kerne wahrnehmen kann. Das bei den peripheren Epidermisfortsiitzen noch zu beobachtende Stratum corneum verliert sich zum Teile vollst~indig, zum Teile bleibt es erhalten, so dab oft der gesamte Epithelfortsatz frei zutage liegt. Oft fehlt der suprapapill~re Tell der Epidermis und wird durch eine Kruste ersetzt~ oft aber tritt sie wieder in m~ehtigem Lager auf. Das die kolbigen Epidermisforts~tze bedeckende Epithel zeigt einen allmiihlichen ~bergang der normalen Deckepithelzellen zu den immer diehter werdenden protoplasma~irmeren Epithelzellen, die, wie friiher beschrieben~ hnlagen zu Haufenbildungen zeigen. An den zentralen Anteilen des Pr~parates findet man eine sehr diinn% deutliche parakeratotische Hornlage und sehr diinne Epidermislage. Unterhalb dieser Epidermis findet man ein N e t z y o n S t r ~ n g e n , d i e m e i s t aus sehrdichtaneinandergefiigtenZellreihen und aus protoplasmaarmen Zellen mit bl~schenf5rmig e n K e r n e n b e s t e h e n . In den Maschen dieses Netzes finder sich ein ungef/irbtes, sehr feines, fibrillKres Netz, in das feine, Ningliehe, spindelfSrmige Zellen eingelagert erscheinen. Die Kutis unterhalb der besehriebenen, kolbigen EpidermisfortsKtze ist sehr stark infiltriert, die Zellen zum Teile spindelig, zum Teile fund und lagern sich sowohl unterhalb der Epidermisfortsi~tze als zwischen denselben. Dabei hat sich das Epithel an vielen Stellen yon der Unterlage abgehoben und die so entstandenen HShlen sind oft yon Fibringerinnsel und isolierten Zellen durchsetzt. Die Blutgef~il~e sind erweitert, yon Zellm~inteln ums~umt. Unterhalb des Netzes der Zellstr~nge ist die Kutis aus dichtgeffigten, spindelfSrmigen Zellen zusammengesetzt. Wie man also aus dieser Beschreibung ersehen kann, ist die zentrale Stelle mit Leichtigkeit als ein Epitheli0m im Sinne eines B a s a 1z e 11 e n e p i t h e 1i o m s anzusehen. Was jedoch den peripheren Anteil betrifft, so w~re derselbe mit Riieksicht auf das klinische Aussehen nicht leicht als ein Epitheliom anzusehen. Die verl~ingerten und verbreiterten Epithelforts~itze und das starke Infiltr~t in der Kutis und auch sp~rliche Rundzellen 476 Weidenfeld. in der Epidermis deuten auf starke Entziindungsvorgiinge him wie dieselbe auch in der Abhebung der Epidermis an einer und der anderen Stelle ihren Ausdruck findet. Man kSnnte einen Moment an Psoriasis denken, wenn nicht das Epithel sofort in das zentral gelegene Epitheliom iibergehen wiirde. Das exzidierte Stfick aus der Geschwulst an der Kopfhaut zeigt folgendes Bild. An dem Pr~parat finder man an einer Stelle die Epidermis sehr dfinn, aus 2--4 Zellagen nur bestehend, die Zellen platt gedriickt, ~ihnlich wie bei einer Parakeratose, die Epidermisforts~tze sehr spitz, gleichfalls zellarm, einzelne yon diesen Fortsiitzen ziehen sich nach der Tiefe, wobei sie in diinne Stri~nge sich fortsetzen, die aus 2--3 Zellreihen bestehen und mitunter horizontal unterhalb des Stratum papillare verlaufen. Das Stratum papillare ist yon grol~en Gef~en durehzogen, sehr stark 5dematSs und ziemlieh zellreich. Nach der einen Seite zu geht diese Epidermisdecke in eine massige, lappige, miichtige, dicke~ aus Epithelzellen bestehende Zellmasse fiber, in dem deutlich der ~bergang in der Weise erfolgt, in der die friiher beschriebenen, nach der Tiefe zu wachsenden Epidermisiorts~itze immer dicker und dicker werden und die dazwiscben liegende Kutis immer kleinere Inseln bilden. Nach der anderen Seite jedoch verliert sieh die Epidermis ganz und es tritt ein Gewebe zutage, das aus Zellstr~ngen besteht. OberflKchlich jedoch ist es vielfach zerkliiftet, indem Epidermistriimmer naeh aul~en zu sich ablSsen, oder aber indem eine mit zugrunde gegangenen Zellen sehr stark durchsetzte, tingierte, aber undifferenzierte, scbollige Masse sich auflagert, die sieh yon der Unterlage nicht sehr scharf abhebt. Diese vielfach gelappte Epithelmasse erreicht eine Zellage yon 50--100 Reihen, deren Zellen deutlich die Charaktere des Epithels zeigen (bliischenf6rmigen Kern und gut differenziertes Protoplasma), nach aul~en jedoch yon keinem Stratum corneum begrenzt erscheint, gegen das Bindegewebe ein mehr oder weniger gut ausgebildetes Stratum cylindricum aufweist. Zwischen diesen Lappen und innerhalb der Epithelmasse finden sich L~ngsund Quersehnitte yon papillenKhnliehen Gebilden, yon sehr verschiedener, weir aber normale Papillendurchschnitte iiberwagender GrSBe, die aus deutlichen, fibrill~ren Bindegewebsfasern bestehen Uber generalisierte multiple Epitheliome der Haut. 477 und auch deutliche Gefiifl-, L~ngs- und Querschnitte zeigen, w~hrend eine Zellanhiiufung ~:icht hervortritt. Unterhalb dieser Decke erscheint die Kutis yon zahlreichen Zellen durchsetzt, wiihrend die fibrill~re Struktur sehr in den Hintergrund gedr~ngt wird, r finden sich grol~e und miichtige, mit mehr oder weniger sp~rlichen Inhalte erffillte Gef~]3e. Die tieferen Anteile zeigen wieder ein anderes Bild~ indem das ganze Gewebe yon soliden Schl~uchen und Str~ngen durchsetzt erseheint, die aus 3--10--12 Zellreihen bestehen, bald parallel ]aufen, bald aber auch durch Querschnitte yon solehen unterbrochen werden, wodureh ein Netz entsteht, dessen Maschen yon fibrilliirem Bindegewebe ausgefiillt erscheint. Sie bestehen aus deutlichen Epithelzellen mit gut gefiirbtem Protoplasma und gut geF~irbten Zellkernen. Zwischen diesen einzelnen Schliiuchen finder sich das Bindegewebe in nur zarten Fibrillen angedeutet oder rarefiziert und durch runde oder spindelige Zellen ersetzt, abet auch diese in sehr beschr~nkter und m~fliger Zahl, so daft diese Sehl~iuche mitunter so nahe aneinander riicken, da~ ihre Kontur nur yon einem sehr diinnen bindegewebigen Strang ums~umt wird. Auch die exzidierten Ste]len an der Fuflsohle zeigen die deutlichen Charaktere eines Plattenepithelioms (Prof. L a n d s t e i n e r nahm die Untersuchang vor), wie der oben beschriebene Tumor an der Kopfhaut, wobei genau dieselben Erscheinungen wie in dem obigen Priiparat gefunden wurden. Bei diesem Falle handelt es sich also um eine Erkrankung, bei der es zur Bildung multipler Epitheliome an verschiedenen KSrperstellen kommt, wie es leicht aus der klinisehen und mikroskopischen Untersuchung zur Geniige hervorging, neben welchen die gleiehfalls sehr starke Hyperkeratosis der Planta pedis und die geringere der Palma manus, und die in ihr eingesprengten, verrukSsen, sehr derben spitzen Effloreszenzen in der approximativen Zahl yon 50--60 an den Fii•en, 20--25 an den Hiinden, am meisten ins Auge springt. Man kann diese Epitheliome nach der GrSl~e in drei Gruppen einteilen. Es finden sieh : 1. kleinste Formen bis zu LinsengrS~e, in der Anzahl yon 60--70, abgereehnet die verrukSsen Formen an der Planta pedis and Palma manus. 478 Weidenfeld. 2. Formen bis KreuzergrSBe, 4--5. 3. Formen his Walnu6grSBe oder KleinhiihnereigrSBe, in der Anzahl yon zwei. Von diesen GrSBen finden sich nun fiber das Hautniveau ~orragend linsen- his hellergroBe Stellen 8--10, hfihnereigroBe 2 SteUen, es finden sich aber auch Formen, die im Hautniveau liegen, und zwar yon Linsen- bis 1/2-HellergrSBe, die die andere grSl~ere Menge ausmaehen. Es kSnnte daraus folgen, dab immer neue Effloreszenzen aufsehieBen, dab aber verhiiltnismiil~igwenige sich naeh der HShe und Breite ausdehnen. Die grSBten Effloreszenzen finder sieh am Kopf und FuBe, die aueh prominieren dann an der Ferse und an den Zehen (Ulzerationen) und an den H~nden. Einzelne grSI]ere Effloreszenzen, die aber nicht ulzeriert sind, finder sich in der Klavikulargegend rechts, Riicken-, Lenden- und Kreuzbeingegend, dann an den Ober- und Unterschenkeln und H~nden und Fiigen. VerrukSse Formen in der N~he des Nabels, Unterschenkels. Was die H~ufung dieser Effloreszenzen betrifft, muB man die P~lma manus~ die Lenden~nd Kreuzbeingegend als am dichtesten befallen ansehen, ebenso dicht oder fast so die Fu[~sohlen, weniger schon die Handriicken und die Extremitiiten, wobei die Unterschenkel mehr Effloreszenzen Ms die Obersehenkel aufweisen, am sp~rlichsten jedoch der Rumpf und der Riicken. Man sieht weiterhin, dab das Aussehen dieser Etfloreszenzen ein m u l t i f o r m e s ist, indem sowohl s q u a m S s e , v e r r u k i i s e , u l z e r S s e und n o d S s e Formen sich vorfinden. Die Form ist h~ufig rund, selten unregelm~Ng oder polygonal, meist bei den squamSsen Formen. Was die Tiefe der Ulzerationen betrifft, so erreieht sie kaum 1/~--21/~ r a m . Die Farbe der Effloreszenzen ist bei den squamSsen Formen eine rStliche oder graubraune, bei den verrukSsen eine mehr graubraune, w~,hrend die Basis der Ulzerationen sowohl als aueh die Geschwiilste lebhaft rot erscheinen. Es mull hinzugefiigt werden, daB, wie die Patientin bestimmtest angibt, die Effloreszenzen sich, wenn auch erst nach jahrelanger Dauer, in ihren Formen ~,ndern und dab aus seichten Ulzerationen die tumorartigen Formen entstanden, und erst naeh Reizen oder nach den Injektionen mit hrsazetin sieh ~lber generalisierte multiple Epitheliome der Haut. 479 entwickelten, ein Vorkommnis, das ja auch anderweitig gesehen und beobachtet wurde ( C l a n et). AuBer diesen Formen der Eifloreszenzen, die durch ihr Aussehen bald in die Gruppe der Entziindungen~ bald in die der Geschwiilste eingereiht werden kann, zwischen denen aber nut klinisch, nicht pathologisch anatomisch ein Untersehied besteht~ indem im selben Bride ~)berg~nge yon den dutch Entziindung noeh erkl~irlichen hyperplastischen Bildungen der Epidermis zum sicheren Epitheliom sich finden, interessiert ferner die anamnestisch yon der Patientin angegebene Steigerung des Prozesses und Wucherung tier Effloreszenzen durch die Injektion yon Arsazetin. Zugleich fitllt auch anamnestisch die dutch Jahre fortgesetzte interne Arseniktherapie auf. Vom 14. Jahre angefangen, nahm sie bis zu ihrem 24.--25. Lebensjahr mit kiirzeren oder l~ngeren Unterbreehungen bald Roneegnowasser, bald Solutio arsenicalis Fowleri, bald Kreosotpillen. In ihr 25. Lebensjahr nun muff der Beginn des jetzigen Leidens verlegt werden, da die Patientin mit grSBter Bestimmtheit angibt, da~ nach Verletzung durch einen Kistennagel am FuBe~ die groBe Gesehwulst an der SoMe ihren Ausgang nahm. W~ihrend flit das Auftreten dieser Geschwulst an der Sohle eine Verletzung als Ausgangspunkt die Patientin angibt, ist fiir das Auftreten anderer Effloreszenzen die Erkl~rung nicht mehr zu erbringen. Es darf aber nicht unterlassen werden, die Aussage der Patientin anzufiihren, wonaeh sich die Effioreszenzen am Abdomen innerhalb yon Pigmentierungen bildeten, die im weiteren Verlaufe sich spurlos riickbildeten, aber yon ihr mit Sicherheit als Ausgangspunkt fiir die sparer entstandenen Effloreszenzen zugegeben wurden. Aus den hngaben der Patientin folgt aber weiterhin, dab die Effloreszenzen seit zirka 14 Jahren in kontinuierlicher Folge entstehen und an Zahl sparer zunehmen. Fiir das Verst~indnis der .~tiologie dieses Falles ist die Entstehung der Tatsaehe yon Wiehtigkeit, dab die jetzt persistierenden schwieligen Verdiekungen an den FuBsohlen und H~nden friiher oft auftraten und immer wieder verschwanden, indem die Patientin behauptet, friiher ganz feine Hiinde gehabt zu haben, die im Laufe der letzten Jahre mehrmals rauh 480 Weldeni'eld. wurden, welche Erscheinung verschwand, rezidivierte und end]ich dauernd zuriickblieb. Die Erkrankung besteht nunmehr seit 14 Jahren, geht mit Bildung yon schuppenden, warzenartigen und epitheliomatSsen Schwielenbildungen an Palma und Planta einher, zeigt also ein eminent chronisches, auf das Epithel sicher, aber wahrseheinlich auch auf den PapillarkSrper besehr~nktes Krankheitsbild, bei dem die histologischen Untersuchungen das kliaische Krankheitsbild best~tigen, indem es sich auch nach diesem um ein sieheres Epitheliom oder um beginnende hyperplastische und zum Epitheliom neigende Ver~nderungen der Epidermis handelt. Wenn man sich die Frage vorlegt, welcher Kranl~heitsgruppe dieses Bild subsumiert werden kann, so mul~ vor allem mit Riicksicht auf die grol]e Anzahl der Epitheliome auf jene Krankheitsbilder rekurriert werden, bei welchen eben eine grol]e Anzahl yon Epitheliomen charakteristisch ist. Multiple Epitheliome finder man bekanntlich beim Xeroderma pigmentosum und im spiLteren Alter. Das Xeroderma pigmentosum wird als eine Erkrankung der Haut betrachtet, die yon vielen Autoren als durch die Einwirkung yon Sonnenstrahlen hervorgerufen angesehen wird und charakterisiert sieh dutch StSrungen versehiedener Hautsysteme. Man findet Pigmentanh~ufungen in Form yon Epheliden, Lentigones, dann t)igmentverluste in Form yon weiBem Leukodermaund Viteligo-i~hnlichen, punktfSrmigen oder linsengrol]en Flecken. Dazu kommen Atrophien, Gef~l]stSrungen im Sinne yon Teleangiektasien, Abschilferung der Epidermis. Im Gegensatze dazu kommt es aber auch zu Hypertrophien im Sinne yon kleinen und grSI]eren Bindegewebsgeschwiilsten, Myomen oder auch yon multiplen Epitheliomen. Die Epitheliome kSnnen oberfl~iehlich und relativ gutartig sein oder kSnnen auch in bSse Formen iibergehen, indem sie destruierend nach der Tiefe zu wachsen. Auch die Altershaut zeigt ein ganz iLhnliches Verhalten. Man findet aueh bei ihr Pigmentanhiiufungen, Pigmentatrophie, Lentigones und Epheliden, weif]e, narben~hnliehe, atrophische Stellen, Teleangiektasien~ warzige Effloreszenzen und auch hier oberfliiehlich und tief greifende Epitheliome. In sehr seltenen Fiillen findet man diese Erscheinungen auch bei jiingeren Indi- Uber generalisierte multiple Epitheliome der ttaut. 481 ~iduen. •icht uninteressant ist die Tatsache, dal] der Sitz der Epitheliome am h~ufigsten im Gesichte ist, w~hrend an allen anderen Stellen derselbe viel seltener ist. Aul]er Epitheliomen finder man auch andere Geschwulstformen sowohl bei der Altershaut als beim Xeroderma pigmentosum. So land K r e i b i c h beim Xeroderma pigmentosum Karzinome, Sarkome und ]~ibrome an der ~ul]eren Haut und Fibrome auch an der Zunge. An der Konjunktiva traten in den yon K r e i b i e h publizierten F~llen gleichfalls Geschwiilste auf. Es fo]gt daraus, dal] beam Xeroderma pigmentosum nicht nur auf der iiul]eren Decke, sondern auch an den Schleimh~iuten (Konjunktiva und Mundschleimhaut) Bindegewebsgesehw[ilste auftreten kSnnen, w~hrend Epitheliome der Schleimhaut wieder und nut bis jetzt an der ~iul~eren Haut beobachtet wurden. H~ilt man sieh vor Augen, dal] das Xeroderma pigmentosum als Resultat der Einwirkung des Sonnenliehts aufgefa•t wird, so wird die Analogie mit den RSntgenstrahlen um so uaheliegender, als man auch naeh den Einwirkungen dieser Yeriinderungen an der ttaut wahrnimmt, die in dem einen Falle zur Aplasie, im anderen Falle zur Hyperplasie der verschiedenen Gewebssysteme an den bestrahlten Hautpartien fiihrt. ~ieht unerw~ihnt daft gelassen werden, da~ der Einwirkung des Sonnenlichts aueh der Friihjahrskatarrh, das Erythema solare, das Ekzema solare, die Hydroa vaceiniformae~ die Prurigo aestivalis die Entstehung verdankt, yon denen sich fast alle auf das Gesicht lokalisieren. Dureh neue Untersuchungen ist es auch klar geworden, daI} bei der Hydroa vaeciniformae nur jene Individuen yon ihr befallen werden~ die an H~imatoporphyrie ( H a u s m a n n , E h r m a n n , Grol]) leiden. Auch bei kiinstlieh erzeugter tt~imatoporphyrie bei Tieren treten prompt naeh Bestrahlung ~ihnliehe Erscheinungen auf. ]qimmt man noeh dazu, da] M e y r o w s k y dureh Einwirkung der W~rme und des Lichts Pigmentveriinderungen der Haut nachweisen konnte, so wird die photochemisehe Wirkung als eine wichtige Komponente bei der Entstehung des Xeroderma und auch der Geschwiilste derselben angesehen werden miissen. Die zweite Komponente jedoeh, die diese iEinwirkung protegiert und welche bei der Hydroa vacciniformae niiher definiert ist, ist freilieh beim Xeroderma pigmentosum bis jetzt unbekannt, mull aber vorausgesetzt werden. 482 Weidenfeld. Es ist nicht ausgeschlossen, dal] in ~hnlicher Weise wie beim Xeroderma pigmentosum auch bei der Altershaut Vorg~nge sieh abspielen, die die Haut in ~hnlicher Weise ver~ndern, wie auch natiirlieh bier die ~uferen Seh~dlichkeiten, wie Licht und meehanische Einwirkungen als zweite Komponen'~e mit in Erw~igung zu ziehen sind. Diesen wohlcharakterisierten Hautver~nderungen, deren Ursache zum Tell bekannt ist, ist es naheliegend, auch die Hautver~nderungen nach Arsenik zuzurechnen. W~hrend aber bei dem Xeroderma pigmentosum, bei den RSntgenhypertrophien und RSntgenepitheliom ~u]ere Seh~dlichkeiten wohlbekannter Natur die Ver~nderungen hervorrufen, miissen die Hautver~nderungen nach Arsenikgebraueh h~matogenen Ursprunges sein, iudem hier die Sch~dlichkeit nut durch das B h t zugefiihrt werden kann. Da aber das Arsenik in irgend einer Form in der Haut sich speichert, so kann es vielleicht direkt die Ver~nderungen hervorrufen. Bekanntlich unterscheidet man zwei Formen d e s A r s en i z i s m u s , eine a k u t e und eine c h r o n i s c h e . Bei der a k u t e n Form treten an tier Haut Erytheme auf, u. zw. mit besonderer Vorliebe an der Palma manus und Planta pedis~ dann an der Priiaurikulargegend und selten auch auf dem Kinn. Diese Erytheme sind blaurot~ scharf begrenzt~ zeigen unregelm~l]ige Formen. Es daft natiirlich nicht Wunder nehmen, daft auch Blasen oder Epithelverdickungen auf solehen Erythemen auf schie~en. Man sieht diese Erscheinungen schon nach geringem Gebrauch yon Arsenik. Innerhalb yon schon vornherein bestehenden Entziindungsherden, wie Lichen ruber planus, Psoriesis~ Ekzem, treten sie mit grol~er Vorliebe auf, bei l~ngerem Gebrauche jedoch treten sie dann auch auf diesen Entziindungsherden mehr oder weniger benachbarter Hautstellen auf, und zwar entweder in Form yon mEchtigen Verdickungen tier Epidermis im Sinne yon rauhen, schwarzbraun gefErbten, festhaftenden Schuppen, oder nicht selten als aus einzelnen Follikelmiindungen hervorragende Stacheln, die, wenn sie aus vielen benachbarten hervorragen und in Gruppen gestellt sind, den Eindruck eines L i c h e n s p i n o 1 o s u s erwecken. Der c h r o n i s c h e A r s e n i z i s m u s erscheint amhEufigsten unter der bekannten Erscheinung der Arsenmelanose. Die- ~ber generalisierte multiple Epitheliome der Haut. 483 selbe zeigt aber verschiedene Formen, oft findet man das gesamte Integument sehwarzbraun verf~irbt, h~ufig jedoch sieht man, dab die diffuse Verfiirbung eine scheinbare ist und sich aus schwarzbraunen, yon weil~en unterbrochenen Fleckchen zusammensetzt. Am dichtesten finden sich diese Fleckchen am Stamme, weniger dicht an den Extremit~ten. Nicht gar so selten finder man Teleangiektasien innerhalb weil~er Fleckchen, sowohl am Stamme und Extremit~ten. ~eben diesen Erscheinungen besteht oft diffuse Abschilferung der Hunt, wobei die Schuppen kleienfSrmig sind, oft im Zentrum anhaftend, also godellt erscheinen, so dal~ man an eine Ichthyosis serpentina gemahnt wird. Als eine weitere Erscheinung des chronisehen Arsenizismus tritt die wohlbekannte Hyperkeratosis palmarum et plantarum auf~ die bekanntlich bald rasch voriibergehen, bald aber auch sehr lunge persistieren kann. Dabei kommt es innerhalb dieser Hyperkeratosen zur Bildung yon derben spitzen Warzen, die vielleicht an der Hohlhand das Analogen zum Lichen spinolosus der iibrigen Haut nach Arsenikgebraueh abgeben. Wie die Warzen entstehen und wo sie den Sitz haben, konnte ich nicht aus begreiflichen Griinden feststellen, vermutungsweise bilden sie sich an den AusfiihrungsSffnungen der Schweil~driisen, ~ihnlich wie der Lichen spinolosus nach Arsenikgebrauch an den husfiihrungen der Talgdriisen sich lokalisiert. Resumiert man die Erscheinungen des Arsenizismus, so kann man sagen, dal~ man bei diesen Entziindungvorg~nge, Atrophien, Teleangiektasien, Hyperatrophien vorfindet, wobei die hyperplastischen Ver~inderungen sich fast aussehlie~lich auf die Epidermis, die hypoplastischen sowohl auf die Kutis als auch auf die Epidermis beziehen. In ganz ~hnlieher Weise wie beim Xeroderma pigmentosum, der Altershaut und nach RSntgen, findet man nun auch nach Arsenikgebrauch Epitheliome. U l l m a n n in Wien hat einen solchen Fall beschrieben, ein Vorkommnis, dessen die englische Literatur 5fters Erw~hnung tut. Untersucht man die Priidilektionen der bei diesem Krankheitsbilde vorkommenden Hyperplasien (an Palma manus und Planta pedis), so ist es naheliegend, auf die hier yon vorneherein bestehende dicke Epidermis, als auch vornehmlich auf 48~ Weidenfeld. die hier wirkenden Traumen zu rekurrieren. Welters muff noch ins Auge gefal]t werden, da~ die Wirkung des hrseniks sich besonders an denjenigen Stellen bemerkbar macht, die durch verschiedene pathologische Prozesse, wie z. B. Psoriasis, Lichen ruber planus und acuminatus, Lichen simplex Vidal, Ekzem u. a. yon vorneherein ver~ndert erscheinen. Hier sieht man nicht allein Pigmentanh~ufungen, sondern auch m uumittelbarer N~he der Effloreszenzen Epidermiswucherungen in Form yon Schuppen oder Schwielen. Neben dieser lokalen, durch bestehende Entziindungen eben n~her charakterisierten Priidisposition der Haut, miissen aber auch flit die allgemeinen Ver~nderungen der Haut eine allgemeine Predisposition angenommen werden, weil dafiir die Tatsachen sprechen, dal] verschiedene Individuen bei derselben Darreiehung den Arseniks in so manigfaltiger Weise, meistens gar nicht auf Arsenik, auch nach jahrelangem Gebrauehe, nicht reagieren. Wiihrend man yon der hyperplastischen Komponenie des Arseniks fast gar keinen Gebrauch macht~ wenn man nicht hiezu die Wirkung auf die Haare, auf den Glanz der Haut, das Abfallen der Schuppen und die Hyperpigmentierung chronischer Plaques, die Inokulation uud Hyperpigmentierang der Lichen ruber-Etfioreszenzen dazu rechnen will, wird yon der hypoplastischen Wirkung derselben yon der Therapie ausgiebig Gebrauch gemacht. So sieht man naeh innerem Gebrauch Abfalleu yon Warzen~ Heilung oder mehr oder weniger starke Riickbildung yon Epitheliomen, Sarkomen. Vielleicht gehSrt auch hierher die yon mir nach Salvarsan nachgewiesene Vermehrung der Wachstumsgeschwindigkeit bei Uberh~utung yon Substanzverlusten. Wenn man sich nach diesen Auseinandersetzungen die Frage vorlegt~ wo der Angrifi[spunkt fiir die Arsenikwirkung zu suchen ist, so wird man nicht fehlgehen, diesen an die Hunt selbst zu verlegen. Dafiir spricht vor allem die Tatsache, dal] yon S c h i f f in Wien, dann aus der Klinik N e i s s e r direkt in Haaren und Epidermis Arnenik nachgewiesen werden konnte. Naheliegend ist es, hier auf die grol3e Xhnlichkeit in der Wirkung der Licht- und vieUeicht auch der Wiirmestrahlen hinzuweinen. Aueh durch dan Licht ~ehen wit helm Xeroderma I)ber generalisierte multiple Epitheliome der Haut. 485 pigmentosum Hypo- und Hyperplasie in Erscheinung treten; in gleicher Weise wird auch auf die Wirkung des Lichtes, hypoplastisch zu wirken, bei der Heilung yon Tumoren reflektiert, wie aueh auf die der RSntgenstrahlen, fiber deren in die Haut selbst verlegten Wirkungen g~r kein Zweifel besteht. Auch an der mit X-Strahlen bestrahlten Haut sieht man nach l~ngerer oder kiirzerer Einwirkung intensiv pigmentierte~ weil3e atrophische Stellen, Teleangiektasien auftreten, dabei die Epidermis gl~nzend, diinn werdend, also Symptome, die auf eine Hypoplasie hindeuten. Daneben sieht man aber auch ~rerdickungen der Epidermis im Sinne yon warzenartigen, schwieligen Veriinderungen, die auf das Konto hyperplastischer Vorg~nge zu setzen ist, die dem Gesamtbild eine .~hnlichkeit mit Xeroderma pigmentosum erteilen und genau wie bei allen friiher genannten Erkrankungen der Haut, treten auch bier neben den Hypo- und Hyperplasien oder innerhalb derselben Epitheliome auf, deren histologisehe Untersuehung C 1n n e t (C 1u n e t : Recherches experimental sur les tameurs malignes, These 1910) in jiingster Zeit vorgenommen hat. An der Hand dieser Ausfiihrungen kann man in unserem Falle bei Bestehen hyperplastiseher Prozesse in so groiser Zahl und in der Ge~eralisierung fiber die ganze Haut nur an Ursaehen denken, die auf die ganze Haut einwirken. Xeroderma pigmentosum kann ausgesehlossen werdeni sehon mit Riicksicht darauf~ dab der Prozel~ im sp~iteren Alter und nicht yon Kindheit an begann. Freilieh daft nicht vergessen werden, daiS aueh im sp~teren Alter Xeroderma pigmentosum beobachtet wurde, aber nie fehlten die begleitenden klfl~ischen Symptome. DaiS es sieh um Erscheinungen auf einer gewShnliehen Altershaut handelt, kann bei der relativen Jugendliehkeit der Patientin und bei der jugendlichen glatten Haut mit aller Sicherheit ausgeschlossen werden. Es ist deswegen naheliegend, diese Ver~nderungen mit der grSiS[en Wahrscheialiehkeit auf den langi~hrigen Gebraueh des Arseniks zuriickzufidhren. Wenn aueh an der Haut der Patientin keine Pigmentversehiebungen, keine Melanose und ]~eine vitiligSsen Stellen gefunden wurden, so kSnnen doch die anderen Erseheinungen mit Leichtigkeit als Folgeerseheinungen der Arsenikmedikation angesehen werden. Die hyperkerato- 486 Weidoafeld. tischen Verdiekungen der Planta und Palma, die in diesem eingesprengten verrukSsen, sehr derben Effloreszenzen lassen vor allem keine andere _~tiologie zu. Aber auch die braunen, kleinen, flachen und roten Scheibchen, die zum Tell mit Sehuppen bedeekt sind, dann die seichten und tiefen Ulzeratiohen und die aus diesen hervorwachsenden papillomatSsen Effloreszenzen und knotenfSrmigen Tumoren, die alle als Epitheliome naehgewiesen wurden, sind gentigende Symptome, die fiir eine Arsenwirkung sprechen. Weiters spricht fiir diese Anschauung, daff tats~chlich w~hrend langer ffahre Patientin Arsenik zu sich genommen hat und daff sie eigentlich sich w~hrend der Arsenmedikation hiereals wohlbefunden hat, indem sie w~hrend dieser Zeit meistens abnahm. Auch die Tatsache, daff, w~ihrend die Symptome ihrer jetzigen Erkrankung manifest waren, nach Injektionen yon Arsazetin die Effloreszenzen alle an GrSBe zunahmen, spricht mit aller Entschiedenheit fiir die Abh~ngigkeit dieser Erkrankung vom ArseniL AuffaUend jedoeh ist das v e r s p ~ t e t e E i n s e t z e n der jetzigen Symptome, indem die manifesten Symptome einige Jahre nach der forzierten Arsenikmedikation auftraten. Freilich gibt Patientin an, dal~ oft ihre H~nde Schwielen zeigten, die dann vergingen. GewShnlich sieht man die Folgezust~nde der Arsenikmedikation w~hrend oder kurze Zeit nachher auftreten. Die Erscheinungen kSnnen akute und voriibergehende sein, oder chronische und jahrelang persistierende, aber immer treten sie wzihrend oder nach der Arsenikdarreichung auf. Die chronische Form jedoch beweist, daft der hrsenik lange Zeit entweder in der [taut selbst oder anderswo aufgestapelt werden muff, und daft es yon dort aus kontinuierlich jene charakteristischen Wirkungen im Sinne einer Hypo- oder Hyperplasie ausiibt. Man kSnnte also auch in unserem Falle an eine Speieherung an irgend einem Organe der Patientin denken, yon wo aus in kleinen, aber kontinuierlichen Schiiben der Arsenik in die Bhtbahn gelangt und seine Wirkungen auf die Haut entfaltet. Diese Annahme findet ferner auch ihre Stiitze in den jetzigen Erfahrungen nach Darreichung yon Salvarsan. Aber der Nachweis des Arseniks in Haut und Haaren l~ft auch die Annahme Uber generalisierte multiple Epitheliome der Haut. 487 einer Speieherung in der Haut zu. Aueh yon anderen anorganischen Verbindungen, wie Jod und Brom, ist es bekannt, daft sie in der Haut gespeichert werden und dort die pathologischen VerKnderungen hervorrufen. Denkbar ist natiirlieh, dab genau wie nach RSntgenbestrahlung hypoplastische Wirkungen noch iahre]ang immer wieder auftreten und immer neue Systeme ergreifen, weil eben die Gewebe funktionell mit einander in Verbindung stehen und die StSrung des einen Gewebes erst bei einer gewissen Intensit~it und nach einer gewissen Zeit die funktionell abhi~ngigen Gewebe in Mitleidenschaft zieht. Wenn aber eine einmalige oder kurzdauernde Sch~digung yon vornherein sehr gering ist, so kann natiirlich eine l~ingere Zeit nStig sein, bis sie kliniseh in Erseheinung tritt. Diese Erkliirung kann natiirlich nur fiir die hyperplastischen Bildungen gelten. Unverst~ndlieh w~ren jedoch die hyperplastischen Erseheinungen ohne die Annahme einer Speicherung des Arseniks, entweder in der Haut selbst oder in dem einen oder anderen Organ, yon dem aus die fortw~hrenden Reize ausgehen, vielleieht dadureh, dal~ kleine Arsenikmengen i m m e r w i e d e r in die Blutbahn hineingeworfen werden. Dadurch w~ire eine Analogie hergestellt mit der hyp~rplastischen Wirkung der RSntgenstrahlen, die aueh bei kleiner aber 5fterer Anwendung Hyperplasien der Kutis und Epidermis und aueh Epitheliom bilden. Es ist vielleieht aueh bier der 0 r t die Beobachtung Cl:u n e t s zu erwahnen, dab maligne Tumoren unter geringer RSntgenbestrahlung zur st~rkeren Wirkung angeregt wurden und dal3 er naeh 5fterer, aber immer nur geringer Bestrahlung kiinstlich Hyperplasien, vielleicht auch sogar Tumoren, wenn man die erhaltene Gewebszunahme als solche deuten will, erhalten hat. H~lt man diese Anschanung fiir richtig~ so ist das Fehlen deutlicher, hypoplastischer Erscheinungen und das sp~te Auftreten der hyperplastischen Erscheinungen kein Gegengrund fiir die Annahme, dal~ wir es in unserem Falle mit einer Form des Arsenizismus zu tun haben. Fassen wir in Kiirze die husfiihrungen zusammen, so mug angenommen werden, dab wir in den Lichtstrahlen und RSntArch. f. D e r m a t . u. Syph. Bd. CXI. 31 4:88 Weidenfeld. genstrahlen und im Arsenik Reize vor uns haben, die die F~ihigkeit besitzen, hypoplastische und hyperplastische u in der tIaut einzaleiten. Naheliegend erschien es anzunehmen, daft die hypoplastischen Vorg~nge durch eine rasehe Sch~digung zafolge einmaliger oder weniger Einwirkungen der betreffenden Agentien erfolgt, und dab die hyperplastischen Erseheinungen 5fteren, kleineren und kurzdauernden Reizen ihr Entstehen verdaDken. Hypoplastisehe und hyperpiastisehe Vorg~nge kSnnen durch dieselben Sch~digungen hervorgerufen werden, wobei vielleieht lediglich die Art der Einwirkung in Frage kommt. Man kann die Ursaehen fiir diese u in i n n e r e und ~ u B e r e e i n t e i l e n ; zu den inueren gehSren, wie wir gesehen haben, Arsenik. Welters ist bel~annt, dab aueh andere Gifte, die zum Teil dem Stoffwechsel, zum Teil Krankheitsprodukten oder selbst yon Krankheitserregern abstammen, iihnliche Hypo- und Hyperplasien erzeugen. Ieh will bier nut auf die Syphilis hindeuten, die Atrophie und Hyperplasie (Leukoplakia, Psoriasis mueosa oris), auf den Lupus erythematosus~ der Atrophien und Hyperplasien der Epidermis erzeugt, dann auf den Lupus vulgaris, yon dem aueh eiue Abaft als Lupus verrueosus bekannt ist. Von den zuletzt genannten Krankheiten kann natiirlieh die fragliehe Ursache fiir die hypo- und hyperplastischen Vorg~nge lokal bestehen, es kann aber auch angenommen werden, wie bei der Lues, dab aueh im Blute kreisende Gifte die Hyperplasien erzeugen, wie bei der Arsenikwirkung. Andererseits fiihren aber die genannten Krankheiten auf die oben erw~hnten7 lokalen Ursaehen hiniiber~ zu welchem wir die Hypo- und Hyperplasien nach Lieht- und RSntgenstrahlen oder anderen mechanisehen oder chemisehen Agentien zuzureohnen haben. W~hrend die a l l g e m e i n e n U r s a c h e n mit Beziehung auf i h r e g e n e r a l i s i e r t e Wirkung multiple Lokalisationen in d e r R e g e l a u f w e i s e n , w e r d e n lokale Ursachen vermutlieh beschr~inkte Herde nur hervorrufen. Aber auch die Lokalisation der ersten Form wird durch sekund~ire Vorg~tnge zum Tell bestimmt. Wie schon oben beim Arsenizismus angefiihrt, fibt der in den KSrperkreislauf aufgenommene Arsenik 1. an den Krankheitsherden ~ber generalisierto multiple Epitheliome der Haut. 489 seine Wirkung aus, 2. in der Nachbarschaft, und 3. an Stellen, die mechanischen Traumen ausgesetzt sind. Da die letzten Momente sich der sichtbaren Kontrolle entziehen, spricht man yon Priidilektionsstellen des Arsenizismus. Man kann deswegen ira allgemeinen sagen, dab iiberall dort, wo durch verschiedene Momente, entweder durch st~rkeren ZufluB oder gehemmten AbfluB, wie in Narben, StSrungen in der Zirkulation bestehen, sich die Arsenikwirkung, also der Arsenizismus, manifestieren wird und Hypo- und Hyperplasien entstehen werden. Man sieht also, dab Kombinationen verschiedener Ursaehen nSiig sind, um die geaanate Wirkung zu erzielen, gleichwie auch fiir die lokal wirkenden Sch~digungen zu ihrer Wirkung, wie oben angefiihrt, primate Yer~inderungen des Organismus vorausgesetzt werden miissen. Sicher geniigt es aber in vielen F~llen, dab die Haut allein durch irgendwelche Sch~idigung ver~ndert ist wodurch dann die veranlassenden ~iul]eren Ursachen den geeigneten Boden finden, hypo- oder hyperplastisch zu wirken. In der Art der Zufiihrung tier beschriebenen Sch~idigungen (Licht, RSntgenstrahlen, Radiumstrahlen, Arsenik u. a.) liegt aber die weitere Unterseheidung ihrer Wirkung, d. h. ob sie hypo- oder hyperplastisch wirksn. Hat in unserem Falle die Generalisierung und die Multiplizit~it der Krankheitssymptome mit einer gewissen Sicherheit auf eine allgemeine Ursache hingewiesea, so kann aber der Einwand nicht yon der Hand gewiesen werden, dab die Propagierung auch auf andere Weise erkliirt werden kann. In: unserem Falle seben wir innerhalb seheinbar ganz normaler Haut bald isolierte, bald in Haufen gestellte Epitheliome in allen Stadien auftreten. Die zumeist befallene Lokalisationen sind die Lendengegend, die Kreuzbeingegend, die Palma manus nnd Planta pedis, dann das Dorsum manus und die Zehenbeeren, wo sie gegen die benachbarten Zehen driicken, dann die Unterschenkel and Obersehenkel und der Kopf. Oft finden sich auch an diesen Stellen sonstige Hyperplasien zum Teil in Form yon Sehwielen und yon kleinen braunen schuppenden Effloreszenzen. Ein Nick auf die genannten Lokalisationen zeigt, dab es besonders Stellen sind, die besonders traumatisehen Reizen stark ausgesetzt sind. Fiir die Entstehnng des Karzinoms 31" 490 Weidenfeld. an der Ful]sohle gab Patientin n~it Bestimmtheit das Trauma dutch Stich eines Nagels an, fiir das Kopfepitheliom an der rechten Scheitelgegend kann ein solches Traunm durcll die Hutnadel gleichfalls mit Leichtigkeit angenommen werden; fiir die anderen Stellen jedoch ist die Reibung durch die Kleider, vielleicht auch Kratzen als ~hnliche Momente nicht yon tier Hand zu weisen. In diesen supponierten Traumen wgre nur ein provozierendes Moment zu sehen, nicht etwa die Ursache selber. Die dutch Arsenik veriinderte Haut wiirde unter der Einwirkung dieser Traumen mit Hyperplasie und Epitheliombildung reagieren, wodurch sich auch bei genereller VerteJlung solcber mechanischen Reize die Generalisierung der Hyperplasie und Epitheliome erkliiren kSnnten. Man kSnnte abei auch versucht seth, vielleicht in diesem Falle an iihnliche Ursaehen fiir die Weiterverbreitung zu denken, wie z. B. bet Warzen, bet welchen es bekannt ist, daft durch 0bertragung yon Stelle zu Stelle eine Propagation erfolgt. Ieh selbst babe durch nicht publizierte Versuche feststellen kSnnen, da$ in I-Iautschnitte eingeriebene, durch Zermalmen verkleinerte Warzenteile nach einiger Zeit (5--6 Wochen) kleine Wiirzchen, gewShnlich in Rosenkranzform, eder aueh bandartig zum Vorschein kommen. Filtrierte ich den Brei durch gewShnliches Filtrierpapier, wobei mikroskopisch ganze Zellen, mitunter aber Zelltriimmer im Filtrat erschienen, so entstanden unter sonst gleichen Yersuchsanordnungen und nach derselben Zeit Warzen. Diese Versuche stellte ich nicht nut an mir selbst an, sondern auch an anderen Kollegen, die sonst gar keine Warren batten. W~hrend nun diese W~irzchen bet mir und den Kollegen ganz abortiv verliefen, wnchsen sie bet den Patienten, yon welchen die Warzen stan~men, also die an Warzen litten, in derselben Zeit zu stattlichen Warzen heran. Wie diese, aueh yon anderen Autoren (J a d a s s o h n) unter gleichen Bedingungen ~ihnlich erhaltenen l~esultate ange~ehen werden mSgen, ob Infektion oder Transplantation, ist fiir die Frage gleiehgiiltig, sicher ist, dal~ Warzen durch mecharisehe Einwirkungen auf die fibrige Haut propagiert werden kSnnen. Uber generalisierte multiple Epitheliome der Haut. 491 Diese Art der Propagierung erschien mir aber im ersten Augenblick tats~chlich bei der Patientin vorzuliegen, indem ich mir vorstellte, dal] Patientin mit ihren Fingern~igeln Teilchen des gro]en fungSsen Tumors der Ful]sohle iiberall hin verbreitete. Gegen die Ahnlichkeit der Ausbreitung yon Warzen sprach vor allem der gewichtige Umstand, dab gerade in der N~ihe des Tumors~ Dorsum pedis gar keine Effioreszenzen bestanden. Welters sprach auch dagegen, dal] schon kleinere Effloreszenzen bestanden, als das Epitheliom an der Ful~sohle noch sehr klein war und einem Hiihnerauge glich und zweitens, daft sich auch Effioreszenzen vorfanden, wo Patientin mit ihren Fingern nicht zukommen konnte -- zwischen den Schulterbl~ttern. Nach der Exstirpation des Tumors an der Fu~sohle und am Kopfe und vollst~ndiger Heflung der Wunden, dann nach Bestrahlung aller anderen Stellen und Heilung derselben, traten jedoch immer neue Effloreszenzen und zwar~ wie erw~hnt, in grSl]erer hnzahl an verschiedenen KSrperstellen wie vordem auf. In jiingster Zeit entstand wieder ein Tumor auf der anderen Ful]sohle, der sieh sehr rasch vergrSflerte und angeblich nach einer RSntgenreaktion auftrat. Es folgt aber aus dieser Tatsache, dal3 nach Exstirpation des ganzen Tumors noch immer in ungeschw~chter St~rke Epitheliome auftreten, dal] eine Propagierung dutch Ubertragung in diesem Falle nicht leicht angenommen werden kann, in anderen F~llen jedoe h kann sie nicht yon der Hand gewiesen werden, wie ja die zahlreichen Uberimpfungen am selben und anderen Tieren (E h r 1i c h A p o l a n t , Le~in~ H a r l a n d , C l u n e t u. a.) beweisen. Ich gebe im folgenden eine histologische Beschreibung eines kleinen Tumors yore rechten Unterschenkel, der erst in letzter Zeit zum Vorschein kam, um den Beweis zu erbringen, dab auch linsengrol]e Stellen schon das fertige Epitheliom zum Ausdrucke bringen. An dem ausgeschnittenen Pr~iparat am Untersehenkel, das sich klinisch als ein linsengrol~es, etwa fiber das Hautniveau erhabenes KnStchen erweist, findet man die Oberfl~iche mit einer sehr verdickten Hornschichte bedeckt, die yon der Peripherie nach dem Zentrum immer dicker wird, an der Peripherie in normale Haul mit normaler Schichtung fibergeht. Die Horn- 492 Weidenfeld. haut setzt sich auch trichterfSrmig gegen die Tlefe fort und bildet zwischen sich Perlen yon Hornhautlamellen, die sich in das[tiefere Gewebe hinein fortsetzen, so daft viele Hohlr~ume mit Hornperlen ausgefiillt mit der Hornhaut direkt in Verbindung stehen. Es sigd das wahrscheinliche Reste yon Follikeltrichtern, die nur an einzelnen Stellen sich finden, an anderen aber nicht vorhanden sind. Die normale Haut erscheint gleichfalls etwas verdickt, die Follikel aber yon normalem Aussehen. An einem solchen Follikel angrenzend, beginnt nun der Neubildungsherd einzusetzen, der die Tiefe der Follikel erreicht, und sie sogar iiberschreitet. Beim n~iheren Zusehen jedoch sieht man, dab die angrenzenden Epithelforts~itze der normalen Haut auswachsen und in der Tiefe ihren Zellcharakter [indern, abbiegen und sich mit der Masse des Tumors zu vereinigen scheinen. Der Herd besteht aus Epithelzellen mit grol]en Kernen und setzt sich gegen das Bindegewebe ziemlich scharf ab. Hier finden sich erweiterte, yon einer geringen Infiltration umgebene Blutgef~l~e. Innerhalb dieses Tumors nun sieht man ~ester und Querscbnitte, auch L~ingsschnitte yon Bindegewebe, die grol~en Papillen ~ihnlich sehen, wenig zellreich erscheinen und yon grol]en Quer- und L~ngsschnitten yon Ge/~l]en durchsetzt erseheinen. Hie und da finder sich im Innern der Blutgeffil~e Blur; die Papillen sind 5dematSs. Aul]er diesen Querschnitten finden sich noch verschiedene zwiebelschalenartige Bildungen ~on Zellanh[iufungen (Epithelperlen). Bei st~rkerer YergrSBerung erweist sieh das verdickte Stratum corneum aus zum Teil aus m~chtigen Platten oder Massen gebfldet. Unterhalb dieser Schicht finden sich deutliche massenha/te Anh~ufungen yon Zellkernen yon rundlichem Aussehen, ohne sichtbaren protoplasmatisehen Zelleib. Erst allm~ihlieh in den hSheren Lagen verlieren sich die Kerne, das Ganze wandelt sieh in die massigen Schollen oder Platten des Stratum eorneum urn. Der Ubergang yore Stratum corneum in die Tumormasse ist ein allm~ihlicher, indem die Zellen ihr Protoplasma und ihren Kontur allm~thlich erhalten und dann deutlich Epithelzellen ~hnlich werden. Bei der starken VergrSl]erung sieht man auch, dal] man mehrere Formen yon kleinen Epithelzellen mit deutlich ausgebildeter Protoplasmafaserung und groBe Epithelzellen unterseheiden kann, Uber generalisierte multiple Epitheliome der Haut. 493 die sich in Herden finden, welche streifenfSrmig durch den Tumor ziehen und zwischen sich I-lornbildungen in Formen won Hornperlen zeigen. Der Tumor setzt sich an der Grenze des ziemlich zellreichen Bindegewebes sehr scharf ab. Mitunter fehlen aber auch jede Anzeichen einer Infiltration. Es handelt sich also hier sicher um ein kleines Epitheliom der Haut, dessen Aufbau sehr ~hnlich dem der grol]en Tumorea ist, nur fehlt bier einstweilen die Umwandlung in die schlauchartige Variet~t. b[och an zwei kleineren, mit Schuppen bedeckten Lentigones habe ich die histologische Untersuchung vorgenommen, jedoch sind vorderhand die Studien noch nicht abgeschlossen, Man kann also aus diesen histologischen Untersuchungen den Schlul] ziehen, dab tatslichlich alle Effloreszenzen Epitheliome in verschiedenen Stadien der Entwicklung sind. Aus dem ~orangehenden folgt also, dal] w i r e s bier mit einem Fall zu tun haben, der lange Zeit Arsenik nahm; nachher Schwielen an den Hiinden und dann nach Jahren zuerst einzelne sehr flache, spiiter aber immer zahlreichere, am ganzen Stamm und an den Extremitiiten sich verbreitende Epitheliome yon verschiedener GrSl]e und Form bekam, wobei im Laufe der Zeit die ersten Tumoren sich bis zu Nu~}grSi]e vergrSl~erten. Der Propagierung durch ~bertragung klinischer Geschwulstmassen kann auch durch diese Tatsache, dal] noch immer l~leinste Effloreszenzen nach sehr langer Zeit auftreten, eine weitere Stiitze entzogen werden. Dal] aber nach der Exstirpation der grol]en Tumoren neue, kleinere Effloreszenzen aufschiel~en, kSnnte an die Vorstellung E h r l i c h s erinnern, die der Tatsache zugrunde liegt~ da~ bei Gegenwart eines Tumors neue Inplantationen yon Tumoren am selben Individuum nicht haften, nach Exstirpation aber wieder haften uncl zu deren Erkl~rung er Erniihrungs- und ErschSpfungszust~nde annimnt~ die er als Athrepsie bezeichnet. Ob ~ihnliche u bier bestehen, soll dahingestellt bleiben, es kann sich auch um eine Vermehrung innerhalb der langen Beobachtungszeit (2 Jahre) handeln, die aus anderen inneren Ursachen, gleichwie die Entstehung der ersten Tumoren entspringt. Zwischen diesem Falle und den F~illen yon Xeroderma pigmentosum~ dann solchen nach RSntgenbestrahlungen be- 494 Weidenfeld. stehen, wie oben angefiihrt, weitgehende Analogien. Bei ihnen konstatierte man neben den Hyper- und Hypoplasien auch das Auftreten yon Epitheliomen. Auch in unserem Falle treten zuerst Hyperkeratosen an den II~inden und Ful]sohlen auf. Innerhalb dieser schwieligen Verdickungen bildeten sich die Epitheliome und jene warzenartigen Exkreszenzen innerhalb der hyperkeratotisch verdickten Haut der Handteller und FuJ]sohle, die in der Krankengeschichte des N~iheren beschrieben wurden. Schon durch den gleichartigen Sitz ist es nicht yon der Hand zu weisen, die Hyperplasien als Vorboten der Epitheliome anzusehen wobei zwischen beiden nur graduelle Unterschiede bestehen, die aber in der geringeren oder grSLJeren Szh~digung der Haut oder Epidermis ihren weiteren Grund haben. Wie schon oben angefiihrt, zeigt schon die Beobachtung allein~ da~ die Hyperkeratosis an der Hohlhand und Ful]sohle, die in zwei Formen (einer diffusen und einer zirkumskripten warzenartigen) auftritt, besonders in ihrer zweiten Form sich sich in klinisch gut charakterisierte Epitheliome umwandelt, die bald framboesief6rmig werden oder nach Abfallen der Oberfl~che seiehte Ulzerationen bilden. Eine ~hnliche Sch~digung der gesamten Haut mull angenommen werden~ wenn auch klinisch nur geringe Symptome~ wie Schuppen auf erythematosen Stellen, Pigmentierungen, nachweisbar sind, damit fiberall, wo nur kleine Ursachen noch hinzukommen, wie traumatische Reize, die Epidermiszellen sich in Epitheliomzellen umbilden. Gerade mit Riieksicht auf diese Vorstellung babe ich es wert gefunden, diesen Fall der VerSffentlichung zuzufiihren. Das multiple und generalisierte Auftreten der Epitheliome reiht sich in unsere Vorstellung leichter ein, als das isolierte Auftreten eines Epithelioms. H~tlt man sich jedoeh die Entstehung eines Epithelioms nach RSntgenbestrahlung vor Augen, so kann das isolierte Auftreten yon Epitheliomen nach unseheinbaren~ ~ul]eren Reizen, besonders wenn eine Veriinderung der Haut durch vorausgegangene Einwirkung verschiedener Gifte vorliegt, sich leichter in unseren Vorstelhngskreis einffigen. Wie die Sdh~.digungen die Zellen ver~ndern, ist unbekannt, sicher jedoch scheint es zu sein, dal] sie in kleinen~ aber 5fteren Dosen naeh l~ngerer Zeit die Hyperplasie und diese die Bildung der Epitheliome veranlassen. Durch Kombination yon chemischen und Lichtreizen kSnnten vielleicht doch Anhaltspunkte fiir die pathologisehe Umwandlung des Epithels gewonnen werden, wofiir einzelne Versuche yon C l u n e t zu sprechen ~zheinen.
https://openalex.org/W3157148235	https://www.e3s-conferences.org/articles/e3sconf/pdf/2021/30/e3sconf_farba2021_08002.pdf	English	null	Influence of feed additive on the biological value of broiler chickens’ white meat protein in technological stress conditions	E3S web of conferences	2,021	cc-by	2,284	* Corresponding author: ernest_saif@mail.ru Federal State Budgetary Educational Institution of Higher Education “South Ural State Agrarian University” Troitsk, Russia Federal State Budgetary Educational Institution of Higher Education “South Ural State Agrarian University” Troitsk, Russia Federal State Budgetary Educational Institution of Higher Education “South Ural State Agrarian University” Troitsk, Russia Abstract. The aim of the research was to study the effect of feed additive on the biological value of broiler chickens' white meat protein under pre- slaughter stress conditions. The experiment was carried out on Arbor Acres broiler chickens in the conditions of an industrial poultry farm with floor housing technology. The main diet of poultry in the I experimental group was introduced with feed additive at a 1269 g/t feed dose, II - at a dose of 1693 g/t. The control group poultry received only the main diet. As a result, the white meat of broiler chickens of experimental groups contained 1.5-6.8% more essential amino acids, substituted by 1.3-5.5% compared to control. In the amino acid composition of the broiler chickens' meat of the experimental group I, the most pronounced changes in the content of essential and dispensable amino acids were revealed; their level was above the control by an average of 3.7 and 2.9% respectively. White meat proteins of broiler chickens of the I experimental group were more biologically valuable; their amino acid score was above control by an average of 4.5%. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 254, 08002 (2021) FARBA 2021 E3S Web of Conferences 254, 08002 (2021) FARBA 2021 https://doi.org/10.1051/e3sconf/202125408002 Influence of feed additive on the biological value of broiler chickens' white meat protein in technological stress conditions V. Miftakhutdinov, E.R. Saifulmulyukov*, and E.A. Nogovitsina 1 Introduction The productivity of broiler chickens is directly related to the production intensification and, accordingly, industrial stresses. Stress factors affect the nervous and endocrine systems and metabolic processes occurring in the poultry body, while reducing production rates of operation and overall efficiency of broiler poultry farming. Many scientists agree that technological stresses in industrial poultry farming cause a decrease in weight increment, poultry safety, meat productivity and, as a result, the quality of meat. And the greatest productivity loss is noted precisely during the pre-slaughter period. Mortality rates during the pre-slaughter period depend on many factors: poultry immunity, catching method, placement density, ambient temperature, conditions and duration of transport, distance to the slaughter point and conditions of preslaughter care [1, 2, 3, 4]. The most frequent causes of broilers' death during the pre-slaughter period is the so-called “sudden death syndrome” and injuries, including fractures and liver ruptures [5]. E3S Web of Conferences 254, 08002 (2021) E3S Web of Conferences 254, 08002 (2021) FARBA 2021 https://doi.org/10.1051/e3sconf/202125408002 The catch stage leads to a significant increase in production losses: poultry gets injured, thereby increasing the level of carcasses defect and mortality increase [6]. It is noted that the longer the poultry remains in the catcher's hands, the more chances of various kinds of injuries and subsequent carcasses defects [7, 8]. High ambient temperature during transportation is also a strong poultry stressor [9]. Higher transportation duration increases the risk of poultry feathering contamination with waste products, which leads to a decrease in the sanitary state of carcasses [10]. Prior to slaughter, 3 hours of stress leads to changes in the chemical composition and pH of poultry meat, manifestation of PSE signs [11]. At the same time, the main problem is associated with the decrease in the sensor characteristics of meat, which lead to an increase in technological losses when processing for meat products [12]. Stress affects the metabolism of amino acids, usually reducing their digestibility from feed [13]. The effectiveness of the stress protectors' use in pre-slaughter period for increasing the safety and meat productivity of poultry [14, 15, 16] was proved. In view of the above, the aim of the research was to study the impact of feed additive on the biological value of broiler chickens' white meat protein under pre-slaughter stress conditions. 2 Materials and methods The experiment on “Pik-Antistress” feed additive application was carried out on broiler chickens of final cross hybrid Arbor Acres in the conditions of industrial poultry farm with floor management technology. The poultry was divided into three groups of 6,000 chicks each and kept in one room in separate sections. The scheme of feed additive application is presented in Table 1. Table 1. Scheme of feed additive application Control group Experimental group I Experimental group II Basic diet (BD) BD+PIK-AS (1269 g/t feed for 5 days before slaughter) BD+PICK-AS (1693 g/t feed for 5 days before slaughter) Slaughter at 38 days The amino acid composition of white meat was determined via capillary electrophoresis method in the laboratory of the innovative research center of FSBEI HE South Ural State University. Statistical data processing was performed in the STATISTICA 12 program. Table 1. Scheme of feed additive application Control group Experimental group I Experimental group II Basic diet (BD) BD+PIK-AS (1269 g/t feed for 5 days before slaughter) BD+PICK-AS (1693 g/t feed for 5 days before slaughter) Slaughter at 38 days Table 1. Scheme of feed additive application Control group Experimental group I Experimental group II Basic diet (BD) BD+PIK-AS (1269 g/t feed for 5 days before slaughter) BD+PICK-AS (1693 g/t feed for 5 days before slaughter) Slaughter at 38 days Table 1. Scheme of feed additive application The amino acid composition of white meat was determined via capillary electrophoresis method in the laboratory of the innovative research center of FSBEI HE South Ural State University. Statistical data processing was performed in the STATISTICA 12 program. 3 Results and discussion The analysis of the amino acid content in poultry white meat protein gives a detailed assessment of the change in its biological value under pre-slaughter stress conditions (Fig. 1-3). 2 2 E3S Web of Conferences 254, 08002 (2021) FARBA 2021 E3S Web of Conferences 254, 08002 (2021) https://doi.org/10.1051/e3sconf/202125408002 Fig. 1. The content of essential amino acids in white meat protein of broiler chickens, mg/100 g (n=9) Fig. 1. The content of essential amino acids in white meat protein of broiler chickens, mg/100 g (n= Estimating the content of essential amino acids in the broiler chickens' white meat of experimental groups, we noted that the level of lysine and methionine was on average higher by 1.5-1.7%; valine, leucine, isoleucine and phenylalanine - by 2.1-2.2%, threonine - by 2.7%. The pronounced changes were in the tryptophan content; on average, its level was higher than the control group by experimental ones by 6.8%. Fig. 2. Content of dispensable amino acids in broiler chickens' white meat protein, mg/100 g (n=9) Fig. 2. Content of dispensable amino acids in broiler chickens' white meat protein, mg/100 g (n=9) As for the content of dispensable amino acids in the experimental groups, on average in relation to control, an increased content of glutamic, aspartic acids, glycine and serine by 1.3-1,7%, arginine, histidine and alanine by 1.7-2.0%, tyrosine and proline by 2.5-2.7%, cystine by 5.5% was noted. As for the content of dispensable amino acids in the experimental groups, on average in relation to control, an increased content of glutamic, aspartic acids, glycine and serine by 1.3-1,7%, arginine, histidine and alanine by 1.7-2.0%, tyrosine and proline by 2.5-2.7%, cystine by 5.5% was noted. 3 E3S Web of Conferences 254, 08002 (2021) FARBA 2021 https://doi.org/10.1051/e3sconf/202125408002 Fig. 3. The sum of essential and dispensable amino acids in the broiler chickens' white meat protein, mg/100 g (n=9) Fig. 3. The sum of essential and dispensable amino acids in the broiler chickens' white meat protein, mg/100 g (n=9) In terms of the sum of essential amino acids, the control group in general was on average inferior to experimental ones by 2.1%, in terms of dispensable amino acids - by 1.8%. The most pronounced changes in the amino acid composition of meat were identified in the I experimental group, where the content of essential amino acids on average was higher by 3.7%, dispensable — 2.9%, in relation to control. 4 Conclusions 1. In white meat of experimental groups' broiler chickens, the content of essential amino acids was higher on average by 1.5-6.8%; dispensable - by 1.3-5.5%, compared to control. 2. In the amino acid composition of the broiler chickens' meat of the experimental group I, the most pronounced changes in the content of essential and dispensable amino acids were revealed; their level was above the control by an average of 3.7 and 2.9% respectively. 3. Amino-acid score of white meat proteins of experimental groups' broiler chickens was above the control for lysine and valine by 1.5%, leucine and isoleucine - by 1.7%, methionine and cystine - 2.1, threonine, phenylalanine, and tyrosine — 2.2 and tryptophane — 7.3%. White meat proteins of broiler chickens of the I experimental group were more biologically valuable; their amino acid score was above control by an average of 4.5%. 1. In white meat of experimental groups' broiler chickens, the content of essential amino acids was higher on average by 1.5-6.8%; dispensable - by 1.3-5.5%, compared to control. more valuable. The established differences are consistent with the results of studies obtained by other authors, who noted the positive effect of similar feed additives on the poultry meat amino acid composition. When using the preparation of succinic acid on broiler chickens, an increase in the essential amino acids content in the muscular tissue proteins of experimental groups' chicks in relation to control indicators was observed [17]. Supplements of betaine in the broiler chickens' diet affected the increase in the essential amino acids content in meat [18]. Carnitine introduction to the diet protects branched chain amino acid against oxidation in tissues [19]. Thus, for industrial poultry farming, the pre-slaughter period involves many stress factors, in response to which the poultry organism actively consumes the energy produced by using endogenous nutrients. Due to metabolic disorders, the biological value of meat decreases. The use of feed additives allows to save energy and plastic body resources. 3 Results and discussion The content of essential amino acids in the II experimental group was on average higher by 0.5%, compared to control. The dispensable amino acid content was above control at between 0.3-0.8%, except for cystine, the level of which was 3.1% higher. The protein's amino acid score reflects the approximation of poultry meat's biological value to the reference sample (see table 2). Table 2. Amino acid score of broiler chickens' white meat protein, % Table 2. Amino acid score of broiler chickens' white meat protein, % Indicator Group control experimental I experimental II Leucine + Isoleucine 121.5 124.8 121.6 Lysine 181.7 183.9 182.6 Methionine + cystine 101.5 104.0 103.0 Phenylalanine + Tyrosine 128.1 131.7 128.9 Threonine 112.3 116.1 112.8 Tryptophan 120.8 134.1 122.1 Valin 106.2 109.1 106.4 Amino-acid score of broiler chickens' white meat protein in experimental groups was higher by 1.5% for lysine and valine, leucine, and isoleucine - 1.7, methionine and cystine - 2.1, phenylalanine and tyrosine - 2,2, threonine - 2.2 and tryptophane by 7.3%, relative to control. Despite the trend of increasing amino acid content in the broiler chickens' meat of both experimental groups, the white meat proteins of the I experimental poultry group were 4 4 E3S Web of Conferences 254, 08002 (2021) FARBA 2021 E3S Web of Conferences 254, 08002 (2021) https://doi.org/10.1051/e3sconf/202125408002 more valuable. References 1. C. Grilli, A. R. Loschi, S. Rea, R. Stocchi, L. Leoni, F. Conti British Poultry Science, 56(1), 1 (2015) 2. F.M.C. Vieira, M. Deniz, I.J.O. da Silva, J.A.D. Barbosa, A.M.C. Vieira, F.S. Goncalves Semina-Ciencias Agrarias, 36(6), 3887 (2015) 3. L. Jacobs, E. Delezie, L. Duchateau, K. Goethals, F. A. M. Tuyttens, Poultry Science. 96(2), 266 (2017) 4. G. Di Martino, K. Capello, E. Russo, M. Mazzucato, P. Mulatti, N. Ferre, A. Garbo, M. Brichese, S. Marangon, L. Bonfanti, Animal, 11(12), 2295 (2017) 5. K.E. Kittelsen, E.G. Granquist, O. Kolbjornsen, O. Nafstad, R.O. Moe, Poultry Science, 94(11), 2622 (2015) 6. M.L. d. V. Queiroz, J.A.D. Barbosa Filho, L.M. Duarte, D. d. Brasil, C.R.F. Gadelha, Brazilian Journal of Poultry Science, 17(1), 37 (2015) 7. N. Langkabel, M.P.O. Baumann, A. Feiler, A. Sanguankiat, R. Fries, Poultry Science, 94(8), 1735 (2015) 8. K. E. Kittelsen, E. G. Granquist, G. Vasdal, E. Tolo, R. O. Moe, Animal Welfare, 24(4), 387 (2015) 9. R.S. Spurio, A.L. Soares, R.H. Carvalho, V. Silveira, M. Grespan, A. Oba, M. Shimokomaki, Animal Science Journal, 87(2), 277 (2016) 5 5 https://doi.org/10.1051/e3sconf/202125408002 E3S Web of Conferences 254, 08002 (2021) FARBA 2021 E3S Web of Conferences 254, 08002 (2021) FARBA 2021 10. L. Jacobs, E. Delezie, L. Duchateau, K. Goethals, F. A. M. Tuyttens, Poultry Science, 96(2), 259 (2017) 11. R.T.V. Fernandes, A.M.V. de Arruda, A.D. Melo, J.B.M. Marinho, R.T.V. Fernandes, L.C. de Figueiredo, Journal of Animal Behaviour and Biometeorology, 4(4), 93 (2016) 12. P. M. Groff-Urayama, J. B. Padilha, J. Pia, S. E. Takahashi, Veterinary and Zootechnics, 12(2), 33 (2018) 13. Walid Habashy, M. Milfort, Kwaku Adomako, Youssef Attia, Romdhane Rekaya, Samuel Aggrey, Poultry Science, 96 (2017) 14. V.I. Fisinin, A.V. Miftakhutdinov, D.E. Anosov, Russian Agricultural Sciences, 42(1), 97 (2016) 15. A.V. Miftakhutdinov, E.R. Saifulmulyukov, E.A. Nogovitsina, E.A. Miftakhutdinova, IOP Conference Series: Earth and Environmental Science, AgroCON-2019, 012050 (2019) 16. N.A. Zhuravel, A.V. Miftakhutdinov, S.F. Suchanova, IOP Conference Series: Earth and Environmental Science. AgroCON-2019, 012056 (2019) 17. T. Kurmakaeva, Y. V. Petrova, A. Avdeyenko, Herald of the NI Vavilov Saratov State Agrarian Univ, 25 (2013) 18. M. Nutautaitė, S. Alijošius, S. Bliznikas, V. Šašytė, V. Vilienė, A. Pockevičius, A. Racevičiūtė-Stupelienė, Italian Journal of Animal Science, 19(1), 621 (2020) 19. Shahram G. Adabi, Ross Cooper, Necmettin Ceylan, Muzaffer Corduk, World's Poultry Science Journal, 67, 277 (2011) 6
https://openalex.org/W2073836327	https://discovery.ucl.ac.uk/id/eprint/1405641/1/journal.pgen.1004383.pdf	English	null	Bayesian Test for Colocalisation between Pairs of Genetic Association Studies Using Summary Statistics	PLOS genetics	2,014	cc-by	13,445	Abstract Genetic association studies, in particular the genome-wide association study (GWAS) design, have provided a wealth of novel insights into the aetiology of a wide range of human diseases and traits, in particular cardiovascular diseases and lipid biomarkers. The next challenge consists of understanding the molecular basis of these associations. The integration of multiple association datasets, including gene expression datasets, can contribute to this goal. We have developed a novel statistical methodology to assess whether two association signals are consistent with a shared causal variant. An application is the integration of disease scans with expression quantitative trait locus (eQTL) studies, but any pair of GWAS datasets can be integrated in this framework. We demonstrate the value of the approach by re-analysing a gene expression dataset in 966 liver samples with a published meta-analysis of lipid traits including .100,000 individuals of European ancestry. Combining all lipid biomarkers, our re-analysis supported 26 out of 38 reported colocalisation results with eQTLs and identified 14 new colocalisation results, hence highlighting the value of a formal statistical test. In three cases of reported eQTL-lipid pairs (SYPL2, IFT172, TBKBP1) for which our analysis suggests that the eQTL pattern is not consistent with the lipid association, we identify alternative colocalisation results with SORT1, GCKR, and KPNB1, indicating that these genes are more likely to be causal in these genomic intervals. A key feature of the method is the ability to derive the output statistics from single SNP summary statistics, hence making it possible to perform systematic meta-analysis type comparisons across multiple GWAS datasets (implemented online at http://coloc.cs.ucl.ac.uk/coloc/). Our methodology provides information about candidate causal genes in associated intervals and has direct implications for the understanding of complex diseases as well as the design of drugs to target disease pathways. Citation: Giambartolomei C, Vukcevic D, Schadt EE, Franke L, Hingorani AD, et al. (2014) Bayesian Test for Colocalisation between Pairs of Genetic Association Studies Using Summary Statistics. PLoS Genet 10(5): e1004383. doi:10.1371/journal.pgen.1004383 Editor: Scott M. Williams, Dartmouth College, United States of America Received July 3, 2013; Accepted April 2, 2014; Published May 15, 2014 Copyright: 2014 Giambartolomei 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: CG is supported by a PhD studentship from the British Heart Foundation. Abstract VP is partly supported by the UK Medical Research Council (G1001158) and by the National Institute of Health Research (NIHR) Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. CW is funded by the Wellcome Trust (089989). The Diabetes and Inflammation Laboratory is funded by the JDRF, the Wellcome Trust (091157) and the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre. The Cambridge Institute for Medical Research (CIMR) is in receipt of a Wellcome Trust Strategic Award (100140). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: claudia.giambartolomei.10@ucl.ac.uk results. This strategy has been explored before and allowed the identification of the genes and regulatory variations that are important for several diseases (reviewed in [4]). Claudia Giambartolomei1, Damjan Vukcevic2, Eric E. Schadt3, Lude Franke4, Aroon D. Hingorani5, Chris Wallace6, Vincent Plagnol1 1 UCL Genetics Institute, University College London (UCL), London, United Kingdom, 2 Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Australia, 3 Department of Genetics and Genomics Sciences, Mount Sinai School of Medicine, New York, New York, United States of America, 4 Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands, 5 Institute of Cardiovascular Science, University College London, London, United Kingdom, 6 JDRF/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge, Institute for Medical Research, Department of Medical Genetics, NIHR, Cambridge Biomedical Research Centre, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom Citation: Giambartolomei C, Vukcevic D, Schadt EE, Franke L, Hingorani AD, et al. (2014) Bayesian Test for Colocalisation between Pairs of Genetic Association Studies Using Summary Statistics. PLoS Genet 10(5): e1004383. doi:10.1371/journal.pgen.1004383 PLOS Genetics \| www.plosgenetics.org Author Summary Genome-wide association studies (GWAS) have found a large number of genetic regions (‘‘loci’’) affecting clinical end-points and phenotypes, many outside coding inter- vals. One approach to understanding the biological basis of these associations has been to explore whether GWAS signals from intermediate cellular phenotypes, in particular gene expression, are located in the same loci (‘‘colocalise’’) and are potentially mediating the disease signals. Howev- er, it is not clear how to assess whether the same variants are responsible for the two GWAS signals or whether it is distinct causal variants close to each other. In this paper, we describe a statistical method that can use simply single variant summary statistics to test for colocalisation of GWAS signals. We describe one application of our method to a meta-analysis of blood lipids and liver expression, although any two datasets resulting from association studies can be used. Our method is able to detect the subset of GWAS signals explained by regulatory effects and identify candidate genes affected by the same GWAS variants. As summary GWAS data are increasingly available, applications of colocalisation methods to integrate the findings will be essential for functional follow-up, and will also be particularly useful to identify tissue specific signals in eQTL datasets. These limitations motivate the development of novel method- ologies to test for colocalisation between pairs of traits. Here, we derive a novel Bayesian statistical test for colocalisation that addresses many of the shortcomings of existing tools. Our analysis focuses on a single genomic region at a time, with a major focus on interpreting the pattern of LD at that locus. Our underlying model is closely related to the approach developed by Flutre et al. [10], which considers the different but related problem of maximising the power to discover eQTLs in expression datasets of multiple tissues. A key feature of our approach is that it only requires single SNP p-values and their minor allele frequencies (MAFs), or estimated allelic effect and standard error, combined with closed form analytical results that enable quick comparisons, even at the genome-wide scale. Our Bayesian procedure provides intuitive posterior probabilities that can be easily interpreted. A main application of our method is the systematic comparison between a new GWAS dataset and a large catalogue of association studies in order to identify novel shared mechanisms. Overview of the method Nica et al. [5] proposed a methodology to rank the SNPs with an influence on two traits based on the residual association conditional on the most associated SNP. By comparing the GWAS SNP score with all other SNPs in the associated region, this method accounts for the local LD structure. However, this is not a formal test of a null hypothesis for, or against, colocalisation at the locus of interest. A formal test of colocalisation has been developed in a regression framework. This is based on testing a null hypothesis of proportionality of regression coefficients for two traits across any set of SNPs, an assumption which should hold whenever they share causal variant(s) [12,13]. No assumption is made about the number of causal variants, although the method does assume that in the case of multiple causal variants, all are shared. Both the ranking method and proportionality testing share the drawback of having to specify a subset of SNPs to base the test on, and Wallace [14] shows that this step can generate significant biases. The main sources of bias are overestimation of effect sizes at selected SNPs (termed ‘‘Winner’s curse’’), and the fact that, owing to random fluctuations, the causal variant may not always be the most strongly associated one. These factors lead to rejection of colocalisation in situations where the causal SNP is in fact shared. Although this can be overcome in the case of proportion- ality testing by averaging over the uncertainty associated with the best SNP models [14], perhaps the greatest limitation is the requirement for individual level genotype data, which are rarely available for large scale eQTL datasets. We consider a situation where two traits have been measured in two distinct datasets of unrelated individuals. We assume that samples are drawn from the same ethnic group, i.e. allele frequencies and pattern of linkage disequilibrium (LD) are identical in both populations. For each of the two samples, we consider for each variant a linear trend model between the outcome phenotypes Y and the genotypes X (or a log-odds generalised linear model if one of the two outcome phenotypes Y is binary): Y~mzbXze We are interested in a situation where single variant association p- values and MAFs, or estimated regression coefficients ^b and their estimated precisions var(^b), are available for both datasets at Q variants, typically SNPs but also indels. Bayesian Test for Colocalisation basis of summary statistics. With these advantages in mind, He et al. [7] developed a statistical test to match the pattern of gene expression with a GWAS dataset. This approach, coded in the software Sherlock, can accommodate p-values as input. However, their hypothesis of interest differs from the question of colocalisa- tion, with the focus of the method being on genome-wide convergence of signals, assuming an abundance of trans eQTLs. In particular, SNPs that are not associated with gene expression do not contribute to the test statistic. Such variants can provide strong evidence against colocalisation if they are strongly associated with the GWAS outcome. Author Summary We demonstrate the value of the method by re- analysing a large scale meta-analysis of blood lipids [15] in combination with a gene expression study in 966 liver samples [16]. However, identifying the traits that share a common association signal is not a trivial statistical task. Visual comparison of overlaps of association signals with an expression dataset is a step in this direction (using for example Sanger tool Genevar http://www. sanger.ac.uk/resources/software/genevar/), but the abundance of eQTLs in the human genome and across different tissues makes an accidental overlap between these signals very likely [2]. Therefore visual comparison is not enough to make inferences about causality and formal statistical tests must be used to address this question. Introduction In the last decade, hundreds of genomic loci affecting complex diseases and disease relevant intermediate phenotypes have been found and robustly replicated using genome-wide association studies (GWAS, [1]). At the same time, gene expression measurements derived from microarray [2] or RNA sequencing [3] studies have been used extensively as an outcome trait for the GWAS design. Such studies are usually referred to as expression quantitative trait locus (eQTL) analysis. While GWAS datasets have provided a steady flow of positive and replicable results, the interpretation of these findings, and in particular the identification of underlying molecular mechanisms, has proven to be challeng- ing. Integrating molecular level data and other disease relevant intermediate phenotypes with GWAS results is the natural step forward in order to understand the biological relevance of these In this context, a natural question to ask is whether two independent association signals at the same locus, typically generated by two GWAS studies, are consistent with a shared causal variant. If the answer is positive, we refer to this situation as colocalised traits, and the probability that both traits share a causal mechanism is greatly increased. A typical example involves an eQTL study and a disease association result, which points to the causal gene and the tissue in which the effect is mediated [5–7]. In fact, looking for overlaps between complex trait-associated variants and eQTL variants has been successfully used as evidence of a common causal molecular mechanism (e.g., [5,8]). The same questions can also be considered between pairs of eQTLs [9,10], or pairs of diseases [11]. May 2014 \| Volume 10 \| Issue 5 \| e1004383 1 PLOS Genetics \| www.plosgenetics.org Bayesian Test for Colocalisation PLOS Genetics \| www.plosgenetics.org Overview of the method We make two additional assumptions and discuss later in this paper how these can be relaxed. Firstly, that the causal variant is included in the set of Q variants, either directly typed or well imputed [17–19]. Secondly, that at most one association is present for each trait in the genomic region of interest. We are interested in exploring whether the data support a shared causal variant for both traits. While the method is fully applicable to a case-control outcome, we consider two quantitative traits in this initial description. The success of GWAS meta-analyses has shown that there is considerable benefit in being able to derive association tests on the SNP causality in a region of Q variants can be summarised for each trait using a vector of length Q of (0, 1) values, where 1 means May 2014 \| Volume 10 \| Issue 5 \| e1004383 May 2014 \| Volume 10 \| Issue 5 \| e1004383 PLOS Genetics \| www.plosgenetics.org 2 Bayesian Test for Colocalisation Figure 1. Example of one configuration under different hypotheses. A configuration is represented by one binary vector for each trait of (0,1) values of length n = 8, the number of shared variants in a region. The value of 1 means that the variant is causally involved in disease, 0 that it is not. The first plot shows the case where only one dataset shows an association. The second plot shows that the causal SNP is different for the biomarker dataset compared to the expression dataset. The third plot shows the configuration where the single causal variant is the fourth one. doi:10.1371/journal.pgen.1004383.g001 Figure 1. Example of one configuration under different hypotheses. A configuration is represented by one binary vector for each trait of (0,1) values of length n = 8, the number of shared variants in a region. The value of 1 means that the variant is causally involved in disease, 0 that it is not. The first plot shows the case where only one dataset shows an association. The second plot shows that the causal SNP is different for the biomarker dataset compared to the expression dataset. The third plot shows the configuration where the single causal variant is the fourth one. doi:10.1371/journal.pgen.1004383.g001 SNPs associated with each trait. In contrast, if PP4 is large, the data support a single variant affecting both traits. Overview of the method An illustration of the method is shown in Figure 2 for negative (Figure 2A–B, FRK gene and LDL, PP3 .90%) and positive (Figure 2C–D, SDC1 gene and total cholesterol, PP4 .80%) colocalisation results. that the variant is causally associated with the trait of interest and at most one entry is non-zero. A schematic illustration of this framework is provided in Figure 1 in a region that contains 8 SNPs. Each possible pair of vectors (for traits 1 and 2, which we refer to as ‘‘configuration’’) can be assigned to one of five hypotheses: N H0: No association with either trait While the method uses Approximate Bayes Factor computa- tions (ABF, [20], and Methods), no iterative computation scheme (such as Markov Chain Monte Carlo) is required. Therefore, computations are quick and do not require any specific computing infrastructure. Precisely, the computation time behaves as Qd, where Q is the number of variants in the genomic region and d the number distinct associations (typically d = 2, assuming two traits and at most one causal variant per trait). N H1: Association with trait 1, not with trait 2 N H2: Association with trait 2, not with trait 1 N H3: Association with trait 1 and trait 2, two independent SNPs N H4: Association with trait 1 and trait 2, one shared SNP In this framework, the colocalisation problem can be re- formulated as assessing the support for all configurations (i.e. pairs of binary vectors) in hypothesis H4. Importantly, the use of ABF enable the computation of posterior probabilities from single variant association p-values and MAFs, although the estimated single SNP regression coefficients ^b and their variances or standard errors are preferred for imputed data. Our method is Bayesian in the sense that it integrates over all possible configurations. This process requires the definition of prior probabilities, which are defined at the SNP level (Methods). A probability of the data can be computed for each configuration, and these probabilities can be summed over all configurations and combined with the prior to assess the support for each hypotheses (H)5 1. The result of this procedure is five posterior probabilities (PP0, PP1, PP2, PP3 and PP4). A large posterior probability for hypothesis 3, PP3, indicates support for two independent causal Sample size required for colocalisation analysis Given the well-understood requirements for large sample size for GWAS data, we used simulations to investigate the power of May 2014 \| Volume 10 \| Issue 5 \| e1004383 May 2014 \| Volume 10 \| Issue 5 \| e1004383 PLOS Genetics \| www.plosgenetics.org 3 Bayesian Test for Colocalisation Figure 2. Illustration of the colocalisation results. Negative [SPACE] (A–B, FRK gene and LDL, PP3 .90%) and positive (C–D, SDC1 gene and total cholesterol, PP4 .80%) colocalisation results. 2log10(p) association p-values for biomarker (top, A and C) and 2log10(p) association p-values for expression (bottom, B and D) at the FRK (A, B) and SDC1 locus (C, D), 1Mb range. doi:10.1371/journal.pgen.1004383.g002 Figure 2. Illustration of the colocalisation results. Negative [SPACE] (A–B, FRK gene and LDL, PP3 .90%) and positive (C–D, SDC1 gene and total cholesterol, PP4 .80%) colocalisation results. 2log10(p) association p-values for biomarker (top, A and C) and 2log10(p) association p-values for expression (bottom, B and D) at the FRK (A, B) and SDC1 locus (C, D), 1Mb range. doi:10.1371/journal.pgen.1004383.g002 May 2014 \| Volume 10 \| Issue 5 \| e1004383 Consequence of limited variant density and non-additive associations The y axis shows the median, 10% and 90% quantile of the distribution of PP4 values (which supports a shared common variant). doi:10.1371/journal.pgen.1004383.g003 Figure 3. Simulation analysis with a shared causal variant between two studies. The two datasets used are one eQTL (sample size 966 samples, 10% of the variance explained by the variant) and one biomarker (such as LDL). The variance explained by the biomarker is colour coded and the x-axis shows the sample size of the biomarker study. The y axis shows the median, 10% and 90% quantile of the distribution of PP4 values (which supports a shared common variant). doi:10.1371/journal.pgen.1004383.g003 Statistical power may also be affected by the mode of inheritance of the causal variant. To address this, we simulated cases under a recessive pattern of inheritance. Our results show that if the true model is recessive, but the eQTL signal is nonetheless analysed using the trend test, then we will often also successfully detect a colocalised signal (Figure S9). and Methods). We then selected only the subset of variants present in the Illumina 660K genotyping array. We simulated data under the assumption of a shared causal variant, with 4,000 individuals in the biomarker dataset. We then computed the PP4 statistic with and without restricting the SNP set to the Illumina 660K Chip SNPs (Figure 4). We also considered two different scenarios, with the causal SNP included/not included in the Illumina 660W panel (Figures S1 and S2 for more exhaustive simulations). and Methods). We then selected only the subset of variants present in the Illumina 660K genotyping array. We simulated data under the assumption of a shared causal variant, with 4,000 individuals in the biomarker dataset. We then computed the PP4 statistic with and without restricting the SNP set to the Illumina 660K Chip SNPs (Figure 4). We also considered two different scenarios, with the causal SNP included/not included in the Illumina 660W panel (Figures S1 and S2 for more exhaustive simulations). Consequence of limited variant density and non-additive associations our approach. We generated pairs of eQTL/biomarker datasets assuming a shared causal variant. We varied two parameters: the sample size of the biomarker dataset and the proportion of the biomarker variance explained by the shared genetic variant. We set the proportion of the eQTL variance explained by the shared variant to 10% and we used the original sample size of the liver eQTL dataset described herein [16]. Text S1 contains a description of the simulation procedure. Until recently the assumption that, for a given GWAS signal, the causal variant in that interval had been genotyped was unrealistic. However, the application of imputation techniques [17–19] can provide genotype information about the majority of common genetic variants. Therefore, in situations where a common variant drives the GWAS signal, it is now plausible that, in imputed datasets, genotype information about this variant is available. Nevertheless, limited imputation quality can invalidate this hypothesis. This prompted us to investigate the implication of not including the causal variant in the genotype panel. Results are shown in Figure 3. We find that given a sample size of 2,000 individuals for the biomarker dataset, the causal variant needs to explain close to 2% of the variance of the biomarker to provide reliable evidence in favour of a colocalised signal (lower 10th percentile for PP4 .80%). To address this question, we used Illumina MetaboChip data and imputed the genotyped regions using the Minimac software ([19] May 2014 \| Volume 10 \| Issue 5 \| e1004383 PLOS Genetics \| www.plosgenetics.org 4 Bayesian Test for Colocalisation Figure 3. Simulation analysis with a shared causal variant between two studies. The two datasets used are one eQTL (sample size 966 samples, 10% of the variance explained by the variant) and one biomarker (such as LDL). The variance explained by the biomarker is colour coded and the x-axis shows the sample size of the biomarker study. The y axis shows the median, 10% and 90% quantile of the distribution of PP4 values (which supports a shared common variant). doi:10.1371/journal.pgen.1004383.g003 Figure 3. Simulation analysis with a shared causal variant between two studies. The two datasets used are one eQTL (sample size 966 samples, 10% of the variance explained by the variant) and one biomarker (such as LDL). The variance explained by the biomarker is colour coded and the x-axis shows the sample size of the biomarker study. PLOS Genetics \| www.plosgenetics.org Comparison with existing colocalisation tests We compared the behaviour of our proposed test with that of proportional colocalisation testing [12,14] in the specific case of a biomarker dataset with 10,000 samples (Figure 5, and also Figures S3 and S4). Broadly, in the case of either a single common causal variant or two distinct causal variants, our proposed method could infer the simulated hypotheses correctly (PP4 or PP3 .0.9) with good confidence, and PP3 .0.9 slightly more often than the proportional testing p-value ,0.05. A key advantage in our Bayesian approach is the ability to distinguish evidence for colocalisation (i.e. high PP4) from a lack of power (i.e. high PP0, PP1 or PP2). In both of these cases (high PP4 or high PP0/PP1/ PP2), the use of the proportional approach leads to failure to reject the null even though the interpretation of these situations should differ. Our results show that when the causal variant is directly genotyped by the low density array, the use of imputed data is not essential (Figure 4A). However, in cases where the causal variant is not typed or imputed in the low density panel, the variance of PP4 is much higher (Figure 4B). In this situation, the resulting PP4 statistic tends to decrease even though considerable variability is observed. Inspection of simulation results in Figure 5 (bottom row for tagging SNP, leftmost graph for shared causal variant) shows that while PP4 tends to be lower than for its counterpart with complete genotype data (top row, leftmost graph), PP3 remains low. This indicates that more probability is given to PP0, PP1 and PP2, which can be interpreted as a loss of power rather than misleading inference in favour of distinct variants for both traits. May 2014 \| Volume 10 \| Issue 5 \| e1004383 PLOS Genetics \| www.plosgenetics.org 5 Bayesian Test for Colocalisation Figure 4. Simulation analysis with a shared causal variant between two studies. The two datasets used are one eQTL (sample size 966 samples) and one biomarker (sample size of 4,000 samples). The variance explained by the biomarker and the expression is the same and is colour coded. The x-axis shows the estimated PP4 for 1,000 simulations using data imputed from metaboChip Illumina array. The y-axis uses the same dataset restricted to variants present on the Illumina 660W genotyping array to assess the impact of a lower variant density. A. The causal variant is included in the Illumina 660W panel. Comparison with existing colocalisation tests B. The causal SNP not included in Illumina 660W panel. doi:10.1371/journal.pgen.1004383.g004 Figure 4. Simulation analysis with a shared causal variant between two studies. The two datasets used are one eQTL (sample size 966 samples) and one biomarker (sample size of 4,000 samples). The variance explained by the biomarker and the expression is the same and is colour coded. The x-axis shows the estimated PP4 for 1,000 simulations using data imputed from metaboChip Illumina array. The y-axis uses the same dataset restricted to variants present on the Illumina 660W genotyping array to assess the impact of a lower variant density. A. The causal variant is included in the Illumina 660W panel. B. The causal SNP not included in Illumina 660W panel. doi:10.1371/journal.pgen.1004383.g004 It has been proposed that gene expression may be subject to both global regulatory variation which acts across multiple tissues and secondary tissue specific regulators [21]. Neither approach covers this case explicitly in its construction, but it is instructive to examine their expected behaviour. The proportional approach tends to reject a null of colocalisation, suggesting that a single distinct causal variant can be sufficient to violate the null hypothesis of proportional regression coefficients. In contrast, the Bayesian approach tends to favour the shared variant in the cases covered by our simulations (median PP4 . median PP3), and either hypotheses H3 or H4 can potentially have strong support (PP4 .0.9 in close to 50% of simulations, and PP3 .0.9 in around 25% of simulations). Of course, the ultimate goal should be to extend these tests to cover multiple causal variants, but in the meantime, it can be useful to know that a high PP4 in our proposed Bayesian analysis indicates strong support for ‘‘at least one causal variant’’ and that rejection of the null of proportionality of regression coefficients indicates that the two traits do not share all causal variants, not that they cannot share one. independent associations that explain a similar proportion of the variance of the trait (Figure S8). The natural and statistically exact modification of our approach would compute, for each trait, Bayes factors for sets of SNPs rather than single SNPs (up to N SNPs jointly to accommodate for N distinct associations per trait). However, this approach has two drawbacks. Firstly, the interpretation of the resulting posterior probabilities is more challenging in situations where some but not all of the variants are shared across both traits. Dealing with several independent associations for the same trait We have so far assumed that each trait is associated with at most one causal variant per locus. However, it is not unusual to observe two or more independent associations at a locus for a trait of interest [22]. In the presence of multiple independent associations, the assumption of a single variant per trait prompts the algorithm to consider only the strongest of these distinct association signals. Hence, the presence of additional associations that explain a smaller fraction of the variance of the trait, for example additional and independently associated rare variants, have a negligible impact on our computations. Comparison with existing colocalisation tests More importantly, the typical approach consists of publishing single variant summary statistics, which would prevent the use of standard summary statistics, a key feature of our approach. Owing to the focus of our algorithm on the strongest association signal, an alternative approach to deal with multiple associations consists of using a stepwise regression strategy, which would then reveal the secondary association signals. Our colocalisation test can then be run on using the conditional p-values. We find this approach to be the most practical and illustrate below an application for a locus that contains several independent eQTL associations (Figure 6). In situations where only single SNP summary statistics are available, the approximate conditional meta-analysis framework proposed by Visscher et al. [23] can be used to obtain conditional p-values. Application to a meta-analysis of blood lipids combined with a liver expression dataset Each plot shows a different scenario, the total number of causal variants in a region is indicated by number of circles in the plot titles with causal variants affecting both traits, the eQTL trait only, or the biomarker trait only, indicated by full circles, top-shaded circles and bottom-shaded circles respectively. In the top row the causal variant is typed or imputed, whereas only tag variants are typed/imputed in the bottom row. For proportional testing (under the BMA approach), we show the proportion of simulations with posterior predictive p-value ,0.05 (black horizontal line) while for our Bayesian analysis we plot the proportion of simulations with the posterior probability (PP3 or PP4) of the indicated hypothesis .0.9. Error bars show 95% confidence intervals (estimated based on an average of 1,000 simulations per scenario). In all cases, for the eQTL sample size is 1,000; genetic variants explain a total of 10% of eQTL variance; for the biomarker trait, the sample size is 10,000. doi:10.1371/journal.pgen.1004383.g005 Figure 5. Summary of proportional and Bayesian colocalisation analysis of simulated data. Each plot shows a different scenario, the total number of causal variants in a region is indicated by number of circles in the plot titles with causal variants affecting both traits, the eQTL trait only, or the biomarker trait only, indicated by full circles, top-shaded circles and bottom-shaded circles respectively. In the top row the causal variant is typed or imputed, whereas only tag variants are typed/imputed in the bottom row. For proportional testing (under the BMA approach), we show the proportion of simulations with posterior predictive p-value ,0.05 (black horizontal line) while for our Bayesian analysis we plot the proportion of simulations with the posterior probability (PP3 or PP4) of the indicated hypothesis .0.9. Error bars show 95% confidence intervals (estimated based on an average of 1,000 simulations per scenario). In all cases, for the eQTL sample size is 1,000; genetic variants explain a total of 10% of eQTL variance; for the biomarker trait, the sample size is 10,000. doi:10.1371/journal.pgen.1004383.g005 with caution owing to the extensive polymorphism in the major histocompatibility complex region. found 38 SNP-to-gene eQTLs in liver (Supplementary Table 8 of [15]). Table S1 shows our results for these 38 previously reported colocalisations. A complete list of all our identified colocalisations (independently of previous reports) is provided in Tables S2, S3, S4, S5 (broken down by lipid traits). Application to a meta-analysis of blood lipids combined with a liver expression dataset Teslovich et al. [15] reported common variants associated with plasma concentrations of low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL) and triglyceride (TG) levels in more than 100,000 individuals of European ancestry. They then reported the correlations between the lead SNPs at the loci they found and the expression levels of transcripts in liver. For the lipid dataset we have access only to summary statistics. The liver expression dataset used in this analysis is the same as the one used in [15]. In Teslovich et al., regions are defined within 500 kilobases of the lead SNPs, and the threshold for significance is 5\|10{8. At this threshold, they To illustrate this situation, we simulated datasets with two causal variants: one colocalised eQTL/biomarker signal plus a secondary independent ‘‘eQTL only’’ signal (Figure S8). These simulations confirm that the PP4 statistic is only affected in the presence of two May 2014 \| Volume 10 \| Issue 5 \| e1004383 PLOS Genetics \| www.plosgenetics.org 6 Bayesian Test for Colocalisation Figure 5. Summary of proportional and Bayesian colocalisation analysis of simulated data. Each plot shows a different scenario, the total number of causal variants in a region is indicated by number of circles in the plot titles with causal variants affecting both traits, the eQTL trait only, or the biomarker trait only, indicated by full circles, top-shaded circles and bottom-shaded circles respectively. In the top row the causal variant is typed or imputed, whereas only tag variants are typed/imputed in the bottom row. For proportional testing (under the BMA approach), we show the proportion of simulations with posterior predictive p-value ,0.05 (black horizontal line) while for our Bayesian analysis we plot the proportion of simulations with the posterior probability (PP3 or PP4) of the indicated hypothesis .0.9. Error bars show 95% confidence intervals (estimated based on an average of 1,000 simulations per scenario). In all cases, for the eQTL sample size is 1,000; genetic variants explain a total of 10% of eQTL variance; for the biomarker trait, the sample size is 10,000. Fi 5 S f ti l d B i l li ti l i f i l t d d t E h l t h diff t i th t t l Figure 5. Summary of proportional and Bayesian colocalisation analysis of simulated data. Application to a meta-analysis of blood lipids combined with a liver expression dataset B: 2log10(p) association p-values for SYPL2 expression in 966 liver samples. C: 2log10(p) association p-values for SYPL2 expression conditional on the top eQTL associated SNP at this locus (rs2359653). doi:10.1371/journal.pgen.1004383.g006 Table 2 lists the 14 colocalised loci (15 genes) that were not reported by Teslovich et al. (or in Global Lipids Genetics Consortium [24] for the gene NYNRIN), but for which our method finds strong support for colocalisation (PP4 .75%). Figure S7 shows the LocusZoom plots for these colocalisation results. Eleven of these 15 genes are strong candidates for involvement in lipid metabolism and/or have been previously suggested as candidate genes: SDC1, TGOLN2, INHBB, UBXN2B, VLDLR, VIM, CYP26A1, OGFOD1, HP, HPR, PPARA. See Text S2 for a brief overview of the function of these genes. Four others genes have a less obvious link: CMTM6, C6orf106, CUX2, ENSG00000259359. Three previously reported genes (SYPL2, IFT172, TBKBP1) which, based on our re-analysis, do not colocalise with the lipid traits, have a nearby gene with a high probability of colocalisation (respectively, SORT1, GCKR, KPNB1). This suggests that these genes are more likely candidates in this region. To explore the possibility that secondary signals may colocalise, we applied the stepwise regression strategy described above to deal with several independent associations at a single locus. We performed colocalisation test using eQTL results conditional on the top eQTL associated variant. Two of the loci (SYPL2/LDL or TC, APOC4 and TG) showed evidence of colocalisation with expression after conditional analysis (Table 1). An example of this stepwise procedure for the gene SYPL2 and LDL is provided in Figure 6. We find that the top liver eQTL signal is clearly discordant with LDL association (Table 1 and Figure 6). However, conditioning on the top eQTL signal reveals a second independent association for SYPL2 expression in liver. This secondary SYPL2 eQTL colocalises with the LDL associ- ation (PP4 .90%, Figure 6). Application to a meta-analysis of blood lipids combined with a liver expression dataset Using the coloc web server for this analysis with a PP4 .75, it took 1 minute to complete chromosome 1 and approximately 7 minutes to analyse the entire imputed genome-wide data on a laptop. For only one locus (CEP250), we did not find a significant eQTL signal, pointing to potential differences in bioinformatics processing and/or imputation strategy. In such a situation, both PP3 and PP4 are low and PP0, PP1 and PP2 concentrate most of the posterior distribution. Three loci (TMEM50A, ANGPTL3, PERLD1/PGAP3) do not have enough evidence to strongly support either colocalisation or absence of colocalisation (Table S1) and these should remain marked as doubtful. The majority of our results are consistent with the findings of Teslovich et al., with 26 out of 38 loci having PP4 §50%. To assess the role of the prior, we varied the critical parameter p12, which codes for the prior probability that a variant is associated with both traits. Here we report the results using the p12~10{6. The complete list of results is provided in Table S1. One of these genes, ANGPTL3 is noteworthy. Examining this locus (Figure S6), it is clear that the pattern of association p-values is consistent between LDL and ANGPTL3 expression. However, the extent of LD is strong, with 98 strongly associated variants. In such a situation, there is uncertainty as to whether the data support a shared causal variant for both traits, or two distincts variants for eQTL/LDL. Because the data are consistent with both scenarios, the choice of prior becomes determinant. Accordingly, PP4 drops from 91% to 49% if one uses p12~10{6 instead of p12~10{5. Table 1 lists the previously reported lipid-eQTL for which we find strong support against the colocalisation hypothesis (PP3 . 75%). The LocusZoom association plots for each of these loci can be found in Figure S5. In addition to the loci listed in Table 1, we found strong evidence of distinct signals between HLA-DQ/ HLA-DR and TC (Table S1) but these results must be interpreted May 2014 \| Volume 10 \| Issue 5 \| e1004383 7 PLOS Genetics \| www.plosgenetics.org Bayesian Test for Colocalisation Figure 6. LDL association and eQTL association plots at the SYPL2 locus. The x-axis shows the physical position on the chromosome (Mb) A: -log10(p) association p-values for LDL. The p-values are from the Teslovich et al published meta-analysis of . 100,000 individuals. Web based resource We developed a web site designed for integration of GWAS results using only p-values and the sample size of the datasets (http://coloc.cs.ucl.ac.uk/coloc/). The website was developed using RWUI [25]. Results include a list of potentially causal genes with the associated PP4 with their respective plots and ABF, and can be viewed either interactively or returned by email. Researchers can request a genome-wide scan of results from a genetic association analysis, and obtain a list of genes with a high probability of mediating the GWAS signals in a particular tissue. The tool also allows visualisation of the signals within a genetic region of interest. The database and browser currently include the possibility of investigating colocalisation with liver [15] and brain [26,27] expression data, however the resource will soon be extended to include expression in different tissues. This method, as well as alternative approaches for colocalisation testing [12,14], are also available with additional input options in an R package, coloc, from the Comprehensive R Archive Network (http://cran.r- project.org/web/packages/coloc). Discussion Secondary signals are reported only when there is a secondary eQTL at a p value greater than 10{4 Colocalisation tests are computed using the expression data conditioned on the listed SNP Other genes in the same region as the gene listed that colocalise using our method are reported Our results show that to provide an accurate answer to the colocalisation problem, high-density genotyping and/or accurate use of imputation techniques are key. The quality of the imputation is another important parameter. Indeed, while the variance of the regression coefficient can be estimated solely on the basis of the minor allele frequency for typed SNPs and sample size (and the case control ratio in the case of a binary outcome) [17,28], this ignores the uncertainty due to imputation. Filtering out poorly imputed SNPs partially addresses this problem, with the drawback that it may exclude the causal variant(s). Hence, providing estimates of the variance of the MLE, together with the effect estimates, will result in greater accuracy. This additional option is available on the coloc package in R (http://cran.r- project.org/web/packages/coloc). We currently assume that each genetic variant is equally likely a priori to affect gene expression or trait. A straightforward addition to our methodology would consider location specific priors for each variant, which would depend for example on the distance to the gene of interest, or the presence of functional elements in this chromosome region [29]. Our computation of the BF also assumes that, under H4, the effect sizes of the shared variant on both traits are independent. This could be modified if, for example, one compares eQTLs across different tissue types, or the same trait in two different studies. [30] has proposed a framework to deal with correlated effect sizes, and these ideas could potentially be incorporated in our colocalisation test. Another related issue is the choice of prior probabilities for the various configurations. For the eQTL analysis, we used a 10{4 prior probability for a cis-eQTL. A more stringent threshold may be better suited for trans-eQTLs where the variants are further away from the gene under genetic control. We also used a prior probability of 10{4 for the lipid associations. Although our knowledge about this is still lacking, this estimate has been suggested in the literature in the context of GWAS [20,31,32]. We assigned a prior probability of 1\|10{6 for p12, which encodes the probability that a variant affects both traits. Discussion Figure 6. LDL association and eQTL association plots at the SYPL2 locus. The x-axis shows the physical position on the chromosome (Mb) A: -log10(p) association p-values for LDL. The We have developed a novel Bayesian statistical procedure to assess whether two association signals are colocalised. Our method PLOS Genetics \| www.plosgenetics.org PLOS Genetics \| www.plosgenetics.org May 2014 \| Volume 10 \| Issue 5 \| e1004383 8 Bayesian Test for Colocalisation reported as having a probable shared variant but not supported by our method based on PP3 (posterior probability for distinct signal values) .75%. Secondary signals are reported only when there is a er than 10{4. Colocalisation tests are computed using the expression data conditioned on the listed SNP. Other genes in the same region as the gene listed that colocalise using our method are reported. 3.t001 is best suited for associations detected by GWAS, which are likely to reflect common, imputable, variations with small effects, or a rare variants with large effect sizes. Our aim differs from a typical fine-mapping exercise in the sense that we are not interested in knowing which variant is likely to be causal but only whether a shared causal variant is plausible. The strength of this approach lies in its speed and analytical forms, combined with the fact that it can use single variant p-values when only these are available. Table 1. Loci previously reported to colocalise with liver eQTL, but not supported by our analysis. Chr Region Gene Trait Biom pval Biom SNP eQTL pval eQTL SNP Primary signal Secondary signal Other genes colocalising in region (PP4 .75%) PP3 (%) PP4 (%) PP4 (%) conditional SNP 1 109824678:110224737 SYPL2 LDL 9.7e–171 rs629301 7.1e–103 rs2359653 .99 ,1 99 rs2359653 SORT1/CELSR2/PSRC1/PSMA5 TC 8.0e–52 rs672569 7.1e–103 rs2359653 .99 ,1 99 rs2359653 SORT1/CELSR2/PSRC1/PSMA5 2 27467244:27867303 IFT172 TG 5.7e–133 rs1260326 1.7e–130 rs704791 .99 ,1 C2orf16/GCKR TC 7.3e–27 rs1260326 1.7e–130 rs704791 .99 ,1 C2orf16/GCKR 6 116062804:116462863 FRK LDL 2.9e–09 rs11153594 6.6e–15 rs195517 99 1 TC 1.7e–10 rs9488822 6.6e–15 rs195517 94 6 17 45589357:45989416 TBKBP1 LDL 1.1e–07 rs8072100 2.1e–21 rs9913503 87 9 KPNB1 TC 1.8e–07 rs8072100 2.1e–21 rs9913503 92 2 KPNB1 19 45248464:45648523 APOC4 TG 1.1e–30 rs439401 1.1e–299 19:45452692:A_AG .99 ,1 96 19:45452692:A_AG 20 34013995:34414054 CPNE1 TC 3.8e–10 rs2277862 7.3e–110 rs6060524 .99 ,1 Gene/eQTL associations previously reported as having a probable shared variant but not supported by our method based on PP3 (posterior probability for distinct signal values) .75%. Discussion It has been shown that SNPs associated with complex traits are more likely to be eQTLs compared to other SNPs chosen at random from GWAS platforms [33], and a higher weighting for these SNPs has been proposed when performing Bayesian association analyses [34,35]. Also, eQTLs have been shown to be enriched for disease-associated SNPs when a disease-relevant tissue is used [9,36]. Our sensitivity analysis for the p12 parameter showed broadly consistent results (Table S1). In cases where GWAS data are available for both traits, [10] show that it is possible to estimate these parameters from the data using a hierarchical model. This addition is a possible extension of our approach. The interpretation of the posterior probabilities requires caution. For example, a low PP4 may not indicate evidence against colocalisation in situations where PP3 is also low. It may simply be the result of limited power, which is evidenced by high values of PP0, PP1 and/or PP2. Moreover, a high PP4 is a measure of correlation, not causality. To illustrate this, one can consider the relatively common situation where a single variant appears to affect the expression of several genes in a chromosome region (as observed, for example, in the region surrounding the May 2014 \| Volume 10 \| Issue 5 \| e1004383 May 2014 \| Volume 10 \| Issue 5 \| e1004383 9 Bayesian Test for Colocalisation Table 2. Novel loci not previously reported to colocalise with liver eQTL, but colocalising based on our analysis. Imputation of genetic data Quality control filters were applied both before and after imputation. Before imputation, individuals with more than 10% missing genotypes were removed, and SNPs showing a missing rate greater than 10%, a deviation for HWE at a p-value less than 0.001 were dropped. After imputation, monomorphic SNPs were excluded from analyses. Importantly, GWAS signals can be explained by eQTLs only when the causal variant affects the phenotype by altering the amount of mRNA produced, but not when the phenotype is affected by changing the type of protein produced, although the former seems to be the most common [33]. Furthermore, since many diseases manifest their phenotype in certain tissues exclusively [2,21,37,38], colocalisation results will be dependent on the expression dataset used. In addition to identifying the causal genes, the identification of tissue specificity for the molecular effects underlying GWAS signals is a key outcome of our method. We anticipate that building a reference set of eQTL studies in multiple tissues will provide a useful check for every new GWAS dataset, pointing directly to potential candidate genes/ tissue types where these effects are mediated. To speed up the imputation process, the genome was broken into small chunks that were phased and imputed separately and then re-assembled. This was achieved using the ChunkChromo- some tool (http://genome.sph.umich.edu/wiki/ChunkxChromo some), and specifying chunks of 1000 SNPs, with an overlap window of 200 SNPs on each side, which improves accuracy near the edges during the phasing step. Each chunk was phased using the program MACH1 with the number of states set to 300 and the number of rounds of MCMC set to 20 for all chunks. Phased haplotypes were used as a basis for imputation of untyped SNPs using the software Minimac with 1000 Genomes European ancestry reference haplotypes (phase1 version 3, March 2012) to impute SNPs not genotyped on the Illumina array. Variants with a MAF less than 0.001 were also excluded post-imputation. The data was then collated in probability format that can be used by the R Package snpStats [39]. While this report focuses on finding shared signals between a biomarker dataset and a liver expression dataset, we plan to utilise summary results of multiple GWAS and eQTL studies, for a variety of cell types and traits. In fact, our method can utilise summary results from any association studies. Discussion Chr Region Gene Trait Biompval BiomSNP eQTLpval eQTLSNP PP3 PP4 Reference 2 20201795:20601854 SDC1 TC 1.23E-07 2:20368519 6.66E-09 2:20371380 17 82 [41] 2 85349026:85749085 TGOLN2 HDL 1.01E-07 2:85546192 2.83E-80 2:85553784 17 83 [42] 2 120908798:121308857 INHBB LDL 1.43E-06 2:121305771 4.88E-21 2:121306440 7 77 [43] 3 32322873:32722932 CMTM6 TC 4.66E-06 3:32533010 2.73E-07 3:32523287 8 77 6 34355095:34755154 C6orf106 TC 4.68E-11 6:34546560 4.48E-09 6:34616322 15 85 8 59158506:59558565 UBXN2B LDL 3.86E-09 8:59311697 3.46E-10 8:59331282 13 87 [44] TC 8.79E-13 8:59311697 3.46E-10 8:59331282 15 85 9 2454062:2854121 VLDLR LDL 8.05E-06 9:2640759 1.36E-07 9:2640759 1 91 [45] 10 17079389:17479448 VIM TC 7.22E-07 10:17259642 9.84E-09 10:17260290 5 93 [46] 10 94637063:95037122 CYP26A1 TG 2.38E-08 10:94839642 3.51E-06 10:94839724 3 95 [47] 12 111508189:111908248 CUX2 HDL 4.38E-06 12:111904371 2.81E-16 12:111884608 2 89 LDL 1.73E-09 12:111884608 2.81E-16 12:111884608 2 98 TC 2.36E-11 12:111904371 2.81E-16 12:111884608 2 98 15 96517293:96917352 ENSG00000259359 HDL 8.04E-06 15:96708291 5.50E-13 15:96708291 2 87 16 56310220:56710279 OGFOD1 TC 3.19E-06 16:56490549 3.36E-11 16:56493573 7 84 [48] 16 71894416:72310900 HP LDL 1.75E-22 16:72108093 2.15E-06 16:72108093 1 97 [49] TC 3.22E-24 16:72108093 2.15E-06 16:72108093 1 97 TG 5.66E-06 16:72108093 2.15E-06 16:72108093 2 75 HPR LDL 1.75E-22 16:72108093 4.18E-08 16:72108093 1 99 [50] TC 3.22E-24 16:72108093 4.18E-08 16:72108093 1 99 TG 5.66E-06 16:72108093 4.18E-08 16:72108093 2 89 22 46433083:46833138 PPARA TC 3.59E-06 22:46627603 5.96E-08 22:46632994 10 81 [51] Signals previously not reported as having a probable shared variant but supported by our method based on PP4 (posterior probability for a shared signal) .75% for colocalisation between the liver eQTL dataset and the Teslovic et al. meta-analysis of LDL, HDL, TG, TC, using the strict prior p12~10{6. For 11 genes with strong candidate status for lipid metabolism, we list a key reference that describes their function (see Text S2 for more details of gen functions). May 2014 \| Volume 10 \| Issue 5 \| e1004383 May 2014 \| Volume 10 \| Issue 5 \| e1004383 10 Bayesian Test for Colocalisation SORT1 gene). Several eQTLs will be colocalised, both between them and with the biomarker of interest. In this situation one would typically expect that a single gene is causally involved in the biomarker pathway but the colocalisation test with the biomarker will generate high PP4 values for all genes in the interval. high concordance between data generated using the same array platforms has been previously reported. Expression dataset We used in our analysis gene expression and genotype data from 966 human liver samples. The samples were collected post- mortem or during surgical resection from unrelated European- American subjects from two different non-overlapping studies, which have been described in [16]. The cohorts were both genotyped using Illumina 650Y BeadChip array, and 39,000 expression probes were profiled using Agilent human gene expression arrays. All of the expression data has been normalised as one unit even though they were part of different studies, since The regional association plots for the eQTL and Biomarker datasets were created using LocusZoom [40] (http://csg.sph. umich.edu/locuszoom/). eQTL analysis eQTL p-values, effect sizes, and standard errors were obtained by fitting a linear trend test regression between the expression of each gene and all variants 200 kilobases upstream and downstream from each probe. After filtering out the variants with MAF ,0.001, monomorphic SNPs, multi-allelic SNPs (as reported in 1000 Genomes or in the Ensembl database) and variants not sufficiently well imputed (Rsq ,0.3, as defined by minimac http://genome. sph.umich.edu/wiki/minimac) between both datasets, we applied our colocalisation procedure. We conducted conditional analysis on SNPs with p-values v10{4 for the expression associations, and repeated the colocalisation test using expression data conditioned on the most significant SNP. The aim of this analysis is to explore whether additional signals for expression other than the main one are shared with the biomarker signal. Imputation of genetic data Disease/disease, (cis or trans) eQTL/disease or disease/biomarkers comparisons are all of biological interest and use the same statistical framework. We expect that the fact that the test can be based on single SNP summary statistics will be key to overcome data sharing concerns, hence enabling a large scale implementation of this tool. The increasing availability of RNA-Seq eQTL studies will further increase the opportunity to detect isoform specific eQTLs and their relevance to disease studies. Owing to the increasing availability of GWAS datasets, the systematic application of this approach will potentially provide clues into the molecular mechanisms underlying GWAS signals and the aetiology of the disorders. Ethics statement This paper re-analyses previously published datasets. All samples and patient data were handled in accordance with the policies and procedures of the participating organisations. Biomarker dataset The biomarker p-values from the meta-analyses (with genomic control correction) were obtained from a publicly available re- pository (http://www.sph.umich.edu/csg/abecasis/public/lipids 2010/). Discussion Probe sequences were searched against the human reference genome GRCh37 from 1000 Genomes using BLASTN. Multiple probes mapping to one gene were kept in order to examine possible splicing. The probes were kept and annotated to a specific gene if they were entirely included in genes defined by Ensembl ID or by HGNC symbol using the package biomaRt in R [39]. After mapping and annotating the probes, we were left with 40,548 mapped probes covering 24,927 genes. We show that we can use conditional p-values to deal with multiple independent associations with the same trait at one locus. While we found this solution generally effective, Wallace [14] points out that this top SNP selection for the conditional analysis can create biases, although the bias is small in the case of large samples and/or strong effects. For difficult loci with multiple associations for both traits and available genotype data, it may be more appropriate to estimate Bayes factors for sets rather than single variants in order to obtain an exact answer. This extension would avoid the issue of SNP selection for the conditional analysis. Supporting Information The first ratio inside the sum in this equation is a Bayes Factor (BF) for each configuration, and the second ratio is the prior odds of a configuration compared with the baseline configuration S0. The BF can be computed for each variant from the p-value, or estimated regression coefficient ^b and variance of ^b, using Wakefield’s method. By summing over all configurations in Sh we are effectively comparing the support in the data for one alternative hypothesis versus the null hypothesis. An in-depth description of the method making use of the current assumptions can be found in Text S1. Figure S1 Simulation analysis with a shared causal variant between two studies, comparing results using imputed versus not imputed data where the causal SNP is included in both the cases. The two datasets used are one eQTL (sample size 966 samples) and one biomarker, and each plot shows different sample sizes for the biomarker dataset. The variance explained by the causal variant for both the traits is colour coded. The x- axis shows the estimated PP4 for 1,000 simulations using data imputed from metaboChip Illumina array (Methods). The y- axis uses the same dataset restricted to variants present on the Illumina 660W genotyping array to assess the impact of a lower variant density. The causal variant is included in the Illumina 660W panel. (TIF) Posterior Computation X S[Sh P(DjS)P(S) ð1Þ ð1Þ where P(S) is the prior probability of a configuration, P(DjS) is the probability of the observed data D given a configuration S, and the sum is over all configurations S which are consistent with a given hypothesis Hh, where h = (1,2,3,4). Thus, the probability of the data given a configuration is weighted by the prior probability of that configuration. where P(S) is the prior probability of a configuration, P(DjS) is the probability of the observed data D given a configuration S, and the sum is over all configurations S which are consistent with a given hypothesis Hh, where h = (1,2,3,4). Thus, the probability of the data given a configuration is weighted by the prior probability of that configuration. PP4 ~P(H4jD) ð2Þ The ratios in the numerator and denominator of equation 2 are: The ratios in the numerator and denominator of equation 2 are: To compute the ABF, we also needed to specify the standard deviation for the prior, and we set this to 0.20 for binary traits and 0.15 for quantitative traits (more details in Text S2). P(HhjD) P(H0jD) ~ X S[Sh P(DjS) P(DjS0) \| P(S) P(S0) ð3Þ ð3Þ Choice of priors Next, to avoid computing the proportionality constant in Equation 1, we can reformulate the posterior probability for each hypothesis by writing this quantity as a ratio. For example, the posterior probability under hypothesis 4, dividing each of these terms by the baseline P(H0jD), is: Prior probabilities are assigned at the SNP level and correspond to mutually exclusive events. We assigned a prior of 1\|10{4 for p1 and p2, the probability that a SNP is associated with either of the two traits. Since all SNPs are assumed to have the same prior probability of association, this prior can be interpreted as an estimate for the proportion of SNPs that we expect to be associated with the trait in question. We also assigned a prior probability of 1\|10{6 for p12, the probability that one SNP is associated with both traits. This probability can be better understood when it is re-expressed as the conditional probability of a SNP being associated with trait 2, given that it is associated with trait 1. So assigning a probability of 1\|10{6 means that 1 in 100 SNPs that are associated with trait 1 is also associated with the other. As a sensitivity analysis, we ran the comparison with Teslovich et al. using two other prior probabilities for p12, 2\|10{6 which means 1 in 50 SNPs that are associated with one trait is also associated with the other; and 10{5 which means 1 in 10 SNPs. PP4 ~P(H4jD) Posterior Computation We call a ‘‘configuration’’ one possible combination of pairs of binary vectors indicating whether the variant is associated with the May 2014 \| Volume 10 \| Issue 5 \| e1004383 PLOS Genetics \| www.plosgenetics.org 11 Bayesian Test for Colocalisation selected trait. We can group the configurations into five sets, S0, S1, S2, S3, S4, containing assignments of all SNPs Q to the functional role corresponding to the five hypothesis H0, H1, H2, H3, H4. We can compute the posterior probabilities given the data for each of these 5 hypothesis by summing over the relevant configurations: where Z~^b= ﬃﬃﬃﬃ V p is the usual Z statistic and the shrinkage factor r is the ratio of the variance of the prior and total variance (r~W=(VzW)). Assuming a normal distribution, the p-value of each SNP can be converted to standard one-tailed Z-score by using inverse normal cumulative distribution function. So for a SNP, all that it is needed are the p-values from a standard regression output, and ﬃﬃﬃﬃﬃﬃ W p , the standard deviation of the normal prior N(0,W) on b. The variance of the effect estimate, V, can be approximated using the MAF and sample size. However for imputed data it is preferable to use the variance outputted in standard regression analysis directly in the ABF equation. For the expression dataset used here, the variance and effect estimates from the regression analysis were used for computation of ABFs (see Text S1 for more details). where Z~^b= ﬃﬃﬃﬃ V p is the usual Z statistic and the shrinkage factor r is the ratio of the variance of the prior and total variance (r~W=(VzW)). Assuming a normal distribution, the p-value of each SNP can be converted to standard one-tailed Z-score by using inverse normal cumulative distribution function. So for a SNP, all that it is needed are the p-values from a standard regression output, and ﬃﬃﬃﬃﬃﬃ W p , the standard deviation of the normal prior N(0,W) on b. The variance of the effect estimate, V, can be approximated using the MAF and sample size. However for imputed data it is preferable to use the variance outputted in standard regression analysis directly in the ABF equation. For the expression dataset used here, the variance and effect estimates from the regression analysis were used for computation of ABFs (see Text S1 for more details). P(HhjD)! Bayes factor computation A Bayes Factor for each SNP and each trait 1 and 2 was computed using the Approximate Bayes Factor (ABF, [20]). Wakefield’s method yields a Bayes factor that measures relative support for a model in which the SNP is associated with the trait compared to the null model of no association. Figure S2 Simulation analysis with a shared causal variant between two studies, comparing results using imputed versus not imputed data where the causal SNP is not included in one of the datasets. The two datasets used are one eQTL (sample size 966 samples) and one biomarker, and each plot shows different sample sizes for the biomarker dataset. The variance explained by the causal variant for both the traits is colour coded. Column and row Figure S2 Simulation analysis with a shared causal variant between two studies, comparing results using imputed versus not imputed data where the causal SNP is not included in one of the datasets. The two datasets used are one eQTL (sample size 966 samples) and one biomarker, and each plot shows different sample sizes for the biomarker dataset. The variance explained by the causal variant for both the traits is colour coded. Column and row The equation used is the following: ABF~ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1{r p \|exp Z2 2 \|r ð4Þ ð4Þ May 2014 \| Volume 10 \| Issue 5 \| e1004383 PLOS Genetics \| www.plosgenetics.org PLOS Genetics \| www.plosgenetics.org May 2014 \| Volume 10 \| Issue 5 \| e1004383 12 Bayesian Test for Colocalisation headings are the same as in previous figure. The causal SNP is not included in Illumina 660W panel. behaviour of the statistic in the presence of an additional causal variant affecting the variance explained of the eQTL trait. In all scenarios, the first causal variant explains 10% of the variance of the eQTL trait. The second causal variant explains 1%, 5%, or 10% of the eQTL trait. We show the proportion of simulations with the posterior probability (PP3 or PP4) of the indicated hypothesis .0.9. Error bars show 95% confidence intervals (estimated based on an average of 1,000 simulations per scenario). In all cases, for the eQTL sample size is 1,000; for the biomarker trait, the sample size is 10,000. Figure S3 The relationship between PP4 and the posterior predictive p-value (on a -log10 scale) from proportional testing. Bayes factor computation Proportional testing uses the BMA approach, integrating over all possible two SNP models. Each row shows a different scenario, the total number of causal variants in a region is indicated by number of symbols in the plot titles with the type of causal variant indicated by the symbol: full circle - affects both traits; top only - affects one trait; bottom only- affects other trait. For proportional testing, the grey vertical line indicates the threshold ppp of 0.05. Each column shows the total proportion of trait variance for the biomarker explained by all variants in a region, with variance explained spread equally over all variants. In all cases, for the eQTL trait, n = 1,000, 10% of the variance explained by the variant; for the biomarker trait, n = 10,000. Figure S9 Simulation analysis with a recessive shared causal variant. The two datasets used are one eQTL (sample size 966 samples, 10% of the variance explained by the variant) and one biomarker (sample size 10,000). The variance explained by the biomarker is colour coded and the shape of the dots represent the different mode of inheritance. The simulation procedure and distribution of the statistic are the same as defined in previous figure. Figure S4 The relationship between PP4 and the posterior predictive p-value (on a -log10 scale) from proportional testing, using subset of SNPs which appear on the Illumina HumanOm- niExpress genotyping array. For the eQTL trait, n = 1,000, 10% of the variance explained by the variant; for the biomarker trait, n = 10,000, 1% or 2% of the variance explained by the variant. Column and row headings are the same as in previous figure. (TIF) Figure S4 The relationship between PP4 and the posterior predictive p-value (on a -log10 scale) from proportional testing, using subset of SNPs which appear on the Illumina HumanOm- niExpress genotyping array. For the eQTL trait, n = 1,000, 10% of the variance explained by the variant; for the biomarker trait, n = 10,000, 1% or 2% of the variance explained by the variant. Column and row headings are the same as in previous figure. (TIF) Table S1 Results using reported loci that colocalise with liver eQTL. Published results of loci correlating with both liver expression and one of the four lipid traits (Teslovich et al. (TIF) Figure S5 Regional Manhattan plots corresponding to loci listed in Table 1 of main text. The plots focus on a specific region of the genome with a range of 400 kilobases around the expression probe of the gene specified below each plot. The top plots use the - log10(p-value) from the published meta-analysis with one of the four lipid biomarkers; the bottom plots show the -log10(p-value) computed by fitting a generalised linear model with expression as dependent variable and SNP genotypes as independent variable. Each dot represents one SNP, imputed or directly typed. The value on the top of each plot shows the PP4 from the colocalisation test between the two top SNP of the expression and biomarker associations. Figure S5 Regional Manhattan plots corresponding to loci listed in Table 1 of main text. The plots focus on a specific region of the genome with a range of 400 kilobases around the expression probe of the gene specified below each plot. The top plots use the - log10(p-value) from the published meta-analysis with one of the four lipid biomarkers; the bottom plots show the -log10(p-value) computed by fitting a generalised linear model with expression as dependent variable and SNP genotypes as independent variable. Each dot represents one SNP, imputed or directly typed. The value on the top of each plot shows the PP4 from the colocalisation test between the two top SNP of the expression and biomarker associations. Table S2 eQTL/LDL colocalisation. Positive (PP4 .75%) eQTL/LDL colocalisation results between the liver eQTL dataset and the Teslovich meta-analysis using the most stringent prior for the probability that one SNP is associated with both traits, p12~10{6. The column Signal includes genes that are part of overlapping regions and that colocalise at PP4 .75%; the column Region represents the genomic coordinates for the start and stop of the signal; in the column Tesl, ‘‘Y’’ indicates that this signal with any of the genes included has been reported to be an intermediate for any of the four lipid biomarker associations by Teslovich et al. ; the columns Biom pval and eQTL pval report the lowest p-values found for LDL association and for the expression association respectively, with the corresponding SNP name (Biom SNP and eQTL SNP); the column Best Causal reports the SNP within the region with the highest posterior probability to be the true causal variant. (TIF) The probabilities have been rounded to 1 significant figure. (PDF) Table S2 eQTL/LDL colocalisation. Positive (PP4 .75%) eQTL/LDL colocalisation results between the liver eQTL dataset and the Teslovich meta-analysis using the most stringent prior for the probability that one SNP is associated with both traits, p12~10{6. The column Signal includes genes that are part of overlapping regions and that colocalise at PP4 .75%; the column Region represents the genomic coordinates for the start and stop of the signal; in the column Tesl, ‘‘Y’’ indicates that this signal with any of the genes included has been reported to be an intermediate for any of the four lipid biomarker associations by Teslovich et al. ; the columns Biom pval and eQTL pval report the lowest p-values found for LDL association and for the expression association respectively, with the corresponding SNP name (Biom SNP and eQTL SNP); the column Best Causal reports the SNP within the region with the highest posterior probability to be the true causal variant. The probabilities have been rounded to 1 significant figure. 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Trynka G, Hunt KA, Bockett NA, Romanos J, Mistry V, et al. (2011) Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease. Nature genetics 43: 1193–1201. 43. Johnson MP, Brennecke SP, East CE, Go¨ring HH, Kent Jr JW, et al. (PDF) Figure S6 LDL association and eQTL association plots at the ANGPTL3 locus. The x-axis shows the physical position on the chromosome (Mb) A: 2log10(p) association p-values for LDL. The p-values are from the Teslovich et al published meta-analysis of .100,000 individuals. B: 2log10(p) associ- ation p-values for ANGPTL3 expression in 966 liver samples. Figure S7 Regional Manhattan plots corresponding to loci listed in Table 2 of main text. Row and column headers defined as in previous figure. The genomic range may be greater than *400 kilobases to improve visualisation of the signal. (PDF) Table S3 eQTL/HDL colocalisation. Positive (PP4 .75%) eQTL/HDL colocalisation results between the liver eQTL dataset and the Teslovich meta-analysis. Column and row headings are the same as in previous figure. (PDF) Figure S8 Simulation analysis with multiple shared causal variants. The first plot represents cases with only one causal variant in a region, while the following plots illustrate the May 2014 \| Volume 10 \| Issue 5 \| e1004383 PLOS Genetics \| www.plosgenetics.org 13 Bayesian Test for Colocalisation Table S4 eQTL/TG colocalisation. Positive (PP4 .75%) eQTL/HDL colocalisation results between the liver eQTL dataset and the Teslovich meta-analysis. Column and row headings are the same as in previous figure. Table S4 eQTL/TG colocalisation. Positive (PP4 .75%) eQTL/HDL colocalisation results between the liver eQTL dataset and the Teslovich meta-analysis. Column and row headings are the same as in previous figure. (PDF) Text S1 Supplementary materials. Expanded methods, deriva- tions and analyses. (PDF) Text S2 Overview of gene function of new colocalisation results associated with blood lipid levels and liver expression. (PDF) Table S5 eQTL/TC colocalisation. Positive (PP4 .75%) eQTL/HDL colocalisation results between the liver eQTL dataset Table S5 eQTL/TC colocalisation. Positive (PP4 .75%) eQTL/HDL colocalisation results between the liver eQTL dataset and the Teslovich meta-analysis. Column and row headings are the same as in previous figure. (PDF) References 1. Feero WG, Guttmacher AE, Manolio TA (2010) Genomewide association studies and assessment of the risk of disease. New England Journal of Medicine 363: 166–176. Available: http://sysbio.mrc-bsu.cam.ac.uk/Rwui/tutorial/Technical_Report. pdf. Accessed 22 April 2014. 1. Feero WG, Guttmacher AE, Manolio TA (2010) Genomewide association studies and assessment of the risk of disease. New England Journal of Medicine 363: 166–176. 2. 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Wen X, Stephens M (2011) Bayesian methods for genetic association analysis with heterogeneous subgroups: from meta-analyses to gene-environment interactions. arXiv preprint arXiv:1111.1210. 7. He X, Fuller CK, Song Y, Meng Q, Zhang B, et al. (2013) Sherlock: Detecting gene-disease associations by matching patterns of expression qtl and gwas. The American Journal of Human Genetics 92: 667–680. 31. Author Contributions and the Teslovich meta-analysis. Column and row headings are the same as in previous figure. and the Teslovich meta-analysis. Column and row headings are the same as in previous figure. Conceived and designed the experiments: CG DV CW VP LF. Performed the experiments: CG CW VP. Analyzed the data: CG CW. Contributed reagents/materials/analysis tools: EES LF ADH. Wrote the paper: CG CW DV VP ADH. the experiments: CG CW VP. Analyzed the data: CG CW. Contributed reagents/materials/analysis tools: EES LF ADH. Wrote the paper: CG CW DV VP ADH. reagents/materials/analysis tools: EES LF ADH. Wrote the paper: CG CW DV VP ADH. y p g 48. Saito K, Adachi N, Koyama H, Matsushita M (2010) Ogfod1, a member of the 2-oxoglutarate and iron dependent dioxygenase family, functions in ischemic signaling. FEBS letters 584: 3340–3347. 50. Nielsen MJ, Petersen SV, Jacobsen C, Oxvig C, Rees D, et al. (2006) Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein. Blood 108: 2846–2849. 49. Wassell J, et al. (1999) Haptoglobin: function and polymorphism. Clinical laboratory 46: 547–552. 46. Sarria A, Panini S, Evans R (1992) A functional role for vimentin intermediate filaments in the metabolism of lipoprotein-derived cholesterol in human sw-13 cells. Journal of Biological Chemistry 267: 19455–19463. J g y 47. Hafner M, Rezen T, Rozman D (2011) Regulation of hepatic cytochromes p450 by lipids and cholesterol. Current drug metabolism 12: 173–185. p 51. Staels B, Maes M, Zambon A (2008) Fibrates and future ppara agonists in the treatment of cardiovascular disease. Nature Clinical Practice Cardiovascular Medicine 5: 542–553. References (2012) Genome-wide association scan identifies a risk locus for preeclampsia on 2q14, near the inhibin, beta b gene. PloS ONE 7: e33666. 44. Wang CW, Lee SC (2012) The ubiquitin-like (ubx)-domain-containing protein ubx2/ubxd8 regulates lipid droplet homeostasis. Journal of Cell Science 125: 2930–2939. 23. Yang J, Ferreira T, Morris AP, Medland SE, Madden PA, et al. (2012) Conditional and joint multiple-snp analysis of gwas summary statistics identifies additional variants influencing complex traits. Nature genetics 44: 369–375. 45. Nasarre L, Juan-Babot O, Gastelurrutia P, Llucia-Valldeperas A, Badimon L, et al. (2012) Low density lipoprotein receptor–related protein 1 is upregulated in epicardial fat from type 2 diabetes mellitus patients and correlates with glucose and triglyceride plasma levels. Acta Diabetol 51: 23–30. 24. Consortium GLG, et al. (2013) Discovery and refinement of loci associated with lipid levels. Nat Genet 45: 1274–1283. 25. Newton R, Wernisch L (2007) Rwui: A web application to create user friendly web interfaces for r scripts. New Functions for Multivariate Analysis: 32. May 2014 \| Volume 10 \| Issue 5 \| e1004383 May 2014 \| Volume 10 \| Issue 5 \| e1004383 PLOS Genetics \| www.plosgenetics.org 14 46. Sarria A, Panini S, Evans R (1992) A functional role for vimentin intermediate filaments in the metabolism of lipoprotein-derived cholesterol in human sw-13 cells. Journal of Biological Chemistry 267: 19455–19463. 47. Hafner M, Rezen T, Rozman D (2011) Regulation of hepatic cytochromes p450 by lipids and cholesterol. Current drug metabolism 12: 173–185. 48. Saito K, Adachi N, Koyama H, Matsushita M (2010) Ogfod1, a member of the 2-oxoglutarate and iron dependent dioxygenase family, functions in ischemic signaling. FEBS letters 584: 3340–3347. 49. Wassell J, et al. (1999) Haptoglobin: function and polymorphism. Clinical laboratory 46: 547–552. 50. Nielsen MJ, Petersen SV, Jacobsen C, Oxvig C, Rees D, et al. (2006) Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein. Blood 108: 2846–2849. 51. Staels B, Maes M, Zambon A (2008) Fibrates and future ppara agonists in the treatment of cardiovascular disease. Nature Clinical Practice Cardiovascular Medicine 5: 542–553. Bayesian Test for Colocalisation May 2014 \| Volume 10 \| Issue 5 \| e1004383 PLOS Genetics \| www.plosgenetics.org PLOS Genetics \| www.plosgenetics.org 15
https://openalex.org/W2097174875	https://dash.harvard.edu/bitstream/1/23993629/1/4627330.pdf	English	null	UXT, a novel MDMX-binding protein, promotes glycolysis by mitigating p53-mediated restriction of NF-κB activity	Oncotarget	2,015	cc-by	6,091	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Citation Citation Qi, Min, Suthakar Ganapathy, Weiqi Zeng, Jianglin Zhang, John B. Little, and Zhi-Min Yuan. 2015. “UXT, a novel MDMX-binding protein, promotes glycolysis by mitigating p53-mediated restriction of NF-κB activity.” Oncotarget 6 (19): 17584-17593. Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:23993629 ABSTRACT The importance of stress-induced p53 activation has been extensively investigated and well established. How the basal activity of p53 prevents carcinogenesis, however, remains incompletely understood. We report the identification of a novel p53 inhibitor, UXT, which binds to MDMX and suppresses the basal activity of p53. Interestingly, human TCGA database indicates that the UXT gene is frequently amplified in human sarcoma where p53 mutation is rare. We thus used sarcoma as a model to show that UXT acts as an oncogene promoting cell proliferation in vitro and tumor progression in vivo. A screening of 10 major cellular pathways uncovered that UXT-mediated p53 inhibition results in an activation of NF-κB, leading to induction of glycolysis. While elevated glycolytic metabolism provides growth advantage it also renders UXT expressing sarcoma cells heightened sensitivity to glycolysis inhibition. Altogether, our data demonstrate a crucial role for the basal activity of p53 in restriction of NF-κB. By impeding such an activity of p53, UXT unleashes the oncogenic activity of NF-κB resulting in induction of glycolysis fueling carcinogenesis. Published: May 07, 2015 Published: May 07, 2015 Accepted: April 24, 2015 Min Qi1,, Suthakar Ganapathy2,, Weiqi Zeng3, Jianglin Zhang3, John B. Little2, Zhi-Min Yuan2 1Department of Plastic Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, PR China 2Department of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA 3Department of Dermatology, Xiangya Hospital, Central South University, Changsha, Hunan, PR China *These authors have contributed equally to this work Keywords: p53, NF-κB, MDMX, UXT Received: February 17, 2015 Accepted: April 24, 2015 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Oncotarget, Vol. 6, No. 19 www.impactjournals.com/oncotarget/ INTRODUCTION activity of this inhibitory complex can be oncogenic. Indeed, both MDM2 and MDMX are well-established oncogenes because they are frequently overexpressed in many human cancers where the p53 gene is rarely mutated. Although MDM2 and MDMX are potent p53 inhibitors, DNA damage-induced p53 activation in MDM2 and MDMX overexpressing cells remains most intact (6), implicating a critical importance of downregulating the basal p53 activity to the oncogenic function of MDM2 and MDMX. Interestingly, as one of the most studied proteins, the knowledge regarding to the importance of the basal steady state level of p53 to its role as a tumor suppressor remains limited. In response to diverse stress signals, p53 is readily activated inducing a host of cellular effects including senescence and cell death, amongst others (1). Although the p53-mediated stress responses are important to prevent tumor development, its activity has to be tightly regulated to avoid unnecessary pathological consequences (1). Elaborate mechanisms exist to maintain an appropriate homeostasis of p53 activity. While many players are reported to play a role, abundant evidence indicates that MDM2 and MDMX are the principal regulators keeping p53 at a low basal level (2). MDM2 and MDMX function primarily together as a complex to inhibit p53 by directly blocking the transactivation domain of p53 and by targeting p53 for ubiquitination/degradation (3, 4). Many stress signals impinge on the MDM2/MDMX complex and disable their interaction with p53 resulting in p53 activation (5). Whereas insufficient MDM2/MDMX activity can cause p53-dependent cytotoxicity, excess The transcription factor NF-κB regulates various genes important for the immune response, cell proliferation, and cell survival in response to various cellular stresses such as cytokine activation, oxidative stress, and infectious diseases (7, 8). During the immune response, cells consume large amounts of glucose and primarily use aerobic glycolysis to rapidly produce www.impactjournals.com/oncotarget Oncotarget 17584 first confirming the interaction between UXT and MDMX. 293 cells co-expressing UXT with MDMX or MDM2 were subjected to a reciprocal IP-Western analysis. The result indicated a clear binding between UXT and MDMX (Figure 1B). The IP-Western data were further corroborated by immunostaining, which revealed an overt colocalization of the 2 proteins (Figure 1C), indicative of an association between UXT and MDMX. The association between UXT and MDMX was also observed with endogenously expressed protein (Figure 1D). Protein-protein interaction often affects the protein stability of each binding partner. INTRODUCTION We tested this possibility by coexpression of MDMX with an increasing amount of UXT, which indeed resulted in a dose-dependent increase in MDMX protein abundance (Figure 1E). The data altogether indicated that UXT binds to and stabilizes MDMX. enough energy to meet the bioenergetic demands of cellular proliferation and survival (9). The NF-κB pathway has been shown to stimulate aerobic glycolysis by upregulating the expression of GLUT-3 and HIF1α (10, 11), mediating the metabolic response critical for cell function and survival. The NF-κB pathway is often deregulated in human cancer leading to an excess activity that is largely oncogenic (7). Dynamic crosstalk between the p53 and NF-κB pathways has been widely observed. Although this crosstalk is highly context dependent and has been shown to function either as antagonistic or cooperative between the two pathways, p53 and NF-κB are considered to overall function against one another; pro-death versus pro- survival (12, 13). In the context of cellular metabolism, p53 favors oxidative phosphorylation whereas NF-κB stimulates glycolysis.i In this report, we describe the identification of UXT as a novel MDMX-interacting protein. UXT binds to and stabilizes MDMX resulting in reduction of the basal steady state p53 activity. Of interest is the finding that NF-κB activity was selectively upregulated upon p53 inhibition by UXT. Using a combination of metabolomic and genetic approaches, we demonstrated that NF-κB activation induced glycolytic metabolism fueling cancer cell growth and survival. In support of TCGA data showing that the UXT gene is frequently amplified in human cancers, our study uncovers a novel mechanism of oncogenic role of UXT in suppression of basal p53 activity causing NF-κB- mediated induction of glycolysis and carcinogenesis. UXT negatively regulates p53 activity enhancing cell proliferation Given that MDMX is a negative regulator of p53, UXT-mediated stabilization of MDMX would predict this protein as an inhibitor of p53. We employed methods of over- and under-expression of UXT to test this possibility. siRNA-mediated knockdown of UXT was associated with a considerable increase in p53 abundance (Figure 2A). The use of multiple siRNA sequences of UXT indicated that p53 activation was specifically caused by UXT knockdown. In contrast to the effect of UXT depletion, UXT overexpression was associated with a decrease in p53 level, which seemed to be a result of increased turnover because inhibition of protein degradation by MG132, a proteasome inhibitor, recovered the p53 level (Figure 2B). To examine whether the increased p53 protein abundance correlated with its transcriptional activity. We determined the expression of p21, a target gene of p53 and found a marked induction of p21 expression upon UXT knockdown (Figure 2C). This increase of p21 in UXT- depleted cells was p53-dependent as siRNA-mediated p53 knockdown or E6-mediated p53 degradation completely abolished this effect of UXT (Supplementary Figure 2). Identification of UXT as a novel MDMX binding protein As the principal negative regulators of p53, MDMX and MDM2 form a MDM heterocomplex that works together in p53 control. The MDM complex inhibits p53 either as an E3 ligase targeting p53 for ubiquitination/ degradation or directly masking the transactivation domain of p53. Given the importance of the complex in p53 control, any protein that interacts with either MDM2 or MDMX may affect their ability to inhibit p53. We tested this hypothesis by conducting a yeast 2-hybrid screening to search for MDMX-binding partners. We chose MDMX over MDM2 because the later associates with DNA, which led to numerous false positives (not shown). The screening identified an understudied protein, UXT (Figure 1A). Of interest is that mining of TCGA database revealed UXT as a gene frequently overexpressed in human sarcoma (Supplementary Figure 1) where p53 inactivation is usually caused by a heightened activity of its inhibitors because the p53 gene mutation is rare (1). We hypothesized that UXT might contribute to negative regulation of p53 via its binding to MDMX. We tested this hypothesis by We went on to investigate the biological consequence to UXT-mediated p53 regulation by monitoring its effect on cell proliferation, considering the well recognized growth inhibitory activity of p53. In line with the induction of p21 in response to the expression of UXT siRNA, reduced expression of UXT was associated with a marked decrease of the rate of cell proliferation. Conversely, stable overexpression of UXT led to an increase in cell proliferation. Two separate batches of UXT expressing cell lines exhibited a higher rate of proliferation than control cells (Figure 2D). We went on to carry out mouse xenograft experiments to test the in vivo oncogenic activity of UXT. Consistent with the data of cell proliferation, U2OS_UXT cells developed into significantly larger tumors than U2OS_ www.impactjournals.com/oncotarget Oncotarget 17585 Figure 1: UXT binds to and stabilizes MDMX. A. A result of a yeast 2-hybrid assay indicating a positive clone pull out by a bait of MDMX but not MDM2. B. 293 cells were coexpressed with plasmids encoding a Flag-UXT and MDMX. The cells were harvest 24 h after transfection and subjected to reciprocal immunoprecipitations with an anti-Flag (M2 Sigma) or MDMX (Bethyl lab), with lgG included as a control. The immunoprecipitates were analyzed by Western analysis using anti-Flag or anti-MDMX antibodies. C. U2OS cells were transfected with either GFP-UXT or RFP-MDM alone to together. Identification of UXT as a novel MDMX binding protein The cells were fixed 24 h later. Images were taken under a fluorescent microscope and representative images including overlay ones are shown. D. U2OS cells were subjected to anti-MDMX immunoprecipitation with LgG included as a control. The immunoprecipitates were immunoblotted with either anti-MDMX or anti-UXT. E. 293 cells were transfected with a control vector or 1 μg MDMX with 1 or 3 μg of Flag-UXT. The cells were harvested 24 h later and analyzed by Western blot with the indicated antibodies. Figure 1: UXT binds to and stabilizes MDMX. A. A result of a yeast 2-hybrid assay indicating a positive clone pull out by a bait of MDMX but not MDM2. B. 293 cells were coexpressed with plasmids encoding a Flag-UXT and MDMX. The cells were harvest 24 h after transfection and subjected to reciprocal immunoprecipitations with an anti-Flag (M2 Sigma) or MDMX (Bethyl lab), with lgG included as a control. The immunoprecipitates were analyzed by Western analysis using anti-Flag or anti-MDMX antibodies. C. U2OS cells were transfected with either GFP-UXT or RFP-MDM alone to together. The cells were fixed 24 h later. Images were taken under a fluorescent microscope and representative images including overlay ones are shown. D. U2OS cells were subjected to anti-MDMX immunoprecipitation with LgG included as a control. The immunoprecipitates were immunoblotted with either anti-MDMX or anti-UXT. E. 293 cells were transfected with a control vector or 1 μg MDMX with 1 or 3 μg of Flag-UXT. The cells were harvested 24 h later and analyzed by Western blot with the indicated antibodies. UXT-mediated p53 suppression was associated with NF-κB activation EV cells (Supplementary. Figure 3 & Figure 2E). Immunohistochemistry analysis of Ki67, a commonly used proliferation marker, indicated a higher rate of cell proliferation in U2OS_UXT cells than U2OS_EV cells (Figure 2F). Taken together, our data revealed that UXT inhibits p53 activity via binding to MDMX promoting cancer cell proliferation in vitro and tumor development in vivo. Such an oncogenic role of UXT is consistent with its overexpression in human cancer. Having shown UXT-induced growth stimulation, we explored the underlying mechanism by performing a screening of the 10 cellular signaling pathways via a luciferase-based assay. Interestingly, UXT overexpression was associated with a selective increase in the activity of the NF-κB pathway, whereas the AKT, MAP and other www.impactjournals.com/oncotarget Oncotarget 17586 2: UXT functions as an oncogene via suppression of p53 activity. A. U2OS cells were transfected with either control siRNACtrl) or 2 independent siRNA sequences targeting different region of the UXT gene. The cells were harvested 48 h later yzed for protein expression by Immunoblot using the indicated antibodies. B. UOS cells stably expressing an empty vector (EV) were established. The U2OS_UXT cells were treated with or without MG132 (10 μM) for 6 h prior to harvesting for Western using the indicated antibodies. C. U2OS cells were transfected as in A and harvest 50 h later for Western analysis using the d antibodies. D. U2OS cells transfected with each of siRNA(1, 2 or 3) or 2 different patches of UXT overexpressing U2OS cells or #2) were measured the rate of cell proliferation using a proliferation assay kit (Gibco). E. Balb C nude mice (4–6 weeks) bcutaneously implanted with 2 X 106 U2OS EV or U2OS UXT cells. The tumors were harvested 8 weeks later and the volumes Figure 2: UXT functions as an oncogene via suppression of p53 activity. A. U2OS cells were transfected with either control siRNA (siRNACtrl) or 2 independent siRNA sequences targeting different region of the UXT gene. The cells were harvested 48 h later and analyzed for protein expression by Immunoblot using the indicated antibodies. B. UOS cells stably expressing an empty vector (EV) or UXT were established. The U2OS_UXT cells were treated with or without MG132 (10 μM) for 6 h prior to harvesting for Western analysis using the indicated antibodies. C. U2OS cells were transfected as in A and harvest 50 h later for Western analysis using the indicated antibodies. D. UXT-mediated p53 suppression was associated with NF-κB activation U2OS cells transfected with each of siRNA(1, 2 or 3) or 2 different patches of UXT overexpressing U2OS cells (UXT#1 or #2) were measured the rate of cell proliferation using a proliferation assay kit (Gibco). E. Balb C nude mice (4–6 weeks) were subcutaneously implanted with 2 X 106 U2OS_EV or U2OS_UXT cells. The tumors were harvested 8 weeks later and the volumes were measured. The numbers are averages from a group of 5 mice ± standard deviation. F. The harvested tumor tissues were subjected to immunohistochemistry staining with an anti-Ki67 antibody. The representative images were shown. cell growth promoting pathways were not significantly altered. We further verified the effect of UXT on the NF-κB activity by examining p65 subcellular distribution and p65 phosphorylation, 2 commonly used markers of NF-κB activity (7). Indeed, a clear increase in nuclear localization of p65 was evident when U2OS_UXT expressing cells were compared with the U2OS_EV cells (Figure 3B). In support of increased NF-κB activity, Immunoblot with a phosphor-specific antibody revealed that p65 was markedly phosphorylated in U2OS_UXT but not U2OS_EV cells (Figure 3C). Little change of Akt phosphorylation was detected, consistent with the result obtained from pathway screening indicating a selective stimulation of NF-κB activity in U2OS_UXT cells. We went on to explore the mechanism by which NF-κB is activated by UXT expression. Available information indicates an antagonistic interaction between p53 and NF-κB (11). Given that merely loss of p53 function www.impactjournals.com/oncotarget Oncotarget 17587 Figure 3: UXT-induced p53 inhibition caused activation of NF-B. A. U2OS_UXT cells were subjected to Cignal Finder™ 10-Pathway Reporter Arrays (Qiagen). The results are from 3 independent experiments performed in triplicates. The numbers are means ± SD. B. U2OS_EV or U2OS_UXT cells were analyzed by immunostaining with an anti-p65 (Cell Signaling, MA) co-stained with DAPI. Representative images are shown. C. Cell lysates prepared from U2OS_EV or U2OS_UXT cells were analyzed by Immunoblot with the ndicated antibodies. D. U2OS_EV or U2OS_UXT cells that were transfected with either siRNACtrl or siRNAp53 were treated with or without Nutlin-3a (10 μM 3 h). The cells were harvested and subjected Western analysis using the indicated antibodies. Figure 3: UXT-induced p53 inhibition caused activation of NF-B. A. U2OS_UXT cells were subjected to Cignal Finder™ 10-Pathway Reporter Arrays (Qiagen). The results are from 3 independent experiments performed in triplicates. The numbers are means ± SD. B. UXT-mediated p53 suppression was associated with NF-κB activation U2OS_EV or U2OS_UXT cells were analyzed by immunostaining with an anti-p65 (Cell Signaling, MA) co-stained with DAPI. Representative images are shown. C. Cell lysates prepared from U2OS_EV or U2OS_UXT cells were analyzed by Immunoblot with the indicated antibodies. D. U2OS_EV or U2OS_UXT cells that were transfected with either siRNACtrl or siRNAp53 were treated with or without Nutlin-3a (10 μM 3 h). The cells were harvested and subjected Western analysis using the indicated antibodies. p53 inhibition, consistent with an antagonistic interaction between p53 and NF-κB (12, 13). can lead NF-κB activation (12), we asked whether the compromised function of p53 in U2OS_UXT cells could be responsible for the elevated NF-κB activity. We tested this question using a small molecular p53 activator Nutlin-3a, which disassociates the binding of MDM2 to p53 (5). Treatment of U2OS_UXT cells with Nutlin- 3a resulted in robust p53 activation (Figure 3D, lane 2), as expected. Remarkably, the increased p53 activity was associated with diminished NF-κB activity (Figure 3D, lane 4). This effect appeared to be p53-dependent because the NF-κB activity was not reduced by nulin-3a when the expression of p53 was depleted by siRNA (Figure 3D, lane 6). Together, the results indicated an elevated NF-κB activity in U2OS_UXT cells because of UXT-mediated DISCUSSION In this study, we identified UXT as a novel MDMX interacting protein and the binding of UXT resulted in stabilization of the MDMX protein and consequent inhibition of p53 activity, uncovering a previously unappreciated oncogenic activity of UXT. The biological significance of UXT-mediated p53 suppression is underscored by the finding that the UXT gene is frequently amplified in several types of human cancer according to the TCGA database. Among them, human sarcoma is of particular interest because the p53 gene in this type of cancer is usually not mutated (1), implicating UXT-dependent p53 inhibition as a novel mechanism of p53 inactivation in human sarcoma. Using a combination of loss-of-function and gain-of-function approach, we provided multiple lines of in vitro and in vivo evidence demonstrating an oncogenic role of UXT in sarcoma progression. NF-κB promotes glycolytic metabolism Increased NF-κB activity stimulates glycolysis by inducing the expression of glucose transporter and glycolytic genes, directly or indirectly (9, 10). We asked whether the yellowish media color was caused by increased rate of glycolysis. Measurement of the extracellular concentration of glucose and lactate (the major product of glycolysis) revealed a significant decrease in glucose whereas an increase in the lactate level in UXT expressing cells relative to the control cells (Figure 4A – 4B), consistent with an increased rate of glycolysis. To substantiate this finding we performed glucose flux analysis and indeed detected a significant increase in the rate of glycolytic metabolism, as evidenced by overt accumulation of a number of glycolytic metabolites (Figure 4C). To corroborate the metabolomics data, we measured the level of transcripts of metabolic genes. Interestingly, the expression of multiple glycolytic genes including GLUT-1 & 3, HK-2 & 3, LDHA and ENO was significantly increased in UXT expressing cells when compared with the control cells (Figure 4D). The induction of so many glycolytic genes was unexpected since GLUT- 3 is the only known target gene of NF-κB (7). Previous studies showed that NF-κB could transcriptionally induce the expression of HIF-1α, a master transcription factor for multiple glycolytic genes (9, 10). We thus measured the expression of HIF-1α and detected a considerable induction of this transcription factor at both the transcript and protein levels in UXT expressing cells (Figure 4E). In line with the NF-κB -mediated effect, the increase in HIF-1α was almost completed abrogated when a specific inhibitor of NF-κB (Capaisacin) was added to the cell culture. The result together showed that the increased activity of NF-κB in UXT overexpressing cancer cells induced glycolysis via upregulation of HIF-1α. cells, U2OS_UXT cells exhibited increased resistance to irradiation-induced killing (Figure 5B). Interestingly, inhibition of glycolysis with 2DG considerably sensitized U2OS_UXT cells to irradiation-induced cell death whereas U2OS_EV cells were much less affected (Figure 5B). To substantiate the observation, we measured irradiation- induced γH2AX, a surrogate marker of DNA damage. In line with the cell survival results, U2OS_UXT cells were more resistant to irradiation as evident by significantly fewer numbers of γH2AX-positive cells than U2OS_ EV cells induced by 4Gy-irradiation (Supplementary Figure 5). However, treatment with 2DG was associated with a much greater increase in γH2AX-positive cells in U2OS_UXT cells than U2OS_EV cells (Figure 5C), indicating that inhibition of glycolysis preferentially sensitized U2OS_UXT cells over U2OS_EV cells. NF-κB promotes glycolytic metabolism The transcription factor NF-κB plays an important role in multiple cellular processes, including immune signaling, inflammation, proliferation and survival (7). In cancer cells, NF-κB activation is frequently associated with increased proliferation and survival. Interestingly, we observed that the media color of U2OS_UXT culture turned yellowish much more rapidly than that of U2OS_EV cells (Supplementary. Figure 4), suggesting increased production of acidic metabolites. In line with www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 17588 this observation was the finding that NF-κB plays a key role in regulation of cellular metabolism. Increased NF-κB activity stimulates glycolysis by inducing the expression of glucose transporter and glycolytic genes, directly or indirectly (9, 10). We asked whether the yellowish media color was caused by increased rate of glycolysis. Measurement of the extracellular concentration of glucose and lactate (the major product of glycolysis) revealed a significant decrease in glucose whereas an increase in the lactate level in UXT expressing cells relative to the control cells (Figure 4A – 4B), consistent with an increased rate of glycolysis. To substantiate this finding we performed glucose flux analysis and indeed detected a significant increase in the rate of glycolytic metabolism, as evidenced by overt accumulation of a number of glycolytic metabolites (Figure 4C). To corroborate the metabolomics data, we measured the level of transcripts of metabolic genes. Interestingly, the expression of multiple glycolytic genes including GLUT-1 & 3, HK-2 & 3, LDHA and ENO was significantly increased in UXT expressing cells when compared with the control cells (Figure 4D). The induction of so many glycolytic genes was unexpected since GLUT- 3 is the only known target gene of NF-κB (7). Previous studies showed that NF-κB could transcriptionally induce the expression of HIF-1α, a master transcription factor for multiple glycolytic genes (9, 10). We thus measured the expression of HIF-1α and detected a considerable induction of this transcription factor at both the transcript and protein levels in UXT expressing cells (Figure 4E). In line with the NF-κB -mediated effect, the increase in HIF-1α was almost completed abrogated when a specific inhibitor of NF-κB (Capaisacin) was added to the cell culture. The result together showed that the increased activity of NF-κB in UXT overexpressing cancer cells induced glycolysis via upregulation of HIF-1α. this observation was the finding that NF-κB plays a key role in regulation of cellular metabolism. NF-κB promotes glycolytic metabolism The results altogether support an important role of glycolysis in supporting survival of UXT expressing cells. NF-κB-induced glycolysis contributes to UXT- enhanced cell proliferation and survival Recent studies showed that a diminished p53 function caused a constitutive activation of NF-κB (12). The basal p53 level in UXT expressing cells was considerably down regulated because of increased degradation (Figure 2A), likely caused by the elevated level of MDMX, which forms a heterocomplex with MDM2 targeting p53 for ubiquitination/degradation (3, 4). In line with the antagonistic relationship between p53 and NF-κB, the diminished p53 activity in UXT-expressing cancer cells was associated with an elevated NF-κB activity, which was initially uncovered by the pathway screening and subsequently validated by examining p65 nuclear distribution and phosphorylation. We provided evidence demonstrating that UXT-mediated p53 inhibition was indeed responsible for the increased activity of NF-κB. It has been reported that p53 could also antagonize m-TOR activity (14). We, however, detected little change of AKT activity in UXT cells, indicative of a selective activation of NF-κB. Together with the published work (12), our Given the importance of elevated glycolysis, or “Warburg effect” in cancer cell proliferation and survival, the UXT-mediated induction of glycolysis prompted us to examine its role in cell proliferation. We tested whether UXT-dependent cell proliferation could be affected by inhibition of glycolysis via the use of a specific inhibitor of glycolysis, 2-deoxyglucose. The result indeed revealed an important role of glycolysis in UXT-induced cell proliferation. Addition of 2DG was associated a dose-dependent suppression of cell proliferation and importantly, the inhibitory activity of 2DG was much stronger in UXT overexpressing cells than in the control cells (Figure 5A), consistent with a notion that UXT stimulates cell proliferation via at least in part promoting glycolytic metabolism. We next investigated the contribution of glycolysis to cancer cell sensitivity to cancer therapy. When compared with U2OS_EV www.impactjournals.com/oncotarget Oncotarget 17589 4: UXT induces glycolytic metabolism by NF-κB-dependent upregulation of HIF-1α. Cell culture me from an equal number cells. The concentrations of lactate A. and glucose B. were determined by a colorimetric kit (Bio bers are means ± SD from 3 experiments performed in triplicates. C. U2OS_EV or U2OS_UXT cells were incuba -glucose for 15 min prior to metabolite extraction and targeted LC-MS/MS analysis. The ratio of 13C labeled to unlabe es was measured by LC-MS/MS are presented as mean ± SD over 3 independent samples. Metabolites with P values < comparisons are shown. D. U2OS_EV or U2OS_UXT cells were harvested and mRNA isolated. Transcripts of the i re determined by quantitative RT-PCR. NF-κB-induced glycolysis contributes to UXT- enhanced cell proliferation and survival Data are means ± SD from 3 experiments performed in triplicates. E. U2O XT cells were treated with or without Capsaicin (100 μM 3 h). qRT-PCR or Western determined the expression levels o s and protein respectively Figure 4: UXT induces glycolytic metabolism by NF-κB-dependent upregulation of HIF-1α. Cell culture media were collected from an equal number cells. The concentrations of lactate A. and glucose B. were determined by a colorimetric kit (BioVision). The numbers are means ± SD from 3 experiments performed in triplicates. C. U2OS_EV or U2OS_UXT cells were incubated with [1, 2-13C]-glucose for 15 min prior to metabolite extraction and targeted LC-MS/MS analysis. The ratio of 13C labeled to unlabeled (12C) metabolites was measured by LC-MS/MS are presented as mean ± SD over 3 independent samples. Metabolites with P values < 0.05 for pair-wise comparisons are shown. D. U2OS_EV or U2OS_UXT cells were harvested and mRNA isolated. Transcripts of the indicated genes were determined by quantitative RT-PCR. Data are means ± SD from 3 experiments performed in triplicates. E. U2OS_EV or U2OS_UXT cells were treated with or without Capsaicin (100 μM 3 h). qRT-PCR or Western determined the expression levels of HIF-1α transcripts and protein, respectively. Oncotarget 17590 XT cells depend on glycolysis for cell survival. A. U2OS_EV or U2OS_UXT cell ndicated concentration for 48 h. Cell viability was determined and the data are means ± S es. B. U2OS_EV or U2OS_UXT cells were pre-treated with vehicle (Veh) or 2-DG (2mM ated dose. Cell numbers were determined 72 h after irradiation. Data are means ± SD from 3 S_EV or U2OS_UXT cells were pre-treated with vehicle (Veh) or 2-DG (2mM) for 6 h follo were fixed 1 h after irradiation and stained with γH2AX. The numbers of γH2AX-positive c are means ± SD from 3 experiments performed in triplicates. Figure 5: U2OS_UXT cells depend on glycolysis for cell survival. A. U2OS_EV or U2OS_UXT cells were treated with or without 2-DG at the indicated concentration for 48 h. Cell viability was determined and the data are means ± SD from 3 experiments performed in triplicates. B. U2OS_EV or U2OS_UXT cells were pre-treated with vehicle (Veh) or 2-DG (2mM) for 6 h followed by irradiation at the indicated dose. Cell numbers were determined 72 h after irradiation. Data are means ± SD from 3 experiments performed in triplicates. C. NF-κB-induced glycolysis contributes to UXT- enhanced cell proliferation and survival U2OS_EV or U2OS_UXT cells were pre-treated with vehicle (Veh) or 2-DG (2mM) for 6 h followed by irradiation at a dose of 4Gy. The cells were fixed 1 h after irradiation and stained with γH2AX. The numbers of γH2AX-positive cells were counted from 5 random fields. Data are means ± SD from 3 experiments performed in triplicates. Metabolic flux analysis Flux studies were performed according to a published protocol (18). U2OS-EV or U2OS-UXT cells were washed thoroughly with glucose free medium and Cell culture Human osteosarcoma U2OS cells and 293 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in DMEM (Corning cellgro). All the media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin- Immunoblotting Cells were washed with ice cold phosphate buffered saline and then lysed using lysis buffer (20 mM Tris-HCl, pH 7.5, 5mM EDTA, 150 mM NaCl, 1% Nonidet P-40, I mM Na3VO4, 1mM PMSF, 0.1% protease inhibitor) for 45 minutes on ice. The cell extracts were centrifuged at 12, 000 RPM for 15 minutes at 4°C. Equal amounts of cell lysates were separated by SDS-PAGE. The separated proteins were transferred to nitrocellulose membrane and immunoblotted using indicated antibodies. Antibodies were from BD Biosciences (HIF-1α), Cell Signaling Technology (P-p65, P-Akt), abcam (p53, p21, p65, Akt), Sigma-Aldrich (Flag, β-actin). The protein bands were developed using HRP-conjugated secondary antibodies with ECL-chemiluminescent reagent. www.impactjournals.com/oncotarget Oncotarget 17591 gentomycin (Invitrogen) in a humidified atmosphere at 37°C and 5% O2, 5% CO2. data underscore a crucial role of the basal p53 activity in curbing oncogenic NF-κB activity. P53 inactivation would not only result in impediment of its canonical tumor suppressive activities such as cell cycle arrest, senescence or apoptosis, but also unleashing its restraint on NF-κB leading to the activation this oncogenic transcription factor. Reagents All chemicals were purchased from Sigma-Aldrich (St Louis, MO). SiRNA transfection SiRNAs were purchased from Sigma-Aldrich (St Louis, MO). Multiple sequences of SiRNAs were used for p53 and UXT. SiRNAs were reversely transfected using Lipofectamin RNAiMAX (Invitrogen) as per the manufacturer’s instruction. SiGL2 was used as the negative control. Consistent with the importance of increased glycolytic metabolism or the Warburg effect in cancer cell proliferation and survival, we demonstrated that UXT expressing cells depended on glycolysis for proliferation and survival, displaying heightened susceptibility to glycolytic inhibition. The increased sensitivity by inhibition of glycolysis could be due to reduced DNA damage repair since we recently showed that inhibition of glycolysis in cancer cells resulted in chromatin compaction, impeding the assess of DNA damage repair proteins (13). Given the challenge of targeting NF-κB due to its diverse effects, the elevated glycolysis may serve as a better therapeutic target. RNA isolation and quantitative RT-PCR Total RNA was isolated using Trizol Reagent (Invitrogen) according to the manufacturer’s instruction. 1 μg of total RNA was used to make cDNA (iScript cDNA synthesis kit, Bio-Rad), following the manufacturer’s instruction, which was subsequently used for the amplification by quantitative RT-PCR using Applied Biosystem StepOnePlus in the presence of SYBR Green JumpStart (Sigma-Aldrich, St Louis, MO). Ribosomal RNA 18S was used as endogenous normalization control. The n-fold change in mRNAs expression was determined on the basis of ∆∆Ct value. All assays were performed in triplicate. In summary, we showed that UXT binds to and stabilizes MDMX resulting in p53 inhibition. Using sarcoma as a model, we demonstrated that UXT-mediated inhibition of the basal p53 activity resulted in NF-κB activation, which promoted cell proliferation and survival by stimulating glycolysis. Our study offers molecular mechanistic data supporting an oncogenic role of UXT revealed by human TCGA database. The increased sensitivity of UXT expressing cancer cells to inhibition of glycolysis may carry important therapeutic implication. Immunofluorescence For immunofluorescence, U2OS cells were fixed, permeabilized and blocked followed by the incubation with primary antibodies overnight. The slides were then incubated with, DAPI, rabbit Alexa Flour 488, mouse Alexa Flour 594. Nikon TE2000 microscope and NIS elements software were used for imaging. NF-κB promotes cancer cell proliferation and survival by controlling the expression of a large number of genes, which affect diverse cellular processes (7). We provide compelling evidence linking NF-κB activity to tumor metabolism, or increased aerobic glycolysis observed in UXT-overexpressing cancer cells. Apart from directly upregulating GLUT-3 expression, we showed that NF-κB also induced transcriptionally HIF-1α, a master transcription factor that regulates almost every genes of the glycolytic pathway. Indeed, UXT-expressing cells exhibited increased expression of multiple glycolytic genes, offering genetic evidence for the elevated glycolysis. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 17592 (R01CA085679, RO1CA167814 and RO1CA183074 for ZMY). (R01CA085679, RO1CA167814 and RO1CA183074 for ZMY). incubated the cells with medium containing 10mM 1:1 mixture of D-[1, 2-13C]-glucose and unlabeled D-glucose for 15 min. Metabolites were extracted on dry ice with 80% methanol. The metabolites were dried under nitrogen and re-suspended in 20 μL of water for liquid chromatography- mass spectrometry (LC-MS) analysis. cells per ml of aliqout × Viable cells 1 % 2 = cells per ml of aliqout Total number of cells per ml of aliquot × 100 3. Huang H, Yan Z, Liao X, Li Y, Yang J, Zuo Y, Wang Z, Kawai H, Shadfan S, Ganapathy S, & ZM Yuan. The MDM2/MDMX complex is required for control of p53 activity in vivo. Proc Natl Acad Sci U S A. 2011; 19; 108:12001–6. An annexin V apoptosis kit (Biovision, #K101–100) was used for the FACS-based assay as per manufacturer’s instructions 4. Pant V, Xiong S, Iwakuma T, Quintás-Cardama A, Lozano G. Heterodimerization of Mdm2 and Mdm4 is crit- ical for regulating p53 activity during embryogenesis but dispensable for p53 and Mdm2 stability. Proc Natl Acad Sci U S A. 2011; 19; 108:11995–2000. Animal experiment All the procedures on animals were conducted in accordance with the guidelines for the Institutional Animal Care and Use Committee (IACUC) at Harvard T.H. Chan School of Public Health. Mice used in this study were housed under pathogen-free conditions and maintained in 12-h light/12-h dark cycle, with food and water supplied ad libitum. U2OS cells (3X106 cells mixed with Matrigel, Bedton Dickinson, Bedford, MA) cells in a final volume of 100 μL were injected into the flank region of 4–6 weeks old Balb/cnu/ nu (Harlan laboratories) mice. When tumors reached 0.1 cm, mice were randomized into different groups for treatments. 5. Li Q, Lozano G. Molecular pathways: targeting Mdm2 and Mdm4 in cancer therapy. Clin Cancer Res. 2013; 1; 19:34–41. 6. Christophorou M.A., et al. The pathological response to DNA damage does not contribute to p53-mediated tumor suppression. Nature. 2006; 443:214–7. 7. Ben-Neriah, Y., M. Karin. Inflammation meets cancer, with NF-kappa B as the matchmaker. Nature Immunology. 2011; 12:715–723. 8. Wang, T., C. Marquardt, and J. Foker. Aerobic Glycolysis during Lymphocyte-Proliferation. Nature. 1976; 261:702–705. Histological analysis Tissues were fixed with 10% formalin, embedded in paraffin and sectioned. Hematoxylin and eosin staining were performed according to standard procedure. 9. Rois J, et al. NF-κB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1α. Nature. 2008; 453:807–811. 10. Van Uden P, et al. Regulation of hypoxia-inducible factor-1α by NF-κB. Biochem. J., 2008; 412:477–484. Statistical analysis In-vitro experiments were repeated at least three times. Two-way ANOVA was used for the statistical calculation. Mann-Whitney U-test was used for comparisons between different groups. 11. Tergaonkar V., N.D. Perkins, p53 and NF-kappaB crosstalk: IKKalpha tips the balance. Mol Cell. 2007; 26:158–9. 12. Kawauchi K, Araki K, Tobiume K. Tanaka N. 2008. p53 regu- lates glucose metabolism through an IKK-NF-kB pathway and inhibits cell transformation. Nat Cell Biol. 2008; 10:611–618. All authors declare no conflict of interest. All authors declare no conflict of interest. ACKNOWLEDGMENTS 13. Liu XS, Little JB, Yuan ZM. 2015. Glycolytic metabo- lism influences global chromatin structure. Oncotarget. 28:4214–25. We are grateful to current and former members of the Yuan lab for experimental support, advice and helpful discussions. This work was supported in part by the Morningside Foundation, the Zhu Fund and a grant from the National Natural Science Foundation of China (81301986 for Min Qi), grants from NIH/NCI 14. Demidenko ZN, Korotchkina LG, Gudkov AV, Blagosklonny MV. Paradoxical suppression of cellular senescene by p53. PNAS 2010. 107:9660–4. www.impactjournals.com/oncotarget REFERENCES Cell viability was assessed using the trypan-blue exclusion assay. The percentages of viable cells were counted as follows: 1. Kruse, J.P., W. Gu,. Modes of p53 regulation. Cell. 2009; 137:609–22. 1. Kruse, J.P., W. Gu,. Modes of p53 regulation. Cell. 2009; 137:609–22. Total number of viable Total number of viable Total number of viable 2. Ashcroft, M, K.H. Vousden, Regulation of p53 stability Oncogene. 1999; 18:7637–43. www.impactjournals.com/oncotarget Oncotarget 17593
https://openalex.org/W2171482734	https://europepmc.org/articles/pmc2853485?pdf=render	English	null	A 2cM genome-wide scan of European Holstein cattle affected by classical BSE	BMC genomic data	2,010	cc-by	9,092	© 2010 Murdoch 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. A 2cM genome-wide scan of European Holstein cattle affected by classical BSE Brenda M Murdoch1, Michael L Clawson2, William W Laegreid3, Paul Stothard1, Matthew Settles4, Stephanie McKay1,5, Aparna Prasad1, Zhiquan Wang1, Stephen S Moore1, John L Williams6 Correspondence: stephen.moore@ales.ualberta.ca 1Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada Abstract Background: Classical bovine spongiform encephalopathy (BSE) is an acquired prion disease that is invariably fatal in cattle and has been implicated as a significant human health risk. Polymorphisms that alter the prion protein of sheep or humans have been associated with variations in transmissible spongiform encephalopathy susceptibility or resistance. In contrast, there is no strong evidence that non-synonymous mutations in the bovine prion gene (PRNP) are associated with classical BSE disease susceptibility. However, two bovine PRNP insertion/deletion polymorphisms, one within the promoter region and the other in intron 1, have been associated with susceptibility to classical BSE. These associations do not explain the full extent of BSE susceptibility, and loci outside of PRNP appear to be associated with disease incidence in some cattle populations. To test for associations with BSE susceptibility, we conducted a genome wide scan using a panel of 3,072 single nucleotide polymorphism (SNP) markers on 814 animals representing cases and control Holstein cattle from the United Kingdom BSE epidemic. Results: Two sets of BSE affected Holstein cattle were analyzed in this study, one set with known family relationships and the second set of paired cases with controls. The family set comprises half-sibling progeny from six sires. The progeny from four of these sires had previously been scanned with microsatellite markers. The results obtained from the current analysis of the family set yielded both some supporting and new results compared with those obtained in the earlier study. The results revealed 27 SNPs representing 18 chromosomes associated with incidence of BSE disease. These results confirm a region previously reported on chromosome 20, and identify additional regions on chromosomes 2, 14, 16, 21 and 28. This study did not identify a significant association near the PRNP in the family sample set. The only association found in the PRNP region was in the case-control sample set and this was not significant after multiple test correction. The genome scan of the case-control animals did not identify any associations that passed a stringent genome-wide significance threshold. Conclusions: Several regions of the genome are statistically associated with the incidence of classical BSE in European Holstein cattle. Further investigation of loci on chromosomes 2, 14, 16, 20, 21 and 28 will be required to uncover any biological significance underlying these marker associations. Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Open Access Background which the secondary structure consists mainly of alpha- helices. The disease-associated or misfolded form, PrPRes, has a substantial increase in the beta-pleated sheets con- tent and reduction of the alpha-helices in comparison to the native form [2]. This altered confirmation is asso- ciated with an increased resistance to digestion with pro- teinase K [3]. Furthermore, the presence of PrPRes behaves like a seed that promotes the conversion of further native PrPc to PrPRes via a mechanism that is to date not completely understood [4]. Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that have been identified in a number of mammalian species (humans, cattle, sheep, mice, etc.) [1]. One of the common characteristics of these diseases is the accumulation of abnormally folded prion protein within the central nervous system. The prion protein is a glycosyl-phosphatidylinositol (GPI) anchored protein that has a native form (PrPc) for * Correspondence: stephen.moore@ales.ualberta.ca 1Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada Specific PRNP alleles of non-synonymous polymorph- isms in humans and sheep are associated with acquired Page 2 of 10 Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 TSE susceptibility [5-8]. This is not the case in cattle, although the deletion alleles of a 23 base pair insertion/ deletion (InDel) polymorphism in the bovine PRNP promoter region and of a 12 base pair InDel within intron 1 have been associated with incidence of classical bovine spongiform encephalopathy (BSE) [9-11]. Both of these polymorphisms are contained in a block of high linkage disequilibrium (LD) within PRNP that appears conserved in many cattle populations, and is entirely outside of the coding region [12]. Thus, the full extent of PRNP association with classical BSE is currently not known. Previous genetic studies of BSE cattle have iden- tified putative loci other than PRNP (located on chro- mosome 13 at 47.2 Mb) that are associated with incidence of disease [13,14]. These studies carried out low density whole-genome scans with microsatellite markers approximately every 20 cM in female European Holstein cattle which contracted BSE and unaffected half-sib controls. Family based association testing for BSE incidence Family based association testing for BSE incidence The related sample set (N = 481) was comprised of six paternal half-sib sire families of which 4 were scanned previously with microsatellites [13,14]. Samples were not available from either the sires or the dams; therefore, the sib-TDT analysis method [17] was used. Case-control association testing for BSE incidence Case control association testing for BSE incidence The case-control samples were comprised of 149 BSE case and 184 control animals. The control samples include a least one animal collected from the same farm as each of the BSE cases as well as the controls for, and 15 BSE negative animals. The genotyping data on these animals was analyzed for an association with disease sta- tus using the case-control allelic test within the PLINK software [18]. This analysis (320 animal across 2,872 SNPs after quality control) revealed 20 SNPs with a p < 0.01, 14 SNPs with a p < 0.005 and 6 SNPs with a p < 0.001. In order to determine the number of these SNPs that may have occurred by chance, an empirical p- value for each single SNP and across all SNPs (genome wide) was calculated using the max (T) permutation pro- cedure with 10,000 permutations. Following correction for the false discovery rate no significant associations at p ≤0.05 genome-wide significance were identified in this data set. This was consistent with the use of Bonferroni multi-test correction on this data set, where again none of the SNPs achieved significance of p ≤0.05. To assess significance a threshold was set at p ≤1.7 × 10-5 (Bonfer- roni calculation). Twenty SNPs were identified with p < 0.01 (Table 2), where four SNPs had a p ≤5 × 10-4 and one SNP had a p ≤1 × 10-4. Thus a single SNP had a suggestive association with BSE incidence on chromo- some 14 (p = 7.25 × 10-5). SNPs which did not quite reach this threshold but had a p ≤5 × 10-4 were found on chromosomes 4, 10, 14, and 15. The number of loci on each chromosome, the identity of these specific SNPs and their corresponding p-values are reported in Table 2. Background Addition- ally, PLINK software [18] was used to establish an empirical p value and determine significance. The results for this sib-TDT analysis (412 animals across 2,827 SNPs after quality control) identified 46 SNPs that passed the Bonferroni correction with a p < 0.05 and 27 SNPs that passed the Bonferroni correction with a p < 0.01 (Table 1). In addition, a 10,000,000 permutation test was performed on this data using the PLINK “max T” to establish genome wide empirical p values. The genome wide 10,000,000 permutation identified 31 SNPs with an empirical permutation of p < 0.01. This group of SNPs included the 27 SNPs which passed the Bonfer- roni at p < 0.01 plus four additional SNPs. Many of these SNPs are located in very close proximity [16] to one another and are potentially in LD in the population. The objective of this study was to test loci throughout the bovine genome for an association with classical BSE using markers at 2 cM resolution. This resolution is approximately a ten-fold improvement over previous genome scans of BSE samples. Two animal sample sets were used, allowing for two analysis approaches: a case- control association study and family based sib-transmis- sion disequilibrium test (sib-TDT) study [13,14]. In both sample sets the cattle used were female European Hol- stein cattle which contracted BSE in the late 1980’s and early 1990’s (family analysis) and in the mid-1990’s (case-control analysis) and were identified on commer- cial farms where the most likely source of disease was through consumption of contaminated feed during the United Kingdom’s BSE epidemic. Although the United Kingdom imposed a ruminant feed ban of meat and bone meal in June 1988, it wasn’t until August 1996 that a total ban on bovine meat and bone meal was implemented [15]. Results All other locations denoted by # were either determined from bovine sequence version 2.0, 4.1 or the Maryland sequence assembly. Permuted p values reported here are from a 10,000,000 genome-wide SNP permutation. Locations were determined by blast to bovine sequence version 4.0. All other locations denoted by # were either determined fr 4.1 or the Maryland sequence assembly. Permuted p values reported here are from a 10,000,000 genome-wide SNP permutatio This data set was also subjected to a best fit model test where the standard allelic, trend, dominance, recessive and genotypic association tests were performed, and the test with the lowest p value was reported. All of the SNPs identified above were also identified in the best fit model as either allelic or trend, however additional SNPs with recessive, dominant and genotype associations were also identified. In the best fit model there were a total of fourteen SNPs with a recessive mode of action, seven dominant SNPs and eight genotypic SNPs with a p < 0.01 (see Additional file 1). Using the same thresholds as described above, (i.e. significant with a p ≤10-5 and sug- gestive with a p ≤10-4), one SNP on chromosome 14 had a suggestive association with BSE incidence. present study, clinically healthy animals were used as controls, however, these animals may have been incu- bating disease or may not have ingested enough infec- tious agent to become symptomatic, or alternatively, they may in fact have been resistant to disease. There- fore, the analysis was performed with the realization that phenotypic noise in the controls will have reduced the power to detect associations. Another consideration is that classical BSE is a complex trait which may be more consistent with interactive and possibly subtle effects of multiple contributing loci. Therefore, multiple testing corrections applied to results such as these may be overly prone to type II errors (i.e. discarding real associations). Consequently, it is important to examine the results for supporting evidence of associations between disease and genetic loci, as discussed below. present study, clinically healthy animals were used as controls, however, these animals may have been incu- bating disease or may not have ingested enough infec- tious agent to become symptomatic, or alternatively, they may in fact have been resistant to disease. There- fore, the analysis was performed with the realization that phenotypic noise in the controls will have reduced the power to detect associations. Results Another consideration is that classical BSE is a complex trait which may be more consistent with interactive and possibly subtle effects of multiple contributing loci. Therefore, multiple testing corrections applied to results such as these may be overly prone to type II errors (i.e. discarding real associations). Consequently, it is important to examine the results for supporting evidence of associations between disease and genetic loci, as discussed below. Results The panel of SNP markers used in this study consisted of 3,072 SNPs dispersed across the genome at an approximate average interval of 2 cM [16]. Of these, an average 2,853 passed quality control measures and had an average genotyping success rate of 0.99. The samples used in this study came from two sets of ani- mals with different relatedness that allowed for the use of several statistical analyses to test the association between SNP genotypes with classical BSE incidence. By using both offspring from six sires (sib-TDT) and (case-control) Holstein animals, this study examined within-breed and within-family SNP association with disease incidence. Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Page 3 of 10 Page 3 of 10 Table 1 The results of sib-TDT model analysis using the large family sample set. Location unadjusted permuted SNP ID CHR bp Gene p value p value Bonferroni AAFC02065030 2 37,055,124 1.93E-08 3.61E-05 5.45E-05 rs29020694 4 90,418,076 1.87E-06 4.05E-03 5.29E-03 AAFC02132123_1 4 117,126,683 LOC100138299 9.38E-07 1.98E-03 2.65E-03 AAFC02132123_2 4 117,126,810 LOC100138299 3.38E-06 7.33E-03 9.55E-03 AAFC02012009 5 30,554,866 LOC507184 3.53E-06 7.69E-03 9.98E-03 rs29003193 5 66,501,993 3.12E-08 5.79E-05 8.82E-05 rs29012226 5 67,701,052 ANKS1B 6.63E-07 1.38E-03 1.87E-03 SCAFFOLD106936 6 98,767,642 6.35E-07 1.32E-03 1.80E-03 rs29016161 7 24,604,791 2.45E-06 5.31E-03 6.93E-03 rs29017305 9 62,946,258 BACH2 2.30E-06 4.97E-03 6.49E-03 rs29013631 10 38,725,566 2.25E-06 4.87E-03 6.36E-03 rs29022366 12 80,076,729 LOC786668 2.74E-06 5.95E-03 7.75E-03 rs29010388 14 4,145,186 4.20E-07 8.56E-04 1.19E-03 AAFC02138417 15 70,632,844 3.31E-06 7.19E-03 9.36E-03 rs29010371 16 63,558,950 FAM129A 1.06E-06 2.26E-03 3.00E-03 rs29009572 17# 37,575,810 1.29E-06 2.76E-03 3.65E-03 rs29021871 17 44,213,230 1.06E-06 2.25E-03 2.99E-03 CART 20# 4,922,252 CART 3.57E-08 6.75E-05 1.01E-04 rs29018531 20 38,814,738 1.32E-06 2.82E-03 3.72E-03 AAFC02028192 21 5,702,403 MEF2A 1.24E-07 2.49E-04 3.49E-04 rs29022862 21 12,595,937 1.85E-07 3.73E-04 5.22E-04 rs29009825 21 24,827,395 1.90E-06 4.11E-03 5.38E-03 rs29012664 22 42,415,179 FHIT 5.30E-07 1.10E-03 1.50E-03 NW_930303_1 24# 29,286,364 1.02E-07 2.05E-04 2.90E-04 NW_930303_2 24# 29,286,314 3.06E-07 6.17E-04 8.64E-04 SCAFFOLD176855 28 26,871,628 SLC29A3 1.03E-06 2.18E-03 2.90E-03 SCAFFOLD68962 X 7,641,750 3.15E-06 6.86 E-03 8.91E-03 Locations were determined by blast to bovine sequence version 4.0. All other locations denoted by # were either determined from bovine sequence version 2.0, 4.1 or the Maryland sequence assembly. Permuted p values reported here are from a 10,000,000 genome-wide SNP permutation. Table 1 The results of sib-TDT model analysis using the large family sample set. Locations were determined by blast to bovine sequence version 4.0. PRNP gene Clinical presentation of BSE disease is a difficult pheno- type to test for genetic associations. Animals that have developed BSE are clearly susceptible, however, those which are clinically healthy are difficult to assess. In the PRNP variation was not exhaustively tested for an asso- ciation with classical BSE as the focus of this study was primarily genome wide. Over 380 polymorphisms are Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Page 4 of 10 Table 2 PLINK case-control association results of the case-control sample set. Location unadjusted SNP ID CHR bp Gene p value rs29013431 2 15,312,400 5.99E-03 rs29012194 2 26,095,592 6.28E-03 rs29016537 2 29,654,789 1.30E-03 rs29020907 4 59,252,723 IMP2 4.97E-04 rs29024570 10 4,359,586 4.30E-04 AAFC02107025 10 21,153,203 DHRS1 4.73E-03 rs29024728 13 28,618,368 3.34E-03 PRNP08 13 47,214,453 PRNP 4.38E-03 rs29021171 14 10,106,755 3.84E-03 SCAFFOLD51887 14 43,984,153 7.25E-05* rs29014819 15 35,713,980 7.17E-04 rs29014820 15 35,714,095 6.05E-04 rs29014821 15 35,714,113 4.66E-04 AAFC02014662 16 65,943,749 7.95E-03 AAFC02012500_1 21 11,820,092 MCTP2 1.82E-03 AAFC02012500_2 21 11,820,201 MCTP2 3.93E-03 rs29026011 21 13,254,954 LOC618464 3.00E-03 rs29019629 21 33,209,686 CSPG4 2.01E-03 rs29011202 24 18,493,192 9.08E-03 AJ496776 28 31,495,721 5.74E-03 All locations determined by blast to bovine sequence version 4.0. Threshold of significance is p ≤1.7 × 10-5 and suggestive significance p ≤10-4 is denoted as . Table 2 PLINK case-control association results of the case-control sample set. test developed in 1993 by Spielman [17] is intended to test for linkage between complex diseases and genetic markers. The sib-TDT approach used here does not reconstruct parental genotypes in their absence, but uses marker data from unaffected half siblings instead [17]. The DFAM analysis model fits the structure of the related half-sibling sample set and has been utilized in other species as well [20]. The study presented here used a much larger number of markers than the pre- vious studies and used an analysis approach that is robust to population stratification [21]. The advantage this analysis has over the case-control approach is that with 302 affected animals it has twice the number of affected individuals, and thus, a higher study power and likelihood of detecting markers associated with disease loci. Many of the samples included in the family sample set used in this study were also used by Hernández-Sánchez et al. [13] and Zhang et al. [14], while the case-control sample set was analyzed here for the first time. Hernán- dez-Sánchez et al. PRNP gene [13] also used a TDT approach, how- ever, their analysis method requires heterozygous parents to allow the parent of origin of alleles to be unequivocally determined and as a result many animals had to be disregarded in their analysis. In addition, pro- geny with the same genotype as the predicted genotype of the sires or progeny that were themselves homozy- gous were excluded. As a result, although the TDT method used by Hernández- Sánchez et al. [13] has the potential to be powerful, the use of this approach with microsatellite marker based data was limited by the number of genotypes which could be used and ranged from 92 (in the case of marker BMS1658) to 210 (in the case of INRA36). The analysis method used to localize QTLs by Zhang et al. [14] was a regression approach which does not require the parents to be heterozygous and hence all individuals could be included in the analy- sis. However, the QTL approach is not robust to popu- lation stratification. Moreover, the total number of samples (360) used in the Zhang et al. [14] study was smaller than that of Hernández-Sánchez et al. [13] (530) as well as this study (412). The two previous analyses of overlapping family samples yielded different results: the TDT analysis of Hernández-Sánchez et al. [13] found evidence for associations with BSE incidence on chro- mosomes 5, 10 and 20, whereas the QTL analysis by Zhang et al. [14] identified BSE associated QTL chro- mosomes 1, 6, 13, 17, 19 and X/Yps. SNPs in the regions matching regions found in previous studies are detailed in Table 3. With regards to the family data this study offers a similar power to that of the TDT analysis by Hernández-Sánchez et al. [13], however uses half-sib controls as opposed to inferring the sires genotype. In addition, due to necessary genotypic restrictions (only All locations determined by blast to bovine sequence version 4.0. Threshold of significance is p ≤1.7 × 10-5 and suggestive significance p ≤10-4 is denoted as . All locations determined by blast to bovine sequence version 4.0. Threshold of significance is p ≤1.7 × 10-5 and suggestive significance p ≤10-4 is denoted as . known to reside throughout the coding and non-coding regions of PRNP [12]. Of these, 13 PRNP haplotype tag- ging SNPs (htSNPs) were used in the scans of which 8 were informative for both data sets. PRNP gene The htSNPs used in this study capture a large portion of PRNP haplotype variation observed in a diverse assemblage of U.S cattle, spanning the promoter region into the last exon, how- ever, they do not capture all of it. Additionally, the two InDels previously identified [9-11] as having allele asso- ciations with classical BSE were not genotyped in this study and thus no information is available for this sam- ple set and the InDels PRNP haplotypes. One SNP (PRNP08) had a p value of 4.38 × 10-3 for an association with classical BSE but did not pass multiple test correc- tions. Consequently, no significant associations of PRNP variation (the 8 informative htSNPs) with classical BSE were identified in this single marker analysis. Given that the single marker analysis of the informative htSNPs did not capture all of the htSNPs in the PRNP region or the two InDels it should not be considered an exhaustive analysis of the PRNP gene region. Family-based analysis The location determined by older bovine sequence version 2.0 is denoted by #. All other locations were determined by blast to bovine sequence version 4.0 and are reported in mega bases. The previous studies reported location in cM however for the sake of uniformity the location is reported here, based on marker positions, in Mb. heterozygous genotypes can be used) of the TDT method, the approach used here allowed for a greater number of animals to be included in the analysis. Although the total number of animals used in this ana- lysis includes two smaller families (6 families versus 4 families in the previous studies [13,14]) this analysis method does not infer sire genotypes and therefore the inclusion of the two smaller families do not reduce the overall power of this analysis. heterozygous genotypes can be used) of the TDT method, the approach used here allowed for a greater number of animals to be included in the analysis. Although the total number of animals used in this ana- lysis includes two smaller families (6 families versus 4 families in the previous studies [13,14]) this analysis method does not infer sire genotypes and therefore the inclusion of the two smaller families do not reduce the overall power of this analysis. location of these significant markers can be found in Table 3. Moreover, the significant marker on BTA 6, Scaffold106936 at 98.7 Mb corresponds with the QTL region, 51-72 cM on mouse chromosome 5, previously associated with susceptibility to TSE in mice [22]. Addi- tionally, the homologous region to that identified on BTA 6 was also identified as a QTL modulating scrapie incubation period in sheep [23]. Interestingly, the QTL region on mouse chromosome 5 described by Moreno et al. [22] also corresponds to the location of a signifi- cant SNP identified on BTA 17, 44.2 Mb. Thus these chromosomal regions identified in the present study are also supported by studies in cattle and other species. Comparative locations were determined by using the National Center for Biotechnology information map viewer of the mouse QTL regions (build 37.1), then the human and Btrna were selected and bovine locations were determined. The sib-TDT analysis in this study identified two sig- nificant SNPs on BTA 20 associated with BSE incidence, rs29018531 at 38.8 Mb and a SNP within the cocaine and amphetamine responsive transcript peptide gene, (CART). Family-based analysis Family-based analyses, although in general being less powerful than case-control studies [19], offer robustness to non-random mating. The transmission/disequilibrium Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Page 5 of 10 Page 5 of 10 Table 3 Comparison of SNPs observed in this study with locations indentified in other studies. Analysis Location Previously Peak CHR SNP ID Method p-value Mb Observed Location Mb 1 rs29009859 sib-TDT 2.0 × 10-5 89.7 [14] 106.5 5 AAFC02012009 sib-TDT 9.9 × 10-3 30.6 No 5 E25B16-36408-3 sib-TDT 8.8 × 10-5 66.5 No 5 rs29012226 sib-TDT 1.9 × 10-3 67.7 No 5 rs29024670 sib-TDT 3.0 × 10-5 112.8 [13] 107.0 6 SCAFFOLD106936_12205 sib-TDT 1.8 × 10-3 98.8 No 10 rs29024570 Case-control 4.3 × 10-4 4.4 No 10 rs29013631 sib-TDT 6.5 × 10-3 38.7 No 10 rs29015623 sib-TDT 9.0 × 10-5 48.0 [13] 40.0 17 rs29021871 sib-TDT 3.0 × 10-3 44.2 No 19 rs29027102 sib-TDT 2.0 × 10-5 62.5 [14] 53.5 20 rs29018531 sib-TDT 3.7 × 10-3 38.8 [13] 46.0 20 CART-SNP sib-TDT 1.0 × 10-4 4.9# No X SCAFFOLD68962_9331 sib-TDT 1.0 × 10-5 7.6 No X SCAFFOLD285727_12117 sib-TDT 1.0 × 10-5 85.5 [14] 112.5 Sib-TDT p values are reported as unadjusted unless denoted by which indicates Bonferroni corrected values that pass 10,000,000 permutations. All case control p-values are reported are as unadjusted and suggested significance is denoted as . Only SNPs with appreciable or significant p-values located within the confidence intervals of ref# [14] or within 10 cM of ref# [13] are reported here. The location determined by older bovine sequence version 2.0 is denoted by #. All other locations were determined by blast to bovine sequence version 4.0 and are reported in mega bases. The previous studies reported location in cM however for the sake of uniformity the location is reported here, based on marker positions, in Mb. Table 3 Comparison of SNPs observed in this study with locations indentified in other studies. n of SNPs observed in this study with locations indentified in other studies. Sib-TDT p values are reported as unadjusted unless denoted by * which indicates Bonferroni corrected values that pass 10,000,000 permutations. All case control p-values are reported are as unadjusted and suggested significance is denoted as *. Only SNPs with appreciable or significant p-values located within the confidence intervals of ref# [14] or within 10 cM of ref# [13] are reported here. Case-control analysis Another chromosomal region which harbours signifi- cant markers that were identified in the family-based analyses and was observed but failed to reach significance in the case-control analysis is on chromosome 21. Four SNPs were observed on BTA 21 from the analysis of case-control samples, two SNPs which are located at 11.8 Mb (p = 4.4 × 10-3 and p = 8.3 × 10-3) in the MCTP2 gene, another at 13.3 Mb (p = 2.5 × 10-3) in LOC618464, and an additional SNP at 33.2 Mb (p = 2.0 × 10-3) in the gene CSPG4. The family based sib-TDT analysis identi- fied three significant SNPs, at 5.7 Mb in the MEF2 gene (p = 3.5 × 10-4), 12.6 Mb (p = 5.2 × 10-4), and 24.83 Mb (p = 5.4 × 10-3). When considering both sample sets there appears to be an interval from 11.8 Mb-13.3 Mb with the outer range of approximately 5.7-33.2 Mb for a locus with an effect on BSE. BTA 21 shares conservation of synteny with sheep OAR 16 where QTLs for scrapie susceptibility and scrapie incubation period have been reported [25]. In selecting the analysis approach, it is important to match the appropriate model to the data structure to maximize the power. This study used a case-control approach to analyze the paired control with BSE ani- mals. This approach is powerful in its ability to detect loci linked with disease: however, it has been criticized by geneticists for its lack of robustness for population stratification arising from non-random mating or unknown relationships between individuals [19]. The data presented here was examined for stratification and none was observed (see Additional file 2). However, it is likely that some family relationships exist between some of the cases and controls but the extent and the effect was unable to be determined. Despite the power of the case-control approach, this study was limited by the relatively small number of animals used (149 cases and 184 controls) and no significant results were observed. An increase in the number of cases and controls included in this sample set would have a dramatic effect on the power to detect loci associated with disease [19], as would a higher density scan conducted with an increased number of genome-wide markers. Family-based analysis CART is not currently on the Btau4.0 bovine sequence assembly but was previously mapped to chro- mosome 20 at 38.5cR [15]. In addition, the location of CART was reported on Btau2.0 as 4.92 Mb as well as the Maryland map as 9.78 Mb. Therefore it is unclear from this study if the observed associations identified here are attributable to one locus or two separate loci. The study of Hernández-Sánchez et al. [13] observed an association with marker INRA36 (at 37.9 Mb) on BTA 20 with BSE incidence. This study also identified signifi- cant markers on chromosomes 5, 6, 10, 17 and X asso- ciated with BSE. The study by Hernández-Sánchez et al. [13] also reports associations on BTA 5 and 10 but did not report confidence intervals. The marker identified on BTA 6 in this study (Table 3) is in the same chromo- somal region as the marker described by Zhang et al. [14] and is within the confidence interval. The precise In QTL studies in mice Monero et al., 2008 [22] and Llyod et al., 2002 [24] used a panel of 72 microsatellites to examine 282 F2 and 124 F2 mice respectively. How- ever these mice were inoculated intracerebrally as com- pared to our study where infectious material was orally ingested through contaminated feed. Comparatively these studies have fewer animals and although they use microsatellite markers the information content is limited due to the fact that both studies only use 2 inline bred strains. In addition, it should be noted that single mar- ker association analysis, as reported in this manuscript differs from QTL analysis. These differences make direct Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Page 6 of 10 and the last (p = 1.0 × 10-3) at 29.7 Mb. From the dif- ferent analyses methods and across the two sample sets four markers were identified in the chromosomal region from 15.3 Mb-37.1 Mb on BTA2, with the region of 29- 37 Mb being significant. This data supports an associa- tion between chromosome 2 and BSE disease, however from the data it cannot be determined if this effect is from one locus or several loci spread across the region. comparison rather difficult. Given that neither this study nor the previous studies, even in other species, can really be considered comprehensive genome scans they suffer from the same shortfalls. Case-control analysis Regions containing loci significantly associated with disease status identified on chromosomes 14, 16 and 28 in the sib-TDT analysis of the family-based data were also observed to be of interest, but failed to reach signif- icance, in the case-control study. On chromosome 14 the marker, rs29021171, at 10.1 Mb had a p = 2.3 × 10-3 in the analysis of case-control animals and is in relative close proximity to, rs29010388, at 4.2 Mb which was identified as significant in the sib-TDT analysis (p = 1.2 × 10-3). On chromosome 16 marker AAFC02014662 at 65.9 Mb identified in the case-control (p = 7.95 × 10-3) is in close proximity to rs29010371 at 63.5 Mb, which is in the gene FAM129A, that was identified as significant (p = 3.0 × 10-3) in the sib-TDT analysis. Finally on chromosome 28 marker AJ496776, at 31.5 Mb identified in the case- control sample analysis (p = 9.9 × 10-3) is in close proxi- mity to the marker SCAFFOLD176855 (within SLC29A3 gene) at 26.9 Mb identified as significant (p = 2.9 × 10-3) in the sib-TDT analysis. None of these chromosomal regions have been previously reported as being associated with BSE. Shared regions identified in the sib-TDT family and implicated in the case-control analysis Many of the SNPs included in the panel are in close proximity and are in LD in Holstein [16]. Therefore, it may be more appropriate to consider the results in terms of chromosomal regions instead of individual markers. Linkage disequilibrium will result in the alleles of several closely spaced SNPs being associated with dis- ease status because they all fall on the same haplotype. Thus, it would be expected that several SNPs in LD with a locus involved in disease would show significant associations. Examples of this can be observed with the loci on BTA 15 in Table 2 as well as BTA 4 in Table 1. In addition, if the same regions give significant or a nearly significant association across the different sample sets, this would also increase confidence that the asso- ciation is real. Chromosome 2 is a good example: the most significant marker in this study, AAFC02065030, with a Bonferroni corrected p = 5.5 × 10-5 in the family based analysis is at 37.1 Mb on this chromosome. In the case-control samples set, three markers were identified on Chromosome 2, one at 15.3 Mb, another at 26.1 Mb, Family-based analysis In this study there remains the distinct possibility of a type 2 error which fails to identify a genome location that is associated with disease. Conclusions The large number of SNP markers and the two sets of animals used in this study make it the most comprehen- sive study to date to test genetic loci for an association with classical BSE in European Holstein cattle. The genome-wide scan of half sib families identified an asso- ciation between the genetic loci on 18 chromosomes with BSE incidence in European Holstein cattle, includ- ing a region on BTA 20 associated with BSE incidence that has been reported in previous studies. The identifi- cation of markers at or near statistical significance within the same chromosomal regions in both sets of samples provides independent evidence for the association of those regions and the presence of one or more genes within the regions influencing the incidence of BSE in cattle. However, these results need to be confirmed in additional cattle populations or other species. The data in this study can be made available upon request. Another candidate gene, which is in close proximity to three SNPs on BTA 2 at ~29.3 Mb, with alleles that associated with BSE incidence is B3GALT1. Beta-1,3- galactosyltransferase (B3GALT1), is a transferase poly- peptide gene involved in the biosynthesis of GPI anchors. The involvement of the GPI anchor, with lipid raft and TSE disease has been investigated [32] and it is thought that the GPI anchor may affect the conforma- tion, or the association of the prion protein with specific membrane domains [33]. An additional potential candi- date gene is CART, cocaine and amphetamine regulate transcript. This neuropeptide plays a role in a variety of physiological processes, some of which include: promo- tion of hippocampal neurons by upregulating brain- derived neurotrophic factors [34] and synaptogenesis [35]. In addition, the expression of CART has shown to be down regulated in mouse prion disease [36]. It is worth noting that this study identified a large number of associations with classical BSE disease inci- dence throughout the bovine genome versus one single major locus with a large effect in the bovine genome. This would make it difficult to select cattle that are genetically resistant to classical BSE, however the results give some insight into gene pathways important during disease progression. g p The SNP located on BTA 14 (43.9 Mb), associated with BSE incidence in the case-control analysis, is in close proximity to the gene exostoses (multiple) 1, EXT1. Candidate genes identified in the case-control and/or sib-TDT family analysis The SNPs identified from the family samples on chro- mosomes 4, 5, 9, 12, 16, 20, 21, 22 and 28 are all found within genes; however, the polymorphisms on Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Page 7 of 10 Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 STMN2 and EXT1 are functional and positional gene candidates. chromosomes 4 and 12 are in hypothetical genes. The most notable is the polymorphism on chromosome 5, rs29012226, in the ankyrin repeat and sterile alpha motif domain containing 1B gene (ANKS1B). This gene is also known as amyloid beta protein precursor (APP) intracellular domain associated protein 1 (AIDA-1), and is associated with APP binding [26]. It is well known that APP generates beta amyloid and plays a key role in Alzheimers disease [27-30]. Further cellular prion pro- tein and AIDA-1 has been implicated as a receptor for amyloid-b oligomers [31,26], making ANKS1B a good candidate gene for further study. Conclusions McCormick et al., [37] showed that EXT1 is an endoplasmic reticulum (ER)-resident type II transmem- brane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell sur- face heparan sulfate glycosaminoglycans (GAGs). The N terminus of PrP contains a GAG-binding motif and it is thought that PrP binding of GAG is important in prion disease [37-39]. Additionally, this region contains another candidate gene STMN2, which has been identi- fied in a whole genome association study for genetic risk factors for variant Creutzfeldt-Jakob in humans [40]. Specifically, Mead and others [40] found an asso- ciation with acquired prion diseases, including vCJD (p = 5.6 × 10(-5)), kuru incubation time (p = 0.017), and resistance to kuru (p = 2.5 × 10(-4)), in a region upstream of STMN2 (the gene that encodes SCG10). Superior cervical ganglion 10, SCG10, is a neuronal growth associated protein and may play a role in neu- ronal differentiation in modulating membrane interac- tion with the cytoskeleton during neurite outgrowth. STMN2 is at 39.9 Mb on chromosome 14 in cattle, which is in close proximity to EXT1, making both Statistical analysis h f The PLINK software v1.04 was used to perform the majority of the statistical analysis [18]. The data from the case-control sample set were analyzed using the basic case-control association (c2) test. Whereas the family sample set was analyzed with the DFAM program [18], which is an adjusted family TDT analysis, as described by Spielman [17]. To correct for multiple tests Bonferroni single-step adjusted p-values (BONF) proce- dures were applied. A permutation test was also used in this study, which was max(T)” permutation with 10,000 and 10,000,000 permutations, a procedure that permutes both a point-wise SNP significance and a genome-wide significance. Animal information This study used two sets of samples from cases and controls, but with different structures. The first sample set consisted of female European Holstein collected in the mid 90’s and included 149 BSE case and 184 control animals. The control animals were contemporaries of the BSE cases and collected from the same farms. In addition 15 BSE negative, determined by post-mortem histology, and paired control animals were included in the control set. The second sample set was family based and consisted of 302 BSE affected and 179 unaffected half-sib Holsteins from six sire families. All the BSE affected and unaffected cattle within one family were paternal half sibs from the designated sire but with dif- ferent dams. No DNA samples were available from any of the sires. In both sample sets cattle designated as BSE positive were first examined by qualified veterinarians. BSE sta- tus was subsequently confirmed post-mortem by histol- ogy (by the Veterinary Laboratories Agency, New Haw, Surrey, UK). None of the control animals exhibited any clinical symptoms of disease and were presumed to be free of disease. All of the control animals were age and Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Page 8 of 10 Table 4 The sire identities and the number of half sib offspring analyzed in this study verses the number of animal used two previous studies. Sire Number of BSE Number of control This study [13] [14] This study [13] [14] Barold Rock Seal 33 53 43 11 28 22 Brynhyfryd Cascade 68 93 72 28 56 18 Leighton Workboy 71 88 70 40 44 22 Maybar Juniper 90 124 83 51 44 30 Bowerchalk Polacca 4 0 0 6 0 0 Deri cascade 3 0 0 7 0 0 Total 269 358 268 143 172 92 Table 4 The sire identities and the number of half sib offspring analyzed in this study verses the number of animal used two previous studies. sex matched from the same calving season and from the same farm as the BSE cases. As such, the control ani- mals are assumed to have been exposed to the same environment. DNA isolation and genotyping Genomic DNA from the case-control was isolated from blood using a high salt phenol/chloroform extraction method as described by Sherman et al. [41]. Genomic DNA from the family animal set was isolated from blood samples by phenol and chloroform extraction, as described by Hernández-Sánchez et al. [13]. The genotyping panel was comprised of two oligonu- cleotide pool assays (OPAs) as described by McKay and others in 2007 [16]. Briefly, 5,500 SNP were mapped on the Roslin-Cambridge 3,000 rad bovine-hamster whole genome radiation hybrid panel (WGRH3000) [42] and the minor allele frequency (MAF) was determined on a variety of breeds, including Holstein. Of the original SNPs, 3,072 were selected to give the greatest genome coverage and MAF >0.05 for the genome scan. An Illu- mina GoldenGate assay [43] was performed using the two custom OPAs and genotypes determined using an Illumina BeadScan (Illumina Inc., San Diego, CA) and the Illumina BeadStudio software. Sequences containing SNPs (see additional file 3) were blasted on the bovine assembly (4.0) to determine the SNP locations [16] and, the location for all except 111 SNPs were determined. the PLINK program. Following the removal of SNPs that did not cluster well, 2,904 SNPs were used in the analysis. Of these loci, a further 22 SNPs were removed due to missing data (GENO>0.1), seven duplicated SNPs, and a further 48 SNPs were removed due to low allele frequency (MAF<0.01). The MAFs for each of the SNPs in this data set is provided in additional file 3 and the graphical representation of the MAF for each chro- mosome is shown as in additional file 4. Of the indivi- duals examined, 5 samples failed and 64 were removed because of low genotyping frequency (MIND>0.1), for the 412 remaining individuals (see Table 4) a genotyping success rate of 0.98 was obtained. Additional file 1: The best fit model analysis results (Genotypic, Recessive, and Dominant) of the case-control sample set. Additional file 2: Multi-dimensional Scaling (MDS) of the multiple family sample set and unrelated sample set. Additional file 3: Sequence information for the SNPs and the minor allele frequencies for the family and case-control sample sets in order of position. Additional file 4: Graphical plots of the MAF versus position for each chromosome for the multiple family sample set and unrelated sample set. References 23. Moreno CR, Cosseddu GM, Schibler L, Roig A, Moazami-Goudarzi K, Andreoletti O, Eychenne F, Lajous D, Schelcher F, Cribiu EP, Laurent P, Vaiman D, Elsen JM: Identification of new quantitative trait loci (other than the PRNP gene) modulating the scrapie incubation period in sheep. Genetics 2008, 179(1):723-726. 1. Prusiner SB, Hsiao KK: Human prion diseases. Ann Neuro 1994, 35:385-395. 1. Prusiner SB, Hsiao KK: Human prion diseases. Ann Neuro 1994, 35:385-395. 2. Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE, Prusiner SB: Conversion of a-helices into b-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA 1993, 90:10962-10966. 24. Lloyd SE, Uphill JB, Targonski PV, Fisher EM, Collinge J: Identification of genetic loci affecting mouse-adapted bovine spongiform encephalopathy incubation time in mice. Nuerogenetics 2002, 4(2):77-81. 3. Cronier S, Gros N, Tattum MH, Jackson GS, Clarke AR, Collinge J, Wadsworth JDF: Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin. Biochem J 2008, 416(2):297-305. 25. Laurent P, Schibler L, Vaiman A, Laubier J, Delcros C, Cosseddu G, Vaiman D, Cribiu EP, Yerle M: A 12 000-rad whole-genome radiation hybrid panel in sheep: application to the study of the ovine chromosome 18 region containing a QTL for scrapie susceptibility. Anim Genet 2007, 38(4):358-363. 4. Atarashi R, Moore RA, Sim VL, Hughson AG, Dorward DW, Onwubiko HA, Priola SA, Caughey B: Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein. Nat Methods 2007, 8:645-650. 4. Atarashi R, Moore RA, Sim VL, Hughson AG, Dorward DW, Onwubiko HA, Priola SA, Caughey B: Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein. Nat Methods 2007, 8:645-650. 26. Ghersi E, Noviello C, D’Adamio L: Amyloid -b protein precursor (AbPP) intracellular domain-associated protein-1 proteins bind to AbPP and modulate its processing in an isoform-specific manner. JBC 1990, 279(47):49105-49112. 5. Laegreid WW, Clawson ML, Heaton MP, Green BT, O’Rourke KI, Knowles DP: Scrapie resistance in ARQ sheep. J Virol 2008, 82(20):10318-10320. 6. Alvarez L, Arranz JJ, San Primitivo F: Identification of a new leucine haplotype (ALQ) at codon 154 in the ovine prion protein gene in Spanish sheep. J Anim Sci 2006, 84(2):259-265. 27. Authors’ contributions 19. Risch N, Teng J: The relative power of family-based and case-control designs for linkage disequilibrium studies of complex human diseases I. DNA pooling. Genome Res 1998, 8:1273-1288. BMM, SSM carried out the DNA isolation and genotyping and JLW designed strategies for sampling infected and control animals and provided the samples. BMM, MS, PS, AP and ZW participated in the bioinformatics and statistical analysis. BMM, SSM, JLW, MLC, and WWL participated in the study design and helped with the draft of the manuscript. All authors read and approved the final manuscript. 20. Wiiki A, Wade C, Biagi T, Ropstad E, Bjerkas E, Lindblad-Toh K, Lingaas F: A deletion in Nephronophthisis 4 (NPHP4) is associated with recessive cone-rod dystrophy in standard wire-haired dachshund. Genome research 2008, 18(9):1415-1421. 21. Devlin B, Roeder K, Bacanu SA: Unbiased methods for population-based association studies. Genet Epidem 2001, 21:273-284. Abbreviations BSE: bovine spongiform encephalopathy; cM: centimorgans; cR: centirad; ER: endoplasmic reticulum; GAG: glycosaminoglycans; GPI: glycosyl- phosphatidylinositol; htSNP: haplotype tagging single nucleotide polymorphism; InDel: insertion/deletion polymorphism; LD: Linkage disequilibrium; MAF: minor allele frequency; OPA: oligonucleotide pool assay; PrPc: native prion protein; PrPRes: misfolded prion protein; QTL: quantitative trait loci; Sib-TDT: sibling transmission disequilibrium test; SNP: single nucleotide polymorphism; TSE: transmissible spongiform encephalopathy. 13. Hernández-Sánchez J, Waddington D, Wiener P, Haley CS, Williams JL: Genome-wide search for markers associated with bovine spongiform encephalopathy. Mamm Genome 2002, 13(3):164-168. 14. Zhang C, de Koning DJ, Hernández-Sánchez J, Haley CS, Williams JL, Wiener P: Mapping of multiple quantitative trait loci affecting bovine spongiform encephalopathy. Genetics 2004, 167(4):1863-1872. 15. Anderson RM, Donnelly CA, Ferguson NM, Woolhouse MEJ, Watt CJ, Udy HJ, MaWhinney S, Dunstan SP, Southwood TRE, Wilesmith JW, Ryan JB Hoinville LJ, Hillerton JE, Austin AR, Wells GA: Transmission dynamics and epidemiology of BSE in British cattle. Nature 1996, 382:779-788. Author details 1 1Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada. 2USDA, ARS, US Meat Animal Research Center, Clay Center, Nebraska, USA. 3Department of Veterinary Pathobiology, University of Illinois, Urbana, Illinois, USA. 4Department of Animal Science, Washington State University, Pullman, Washington, USA. 5Divisions of Animal Sciences, University of Missouri, Columbia, Missouri, USA. 6Parco Tecnologico Padano, Via Einstein, Polo Universitario, Lodi, 26900 Italy. 17. Spielman RS, Ewens WJ: A sibship test for linkage in the presence of association: the sib transmission/disequilibrium test. Am J Hum Genet 1998, 62:450-458. 18. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MAR, Bender D, Maller J, Sklar P, de Bakker PIW, Daly MJ, Sham PC: PLINK: A tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007, 81:559-575. Received: 1 June 2009 Accepted: 29 March 2010 Published: 29 March 2010 22. Moreno CR, Lantier F, Lantier I, Sarradin P, Elsen JM: Detection of new quantitative trait Loci for susceptibility to transmissible spongiform encephalopathies in mice. Genetics 2003, 165(4):2085-91. Acknowledgements The authors wish to thank Alberta Bovine Genomics group and PrioNet Canada and Alberta Prion Research Institute (200500696) for their financial support. JLW acknowledges the Cariplo Foundation for their support. 16. McKay SD, Schnabel RD, Murdoch BM, Aerts J, Gill CA, Gao C, Li C, Matukumalli LK, Stothard P, Wang Z, Van Tassell CP, Williams JL, Taylor JF, Moore SS: Construction of bovine whole-genome radiation hybrid and linkage maps using high-throughput genotyping. Anim Genet 2007, 38(2):120-125. Data quality control Quality control analyses were carried out by removing confounding effects prior to the data analysis. For the case-control sample set from the original 3,072 SNPs in the assay, 122 SNPs were excluded based on assay fail- ure, as observed by poor clustering of alleles using the BeadStudio software. The remaining 2,950 SNPs were submitted to the PLINK program [18] where seven duplicated SNPs were removed and 12 SNPs were removed because more than 10% of the samples failed to genotype at that locus (GENO >0.1). Additionally, 13 samples were removed due to low genotyping rate (MIND>0.1). A further 61 SNPs were removed due to a MAF <0.01. The MAFs for each of the SNPs in this data set is provided in additional file 3 and the graphical representation of the MAF for each chromosome is shown in additional file 4. The remaining individuals that were included in the analyses had a mean genotyp- ing rate of 0.995. Population stratification was tested for in this data set and none was observed (data in addi- tional file 2). Additional file 4: Graphical plots of the MAF versus position for each chromosome for the multiple family sample set and unrelated sample set. For the family sample set, transmission disequilibrium analysis was performed using the sib-TDT application in Page 9 of 10 Page 9 of 10 Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 12. Clawson ML, Heaton MP, Keele JW, Smith TPL, Harhay GP, Laegreid WW: Prion gene haplotypes of U.S. cattle. BMC Genetics 2006, 7:51. References Ring S, Weyer SW, Kilian SB, Waldron E, Pietrzik CU, Filippov MA, Herms J, Buchholz C, Eckman CB, Korte M, Wolfer DP, Müller UC: The secreted b- amyloid precursor protein ectodomain APPsa is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP- deficient mice. J Neurosci 2007, 27(29):7817-7826. 7. Collinge J, Sidle KCL, Meads J, Ironside J, Hill AF: Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature 1996, 383:685-690. , 8. Gambetti P, Kong Q, Zou W, Parchi P, Chen SG: Sporadic and familial CJD: classification and characterisation. Br Med Bull 2003, 66:213-239. 28. Li ZW, Stark G, Götz J, Rülicke T, Gschwind M, Huber G, Müller U, Weissmann C: Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells. Proc Natl Acad Sci USA 1996, 93(12):6158-6162. 9. Sander P, Hamann H, Drögemüller C, Kashkevich K, Schiebel K, Leeb T: Bovine prion protein gene (PRNP) promoter polymorphisms modulate PRNP expression and may be responsible for differences in bovine spongiform encephalopathy susceptibility. J Biol Chem 2005, 280(45):37408-37414. 29. Müller U, Cristina N, Li ZW, Wolfer DP, Lipp HP, Rülicke T, Brandner S, Aguzzi A, Weissmann C: Behavioral and anatomical deficits in mice homozygous for a modified b-amyloid precursor protein gene. Cell 1994, 79(5):755-765. 10. Juling K, Schwarzenbacher H, Williams JL, Fries R: A major genetic component of BSE susceptibility. BMC Biol 2006, 4:33. 11. Haase B, Doherr MG, Seuberlich T, Drögemüller C, Dolf G, Nicken P, Schiebel K, Ziegler U, Groschup MH, Zurbriggen A, Leeb T: PRNP promoter polymorphisms are associated with BSE susceptibility in Swiss and German cattle. BMC Genetics 2007, 8:15. 30. Zheng H, Jiang M, Trumbauer ME, Sirinathsinghji DJS, Hopkins R, Smith DW, Heavens RP, Dawson GR, Boyce S, Conner MW, Stevens KA, Slunt HH, Sisoda SS, Chen HY, Ploeg Van der LHT: b-amyloid precursor Page 10 of 10 Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 protein-deficient mice show reactive gliosis and decreased locomotor activity. Cell 1995, 81(4):525-531. protein-deficient mice show reactive gliosis and decreased locomotor activity. Cell 1995, 81(4):525-531. 31. Laurén J, Gimbel DA, Nygaard HB, Gilbert JW, Strittmatter SM: Cellular prion protein mediates impairment of synaptic plasticity by amyloid-b oligomers. Nature 2009, 457(7233):1128-1132. 32. Paulick MG, Bertozzi CR: The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins. References Biochemistry 2008, 47(27):6991-7000. 33. Campana V, Caputo A, Sarnataro D, Paladino S, Tivodar S, Zurzolo C: Characterization of the properties and trafficking of an anchorless form of the prion protein. J Biol Chem 2007, 282(31):22747-22756. 34. Wu B, Hu S, Yang M, Pan H, Zhu S: CART peptide promotes the survival of hippocampal neurons by upregulating brain-derived neurotrophic factor. Biochem Biophys Res Commun 2006, 347(3):656-661. y 35. Abrahám H, Orsi G, Seress L: Ontogeny of cocaine- and amphetamine- regulated transcript (CART) peptide and calbindin immunoreactivity in granule cells of the dentate gyrus in the rat. Int J Dev Neurosci 2007, 25(5):265-274. 36. Sorensen G, Medina S, Parchaliuk D, Phillipson C, Robertson C, Booth SA: Comprehensive transcriptional profiling of prion infection in mouse models reveals networks of responsive genes. BMC Genomics 2008, 9:114. 37. McCormick C, Leduc Y, Martindale D, Mattison K, Esford LE, Dyer AP, Tufaro F: The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate. Nat Genet 1998, 19(2):158-161. 38. Yin S, Pham N, Yu S, Li C, Wong P, Chang B, Kang SC, Biasini E, Tien P, Harris DA, Sy MS: Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans. Proc Natl Acad Sci USA 2007, 104(18):7546-7551. 39. Yu S, Yin S, Pham N, Wong P, Kang SC, Petersen RB, Li C, Sy MS: Ligand binding promotes prion protein aggregation–role of the octapeptide repeats. FEBS J 2008, 275(22):5564-5575. 39. Yu S, Yin S, Pham N, Wong P, Kang SC, Petersen RB, Li C, Sy MS: Ligand binding promotes prion protein aggregation–role of the octapeptide repeats. FEBS J 2008, 275(22):5564-5575. 40. Mead S, Poulter M, Uphill J, Beck J, Whitfield J, Webb TEF, Campbell T, Adamson G, Deriziotis P, Tabrizi SJ, Hummerich H, Verzilli C, Alpers MP, Whittaker JC, Collinge J: Genetic risk factors for variant Creutzfeldt-Jakob disease: a genome-wide association study. Lancet Neurol 2009, 8(1):57-66. 40. Mead S, Poulter M, Uphill J, Beck J, Whitfield J, Webb TEF, Campbell T, Adamson G, Deriziotis P, Tabrizi SJ, Hummerich H, Verzilli C, Alpers MP, Whittaker JC, Collinge J: Genetic risk factors for variant Creutzfeldt-Jakob disease: a genome-wide association study. Lancet Neurol 2009, 8(1):57-66. y 41. Sherman EL, Nkrumah DJ, Murdoch BM, Moore SS: Identification of polymorphisms influencing feed intake and efficiency in beef cattle. Anim Genet 2008, 39(3):225-231. 41. Murdoch et al. BMC Genetics 2010, 11:20 http://www.biomedcentral.com/1471-2156/11/20 References Sherman EL, Nkrumah DJ, Murdoch BM, Moore SS: Identification of polymorphisms influencing feed intake and efficiency in beef cattle. Anim Genet 2008, 39(3):225-231. 42. Williams JL, Eggen A, Ferretti L, Farr CJ, Gautier M, Amati G, Ball G, Caramorr T, Critcher R, Costa S, Hextall P, Hills D, Jeulin A, Kiguwa S, Ross O, Smith AL, Saunier K, Urquhart B, Waddington D: A bovine whole-genome radiation hybrid panel and outline map. Mamm Genome 2002, 13(8):469-474. 43. Oliphant A, Barker DL, Stuelpnagel JR, Chee MS: BeadArray technology: enabling an accurate, cost-effective approach to high-throughput genotyping. Biotechniques 2002, , Suppl 56-8: 60-61. doi:10.1186/1471-2156-11-20 Cite this article as: Murdoch et al.: A 2cM genome-wide scan of European Holstein cattle affected by classical BSE. BMC Genetics 2010 11:20. 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https://openalex.org/W2123953280	https://hal.inria.fr/hal-01571377/document	French	null	Hypercube Neural Network Algorithm for Classification	IFIP advances in information and communication technology	2,011	cc-by	8,644	To cite this version: Dominic Palmer-Brown, Chrisina Jayne. Hypercube Neural Network Algorithm for Classification. 12th Engineering Applications of Neural Networks (EANN 2011) and 7th Artificial Intelligence Appli- cations and Innovations (AIAI), Sep 2011, Corfu, Greece. pp.41-51, 10.1007/978-3-642-23957-1_5. hal-01571377 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01571377 https://inria.hal.science/hal-01571377v1 Submitted on 2 Aug 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 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https://openalex.org/W2919387156	https://europepmc.org/articles/pmc6362198?pdf=render	English	null	Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1	Scientific reports	2,019	cc-by	10,130	Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1 Stacey N. Barnes1, Rick E. Masonbrink2, Thomas R. Maier1, Arun Seetharam2, Anoop S. Sindhu3, Andrew J. Severin 2 & Thomas J. Baum 1 Received: 6 July 2018 Accepted: 13 December 2018 Published: xx xx xxxx Spliced leader trans-splicing (SLTS) plays a part in the maturation of pre-mRNAs in select species across multiple phyla but is particularly prevalent in Nematoda. The role of spliced leaders (SL) within the cell is unclear and an accurate assessment of SL occurrence within an organism is possible only after extensive sequencing data are available, which is not currently the case for many nematode species. SL discovery is further complicated by an absence of SL sequences from high-throughput sequencing results due to incomplete sequencing of the 5’-ends of transcripts during RNA-seq library preparation, known as 5′-bias. Existing datasets and novel methodology were used to identify both conserved SLs and unique hypervariable SLs within Heterodera glycines, the soybean cyst nematode. In H. glycines, twenty-one distinct SL sequences were found on 2,532 unique H. glycines transcripts. The SL sequences identified on the H. glycines transcripts demonstrated a high level of promiscuity, meaning that some transcripts produced as many as nine different individual SL-transcript combinations. Most uniquely, transcriptome analysis revealed that H. glycines is the first nematode to demonstrate a higher SL trans-splicing rate using a species-specific SL over well-conserved Caenorhabditis elegans SL-like sequences. Pre-mRNA splicing is a vital mechanism associated with the expression and regulation of eukaryotic genes. The most widely deployed splicing mechanism is cis-splicing, which enables the removal of intron sequences from mRNA molecules. Trans-splicing is less widespread and results in the fusion of RNA molecules that are tran- scribed from different genomic loci. The most prevalent form of trans-splicing involves the addition of a short, spliced leader (SL) sequence to the 5′ end of mRNA transcripts, referred to as spliced leader trans-splicing (SLTS). SLTS has evolved independently in a diverse set of phyla including Nematoda, Platyhelminthes, Trypanosoma, Cnidaria, Rotifera, Chordata, Arthropoda and Dinoflagellata1–8. f p fl g SLs originate from SL RNA genes, whose transcripts are divided into two parts by a donor splice site: a 5′ exon-like SL region and a 3′ intron-like region9–11. SL RNAs maintain a conserved secondary structure comprised of hairpins and a single-stranded Sm binding site (5′-purine-AU4–6G-purine-3), which allows the SL RNA to interact with proteins that are required for SLTS3,12,13. It is evident that SLTS has a role in resolving polycistonic mRNAs in Caenorhabditis elegans, acting as a pre- requisite for subsequent translation14. In C. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 6 July 2018 Accepted: 13 December 2018 Published: xx xx xxxx 1Plant Pathology & Microbiology Department, Iowa State University, Ames, IA, 50011, USA. 2Office of Biotechnology, Genome Informatics Facility, Iowa State University, Ames, IA, 50011, USA. 3CHS, Inc., Grandin, ND, 58038, USA. Correspondence and requests for materials should be addressed to T.J.B. (email: tbaum@iastate.edu) Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1 Stacey N. Barnes1, Rick E. Masonbrink2, Thomas R. Maier1, Arun Seetharam2, Anoop S. Sindhu3, Andrew J. Severin 2 & Thomas J. Baum 1 elegans, approximately 70% of transcripts are trans-spliced to a 22nt SL: SL1 or SL23,15–17. However, operon resolution is not the sole function of SLTS in C. elegans, as only 17% of C. elegans transcripts originate from operons15,18. It has been hypothesized that SLTS is involved in many transla- tional regulation mechanisms, including the replacement of deleterious sequences in the 5′-untranslated region, addition of translational motifs from within the SL sequence, or by replacing a transcript’s 5′-monomethylated cap with a 5′-hypermodified cap structure18–24. 1Plant Pathology & Microbiology Department, Iowa State University, Ames, IA, 50011, USA. 2Office of Biotechnology, Genome Informatics Facility, Iowa State University, Ames, IA, 50011, USA. 3CHS, Inc., Grandin, ND, 58038, USA. Correspondence and requests for materials should be addressed to T.J.B. (email: tbaum@iastate.edu) Scientific Reports \| (2019) 9:1356 \| https://doi.org/10.1038/s41598-018-37857-0 1 www.nature.com/scientificreports/ Figure 1. MH119144 is SL trans-spliced by a novel SL sequence. (A) Pairwise sequence alignment of C. elegans SL1 (CeSL1) and the first 22 nucleotides of MH119144 using EMBOSS needle with default settings. (\|) = matching nucleotides, (.) = mismatched nucleotides, (−) = gap. (B) Secondary structural prediction of the putative MH119144 SL RNA consensus sequence using RNAfold shows a characteristic 3-hairpin SL RNA structure. (C) RT-PCR using H. glycines gDNA (g), cDNA (c) or no template DNA (NT). All lanes use a gene- specific reverse primer with either a gene-specific (GSP) forward primer or a SL forward primer. Figure 1. MH119144 is SL trans-spliced by a novel SL sequence. (A) Pairwise sequence alignment of C. elegans SL1 (CeSL1) and the first 22 nucleotides of MH119144 using EMBOSS needle with default settings. (\|) = matching nucleotides, (.) = mismatched nucleotides, (−) = gap. (B) Secondary structural prediction of the putative MH119144 SL RNA consensus sequence using RNAfold shows a characteristic 3-hairpin SL RNA structure. (C) RT-PCR using H. glycines gDNA (g), cDNA (c) or no template DNA (NT). All lanes use a gene- specific reverse primer with either a gene-specific (GSP) forward primer or a SL forward primer. Sequence data indicate that all nematode species studied to date utilize SL trans-splicing. In all nematodes, SLs with similarity to SL1 and/or SL2 are found, with an exception of Trichinella spiralis, which uses non-canonical spliced leaders25–32. Interestingly, sequence analysis of the potato cyst nematodes Globodera rostochiensis and G. pallida, identified multiple hypervariable SL sequences in addition to SL1 and SL226,33. Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1 Stacey N. Barnes1, Rick E. Masonbrink2, Thomas R. Maier1, Arun Seetharam2, Anoop S. Sindhu3, Andrew J. Severin 2 & Thomas J. Baum 1 The diversity of SL sequences found in Globodera spp. and the dearth of information regarding their functionality highlights a need to improve our understanding through the investigation of nematode genomic and transcriptomic data. Previous studies have identified SL1 in the soybean cyst nematode, Heterodera glycines, a highly damaging plant parasite closely related to Globodera spp.34. Subsequently, the SL1 sequence has been used to successfully generate H. glycines cDNA libraries (LIBEST_005577; unpublished McCarter, J., Clifton, S., Chiapelli, B., Pape, D., Martin, J., Wylie, T., Dante, M., Marra, M., Hillier, L., Kucaba, T. et al.). y ) In this current study, we utilize the recently assembled H. glycines genome35 and the RNA-seq reads from an early-life stages transcriptome36 to extensively characterize SLs and their usage in H. glycines. Serendipitous observation of variation in the 5′-end of two previously sequenced H. glycines transcripts led to the discovery of a novel SL. Through subsequent bioinformatic approaches utilizing both H. glycines genomic and transcriptomic data, this report shows that H. glycines possesses at least twenty-one SLs, found on a total of 2,532 H. glycines transcripts, which account for approximately one-third of H. glycines genes. Functional analysis of the H. glycines SL trans-spliced transcripts reveals involvement in a variety of biological processes. Interestingly, around 45% of the transcripts are promiscuously trans-spliced by SLs suggesting that there is functional redundancy amongst SL RNA molecules. Furthermore, H. glycines is the first nematode to show a transcriptome-wide preference for a species-specific SL sequence over the well-conserved C. elegans SL-like sequences. Scientific Reports \| (2019) 9:1356 \| https://doi.org/10.1038/s41598-018-37857-0 Results Di The alignment was performed using T-coffee with default settings. () indicates a consensus nucleotide position and (−) denotes a sequence gap. Pink, yellow and green shading indicates good, average and bad nucleotide matches respectively. Figure 2. Sequence alignment of novel hypervariable H. glycines SLs. Multiple sequence alignment of H. glycines HgSL3 RNA blast results with SL RNA-like secondary structural predictions. The alignment was performed using T-coffee with default settings. () indicates a consensus nucleotide position and (−) denotes a sequence gap. Pink, yellow and green shading indicates good, average and bad nucleotide matches respectively. Figure 3. Workflow of the bioinformatic pipeline for SL trans-splicing transcript identification. Figure 3. Workflow of the bioinformatic pipeline for SL trans-splicing transcript identification. (Fig. 1C). Genic structure predictions performed on chorismate mutase indicate that the absence of a band within the gDNA reaction is not due to the primers being located on intron/exon borders. Furthermore, a control PCR amplification with cDNA and gDNA templates was performed using a gene-specific primer pair to verify the presence of the chorismate mutase gene in both DNA samples (Fig. 1C). (Fig. 1C). Genic structure predictions performed on chorismate mutase indicate that the absence of a band within the gDNA reaction is not due to the primers being located on intron/exon borders. Furthermore, a control PCR amplification with cDNA and gDNA templates was performed using a gene-specific primer pair to verify the presence of the chorismate mutase gene in both DNA samples (Fig. 1C). Collectively, three tiers of evidence support the legitimacy of this novel SL, including: mapping of the putative SL and the remainder of the transcript to separate locations within the genome, the similarity of the putative SL RNA sequence to known SL RNAs, and the absence of a SL chorismate mutase PCR product when using gDNA. This novel SL will subsequently be named Heterodera glycines spliced leader 3 (HgSL3) to distinguish it from C. elegans SL1-like and SL2-like sequences in other nematode species. The H. glycines genome contains multiple novel SL sequences. To investigate the existence of pre- viously identified SL sequences in H. glycines, all known SLs from C. elegans and Globodera spp. were mapped to the H. glycines genome. SL1 mapped to 180 loci in the H. glycines genome, twenty-two sequences of which were located within close proximity to a Sm motif13. The only Globodera spp. SL variant present in the H. Results Di Discovery of a novel spliced leader in H. glycines. Exploring the available H. glycines expressed sequence tags on NCBI revealed two transcripts coding for chorismate mutase proteins (AY16022537 & MH119144), which are important enzymes for parasitism in multiple plant-parasitic nematodes37–42. Alignment of the 5′ end of MH119144 and the SL1 primer sequence used to clone AY160225 revealed divergent 5′ ends, suggesting the presence of a novel SL sequence (Fig. 1A). To investigate the putative MH119144 SL sequence, the entire transcript was mapped to the H. glycines genome with BLASTn. All but the first fifteen nucleotides of MH119144 mapped to scaffold_282 (Supplemental Fig. S1). To locate the 5′-end of MH119144 in the H. glycines genome, the twenty-two-nucleotide putative SL was mapped to the genome with BLASTn. The putative SL had four exact hits in the H. glycines genome, all of which mapped within a 2.5 Kb region on scaffold_362, confirming that MH119144 is comprised of two sequences that are located in distinctly separate regions of the genome (Supplemental Fig. S1). y p g g pp g In order for a SL to be functional, transcription must create a distinct non-coding hairpin SL RNA structure with a single-stranded Sm motif3,12,13. To identify the presence of these features, the ninety-eight nucleotides downstream of the four putative SL hits were extracted from the genome. All sequences had 99% sequence iden- tify and displayed the typical secondary structure of functional SL RNAs (Fig. 1B).h y y y y g The validity of the putative SL was tested further using RT-PCR to search for the putative SL chorismate mutase sequence in H. glycines gDNA and cDNA (Fig. 1C). Using the putative SL sequence as a forward primer and a gene-specific reverse primer, a visible band was produced when using a cDNA template, but not gDNA Scientific Reports \| (2019) 9:1356 \| https://doi.org/10.1038/s41598-018-37857-0 2 www.nature.com/scientificreports/ ntificreports/ Figure 2. Sequence alignment of novel hypervariable H. glycines SLs. Multiple sequence alignment of H. glycines HgSL3 RNA blast results with SL RNA-like secondary structural predictions. The alignment was performed using T-coffee with default settings. () indicates a consensus nucleotide position and (−) denotes a sequence gap. Pink, yellow and green shading indicates good, average and bad nucleotide matches respectively. Figure 2. Sequence alignment of novel hypervariable H. glycines SLs. Multiple sequence alignment of H. glycines HgSL3 RNA blast results with SL RNA-like secondary structural predictions. Results Di glycines HgSL12 yes HgSL13 — — 4 G.rostochiensis SL1a no — — 73 G.rostochiensis SL1c no 1 1 49 G.rostochiensis SL1e no 1 — 96 G.rostochiensis SL1g no — 1 2 G.rostochiensis SL1i no — — 28 G.rostochiensis SL2a no SL2c, SL2e, SL2f, SL2g, SL2i 3 — 7 G.rostochiensis SL2b no 1 — 5 G.rostochiensis SL2d no — 3 10 G.rostochiensis SL2h no — — 3 G.rostochiensis SL3a no SL3b — — 2 G.rostochiensis SL3c no SL3d, SL3e — — 1 G.rostochiensis SL3f no — — — G.rostochiensis SL4a no SL4b, SL4c, SL4d, SL4e 12 21 326 G.rostochiensis SL4f no — — 14 187 2215 6350 Table 1. Summary of genomic and transcriptomic findings for truncated C. elegans, H. glycines and G. rostochiensis SLs. Identification of a full-length SL RNA with a proximal Sm motif within the H. glycines genome for each representative truncated SL is defined as yes/no in the second column. Identical truncated SL members for which a full-length sequence and proximal Sm motif were identified in the genome are denoted with an (). The 3 methods of SL trans-spliced transcript identification and the number of SL trans-spliced transcripts identified by each method are under the collective Trinity Transcript header. EST BLAST, Direct BLAST and Read-to-transcript columns respectively indicate the number of sequences identified within the NCBI EST database, H. glycines transcriptome, or illumina trimmed reads that were subsequently mapped to H. glycines transcripts. Nematode Representative truncated SL SL RNA in the H.glycines genome Identical truncated SL members Trinity Transcripts EST BLAST Direct BLAST Read-to- transcript C. elegans SL1 yes SL1b, SL1d, SL1f, SL1h 76 1267 1305 C. elegans SL2.1 no SL2.2, SL2.3, SL2.4 — — — C. elegans SL2.5 no SL2.6, SL2.7, SL2.14, SL2.15, SL2.16 — — — C. elegans SL2.8 no — — 1 C. elegans SL2.9 no SL2.10, SL2.11 — — 1 C. elegans SL2.12 no SL2.12 — — 16 C. elegans SL2.18 no — — — H. glycines HgSL3 yes HgSL5, HgSL6, HgSL7, 80 899 1726 H. glycines HgSL4 yes 7 6 1318 H. glycines HgSL8 yes HgSL9* 6 17 1363 H. glycines HgSL10 yes HgSL11* — — — H. Results Di glycines genome for each representative truncated SL is defined as yes/no in the second column. Identical truncated SL members for which a full-length sequence and proximal Sm motif were identified in the genome are denoted with an (). The 3 methods of SL trans-spliced transcript identification and the number of SL trans-spliced transcripts identified by each method are under the collective Trinity Transcript header. EST BLAST, Direct BLAST and Read-to-transcript columns respectively indicate the number of sequences identified within the NCBI EST database, H. glycines transcriptome, or illumina trimmed reads that were subsequently mapped to H. glycines transcripts. million reads had a terminal SL, the legitimacy of which is supported by SL BLAST hits preferentially locating to the 5′-read ends (Fig. 5). Subsequent mapping of the SL-reads to the H. glycines transcriptome revealed a false positive rate of SL-reads at 88.4%, with 9,927/85,876 reads mapping to the 5′ end of 1,635 unique SL trans-spliced transcripts. Again, a portion of the transcripts appeared to be the target of more than one SL RNA molecule, resulting in 6,350 SL-transcript combinations (Table 1 and Fig. 4). Collectively, these analyses identified in 2,532 unique SL trans-spliced transcripts and 21 functional SLs (Table 1 and Fig. 4). Interestingly, when combining all three analyses, HgSL3 is present on 30.9% of SL trans-spliced transcripts making it the most abundantly used SL, a finding unique to H. glycines. Furthermore, 45.5% of the 2,532 SL trans-spliced transcripts were spliced by two or more SLs, with trans-splicing of five or more different SLs onto 6.8% of these transcripts (Fig. 6). Genomic features of transcripts that possess spliced leaders. To functionally characterize the genes that give rise to SL trans-spliced transcripts, all SL trans-spliced transcripts were mapped to the H. glycines genome using GMAP44. Exonic overlap between H. glycines genes and SL trans-spliced transcripts accounted for approximately one-third of the genes in the genome (9,042/29,959). It is interesting that of the 9,042 SL trans-spliced genes, approximately one-third (3,013) co-align with annotated repeats in the H. glycines genome. The ten most abundant repeats comprised 27.7% of the 3,013 trans-spliced genes. The most abundant functionally annotated repeat is associated with a LINE/CR1 retrotransposon (3.4%), suggesting that a significant portion of SL trans-spliced transcripts are associated with transposons (Table 2). p p p To assess the positioning of SL trans-spliced genes within the H. Results Di glycines HgSL4 yes 7 6 1318 H. glycines HgSL8 yes HgSL9 6 17 1363 H. glycines HgSL10 yes HgSL11* — — — H. glycines HgSL12 yes HgSL13* — — 4 G.rostochiensis SL1a no — — 73 G.rostochiensis SL1c no 1 1 49 G.rostochiensis SL1e no 1 — 96 G.rostochiensis SL1g no — 1 2 G.rostochiensis SL1i no — — 28 G.rostochiensis SL2a no SL2c, SL2e, SL2f, SL2g, SL2i 3 — 7 G.rostochiensis SL2b no 1 — 5 G.rostochiensis SL2d no — 3 10 G.rostochiensis SL2h no — — 3 G.rostochiensis SL3a no SL3b — — 2 G.rostochiensis SL3c no SL3d, SL3e — — 1 G.rostochiensis SL3f no — — — G.rostochiensis SL4a no SL4b, SL4c, SL4d, SL4e 12 21 326 G.rostochiensis SL4f no — — 14 187 2215 6350 Table 1. Summary of genomic and transcriptomic findings for truncated C. elegans, H. glycines and G. rostochiensis SLs. Identification of a full-length SL RNA with a proximal Sm motif within the H. glycines genome for each representative truncated SL is defined as yes/no in the second column. Identical truncated SL members for which a full-length sequence and proximal Sm motif were identified in the genome are denoted with an (). The 3 methods of SL trans-spliced transcript identification and the number of SL trans-spliced transcripts identified by each method are under the collective Trinity Transcript header. EST BLAST, Direct BLAST and Read-to-transcript columns respectively indicate the number of sequences identified within the NCBI EST database, H. glycines transcriptome, or illumina trimmed reads that were subsequently mapped to H. glycines transcripts. Nematode Representative truncated SL SL RNA in the H.glycines genome Identical truncated SL members Trinity Transcripts EST BLAST Direct BLAST Read-to- transcript C. elegans SL1 yes SL1b, SL1d, SL1f, SL1h 76 1267 1305 C. elegans SL2.1 no SL2.2, SL2.3, SL2.4 — — — C. elegans SL2.5 no SL2.6, SL2.7, SL2.14, SL2.15, SL2.16 — — — C. elegans SL2.8 no — — 1 C. elegans SL2.9 no SL2.10, SL2.11 — — 1 C. elegans SL2.12 no SL2.12 — — 16 C. elegans SL2.18 no — — — H. glycines HgSL3 yes HgSL5, HgSL6, HgSL7, 80 899 1726 H. glycines HgSL4 yes 7 6 1318 H. glycines HgSL8 yes HgSL9* 6 17 1363 H. glycines HgSL10 yes HgSL11* — — — H. Results Di glycines HgSL12 yes HgSL13* — — 4 G.rostochiensis SL1a no — — 73 G.rostochiensis SL1c no 1 1 49 G.rostochiensis SL1e no 1 — 96 G.rostochiensis SL1g no — 1 2 G.rostochiensis SL1i no — — 28 G.rostochiensis SL2a no SL2c, SL2e, SL2f, SL2g, SL2i 3 — 7 G.rostochiensis SL2b no 1 — 5 G.rostochiensis SL2d no — 3 10 G.rostochiensis SL2h no — — 3 G.rostochiensis SL3a no SL3b — — 2 G.rostochiensis SL3c no SL3d, SL3e — — 1 G.rostochiensis SL3f no — — — G.rostochiensis SL4a no SL4b, SL4c, SL4d, SL4e 12 21 326 G.rostochiensis SL4f no — — 14 187 2215 6350 Table 1. Summary of genomic and transcriptomic findings for truncated C. elegans, H. glycines and G. rostochiensis SLs. Identification of a full-length SL RNA with a proximal Sm motif within the H. glycines genome for each representative truncated SL is defined as yes/no in the second column. Identical truncated SL members for which a full-length sequence and proximal Sm motif were identified in the genome are denoted with an (). The 3 methods of SL trans-spliced transcript identification and the number of SL trans-spliced transcripts identified by each method are under the collective Trinity Transcript header. EST BLAST, Direct BLAST and Read-to-transcript columns respectively indicate the number of sequences identified within the NCBI EST database, H. glycines transcriptome, or illumina trimmed reads that were subsequently mapped to H. glycines transcripts. Table 1. Summary of genomic and transcriptomic findings for truncated C. elegans, H. glycines and G. rostochiensis SLs. Identification of a full-length SL RNA with a proximal Sm motif within the H. glycines genome for each representative truncated SL is defined as yes/no in the second column. Identical truncated SL members for which a full-length sequence and proximal Sm motif were identified in the genome are denoted with an (). The 3 methods of SL trans-spliced transcript identification and the number of SL trans-spliced transcripts identified by each method are under the collective Trinity Transcript header. EST BLAST, Direct BLAST and Read-to-transcript columns respectively indicate the number of sequences identified within the NCBI EST database, H. glycines transcriptome, or illumina trimmed reads that were subsequently mapped to H. glycines transcripts. Table 1. Summary of genomic and transcriptomic findings for truncated C. elegans, H. glycines and G. rostochiensis SLs. Identification of a full-length SL RNA with a proximal Sm motif within the H. Results Di glycines genome was SL1b, which lacked a proximal Sm motif (Supplemental Table 1). g p pp To search for novel HgSL RNA genes, HgSL3 RNA was queried with BLAST against the H. glycines genome. A total of twenty sequences were identified that also contained single-stranded Sm-binding sites flanked by hair- pins (Supplemental Table 1). Alignment of the first twenty-two nucleotides of the putative HgSL RNA sequences yielded ten additional unique HgSLs, numbered HgSL4–13 (Fig. 2). Splice leaders are promiscuously present on multiple H. glycines transcripts. To assess SLTS in H. glycines, known SLs were truncated to the 3′ most 11nt yielding a total of twenty-six unique sequences (7 from C. elegans, 5 from H. glycines, and 14 from Globodera spp.). The use of truncated SLs has been demonstrated to circumvent the low availability of complete 5′-ends in RNA-seq data25,26.hi The truncated SLs were used as query sequences for three separate BLAST analyses. In the first approach, SLs were queried against the NCBI EST database, in the second approach SLs were queried against a H. glycines transcriptome36. A two-tiered third approach that involved SL queries to trimmed Illumina reads with subsequent mapping to the 5′ end of transcripts (Fig. 3). This third approach circumvents RNA-seq 5′ bias, which may result in the misassembly at the 5′ end of transcripts43. BLAST searches to ESTs and transcripts yielded 187 and 2,215 SL trans-spliced transcripts respectively, with a 40% (74/187) rediscovery rate of ESTs within the transcriptome (Table 1 and Fig. 4). After removing the SLs from the sequences, all ESTs were unique, while only 2076/2,215 transcripts were unique, revealing that in some cases transcripts are not uniquely spliced to one SL (Table 1 and Fig. 4). Using the read-based approach, 85,876 of ~11.4 Scientific Reports \| (2019) 9:1356 \| https://doi.org/10.1038/s41598-018-37857-0 3 www.nature.com/scientificreports/ Nematode Representative truncated SL SL RNA in the H.glycines genome Identical truncated SL members Trinity Transcripts EST BLAST Direct BLAST Read-to- transcript C. elegans SL1 yes SL1b, SL1d, SL1f, SL1h 76 1267 1305 C. elegans SL2.1 no SL2.2, SL2.3, SL2.4 — — — C. elegans SL2.5 no SL2.6, SL2.7, SL2.14, SL2.15, SL2.16 — — — C. elegans SL2.8 no — — 1 C. elegans SL2.9 no SL2.10, SL2.11 — — 1 C. elegans SL2.12 no SL2.12 — — 16 C. elegans SL2.18 no — — — H. glycines HgSL3 yes HgSL5, HgSL6, HgSL7*, 80 899 1726 H. Results Di glycines genome, the genome was parti- tioned into 50 kb bins. Analysis of the 50 kb genomic segments showed that SL trans-spliced genes were dispersed Scientific Reports \| (2019) 9:1356 \| https://doi.org/10.1038/s41598-018-37857-0 4 www.nature.com/scientificreports/ Figure 4. Summary of the H. glycines SL trans-spliced transcript analyses. The Venn diagram represents the overlapping and unique SL trans-spliced transcripts identified through the three-part approach using BLASTn against the NCBI ESTs (EST BLAST), the Trinity transcriptome (Direct BLAST) and the unassembled reads (read-to-transcript). Figure 4. Summary of the H. glycines SL trans-spliced transcript analyses. The Venn diagram represents the overlapping and unique SL trans-spliced transcripts identified through the three-part approach using BLASTn against the NCBI ESTs (EST BLAST), the Trinity transcriptome (Direct BLAST) and the unassembled reads (read-to-transcript). Figure 5. HgSLs are preferentially located at the 5′ end of H. glycines reads. All truncated HgSLs were queried with BLASTn against the H. glycines raw reads, which were also used for the Trinity assembly. The read nucleotide start positions were plotted to show the strong preference for HgSLs to be located at the 5′ end of the read. Figure 4. Summary of the H. glycines SL trans-spliced transcript analyses. The Venn diagram represents the overlapping and unique SL trans-spliced transcripts identified through the three-part approach using BLASTn against the NCBI ESTs (EST BLAST), the Trinity transcriptome (Direct BLAST) and the unassembled reads (read-to-transcript). Figure 5. HgSLs are preferentially located at the 5′ end of H. glycines reads. All truncated HgSLs were queried with BLASTn against the H. glycines raw reads, which were also used for the Trinity assembly. The read nucleotide start positions were plotted to show the strong preference for HgSLs to be located at the 5′ end of the read. Figure 5. HgSLs are preferentially located at the 5′ end of H. glycines reads. All truncated HgSLs were queried with BLASTn against the H. glycines raw reads, which were also used for the Trinity assembly. The read nucleotide start positions were plotted to show the strong preference for HgSLs to be located at the 5′ end of the read. throughout the genome. However, clustering of SL trans-spliced genes was also evident, as 40 of the 2,640 50 kb bins had 14 or more consecutively arranged SL trans-spliced genes (Table 3). Functional analysis of SL trans-spliced transcripts reveals involvement in a variety of biological processes. Results Di In order to gain functional insight into the role of SL trans-splicing in H. glycines, the SL trans-spliced transcripts were annotated with Blast2go45 (Supplemental Table 2). Over half (52%) of the annotated transcripts were involved in metabolic and developmental processes (Fig. 7A), with the top two biological pro- cesses involved in ‘Embryo development ending or egg hatching’ and ‘Nematode larval development’ (Fig. 7B). A complementary GO enrichment analysis was performed on the corresponding genomic genes, revealing a similar profile of functions involved in metabolic processes (Fig. 7C, Supplemental Table 3). Effector transcripts are SL trans-spliced and display an all-or-none relationship with multi-gene copy effectors. To investigate whether spliced leaders could be involved in parasitism, we searched for exon-exon overlap between SL trans-spliced transcripts and effector genes in the genome. Effector genes pro- duce proteins that are secreted by H. glycines during parasitism and are thought to play a major role in altering host cell structure and function. Reviewed by46–49. Within the H. glycines genome there are 80 known bona fide Scientific Reports \| (2019) 9:1356 \| https://doi.org/10.1038/s41598-018-37857-0 5 www.nature.com/scientificreports/ Figure 6. SL trans-spliced transcripts are promiscuously trans-spliced. Bars represent the number of SL- transcript combinations identified when combining the SL trans-spliced transcripts identified across all three BLAST analyses. Figure 6. SL trans-spliced transcripts are promiscuously trans-spliced. Bars represent the number of SL- transcript combinations identified when combining the SL trans-spliced transcripts identified across all three BLAST analyses. Figure 6. SL trans-spliced transcripts are promiscuously trans-spliced. Bars represent the number of SL- transcript combinations identified when combining the SL trans-spliced transcripts identified across all three BLAST analyses. Percent Repeatmodeler repeat Repeat annotation 4.82513 Motif:rnd-4_family-65 Unknown 3.68702 Motif:rnd-4_family-352 Unknown 3.36692 Motif:rnd-4_family-1273 LINE/CR1 3.09425 Motif:rnd-5_family-2862 Unknown 2.48963 Motif:rnd-3_family-228 Unknown 2.37107 Motif:rnd-4_family-1152 DNA/TcMar-Tc2 2.03912 Motif:rnd-4_family-299 Unknown 2.03912 Motif:rnd-4_family-71 DNA/Mule-MuDR 1.97985 Motif:rnd-3_family-757 DNA/MuLE-MuDR 1.82573 Motif:rnd-3_family-42 Unknown Table 2. Repeats associated with trans-spliced, repetitive genes. Percent Repeatmodeler repeat Repeat annotation 4.82513 Motif:rnd-4_family-65 Unknown 3.68702 Motif:rnd-4_family-352 Unknown 3.36692 Motif:rnd-4_family-1273 LINE/CR1 3.09425 Motif:rnd-5_family-2862 Unknown 2.48963 Motif:rnd-3_family-228 Unknown 2.37107 Motif:rnd-4_family-1152 DNA/TcMar-Tc2 2.03912 Motif:rnd-4_family-299 Unknown 2.03912 Motif:rnd-4_family-71 DNA/Mule-MuDR 1.97985 Motif:rnd-3_family-757 DNA/MuLE-MuDR 1.82573 Motif:rnd-3_family-42 Unknown Table 2. Repeats associated with trans-spliced, repetitive genes. Percent Repeatmodeler repeat Repeat annotation 4.82513 Motif:rnd-4_family-65 Unknown 3.68702 Motif:rnd-4_family-352 Unknown 3.36692 Motif:rnd-4_family-1273 LINE/CR1 3.09425 Motif:rnd-5_family-2862 Unknown 2.48963 Motif:rnd-3_family-228 Unknown 2.37107 Motif:rnd-4_family-1152 DNA/TcMar-Tc2 2.03912 Motif:rnd-4_family-299 Unknown 2.03912 Motif:rnd-4_family-71 DNA/Mule-MuDR 1.97985 Motif:rnd-3_family-757 DNA/MuLE-MuDR 1.82573 Motif:rnd-3_family-42 Unknown Table 2. Repeats associated with trans-spliced, repetitive genes. Table 2. Results Di Repeats associated with trans-spliced, repetitive genes. effector proteins, 28 of which originate from multiple gene copies and 51 are single gene effectors, to make a total of 121 currently confirmed effector genes. SL trans-spliced transcripts overlapped with the first exon of 29/121 effector genes, indicating that approximately 24% of the currently known bona fide effector genes are subject to SL trans-splicing (Table 4). Interestingly 23/28 multi-gene copy effectors display an all-or-none relationship with SL trans-splicing. For example, 5/5 genes corresponding to 11A06 are SL trans-spliced, while 0/5 genes are SL trans-spliced for 4D06 and 32E03 (Table 4, Supplemental Table 4). effector proteins, 28 of which originate from multiple gene copies and 51 are single gene effectors, to make a total of 121 currently confirmed effector genes. SL trans-spliced transcripts overlapped with the first exon of 29/121 effector genes, indicating that approximately 24% of the currently known bona fide effector genes are subject to SL trans-splicing (Table 4). Interestingly 23/28 multi-gene copy effectors display an all-or-none relationship with SL trans-splicing. For example, 5/5 genes corresponding to 11A06 are SL trans-spliced, while 0/5 genes are SL trans-spliced for 4D06 and 32E03 (Table 4, Supplemental Table 4). Scientific Reports \| (2019) 9:1356 \| https://doi.org/10.1038/s41598-018-37857-0 Discussionh This study identified and functionally characterized SLs and SL trans-spliced transcripts of the plant-parasitic soybean cyst nematode Heterodera glycines. The recent availability of both the H. glycines genome and transcrip- tome has provided an opportunity to extensively characterize SL use and function in a parasitic nematode.hf This study identified and functionally characterized SLs and SL trans-spliced transcripts of the plant-parasitic soybean cyst nematode Heterodera glycines. The recent availability of both the H. glycines genome and transcrip- tome has provided an opportunity to extensively characterize SL use and function in a parasitic nematode.hf This study was prompted by the discovery of HgSL3 at the 5′-end of a chorismate mutase effector cDNA, leading to the identification of a unique set of hypervariable HgSLs. Novel hypervariable SLs have previously been discovered in the potato cyst nematode G. rostochiensis and the animal-parasitic nematode T. spiralis25,26. Interestingly, despite the high volume of SLs that have been discovered in these three species, genomic data suggest a low interspecies conservation of SLs. Given the parasitic nature of all three species, as well as the per- ceived link between SLs and translational regulation, it is possible that the hyper-variation of SLs is a response to parasitism of different hosts. This study investigated a possible link between SLs and known parasitic molecules, referred to as effectors, and found that 24% (29/121) of bona fide effector genes are subject to SL trans-splicing. Previous hypotheses indicate that species use SL trans-splicing as a form of translational control to respond to changing environments, particularly in response to nutrient availability20. Discussionh Effector Total number of genes in the genome Total number of genes that are SL trans-spliced GLAND5 6 3 11A06 5 5 16A01 5 3 5D08 3 1 10C02 2 2 4F01 2 2 17G06 2 1 33E05 2 1 10A06 1 1 19B10 1 1 21E12 1 1 2D01 1 1 33A09 1 1 5A08 1 1 8A07 1 1 flGSB3 1 1 GLAND11 1 1 GLAND12 1 1 GLAND4 1 1 Table 4. Effector genes giving rise to SL trans-spliced transcripts. Effector Total number of genes in the genome Total number of genes that are SL trans-spliced GLAND5 6 3 11A06 5 5 16A01 5 3 5D08 3 1 10C02 2 2 4F01 2 2 17G06 2 1 33E05 2 1 10A06 1 1 19B10 1 1 21E12 1 1 2D01 1 1 33A09 1 1 5A08 1 1 8A07 1 1 flGSB3 1 1 GLAND11 1 1 GLAND12 1 1 GLAND4 1 1 Table 4. Effector genes giving rise to SL trans-spliced transcripts. Effector Total number of genes in the genome Total number of genes that are SL trans-spliced GLAND5 6 3 11A06 5 5 16A01 5 3 5D08 3 1 10C02 2 2 4F01 2 2 17G06 2 1 33E05 2 1 10A06 1 1 19B10 1 1 21E12 1 1 2D01 1 1 33A09 1 1 5A08 1 1 8A07 1 1 flGSB3 1 1 GLAND11 1 1 GLAND12 1 1 GLAND4 1 1 Table 4. Effector genes giving rise to SL trans-spliced transcripts. Table 4. Effector genes giving rise to SL trans-spliced transcripts. effector transcripts, one SL trans-spliced and one not, may provide H. glycines with a way to mitigate host defense responses through differentially regulating the two subsets of effectors.i effector transcripts, one SL trans-spliced and one not, may provide H. glycines with a way to mitigate host defe responses through differentially regulating the two subsets of effectors.i p gf y g gf To identify H. glycines SL trans-spliced transcripts, SLs were first truncated at the 5′-ends before being queried using BLAST against H. glycines sequences. The use of truncated SLs was previously utilized in G pallida26. Before truncating the SLs in H. glycines, we first verified that this approach was necessary by using the full-length SLs as query sequences against the H. glycines ESTs and transcriptome. Only fifteen sequences, none of which were SL1, were identified across both databases (available in GitHub). Discussionh The failure to recover full-length SL1 supports the lack of 5′-ends within the H. glycines datasets, as SL1 is present in H. glycines and other related nematodes26,27. The read-based approach further verified the lack of complete 5′-ends within the H. glycines transcriptome by showing that the truncated SLs were predominantly located at the first nucleotide of raw reads that were under- represented in mature transcripts. To further complicate transcriptome assembly in SLTS organisms, this study revealed that 45.5% of SL trans-spliced transcripts do not have one unique SL-transcript combination. The pro- miscuous nature of SLs on otherwise identical transcripts may cause high ambiguity in the assembly step, result- ing in 5′ truncations or the assembly of a transcript that reflects only the most abundant SL-transcript while discarding lower expressed SL-transcripts. g p p Analysis of the available H. glycines ESTs, transcriptome and raw reads used in this study concluded that HgSL3 is the most prevalent SL in H. glycines, with 30.9% of the SL trans-spliced transcripts being trans-spliced by HgSL3. The predominant use of a non-SL1 sequence is unique to H. glycines and contrasts with findings in C. elegans and the animal-parasitic nematode Ascaris suum, as well as G. pallida where SL1 and SL1 variants were identified on >90% of the SL-containing G. pallida reads26. i g p C. elegans operon genes, which are resolved into monocistronic transcripts using SL trans-splicing, are upreg- ulated during recovery from growth-arrested states14,50. Operon arrangement is believed to be advantageous in C. elegans during times of limited resources as there are less promoters competing for transcriptional resources50. In the case of H. glycines, SL trans-spliced transcripts were found to be involved in ‘Embryo development ending or egg hatching’ and ‘Nematode larval development,’ suggesting that SL trans-splicing may also be involved in initiating developmental changes in H. glycines. Operon arrangement has not yet been defined in H. glycines, how- ever the clustering of SL trans-spliced transcripts in the genome suggests the presence of operon-like structures.ii To both adapt and improve upon existing SL identification pipelines18,51,52, we developed a SL identification pipeline that utilizes generic RNA-seq, assembled transcripts, and ESTs, rather than requiring SL trapping prior to sequencing53,54. This method provides an alternative to existing pipelines by utilizing the propensity for SLs to be trans-spliced at 5′ ends and avoiding the requirement of unmapped reads having dual genomic mapping18,51. Discussionh The existence of two subsets of Scientific Reports \| (2019) 9:1356 \| https://doi.org/10.1038/s41598-018-37857-0 6 www.nature.com/scientificreports/ Scaffold Number start position Number of consecutive SL trans-spliced genes Scaffold Number start position Number of consecutive SL trans-spliced genes 6 800000 20 4 200000 15 116 100000 19 447 0 15 127 0 19 488 0 15 16 700000 19 5 250000 15 141 100000 18 9 500000 15 466 0 18 10 750000 14 5 800000 18 127 200000 14 116 150000 17 136 150000 14 68 0 17 136 50000 14 95 150000 17 154 50000 14 136 200000 16 160 0 14 192 0 16 171 0 14 2 100000 16 19 400000 14 2 150000 16 210 0 14 21 50000 16 229 0 14 10 100000 15 255 50000 14 123 50000 15 51 450000 14 127 100000 15 7 400000 14 167 50000 15 7 800000 14 345 0 15 8 900000 14 Table 3. SL trans-spliced transcripts are clustered in the H. glycines genome. Table 3. SL trans-spliced transcripts are clustered in the H. glycines genome. Figure 7. Gene Ontology (GO) biological processes for SL trans-spliced transcripts and genes. (A) SL trans-i Figure 7. Gene Ontology (GO) biological processes for SL trans-spliced transcripts and genes. (A) SL trans- spliced transcripts. (B) Specific child terms for SL trans-spliced transcripts. (C) SL trans-spliced genes. Bars represent the number of proteins represented for each GO biological process that was identified using Blast2go (transcripts) or Ontologizer (genes). Figure 7. Gene Ontology (GO) biological processes for SL trans-spliced transcripts and genes. (A) SL trans- spliced transcripts. (B) Specific child terms for SL trans-spliced transcripts. (C) SL trans-spliced genes. Bars represent the number of proteins represented for each GO biological process that was identified using Blast2go (transcripts) or Ontologizer (genes). Scientific Reports \| (2019) 9:1356 \| https://doi.org/10.1038/s41598-018-37857-0 7 www.nature.com/scientificreports/ Effector Total number of genes in the genome Total number of genes that are SL trans-spliced GLAND5 6 3 11A06 5 5 16A01 5 3 5D08 3 1 10C02 2 2 4F01 2 2 17G06 2 1 33E05 2 1 10A06 1 1 19B10 1 1 21E12 1 1 2D01 1 1 33A09 1 1 5A08 1 1 8A07 1 1 flGSB3 1 1 GLAND11 1 1 GLAND12 1 1 GLAND4 1 1 Table 4. Effector genes giving rise to SL trans-spliced transcripts. Materials and Methods Identification and structure prediction of putative SLRNAs. All G. rostochiensis26, C. elegans (PRJNA13758) SL sequences and the 22nt SL sequence from MH119144 were queried to the H. glycines genome with BLASTn V2.4.0 + (E-value 1.0e-3)55. SL hits and the adjacent 3′ 98 nucleotides were extracted using Samtools V1.456. Secondary structure was predicted using RNAfold V2.1.9 with unpaired bases participating in at most one dangling end. All extracted sequences were analyzed for a downstream Sm motif (5′-purine-AU4–6G-purine-3′)57. DNA extraction and amplification. To confirm the functionality of putative SL on transcript MH119144 OP50 H. glycines was propagated on Williams 82 soybean. To isolate mixed-stage nematodes, root tissue was mac- erated with a blender, sieved and separated with a sucrose gradient58. Nematodes were ground in liquid nitrogen and total RNA was extracted using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA). One μg of total RNA was treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA) and cDNA was synthesized using qScript cDNA SuperMix (Quantabio, Beverly, MA, USA). Genomic DNA was also extracted from ground nematode tissue using QIAamp DNA Mini Kit (Qiagen). RT-PCR was performed on a Bio-Rad S1000TM thermal cycler with reactions containing 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, and 1 unit of Taq DNA Polymerase (ThermoFisher Scientific). Thermocycler conditions were: 94 °C for 3 min, 35 cycles of 95 °C for 45 s, 55 °C for 30 s and 72 °C for 1 min, followed by 10 min at 72 °C. Identification of SL trans-spliced transcripts. All putative SL sequences were queried with BLASTn to the H. glycines NCBI EST database, referred to as EST BLAST55,59 and a H. glycines de novo Trinity transcrip- tome assembled from NCBI SRA accession SRP122521, referred to as Direct BLAST36. BLAST hits were filtered to within the first thirteen nucleotides of the transcript, and with a 10nt minimum alignment length. ESTs and trinity transcripts were mapped to the genome using GMAP 20170317, and transcript to gene relationships were identified using exon to exon overlaps with Bedtools intersect V2.26.044,60. Genes were clustered by location using custom bash scripts. Read Analysis. In the method referred to as Read-to-transcript, SLs were truncated to the 11 3′ nucleo- tides and queried with BLASTn V2.4.0+ to Sickle-trimmed (default)61, paired-end reads (word_size 5, -dust no, -task blastn-short) used in generating a H. glycines trinity transcriptome55. Materials and Methods The subject start position for hits was graphed using GraphPad Prism 4. BLAST output was filtered by a 10 bp minimum alignment length and hits within 12 bp of the appropriate read end. Putative SL-containing reads were queried with BLASTn V2.4.0+ to the transcriptome and filtered by 80 bp min alignment length, and within 12 bp of the 5′ transcript end55. Functional Analysis. Functional annotation was performed using Blast2go V4.1. All transcript sequences were searched against the NCBI NR database using Blastx (e-value 1.0–5. Interpro scan was performed using all default applications, and sequences were annotated with an annotation cutoff of 55 and a GO weight of 545,62. GO enrichment for trans-spliced genes was performed using Ontologizer V2.0 with gene functions from the H. glycines genome63. Trans-splicing in Effector and Repetitive Genes. Bedtools intersect V2.26.0 and custom bash scripts were used to identify trans-spliced repetitive genes from a Repeatmodeler V1.0.8 tracks of the genome60,64. Effector genes were mapped to the genome using GMAP 2017031744, and were subjected to bedtools intersect V2.26.0 and custom bash scripts to identify trans-splicing effectors60. Data Availabilityh y The H. glycines expressed sequence tags analyzed during the current study are available through the National Center for Biotechnology Information website. The H. glycines transcriptome data is from the publication https:// doi.org/10.1038/s41598-018-20536-5. The H. glycines genome is publically available on the SCNbase website (https://www.scnbase.org). 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https://openalex.org/W2975399691	https://ieeexplore.ieee.org/ielx7/6287639/8600701/08846691.pdf	English	null	Automatic Modulation Classification Architectures Based on Cyclostationary Features in Impulsive Environments	IEEE access	2,019	cc-by	13,051	Received August 27, 2019, accepted September 12, 2019, date of publication September 23, 2019, date of current version October 4, 2019. Received August 27, 2019, accepted September 12, 2019, date of publication September 23, 2019, date of current version October 4, 2019. Digital Object Identifier 10.1109/ACCESS.2019.2943300 TALES V. R. O. CÂMARA 1, ARTHUR D. L. LIMA1, (Student Member, IEEE), BRUNO M. M. LIMA2, ALUISIO I. R. FONTES 3, ALLAN DE M. MARTINS2, AND LUIZ F. Q. SILVEIRA 1, (Member, IEEE) 1Department of Computer Engineering and Automation, Federal University of Rio Grande do Norte (UFRN), Natal 59078-970, Brazil 2Department of Electrical Engineering, Federal University of Rio Grande do Norte (UFRN), Natal 59078-970, Brazil 3Department of Information, Federal Institute of Rio Grande do Norte (IFRN), Mossoró 59900-000, Brazil C di th T l V R O Câ (t l @ f d b ) is work was supported in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)—Financ de 001, and in part by the High-Performance Computing Center at UFRN (NPAD/UFRN). ABSTRACT Cyclostationary analysis has several applications in communications, e.g., spectral sensing, signal parameter estimation, and modulation classiﬁcation. Most of them consider the additive white Gaussian noise (AWGN) channel model, although wireless communication systems may also be subject to non-Gaussian interference and impulsive noise. In this context, the communication channel can be better modeled by heavy-tailed distributions, such as the non-Gaussian alpha-stable one. Some applications of the cyclostationary approach based on the spatial sign cyclic correlation function (SSCCF), fractional lower-order cyclic autocorrelation function (FLOCAF), and cyclic correntropy function (CCF) demonstrate that these are promising solutions for the analysis of signals in the presence of impulsive non-Gaussian noise. However, the investigation of functions above applied to digital modulation recognition in impulsive environments, and the comparison among them are topics that did not adequately explore yet. This work demonstrates that SSCCF is a particular case of the FLOCAF. Besides, a detailed analysis of the use of the FLOCAF and CCF is presented to obtain cyclostationary descriptors for the recognition of digital modulations BPSK, QPSK, 8-QAM, 16-QAM, and 32-QAM. Automatic modulation classiﬁcation (AMC) architectures, based on the functions mentioned above, are also proposed. Besides, another contribution showed is that both the FLOCAF and CCF allow the symbol rate parameter estimation. The performances of AMC architectures were evaluated in the scenario with modulated signals contaminated with additive non-Gaussian alpha-stable noise. The results demonstrate that both architectures can classify signals in different contamination scenarios. However, the architecture based on the CCF is more efﬁcient than the FLOCAF-based one. INDEX TERMS Additive non-Gaussian alpha-stable noise, correntropy, cyclic correntropy function, cyclo- stationary descriptors, fractional lower-order cyclic autocorrelation function, spatial sign cyclic correlation function, digital modulations, impulsive noise, automatic modulation recognition. INDEX TERMS Additive non-Gaussian alpha-stable noise, correntropy, cyclic correntropy function, cyclo- stationary descriptors, fractional lower-order cyclic autocorrelation function, spatial sign cyclic correlation function, digital modulations, impulsive noise, automatic modulation recognition. INDEX TERMS Additive non-Gaussian alpha-stable noise, correntropy, cyclic correntropy function, cyclo- stationary descriptors, fractional lower-order cyclic autocorrelation function, spatial sign cyclic correlation function, digital modulations, impulsive noise, automatic modulation recognition. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0 Automatic Modulation Classification Architectures Based on Cyclostationary Features in Impulsive Environments TALES V. R. O. CÂMARA 1, ARTHUR D. L. LIMA1, (Student Member, IEEE), BRUNO M. M. LIMA2, ALUISIO I. R. FONTES 3, ALLAN DE M. MARTINS2, AND LUIZ F. Q. SILVEIRA 1, (Member, IEEE) 1Department of Computer Engineering and Automation, Federal University of Rio Grande do Norte (UFRN), Natal 59078-970, Brazil 2Department of Electrical Engineering, Federal University of Rio Grande do Norte (UFRN), Natal 59078-970, Brazil 3Department of Information, Federal Institute of Rio Grande do Norte (IFRN), Mossoró 59900-000, Brazil I. INTRODUCTION mediate stage that exists between reception and signal demodulation. The purpose of automatic modulation classiﬁcation (AMC) is to identify the unknown modulation format of the received noisy signal, in a short period with a hit-rate as high as possible [1], [2]. Therefore, AMC is an inter- AMC applications have arisen in military scenarios, where electronic warfare, surveillance, and threat analysis demand the recognition of signal modulations to identify adver- sary transmitting units, aiming at recovering the inter- cepted signal and jamming. Nowadays, AMC also plays an important role in commercial and civil applications, The associate editor coordinating the review of this manuscript and approving it for publication was Ganesh Naik . 138512 VOLUME 7, 2019 VOLUME 7, 2019 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments The prevalent techniques for the information processing in impulsive non-Gaussian environments are the frac- tional lower-order statistical analysis [35]–[38], corren- tropy measure [39], [40], spatial sign cyclic correlation estimation [41]–[43], fractional lower-order cyclostationary analysis [44], [45], and cyclic correntropy [21], [46], [47]. supporting the development of new paradigms of commu- nication, e.g., cognitive radio, where the transmitter dynam- ically selects the signal modulation type based on channel conditions. AMC algorithms can be classiﬁed into two different categories: likelihood-based (LB) and feature-based (FB) methods [1], [3]. The LB implementation is an optimal solu- tion in terms of the correct classiﬁcation rate. However, it is associated with high computational complexity and demands prior knowledge of statistical information of the received signal, which is usually unavailable in practice. On the other hand, although sub-optimal, the FB is both a blind (without prior information) and more straightforward to implement than the LB-based approach [1], [2], [4]. As far as the authors are aware, approaches based on the spatial sign cyclic correlation estimation and the frac- tional lower-order cyclostationary analysis have not yet been applied to the modulation classiﬁcation issue. Besides, there is no mathematical demonstration that relates both approaches. On the other hand, cyclic correntropy is capable of extract- ing cyclostationary signatures from modulated signals con- taminated by impulsive noise [21], [46], with application in spectral sensing [21]. I. INTRODUCTION However, further investigations still must to be performed in order to evaluate the performance of this technique in recognition of digital modulations in non- Gaussian impulsive environments, such as M-PSK (M-ary phase-shift keying) and M-QAM (M-ary quadrature ampli- tude modulation), which are widely employed in several communication systems [48]–[50]. There are several classiﬁcation approaches based on fea- ture extraction, e.g., instantaneous amplitude, phase, and frequency estimation [5], [6], wavelet transform [7]–[10], use of statistical features such as higher-order moments and cumulants [11]–[14], cyclostationary analysis [2], [15]–[17], and, application of information measures such as correntropy [18]. Particularly, the cyclosta- tionary framework can be highlighted as a powerful method for feature extraction of signals in communication systems. This work aims to analyze the use of the fractional lower- order cyclic autocorrelation function (FLOCAF) and cyclic correntropy function (CCF) to obtain cyclic descriptors that allow the recognition of BPSK, QPSK, 8-QAM, 16-QAM, and 32-QAM modulations. Besides, a classiﬁcation archi- tecture based on such cyclic descriptors is also proposed for the robust classiﬁcation of signals associated with the modulations mentioned above in scenarios with impulsive noise. For over half a century, cyclostationary analysis has been used in several ﬁelds, with emphasis on communication applications [19]. Typical operations in communication sys- tems, e.g., modulation, multiplexing, and coding lead to the manifestation of periodic statistical moments in communi- cation signals, which are then referred to as cyclostationary processes [20]. Classical statistical methods used in signal processing, when applied to cyclostationary signals, only allow performing a limited analysis since they consider the analyzed signal is stationary. However, techniques based on cyclostationary analysis are more suitable to process commu- nication signals [21]. A. CONTRIBUTIONS The main contributions, considering the scope of this work, can be outlined as follows: The main contributions, considering the scope of this work, can be outlined as follows: • A mathematical demonstration is derived to show that the spatial sign cyclic correlation function (SSCCF) is a particular case of the FLOCAF; Cyclostationary analysis allows the extraction of cyclic spectral features from communication signals, also known as cyclostationary signatures, and can be efﬁciently used in Gaussian environments for spectral sensing [22], [23], automatic modulation recognition [23]–[25], and estimation of signal parameters [20], [23], [26]. • A detailed analysis about the cyclic spectrum of BPSK, QPSK, 8-QAM, 16-QAM, and 32-QAM signals obtained by the FLOCAF and CCF is provided, thus evi- dencing that such functions can extract singular descrip- tors capable of distinguishing the modulations above, even in environments contaminated by non-Gaussian alpha-stable noise; Despite being widely used in the literature to model the additive noise in communication, the white Gaussian noise (AWGN) is a limited model when applied to wireless systems, and mobile satellite communications, since such systems are also prone to non-Gaussian inter- ference [27]–[29] and impulsive noise [30]–[32]. In such cases, the alpha-stable distribution appropriately models the communication channels, since it describes the Gaussian and non-Gaussian environments [27], [33], [34]. However, the non-Gaussian alpha-stable distribution does not have deﬁned the second- and higher-order moments. Therefore, in this context, the second- and higher-order cyclostationary analyses are unsuitable for feature extraction from signals in environments modeled by such distribution. • Automatic modulation classiﬁcation architectures are proposed based on analysis of the cyclostationary sig- natures extracted by the FLOCAF and CCF; • An exhaustive investigation on the parameters of the FLOCAF and CCF is presented in order to optimize their respective classiﬁcation architectures; • A performance comparison between the two proposed architectures in impulsive environments is provided; • Numerical results are presented to demonstrate that it is possible to estimate the symbol rate parameter of the sig- nals contaminated by non-Gaussian alpha-stable noise VOLUME 7, 2019 VOLUME 7, 2019 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments FIGURE 1. Probability density functions (fX (X)) in logarithm scale for alpha-stable random variables (X) with different characteristic exponents and β = 0, γ = 1, δ = 0 . 1Particularly, the alpha-stable distribution with (α = 2, β = 0, γ = σ/ √ 2, δ = µ) is reduced to the Gaussian form. from the cyclic descriptors obtained by the FLOCAF and CCF. from the cyclic descriptors obtained by the FLOCAF and CCF. B. PAPER ORGANIZATION This work is organized as follows: initially, Section II intro- duces the main concepts regarding alpha-stable distributions. Section III describes the main cyclostationary analysis func- tions, i.e., CAF, FLOCAF, and CCF. Section IV presents the spectral analysis of the functions mentioned above, applied to investigated modulations; moreover, the spectral analy- sis of the FLOCAF and CCF are extended to modulations contaminated with additive non-Gaussian alpha-stable noise. In Section V, the AMC architectures based on the FLOCAF and CCF are proposed, while Section VI presents the perfor- mance results of these architectures. In Section VII, a method to estimate the symbol rate parameter of the modulated sig- nals, using the FLOCAF and CCF, is described. Finally, the main conclusions and further studies are presented in Section VIII. FIGURE 1. Probability density functions (fX (X)) in logarithm scale for alpha-stable random variables (X) with different characteristic exponents and β = 0, γ = 1, δ = 0 . The lower the value of α, the slower the curve will decay and, therefore, the stronger the noise impulsivity will be. The parameter γ is the dispersion or scaling parameter, whereas β adjusts the distribution symmetry (−1 ≤β ≤1). If β = 0, the distribution is symmetric around its center; if β > 0, the distribution will be asymmetric to the right, whereas it is asymmetric to the left when β < 0. In the particular case where β = 0 and δ = 0, the distributions are called symmetric alpha-stable [54]. The parameter γ is the dispersion or scaling parameter, whereas β adjusts the distribution symmetry (−1 ≤β ≤1). If β = 0, the distribution is symmetric around its center; if β > 0, the distribution will be asymmetric to the right, whereas it is asymmetric to the left when β < 0. In the particular case where β = 0 and δ = 0, the distributions are called symmetric alpha-stable [54]. II. ALPHA-STABLE DISTRIBUTIONS II. ALPHA-STABLE DISTRIBUTIONS Alpha-stable distributions may have Gaussian or non- Gaussian behavior. Non-Gaussian alpha-stable distributions have tails that can be modeled polynomially [51]. In other words, the farthest values from the center of the distribution will occur with a much higher probability in the non-Gaussian case than in the Gaussian one. Due to this heavy-tailed behav- ior, non-Gaussian alpha-stable distributions are commonly used in the modeling of channels subjected to impulsive noise of undetermined variance [26], [29], [52]. Fig. 1 shows the relationship between the impulsivity in symmetric alpha-stable distributions and the tail heaviness in the logarithmic scale. The lower the value assumed by the characteristic exponent (α), the slower the tail will decay and, therefore, the stronger the noise impulsivity will be. Non-Gaussian alpha-stable random variables only present ﬁnite statistical moments of order p when 0 ≤p < α. That is, for a non-Gaussian alpha-stable random variable X, the following properties are valid [55]: The fundamental principles associated with alpha-stable modeling are based on the generalized central limit theo- rem, which demonstrates that the sum of several independent and identically distributed (i.i.d.) random variables, with and without ﬁnite variance, converge to an alpha-stable distribu- tion [34]. This behavior occurs due to the stability property, i.e., the linear combination of i.i.d. random variables whose distributions are alpha-stable result in another alpha-stable random variable [53]. E \|X\|p = ∞, if p ≥α (3) (3) and E \|X\|p < ∞, if 0 ≤p < α. (4) (4) If α = 2, then: If α = 2, then: If α = 2, then: E \|X\|p < ∞, for all p ≥0. (5) (5) Since the most distributions that belong to the alpha-stable family do not have a closed equation to deﬁne the probability density function, they are typically represented by the follow- ing characteristic function [53]: Therefore, alpha-stable distributions have no ﬁnite ﬁrst- or higher-order moments for 0 < α ≤ 1. Otherwise, if 1 < α < 2, they have the ﬁnite ﬁrst-order moment and all the fractional moments of order p < α. In this case, the parameter δ represents the mean value of the distribution. For α = 2, all moments exist, and parameter γ is directly related to the distribution variance. In particular, all non- Gaussian alpha-stable distributions have inﬁnite variance. A. CONTRIBUTIONS The lower the value of α, the slower the curve will decay and, therefore, the stronger the noise impulsivity will be. from the cyclic descriptors obtained by the FLOCAF and CCF. II. ALPHA-STABLE DISTRIBUTIONS φ(ω, α, β, γ, δ)=exp γ α −\|ω\|α + jω2(ω, α, β) +jδω , (1) φ(ω, α, β, γ, δ)=exp γ α −\|ω\|α + jω2(ω, α, β) +jδω , where: 2(ω, α, β) =    β\|ω\|α−1 tan πα 2 α ̸= 1 −β 2 π ln \|ω\| α = 1, (2) (2) For some speciﬁc values of α, an alpha-stable distribution can be reduced to a simpliﬁed form that has a probability density function deﬁned by an exact expression, such as the Gaussian1 (α = 2), Cauchy-Lorentz (α = 1), and Lèvy distributions (α = 0.5). being α the characteristic exponent or distribution stability index (0 < α ≤2). In non-Gaussian alpha-stable distribu- tions (α < 2), this parameter adjusts the impulsivity level of the density function. In turn, the parameter δ is respon- sible for adjusting the location of the distribution center. 1Particularly, the alpha-stable distribution with (α = 2, β = 0, γ = σ/ √ 2, δ = µ) is reduced to the Gaussian form. 138514 VOLUME 7, 2019 VOLUME 7, 2019 VOLUME 7, 2019 VOLUME 7, 2019 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments FIGURE 2. Second-order cyclic autocorrelation function applied to BPSK signal without and with alpha-stable contamination. When contamination exists, it is not possible extract second-order cyclostationary features. In channels that follow a non-Gaussian alpha-stable dis- tribution, the signal-to-noise ratio (SNR) is not used as a channel quality indicator, since this measure will assume a value equal to zero. In this case, the geometric signal-to- noise ratio (GSNR) is usually employed as a quality indicator, which can be calculated as follows [56], [57]: GSNR = A2 2γ 2C 2 α −1 g , (6) (6) where A is the root mean square (RMS) value of the transmit- ted signal; Cg = eCe ≈1.78 is the exponential of the Euler constant, and Ce ≈0.5772 is the Euler constant [33]. In the Gaussian case, the exponential of Euler constant ensures that the GSNR corresponds to SNR. FIGURE 2. Second-order cyclic autocorrelation function applied to BPSK signal without and with alpha-stable contamination. When contamination exists, it is not possible extract second-order cyclostationary features. Although the second-order cyclostationary analysis has several applications, it is not suitable for non-Gaussian alpha- stable process [21], since they have an unlimited second- order moment. A. CYCLIC AUTOCORRELATION FUNCTION A random process x(t) is said cyclostationary in the wide sense when its mean value E[x(t)] and its autocorrelation function Rx(t, τ) are both periodic in t within a period T for any value of τ [20], i.e.: B. FRACTIONAL LOWER-ORDER CYCLIC AUTOCORRELATION FUNCTION Since signals contaminated by additive non-Gaussian alpha- stable noise cannot be analyzed for statistical moments of order p ≥2, the statistical characterization of such signals can be achieved by using lower-order statistical moments, particularly the fractional lower-order covariance (FLOC), which can be deﬁned as [45]: E {x(t + T)} = E {x(t)} , (7) Rx(t + T, τ) = Rx(t, τ), (8) (7) (8) (8) where Rx(t, τ) = E x(t)x∗(t + τ) . (9) (9) Ra,b x (t, τ)=E nh x(t)\|x(t)\|(a−1)i × h x∗(t+τ)\|x∗(t+τ)\|(b−1)io , (13) Thus, in second-order cyclostationary processes, the auto- correlation function can be expanded in terms of the Fourier series as: io (13) where a and b are the fractional lower-order parameters of Ra,b x (t, τ). Unlike second-order moments, FLOC is ﬁnite when applied to symmetric alpha-stable distributions when- ever the following inequality is valid: where a and b are the fractional lower-order parameters of Ra,b x (t, τ). Unlike second-order moments, FLOC is ﬁnite when applied to symmetric alpha-stable distributions when- ever the following inequality is valid: Rx(t, τ) = X ϵ Rϵ x(τ)ej2πϵt, (10) (10) where Rϵ x(τ) represents the Fourier series coefﬁcients given by: 0 ≤a, b < α/2. (14) (14) Rϵ x(τ) ≜1 T Z T/2 −T/2 Rx(t, τ)e−j2πϵtdt. (11) (11) When incorporated into the cyclostationary analysis, FLOC gives rise to the fractional lower-order cyclic autocor- relation function (FLOCAF), which is deﬁned as [45]: Such coefﬁcients deﬁne the cyclic autocorrelation function (CAF), where ϵ is a discrete parameter hereafter called cyclic frequency (ϵ = n/T, ∀n ∈Z). The CAF can be interpreted as an analytic function that veriﬁes if the process x(t) is cyclostationary by evaluating if Rϵ x(τ) is non-null for at least a ϵ ̸= 0. In turn, when Rx(t, τ) has multiple fundamental frequencies, then x(t) is said to be a polycyclostationary process [19]. In this case, the CAF can be expressed by [19]: Rϵ,ab x (τ) ≜1 T Z T/2 −T/2 Ra,b x (t, τ)e−j2πϵtdt. (15) (15) In this case, the FLOCAF makes it possible to verify if a given process x(t) is cyclostationary, even when contaminated by inﬁnite variance noise by evaluating if Rϵ,ab x (τ) is non-null for any ϵ ̸= 0. Rϵ x(τ) ≜ lim T→∞ 1 T Z T/2 −T/2 Rx(t, τ)e−j2πϵtdt. III. CYCLOSTATIONARY ANALYSIS This section introduces the main statistical cyclostation- ary functions, namely the cyclic autocorrelation function, the fractional lower-order cyclic autocorrelation function, and the cyclic correntropy function. The concepts and mathemat- ical basis of the functions are described in detail so that they can be appropriately compared with each other. II. ALPHA-STABLE DISTRIBUTIONS This aspect can be observed in Fig. 2, which shows two cyclostationary signatures of a BPSK signal, being the ﬁrst one without contamination and the second one con- taminated by symmetrical and centralized alpha-stable addi- tive noise with GSNR = 15 dB and α = 1.5. When the BPSK signal is contaminated by non-Gaussian alpha-stable noise of inﬁnite variance, it is not possible to obtain the second-order cyclostationary features. B. FRACTIONAL LOWER-ORDER CYCLIC AUTOCORRELATION FUNCTION (20 T ∞T Z −T/2 (20) (20) V ϵ x (τ) ≜ lim T→∞ 1 T Z T/2 −T/2 Vx(t, τ)e−j2πϵtdt. (27) (27) Equation (20) is also known as the spatial sign cyclic correlation function (SSCCF), being denoted by RS(ϵ, τ) [41], [43]. Using the Gaussian kernel Gσ(·, ·) for the calculation of the cyclic correntropy and considering operator ⟨·⟩, deﬁned by: Thus, the SSCCF can be seen as a particular case of FLOCAF if the parameters a and b of the latter function are both equal to zero. The SSCCF will always converge when applied to alpha-stable distributions, independent of the characteristic exponent (α) because it will always meet the condition established in (14). In other words, this function is more robust to the presence of impulsive noise than any other FLOCAF conﬁguration. However, there is no guarantee that the SSCCF returns the best cyclic descriptors of a signal when considering all other FLOCAF settings in terms of a and b. ⟨·⟩= lim T→∞ 1 2T Z T −T (·) dt, (28) (28) then (27) can be rewritten as: V ϵ x (τ) = D E{Gσ(x(t), x(t + τ))}e−j2πϵtE . (29) (29) Assuming that V ϵ x (τ) is polycycloergodic [19], [21], it is possible to represent (29) as [21]: B. FRACTIONAL LOWER-ORDER CYCLIC AUTOCORRELATION FUNCTION (12) An interesting aspect of the FLOCAF can be seen when its parameters of lower-order statistical moments (a, b) are zero. (12) VOLUME 7, 2019 VOLUME 7, 2019 138515 138515 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments where σ is the kernel size. Thus, it is possible to rewrite (21) as: In this case, FLOC can be represented as follows: as: R0,0 x (t, τ) = E x(t) \|x(t)\| × x∗(t + τ) \|x∗(t + τ)\| . (16) (16) Vx(t, τ) = E {Gσ(x(t), x(t + τ))} . (23) (23) However, in order to avoid discontinuity when x(t) and x(t + τ) are equal to zero, R0,0 x must be represented in terms of the spatial sign function [41], [58] deﬁned by: Let us assume that the correntropy function is periodic in t with period T, i.e., Vx(t + T, τ) = Vx(t, τ). (24) (24) S (x(t)) =    x(t) \|x(t)\| x(t) ̸= 0 0 x(t) = 0. (17) (17) Therefore, it can be expanded in the form of a Fourier series as follows: , as follows: Therefore, (16) can be rewritten as: Therefore, (16) can be rewritten as: Vx(t, τ) = X ϵ V ϵ x (τ)ej2πϵt, (25) (25) R0,0 x (t, τ) = E S (x(t)) S x∗(t + τ) . (18) (18) where V ϵ x (τ) represents the series coefﬁcients, given by: where V ϵ x (τ) represents the series coefﬁcients, given by: Applying R0,0 x (t, τ) to FLOCAF and assuming that x(t) is a polycyclostationary process gives: Rϵ,00 x (τ) = lim T→∞ 1 T Z T/2 −T/2 E S(x(t))S(x∗(t + τ)) e−j2πϵtdt, (19) Rϵ,00 x (τ) = lim T→∞ 1 T Z T/2 −T/2 E S(x(t))S(x∗(t + τ)) e−j2πϵtdt, V ϵ x (τ) = 1 T Z T/2 −T/2 Vx(t, τ)e−j2πϵtdt, (26) (26) (19) which deﬁnes the CCF. which deﬁnes the CCF. and considering that Rϵ,00 x (τ) is polycycloergodic, it is possi- ble to rewrite (19) as: Analogously to the CAF, parameter ϵ represents the cyclic frequencies (ϵ = n/T, ∀n ∈Z). When Vx(t, τ) has multiple fundamental frequencies, then x(t) is said to be a polycyclo- stationary process [21]. In this case, the CCF can be deﬁned as: Rϵ,00 x (τ) ≜ lim T→∞ 1 T Z T/2 −T/2 S(x(t))S(x∗(t + τ))e−j2πϵtdt. C. CYCLIC CORRENTROPY FUNCTION V ϵ x (τ) = D Gσ(x(t), x(t + τ))e−j2πϵtE = 1 √ 2πσ exp −[x(t) −x(t + τ)]2 −2σ 2j2πϵt 2σ 2 . (30) Another approach for the analysis of cyclostationary features of signals contaminated with additive non-Gaussian alpha- stable noise can be deﬁned when using the cyclic correntropy function (CCF). The CCF is based on the correntropy func- tion, which is a nonlinear transformation deﬁned by [39]: (30) Vx(t, τ) = E {kσ(x(t) −x(t + τ))} , (21) (21) Due to the Gaussian kernel exponential decay in (30), the cyclic correntropy function will always converge, even when calculated over a process x(t) subjected to an additive impulsive noise modeled by a non-Gaussian alpha-stable process. This issue ensures robustness of the function to outliers generated by impulsive noise channels, which have less inﬂuence on V ϵ x (τ) since they are further from the Gaussian kernel center. Another interesting feature addressed where kσ(·) is any unimodal, symmetric, and positive deﬁnite function, denominated kernel. In this work, a Gaussian kernel is employed, which is deﬁned by [21]: Gσ (x(t), x(t + τ)) = 1 σ √ 2π exp −[x(t) −x(t + τ)]2 2σ 2 , (22) 138516 VOLUME 7, 2019 mpulsive Environments FIGURE 3. BPSK cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. FIGURE 4. QPSK cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments FIGURE 3. BPSK cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. FIGURE 3. BPSK cyclostationary signatures obtained by the CAF, FLOCAF, to the CCF can be observed while expanding (29) into a Maclaurin series: V ϵ x (τ) = 1 σ √ 2π * ∞ X n=0 (−1)n (2σ 2)nn!E{[(x(t) −x(t + τ))2 V ϵ x (τ) = 1 σ √ 2π * ∞ X n=0 (−1)n (2σ 2)nn!E{[(x(t) −x(t + τ))2 +2σ 22jπϵt]n} + . (31) +2σ 22jπϵt]n} + . (31) (31) From (31), it can be stated that V ϵ x (τ) contains inﬁnite statistical moments information associated with the second- order one, which are strongly related to the kernel size (σ). Larger kernel sizes will contribute to lower-order statistical moments, with a greater inﬂuence of such moments on the calculation of the CCF. C. CYCLIC CORRENTROPY FUNCTION On the other hand, smaller ones will make the high-order moments more relevant in the calcula- tion of V ϵ x (τ). FIGURE 3. BPSK cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. Due to such characteristics, the cyclic correntropy may be able to extract higher-order statistical information from cyclostationary processes that cannot be typically analyzed by the CAF, since the latter function performs a second-order analysis. FIGURE 4. QPSK cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. FIGURE 4. QPSK cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. IV. CYCLIC SPECTRAL ANALYSIS In this section, the cyclic spectral analysis of BPSK, QPSK, 8-QAM, 16-QAM, and, 32-QAM modulations, is pro- vided. First, a comparison among cyclostationary signatures obtained by the CAF, FLOCAF, and, CCF, without noise contamination, is presented. Then, an examination of the FLOCAF and CCF signatures, on the alpha-stable channel, is discussed. 2This value of characteristic exponent was adopted to deﬁne a distribution fairly impulsive [45], [59] A. CAF VERSUS FLOCAF VERSUS CCF WITHOUT CONTAMINATION As treated in the previous section, since the FLOCAF and CCF converge, even under contamination of noise with inﬁ- nite variance, both functions can provide cyclostationary sig- natures robust to impulsive noise contamination. However, a detailed spectral analysis of the cyclostationary signatures for digital modulations, using the functions mentioned above, has not yet been performed in the literature, particularly in the case of M-PSK and M-QAM. FIGURE 4. QPSK cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. Fig. 3 shows the BPSK cyclostationary signatures generated respectively by the CAF, FLOCAF, and CCF. The signature generated by FLOCAF contains all the cyclic descriptors that exist in the CAF signature, but additional cyclic components at ±2fb/fs and ±4fi/fs are evidenced in this case. In turn, a signature generated by the CCF contains all the cyclic components provided by FLOCAF, among which some of them present higher amplitudes, while new cyclic components are presented at (2fi ±2fb)/fs, (4fi ±fb)/fs and (4fi ± 2fb)/fs. Therefore, this section provides an investigation of the ability from the FLOCAF and CCF to extract singular cyclo- stationary features from modulations BPSK, QPSK, 8-QAM, 16-QAM, and, 32-QAM, provided by the FLOCAF and CCF. For a comprehensive comparative analysis, the cyclostation- ary signatures produced by the CAF is also evaluated. Fig. 4 shows the cyclostationary signatures associated with the QPSK modulation. In this case, the signature generated by FLOCAF aggregates all cyclic components provided in the CAF signature, and also cyclic components at ±2fb/fs, ±4fi/fs and ±(4fi ± fb)/fs. On the other hand, the signa- ture generated by the CCF presents all the cyclic compo- nents regarding FLOCAF signature, and also new ones at ±(2fi ± 2fb)/fs and ±(4fi ± 2fb)/fs. The functions are applied to modulated signals with the same intermediate frequency fi, symbol rate fb, and sampling frequency fs to extract the signatures. The cyclic frequency at ϵ = 0 was suppressed in all obtained signatures since it is a common parameter. Besides, this central component is the one that carries the most amount of energy, being respon- sible for hiding the other cyclic frequencies components. The cyclostationary features extracted by each function when applied to noiseless signals are represented in Fig. 3 to Fig. 7. The results in Fig. 5 to Fig. 7 show that the cyclostationary signatures of the 8-, 16- and 32-QAM modulations have 138517 138517 VOLUME 7, 2019 T. V. R. O. A. CAF VERSUS FLOCAF VERSUS CCF WITHOUT CONTAMINATION Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments y y p FIGURE 5. 8-QAM cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. FIGURE 6. 16-QAM cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. FIGURE 7. 32-QAM cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. components presented by the FLOCAF, and also additional cyclic frequency ones. In this case, the signatures obtained with the CCF present some differences between 16-QAM and 32-QAM. Moreover, all the cyclic components presented in the sig- natures generated by the CCF are directly related to the intermediate frequency (fi) and symbol rate (fb) of the ana- lyzed signals. Thus, in addition to robustness to impulsive noise [21], [45], the FLOCAF and CCF are capable of extract- ing unique signatures from BPSK, QPSK, 8-QAM, 16-QAM, and 32-QAM. B. FLOCAF VERSUS CCF ON ALPHA-STABLE CHANNEL The performances of the FLOCAF and CCF for obtaining cyclostationary features from digital modulations, in impul- sive environments, are discussed in this section. For that, an analysis with BPSK, QPSK, 8-QAM, 16-QAM, and, 32 QAM i l i d b ddi i i l h FIGURE 7. 32-QAM cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. FIGURE 5. 8-QAM cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. FIGURE 7. 32-QAM cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. FLOCAF, and CCF. components presented by the FLOCAF, and also additional cyclic frequency ones. In this case, the signatures obtained with the CCF present some differences between 16-QAM and 32-QAM. Moreover, all the cyclic components presented in the sig- natures generated by the CCF are directly related to the intermediate frequency (fi) and symbol rate (fb) of the ana- lyzed signals. Thus, in addition to robustness to impulsive noise [21], [45], the FLOCAF and CCF are capable of extract- ing unique signatures from BPSK, QPSK, 8-QAM, 16-QAM, and 32-QAM. B. FLOCAF VERSUS CCF ON ALPHA-STABLE CHANNEL The performances of the FLOCAF and CCF for obtaining cyclostationary features from digital modulations, in impul- sive environments, are discussed in this section. For that, an analysis with BPSK, QPSK, 8-QAM, 16-QAM, and, 32-QAM signals contaminated by additive symmetric alpha- stable noise, is provided. The contaminated signal can be expressed as: FIGURE 6. 16-QAM cyclostationary signatures obtained by the CAF, FLOCAF, and CCF. similar behavior to those previously described for the BPSK and QPSK modulations. In all cases, the signatures provided by FLOCAF contain all cyclic components presented by the signatures generated by CAF, while also introducing new descriptors. Similarly, the CCF signatures contain all the cyclic components regarding FLOCAF and also present new components in the respective cyclic spectrum. x(t) = s(t) + n(t), (32) (32) where, n(t) is the α-stable noise and s(t) = A(t)cos(2πfi + θ(t)). (33) (33) The signatures generated by CAF shows that it is not effective in extracting cyclic information capable of char- acterizing, properly, each modulation. The cyclic signatures associated with BPSK and 8-QAM shown in Fig. 3 and Fig. 5, respectively, have the same proﬁle. This aspect can also be seen at QPSK, 16-QAM, and 32-QAM, which have identical signatures. The term A(t) is the amplitude of the signal, fi represents an intermediate frequency, and θ(t) is the signal phase. All these parameters are deﬁned according to the modulation format adopted for information transmission. Table 1 presents the sampling frequency, intermediate fre- quency, and symbol rate employed in the generation of the modulated signals, besides the parameters regarding the CCF and FLOCAF setting. The channel was modeled as an alpha- stable distribution with the following parameters: character- istic exponent α = 1.52, symmetry β = 0, location δ = 0, On the other hand, the FLOCAF is capable of extracting singular descriptors for each modulation technique, although the signatures regarding 16-QAM and 32-QAM present slight differences as shown in Fig. 6 and Fig. 7, respectively. The CCF is also able to extract unique signatures for each modulation. The obtained signatures contain all 138518 VOLUME 7, 2019 VOLUME 7, 2019 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments TABLE 1. Simulation parameters. FIGURE 8. Fractional lower-order cyclic autocorrelation function versus cyclic correntropy function (GSNR = 10 dB). B. FLOCAF VERSUS CCF ON ALPHA-STABLE CHANNEL On the other hand, the signatures generated by the CCF present enough cyclostationary information to allow dis- tinguishing the modulations accurately, with less difference between 16-QAM and 32-QAM. Since the signatures, provided by the FLOCAF and CCF, presents dynamic behaviors for the variation of GSNR con- ditions, a more accurate comparing of both functions is pro- vided applying them to automatic modulation classiﬁcation architectures. FIGURE 8. Fractional lower-order cyclic autocorrelation function versus cyclic correntropy function (GSNR = 10 dB). FIGURE 8. Fractional lower-order cyclic autocorrelation function versus cyclic correntropy function (GSNR = 10 dB). Therefore, in Section V, AMC architectures, based on the FLOCAF and CCF, are proposed, and in Section VI the performances of both architectures are compared. V. AUTOMATIC MODULATION CLASSIFICATION ARCHITECTURES As previously demonstrated, the FLOCAF and CCF can extract singular cyclostationary descriptors for each ana- lyzed modulation even in impulsive noise environments. This section describes the AMC architectures based on the func- tions mentioned above. The schematic diagram of the archi- tectures is depicted in Fig . 10. FIGURE 8. Fractional lower-order cyclic autocorrelation function versus cyclic correntropy function (GSNR = 10 dB). FIGURE 9. Fractional lower-order cyclic autocorrelation function versus cyclic correntropy function (GSNR = 0 dB). A. DEFINITION OF MODULATION TEMPLATES The architectures perform a comparison between the cyclic proﬁles of the input signal and the cyclic proﬁles of the noise- less modulation signals available for classiﬁcation, which will be called hereafter by templates. The main difference between the architectures is the cyclostationary function used in the calculation of cyclic proﬁles. The FLOCAF-based architecture uses FLOCAF to generate the templates and the cyclic proﬁle of the input signal. The same approach speciﬁes CCF-based architecture. B. UNKNOWN SIGNAL FIGURE 9. Fractional lower-order cyclic autocorrelation function versus cyclic correntropy function (GSNR = 0 dB). The cyclic proﬁle of the unknown input signal must be gen- erated following the same processes used in the templates. Thus, if the architecture uses FLOCAF, the fractional lower- order moments (a and b) that conﬁgure the function must be the same for both: the templates and the input signal cyclic proﬁle. Similarly, if the architecture uses the CCF, both the templates and the signature of the input signal must be parameterized with the same kernel size (σ). and GSNR equal to 10 dB, and 0 dB. The cyclostationary signatures obtained are shown in Fig. 8 and Fig. 9. Fig. 8 compares the signatures of the modulations gener- ated by the FLOCAF and CCF considering an alpha-stable additive noise contamination with GSNR = 10 dB. In both cases, the functions can obtain unique signatures for each one of the modulations, since the speciﬁc positions and ampli- tudes of the cyclic components are not the same. 3In fact, the Pearson correlation coefﬁcient when negative is said to be inversely correlated. D. CSIG/C16QAM RATIO According to Fig. 6 and Fig. 7, the cyclostationary signatures of 16-QAM and 32-QAM, generated by the FLOCAF and CCF, have few differences. Among them, the most evident one is the cyclic component at the spectral position 4fi that appears in the signatures associated with the 16-QAM, while it is absent in the 32-QAM. Besides that, Fig. 4 shows that the element at the spectral position 4fi, in the QPSK signature, is also the principal difference between that modulation and 16-QAM, whereas this component in the QPSK signature is more signiﬁcant than in the 16-QAM signature. This way, to classify the input signal among QPSK, 16-QAM, and, 32-QAM, was used the ratio between its cyclic component at position 4fi (Csig) and the component of the template of 16-QAM (C16QAM) at the same spectral position 4fi. The criterion applied to choose the class in which the signal belongs consists of comparing the rela- tion Csig/C16QAM with two thresholds, the QPSK threshold (thQPSK) and the 16-QAM threshold (th16QAM). In case that Csig/C16QAM > thQPSK, the input signal will be attributed to the QPSK class. When thQPSK > Csig/C16QAM > th16QAM, the input signal will be classify as 16-QAM. Otherwise, if th16QAM > Csig/C16QAM, the choose class will be 32-QAM. The thresholds are deﬁned based on an extensive investigation of Csig/C16QAM ratio, in a speciﬁc GSNR condition. This process is more detailed in the next section. FIGURE 10. AMC Architecture based on cyclostationary feature extraction. 0.7 < \|ρ\| ≤0.9 indicates strong correlation; \|ρ\| > 0.9 indicates very strong correlation3 [60]. C. CORRELATION COEFFICIENT Fig. 9 represents the cyclostationary signatures for signals contaminated by alpha-stable noise with GSNR = 0 dB obtained by the FLOCAF and CCF. The comparing of both ﬁgures above shows that, as the impulsive noise contamina- tion grows, the degradation of the cyclostationary signatures increases. The second step of the proposed architectures is to compare the unknown signal with the templates. For this, the simi- larity measure, known as the Pearson correlation coefﬁcient, is adopted in this work, which is given by: ρ = COV(X, Y) √VAR(X)VAR(Y). (34) (34) In this context, the signatures generated by FLOCAF are very sensitive to noise contamination. In other words, the cyclic frequency components, of some signatures, when in low GSNR condition, are signiﬁcantly attenuated and even faded, resulting in difﬁculty to distinguish among modulation signatures, e.g., QPSK, 16-QAM, and 32-QAM. Pearson’s correlation coefﬁcient is a technique of low computational complexity that returns values ranging from −1 to 1, which can be classiﬁed as follows: \|ρ\| < 0.3 denotes uncorrelated elements; 0.3 ≤\|ρ\| ≤0.5 denotes poor correlation; 0.5 < \|ρ\| ≤0.7 denotes moderate correlation; VOLUME 7, 2019 138519 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments FIGURE 10. AMC Architecture based on cyclostationary feature extraction. The incidence of false-positives decreases as higher the threshold; however, an elevated threshold also contributes to decreasing of true-positives classiﬁcation, which results in a decrease of classiﬁer hit-rate. Therefore, to establish a suitable hit-rate adjust, and limit the false-positive occur- rences, the chosen threshold was Cth = 0.7, since this value guarantees moderate to strong correlation. In case LCC > Cth, the decision of the input signal class is made. When the LCC is associated with the BPSK or 8-QAM template, the predicted modulation is respectively attributed to BPSK or 8-QAM class. Otherwise, the architec- tures will follow other steps to distinguish QPSK, 16-QAM, and, 32-QAM. VI. AMC EXPERIMENTS In the proposed architectures, the cyclostationary signature of the input signal is correlated with all the templates, where the largest coefﬁcient (LCC) value among them is used to classiﬁcation. The chosen coefﬁcient is compared with a previously deﬁned correlation threshold (Cth). If the largest correlation coefﬁcient is lower than Cth, the input signal will not be recognized. In this section, the performances of the classiﬁcation archi- tectures based on the cyclostationary features, obtained by the FLOCAF and CCF, are evaluated in impulsive environments. In this section, the performances of the classiﬁcation archi- tectures based on the cyclostationary features, obtained by the FLOCAF and CCF, are evaluated in impulsive environments. The environments were characterized by a non-Gaussian additive alpha-stable noise with the following parameters: characteristic exponent α = 1.5, symmetry β = 0, location δ = 0, and GSNR ranging from −10 dB to 10 dB with the step of 2 dB. The modulated signal parameters, i.e., sampling frequency, intermediate frequency, and symbol rate, are the same deﬁned in Table 1 from Section IV-B. The environments were characterized by a non-Gaussian additive alpha-stable noise with the following parameters: characteristic exponent α = 1.5, symmetry β = 0, location δ = 0, and GSNR ranging from −10 dB to 10 dB with the step of 2 dB. The modulated signal parameters, i.e., sampling frequency, intermediate frequency, and symbol rate, are the same deﬁned in Table 1 from Section IV-B. This action contributes to reducing the false-positive prob- lem, that occurs when the predicted modulation is improperly indicated. In other words, the correlation criterion supports the correct modulation classiﬁcation, helping to reduce the biased decision for a speciﬁc class. A. AMC USING THE FLOCAF In the FLOCAF-based AMC architecture test, an exten- sive search is performed to determine the fractional 138520 138520 VOLUME 7, 2019 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments mpulsive Environments FIGURE 12. Average Global classification hit-rate for the considered GSNR range, for distinct fractional lower-order moments, obtained by the FLOCAF-based architecture. FIGURE 13. Definition of thqpsk and th16QAM thresholds. FIGURE 11. Global automatic modulation classification using the FLOCAF for different kernel sizes, after calculating the average of GSNR. FIGURE 12. Average Global classification hit-rate for the considered GSNR range, for distinct fractional lower-order moments, obtained by the FLOCAF-based architecture. FIGURE 11. Global automatic modulation classification using the FLOCAF for different kernel sizes, after calculating the average of GSNR. FIGURE 11. Global automatic modulation classification using the FLOCAF for different kernel sizes, after calculating the average of GSNR. FIGURE 12. Average Global classification hit-rate for the considered GSNR range, for distinct fractional lower-order moments, obtained by the FLOCAF-based architecture. FIGURE 12. Average Global classification hit-rate for the considered FIGURE 11. Global automatic modulation classification using the FLOCAF for different kernel sizes, after calculating the average of GSNR. FIGURE 12. Average Global classification hit-rate for the considered GSNR range, for distinct fractional lower-order moments, obtained by the FLOCAF-based architecture. lower-order parameters (i.e., a and b) that allow optimizing its performance. Thus, the architecture was parameterized for all combinations of a and b assuming the following values: {0; 0.1; 0.3; 0.5}, which are chosen to meet the convergence criterion represented in (14). FIGURE 13. Definition of thqpsk and th16QAM thresholds. FIGURE 13. Definition of thqpsk and th16QAM thresholds. The architecture was tested as follows: ﬁrst, the noisy input signals, which can assume any of the proposed modulations, were generated several times for each value of GSNR and applied to the architecture. Then, the global classiﬁcation rate, which is the average among the classiﬁcation rates of each modulation, was calculated considering 100 iterations per modulation, for each value of GSNR. Finally, different classiﬁcation curves were obtained, changing the fractional lower-order parameters and varying the GSNR. Fig. 11 shows that the FLOCAF-based architecture has distinct behavior, in terms of hit-rate, for each combination of a and b, which also varies with the GSNR. B. AMC USING CCF In the test of the CCF-based AMC architecture, analogously to the case of FLOCAF, an extensive search is performed to determine the best kernel size (σ) that allows optimizing the overall performance. In this case, all values in the range from 0.1 to 1.4 with steps of 0.1 were evaluated. The experiment was conducted as follows: ﬁrst, the noisy input signal, which may assume one of the modulations, was generated several times for each GSNR condition and applied to the architecture. Then, the global classiﬁcation rate was calculated, considering 100 iterations per modulation, for each value of GSNR. Finally, different classiﬁcation curves were obtained for each modulation considering the GSNR variation. The results are represented in Fig. 15. modulations. While for QPSK modulation, the architecture needs GSNR = 4 dB. When GSNR = 8 dB, the architecture achieves a hit- rate close to 100% for all modulations. Since the classiﬁer presents a particular behavior in each GSNR condition, for more detailed analysis, the confusion matrices of some GSNR instances are given in Tables 3 to 5. Fig. 15 shows that the CCF-based AMC architecture has distinct behavior, in terms of hit-rate, for each kernel size, which also varies with the GSNR. Therefore, to determine the value that optimizes the architecture performance for a medium-quality channel, the average of global hit-rate related to the GSNR range was veriﬁed. The summarized results show that, for low GSNR levels, the QPSK modulation is widely confused with 16-QAM. Thus, it is inferred that for a very high noise level, some cyclic components from QPSK modulation are attenuated resulting in a signature that is more approximate of the 16-QAM template than of the QPSK template. However, the matrices also indicate that, as the GSNR increases, the accuracy of QPSK classiﬁcation grows. The results in Fig. 16 denote that, on average, the archi- tecture achieved the highest hit-rate when parameterized with σ = 1.2. Thus, after ﬁnding the best kernel size for the investigated conﬁgurations, more detailed classiﬁer tests, considering 1,000 iterations per modulation, for each GSNR value, can be performed. Similar behavior occurs for the 16-QAM classiﬁcation, for low GSNR levels, this modulation is widely confused with 32-QAM. Just as in the case discussed above, as the GSNR increases, the accuracy of the 16-QAM classiﬁcation also increases. A. AMC USING THE FLOCAF Thresholds of FLOCAF-based AMC architecture. TABLE 4. Confusion Matrix of FLOCAF (a = 0.3, b = 0.5, GSNR = 4 dB). TABLE 4. Confusion Matrix of FLOCAF (a = 0.3, b = 0.5, GSNR = 4 dB). TABLE 2. Thresholds of FLOCAF-based AMC architecture. TABLE 2. Thresholds of FLOCAF-based AMC architecture. TABLE 5. Confusion Matrix of FLOCAF (a = 0.3, b = 0.5, GSNR = 10 dB). TABLE 5. Confusion Matrix of FLOCAF (a = 0.3, b = 0.5, GSNR = 10 dB). slower, which indicates that 16-QAM is more sensitive to noise contamination than other analyzed modulations. These results also indicate that, for the classiﬁcation cri- teria adopted in the proposed architecture, the cyclic signa- tures of BPSK, 8-QAM, and, 32-QAM are the most robust to impulsive noise contamination, once that, even for some negative GSNR values, the classiﬁcation hit-rates for these modulations are elevated. FIGURE 14. Modulation classification curves of FLOCAF-based architecture with a = 0.3 and b = 0.5, and 1,000 interactions per GSNR FIGURE 14. Modulation classification curves of FLOCAF-based architecture with a = 0.3 and b = 0.5, and 1,000 interactions per GSNR. TABLE 3. Confusion Matrix of FLOCAF (a = 0.3, b = 0.5, GSNR = 0 dB). TABLE 3. Confusion Matrix of FLOCAF (a = 0.3, b = 0.5, GSNR = 0 dB). TABLE 3. Confusion Matrix of FLOCAF (a = 0.3, b = 0.5, GSNR = 0 dB). TABLE 3. Confusion Matrix of FLOCAF (a = 0.3, b = 0.5, GSNR = 0 dB). A. AMC USING THE FLOCAF Therefore, to deter- mine the fractional lower-order parameters that optimize the architecture performance for a medium-quality channel in terms of the GSNR, an average hit-rate was calculated con- sidering the GSNR ranging from -10 dB to 10 dB. The result is shown in Fig. 12. FIGURE 13. Definition of thqpsk and th16QAM thresholds. 32-QAM modulations, under the noise contamination corre- sponding to GSNR = 10 dB. For that, each modulation signa- ture was generated 1, 000 times, and the factor Csig/C16QAM was calculated for each one of them. In the particular case when a = b = 0, FLOCAF becomes the SSCCF, and therefore, the cyclostationary descriptors will be less affected by impulsive noise. However, the results in Fig. 12 demonstrate that, on average, the architecture can correctly classify more modulation types when parameterized with a = 0.3 and b = 0.5. This result suggests that the digital modulations evaluated contain cyclostationary information that is better evidenced by such speciﬁc fractional lower- order parameters. The analysis, presented in Fig. 13, shows that the samples associated with 16-QAM are in a particular range of magni- tude that is between QPSK and 32-QAM ranges. Therefore, the thqpsk and th16QAM were respectively established con- sidering three standard deviations above, and three standard deviations below the mean value of 16-QAM samples. The adopted thresholds in the FLOCAF-based AMC architecture, are presented in Table 2. After determining the cyclic function parameters that opti- mize the average performance of the architecture, a more detailed analysis of the classiﬁer can be performed, con- sidering 1,000 iterations per modulation, for each value of the GSNR. After setting the fractional lower-orders parameters (a and b), and adjusting of the thresholds, the architecture was tested, and its classiﬁcation behavior is shown in Fig. 14. Fig. 14 shows that the architecture based on FLOCAF has a distinct classiﬁcation performance for each modulation. The classiﬁcation hit-rates achieves values above of 90% in the GSNR = 0 dB, for BPSK, 8-QAM, and, 32-QAM The thQPSK and th16QAM were deﬁned considering a com- prehensive analysis of signatures of QPSK, 16-QAM, and, VOLUME 7, 2019 138521 VOLUME 7, 2019 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments TABLE 2. Thresholds of FLOCAF-based AMC architecture. FIGURE 14. Modulation classification curves of FLOCAF-based architecture with a = 0.3 and b = 0.5, and 1,000 interactions per GSNR. TABLE 2. B. AMC USING CCF However, the hit-rate of this modulation growing The thQPSK and th16QAM were deﬁned considering an analysis of signatures of QPSK, 16-QAM, and, 32-QAM in 138522 VOLUME 7, 2019 VOLUME 7, 2019 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments FIGURE 15. Global classification hit-rate using the CCF for different kernel sizes, with 100 interactions while varying the GSNR. FIGURE 16. Global automatic modulation classification using the CCF for different kernel sizes, after calculating the average of GSNR. TABLE 6 Thresholds of CCF-based AMC architecture FIGURE 17. Definition of thqpsk and th16QAM thresholds. FIGURE 18. AMC using the CCF with σ = 1.2, and 1,000 interactions while varying the GSNR. FIGURE 15. Global classification hit-rate using the CCF for different kernel sizes, with 100 interactions while varying the GSNR. FIGURE 15. Global classification hit-rate using the CCF for different kernel sizes, with 100 interactions while varying the GSNR. FIGURE 17. Definition of thqpsk and th16QAM thresholds. qpsk 16QAM FIGURE 16. Global automatic modulation classification using the CCF for different kernel sizes, after calculating the average of GSNR. FIGURE 18. AMC using the CCF with σ = 1.2, and 1,000 interactions while varying the GSNR. TABLE 6. Thresholds of CCF-based AMC architecture. TABLE 6. Thresholds of CCF-based AMC architecture. The results in Fig. 18 show that the architecture has dif- ferent performances for each modulation, where the BPSK classiﬁcation is the most accurate, and 16-QAM classiﬁca- tion is the most sensitive to contamination. Even so, in the classiﬁcation of BPSK, QPSK, 8-QAM, and, 32-QAM, the architecture achieves hit-rates close to 100% for GSNRs as of 0 dB. The classiﬁcation of all modulations is approxi- mately 100% when GSNR = 6 dB. an α-stable channel with GSNR = 10 dB. Modula- tion signatures were generated 1, 000 times, and the factor Csig/C16QAM was calculated for each one of them. Fig. 17, shows that the samples associated with 16-QAM are in a particular range of magnitude that is between QPSK and 32-QAM ranges. This way, as in the case of the FLOCAF-based architecture, the thqpsk and th16QAM were established considering three standard deviations above, and three standard deviations below the mean value of 16-QAM samples. The adopted thresholds in the CCF-based AMC architecture are presented in Table 6. Fig. 17, shows that the samples associated with 16-QAM are in a particular range of magnitude that is between QPSK and 32-QAM ranges. This way, as in the case of the FLOCAF-based architecture, the thqpsk and th16QAM were established considering three standard deviations above, and three standard deviations below the mean value of 16-QAM samples. The adopted thresholds in the CCF-based AMC architecture are presented in Table 6. The confusion matrices of different GSNR levels are given in Tables 7 to 9 to provide a detailed analysis of the clas- siﬁer. The matrices show that, for lower GSNR condition, the 16-QAM is confused with 32-QAM. This result indicates that the signature of 16-QAM, under severe noise contami- nation, is more approximate of the 32-QAM template than of the 16-QAM template. After setting the kernel size (σ), and adjusting of the thresholds, the CCF-based architecture was tested, and its classiﬁcation behavior is shown in Fig. 18. Therefore, it can be inferred that the intense contamination over the 16-QAM, contributes to the fading of the cyclic component that differentiates its signature from the 32-QAM 138523 138523 VOLUME 7, 2019 VOLUME 7, 2019 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments FIGURE 19. Cyclostationary signatures of QPSK modulation obtained by the FLOCAF in three different GSNR conditions. FIGURE 20. VII. SYMBOL RATE ESTIMATION As demonstrated in section IV-B, the CCF and FLOCAF are capable of extracting cyclostationary descriptors of the mod- ulations contaminated by alpha-stable non-Gaussian addi- tive noise. Nevertheless, this task becomes harder at very lower GSNR levels, since the intense noise can attenuate the components from cyclostationary signatures. However, some components are more robust than others. TABLE 6. Thresholds of CCF-based AMC architecture. Cyclostationary signatures of QPSK modulation obtained by the CCF in three different GSNR conditions. FIGURE 19. Cyclostationary signatures of QPSK modulation obtained by the FLOCAF in three different GSNR conditions. TABLE 7. Confusion Matrix of CCF (σ = 1.2, GSNR = 0 dB). TABLE 7. Confusion Matrix of CCF (σ = 1.2, GSNR = 0 dB). TABLE 8. Confusion Matrix of CCF (σ = 1.2, GSNR = 4 dB). TABLE 8. Confusion Matrix of CCF (σ = 1.2, GSNR = 4 dB) FIGURE 19. Cyclostationary signatures of QPSK modulation obtained by the FLOCAF in three different GSNR conditions. FIGURE 20. Cyclostationary signatures of QPSK modulation obtained by the CCF in three different GSNR conditions. TABLE 9. Confusion Matrix of CCF (σ = 1.2, GSNR = 10 dB). TABLE 9. Confusion Matrix of CCF (σ = 1.2, GSNR = 10 dB). FIGURE 20. Cyclostationary signatures of QPSK modulation obtained by the CCF in three different GSNR conditions. signature. However, the matrices also indicate that the hit-rate of 16-QAM grows as the GSNR increases. Directly comparing, the AMC CCF-based architecture (Fig.18) performs better than the FLOCAF-based one (Fig.14), since, considering the same channel conditions, and the same modulations, the hit-rate of CCF-based architecture is higher than hit-rate of FLOCAF-based architecture. and, that the symbol rate (fb) of the signal does not exceed half of fi, then fb can be estimated from the signatures by the following method: the symbol rate will assume the value of the spectral position, in the range from 0 to fi/2, of the cyclic component with the most signiﬁcant amplitude. Tests of the symbol rate estimation using the FLOCAF and CCF, over impulsive noise contamination, are provided as follows. VII. SYMBOL RATE ESTIMATION A. SYMBOL RATE ESTIMATION USING THE FLOCAF To demonstrate that the FLOCAF, combined with the method before described in this section, enables the symbol rate esti- mation from modulated signals contaminated by impulsive noise, the following test was performed: ﬁrst, many examples of modulations contaminated with alpha-stable noise were generated for different GSNR conditions ranging from -10 dB to 10 dB with the step of 2 dB. Then, the symbol rate estima- tion was performed for each instance of the signal tested. In the cyclostationary signatures provided by the FLOCAF and CCF, the component associated with the symbol rate of the modulated signal is the most robust to impulsive noise contamination, as demonstrated in Fig. 19 and Fig. 20. Fig. 19 and Fig. 20, show the QPSK signatures generated, with the parameters presented in Table 1 from Section IV-B, by both cyclostationary functions in different GSNR condi- tions: −5 dB, 0 dB, and, 10 dB. In all the cases, the com- ponent associated with the symbol rate remains virtually unaltered, while the other components are fading in lower GSNR conditions. The global symbol rate estimation, which is the average among the estimation hit-rates of each modulation, was cal- culated considering 1,000 iterations per modulation, for each GSNR condition. Fig. 21 shows that using FLOCAF makes it possible to obtain high accuracy levels for the estimation of symbol rates even for low GSNRs. The correct estimation, which begins above 80% in the lowest GSNR condition, increases fast and achieves a hit-rate approximately equal to 100% in GNSR = −2 dB. Thus, since the cyclic component associated with the sym- bol rate is robust to impulsive noise contamination, moreover, considering that the intermediate frequency (fi) is known, 138524 VOLUME 7, 2019 VOLUME 7, 2019 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments FIGURE 21. Global symbol rate estimation using the FLOCAF with a = 0.3 and b = 0.5, for 1,000 interactions per modulation, for each GSNR value. FIGURE 22. Global symbol rate estimation using the CCF with σ = 1.1, applying 1,000 interactions per modulation, for each GSNR value. FIGURE 21. Global symbol rate estimation using the FLOCAF with a = 0.3 and b = 0.5, for 1,000 interactions per modulation, for each GSNR value. A. SYMBOL RATE ESTIMATION USING THE FLOCAF This work also has investigated the inﬂuence of the frac- tional lower-order parameters, a and b, and the kernel size (σ) on the performance of the AMC architectures, since they are free parameters of the FLOCAF and CCF, respec- tively. In this context, the numerical adjustment of FLOCAF parameters shows that the values of a and b that maximize the performance of the FLOCAF-based architecture are non- null, thus indicating that this conﬁguration performs better than a SSCCF-based architecture. Both architectures present satisfactory behavior regarding the AMC task in impulsive environments. Notably, the CCF-based architecture achieved a superior performance than the FLOCAF-based one. In addition to deﬁning new AMC architectures, it has been shown that the FLOCAF and CCF can also provide the symbol rate estimation of the investigated modulations, even with high contamination by impulsive noise. In this context, the estimation based on CCF also achieved a superior perfor- mance than the FLOCAF-based symbol rate estimation. FIGURE 21. Global symbol rate estimation using the FLOCAF with a = 0.3 and b = 0.5, for 1,000 interactions per modulation, for each GSNR value. FIGURE 22. Global symbol rate estimation using the CCF with σ = 1.1, l d l f h l Further studies could include the comparing of the archi- tectures proposed in this work with new CCF-based AMC architectures combined with artiﬁcial neural networks for scenarios subjected to multipath fading and impulsive noise; besides, extend the cyclostationary analysis to higher-order digital modulations like 64-QAM and 128-QAM. Moreover, the symbol rate estimation by CCF could be integrated with an AMC architecture. B. SYMBOL RATE ESTIMATION USING THE CCF where x[m] is a discrete-time signal, M is the number of observations, τ is a discrete-time delay and ϵ is the cyclic frequency. Although it is proven to be effective, the calcu- lation of this estimator is directly related to the number of signal samples. In other words, the higher the value of M, the longer the processing time. Thus, the efﬁcient calculation of CCF can be performed from the following steps: The same scenario, used in symbol rate estimation by FLOCAF, was applied to tests with CCF: the proposed mod- ulations were contaminated with different alpha-stable noise levels, and the symbol rate estimation was performed for each instance of the tested signal. The global symbol rate estimation was calculated considering 1,000 iterations per modulation, in each GSNR condition. Step 1. The input signal x[m] is divided into L blocks with N samples (a given block may contain intersection samples with the previous one); Fig. 22 shows that with the CCF, it is possible to obtain high accuracy for the estimation of the symbol rate even for low GSNRs. The correct estimation, which begins above 97% in the lowest GSNR condition, achieve hit-rates of 100% in GSNR = −8 dB. Step 2. Calculate the correntropy function Vxl(n, τn) from each block of size N, where n = 0, 1, 2, . . . , N −1 and l = 0, 1, 2, . . . , L −1: A directly comparing demonstrates that the estimation of the symbol rate from modulated signals, applying the CCF is more efﬁcient than estimation using FLOCAF (Fig. 21). Vxl(n, τn) = 1 N N−1 X n=0 Gσ(xl[n], xl[n + τn]); (36) (36) APPENDIX A THE CCF IMPLEMENTATION In the systems, the CCF deﬁned in (26) can be calculated using the following estimator: V ϵ x (τ) = 1 M M−1 X m=0 Gσ(x[m], x[m + τ])e−j2πϵm, (35) FIGURE 22. Global symbol rate estimation using the CCF with σ = 1.1, applying 1,000 interactions per modulation, for each GSNR value. (35) REFERENCES 431–436, Apr. 1998. [29] M. L. de Freitas, M. Egan, L. Clavier, A. Goupil, G. W. Peters, and N. Azzaoui, ‘‘Capacity bounds for additive symmetric α-stable noise chan- nels,’’ IEEE Trans. Inf. Theory, vol. 63, no. 8, pp. 5115–5123, Aug. 2017. [7] E. Avci, D. Hanbay, and A. 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Commun., vol. 46, no. 4, pp. VIII. CONCLUSION Step 3. Calculate the mean over the blocks of correntropy function: Step 3. Calculate the mean over the blocks of correntropy function: This work has compared the ability of the FLOCAF and CCF to extract cyclostationary features from BPSK, QPSK, 8-QAM, 16-QAM, and, 32-QAM modulations. The obtained results allow concluding that both functions can extract unique signatures from each modulation format. Vx(n, τn) = 1 L L−1 X l=0 Vxl(n, τn); (37) (37) 138525 VOLUME 7, 2019 T. V. R. O. Câmara et al.: AMC Architectures Based on Cyclostationary Features in Impulsive Environments Step 4. In order to avoid the central component that contains no cyclic information, it is necessary to centralize Vx(n, τn): [15] B. Ramkumar, ‘‘Automatic modulation classiﬁcation for cognitive radios using cyclic feature detection,’’ IEEE Circuits Syst. Mag., vol. 9, no. 2, pp. 27–45, 2nd Quart., 2009. [16] K. Kim, I. A. Akbar, K. K. Bae, J.-S. Um, C. M. Spooner, and J. 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https://openalex.org/W4376610762	https://thrombosisjournal.biomedcentral.com/counter/pdf/10.1186/s12959-023-00500-8	English	null	Clinical trial assessing the safety of edoxaban with concomitant chemotherapy in patients with gynecological cancer-associated thrombosis (EGCAT study)	Thrombosis journal	2,023	cc-by	8,373	Abstract Background Gynecological cancer is one of the highest risk factors for cancer-associated thrombosis (CAT). Although low-molecular-weight heparin (LMWH) is recommended as an anticoagulant for treating CAT, recent studies have shown that direct oral anticoagulants (DOACs) are an acceptable alternative. Patients with cancer require a series of chemotherapies concomitantly with DOAC administration; however, the extent to which these drugs influence DOAC blood concentrations is unknown. In this study, we measured the plasma concentration of edoxaban during chemotherapy for gynecological cancers to determine its safety. Methods Patients histologically diagnosed with ovarian or uterine corpus cancer and CAT were recruited after primary surgery and before the initiation of postoperative adjuvant chemotherapy, including paclitaxel. Patients were administered edoxaban (30 or 60 mg) orally for CAT. The plasma concentrations of edoxaban and active factor Xa were determined and their percentage change before and after chemotherapy was calculated. Additionally, blood coagulation tests were analyzed. Results Sixteen patients with gynecological cancer (12 with ovarian cancer and 4 with uterine corpus cancer) were enrolled. Among these, 15 samples were collected one day after chemotherapy initiation. During chemotherapy, the trough concentration of edoxaban changed from 17.6 ± 10.6 to 20.0 ± 15.6 ng/ml, and the mean percentage change in edoxaban concentration was 14.5%. Therefore, the trough concentrations of edoxaban, which represent excretion capacity, were not significantly increased by chemotherapy with paclitaxel. The area under the plasma edoxaban concentration–time curve and the active factor Xa concentration were also unaffected. Conclusion Patients with CAT and ovarian or uterine corpus cancer administered edoxaban orally showed no signifi- cant increase in the trough concentration of edoxaban while undergoing chemotherapy. This suggests the safety of edoxaban use during the treatment of gynecological cancers. Trial registration EGCAT study; Japan Registry of Clinical Trials, jRCTs051190024. Keywords Cancer associated thrombosis, Edoxaban, DOAC, Gynecological cancer, Trough conce Correspondence: Kenjiro Sawada daasawada@gyne.med.osaka-u.ac.jp Full list of author information is available at the end of the article Correspondence: Kenjiro Sawada daasawada@gyne.med.osaka-u.ac.jp Full list of author information is available at the end of the article © 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. Open Access Open Access © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Clinical trial assessing the safety of edoxaban with concomitant chemotherapy in patients with gynecological cancer‑associated thrombosis (EGCAT study) Tadashi Oride1, Kenjiro Sawada1, Aasa Shimizu1, Yasuto Kinose1, Tsuyoshi Takiuchi1, Michiko Kodama1, Kae Hashimoto1, Eiji Kobayashi1, Eiji Nakatani2 and Tadashi Kimura1 Thrombosis Journal Thrombosis Journal Oride et al. Thrombosis Journal (2023) 21:57 https://doi.org/10.1186/s12959-023-00500-8 Background Taxane agents such as paclitaxel are metabolized in the liver by CYP3A4 [17]. Therefore, the concomitant administration of DOAC and paclitaxel may increase the risk of major bleeding by affecting the blood concentra- tion of DOAC. However, currently there are no studies which have reported the effects of chemotherapeutic agents on the metabolism of DOACs during chemother- apy. CAT is the second leading cause of death in patients with cancer receiving outpatient chemotherapy [3] and its incidence is high in gynecological cancer cases; there- fore, the establishment of treatment strategies for CAT in gynecological cancer cases is clinically important. y g Gynecological cancer has been identified by the Kho- rana Score as one of the highest risk factors for cancer- associated thrombosis (CAT) [5]. Three to ten percent of patients present with CAT at diagnosis, and its incidence increases up to 36% during cancer treatment, including the debulking surgeries and repeated chemotherapies [6, 7]. The risk factors for developing CAT are not only the presence of large pelvic masses which compress iliac veins but as also include comorbidities, immobilization, chemotherapy, targeted therapy (e.g., bevacizumab), surgeries including lymphadenectomy, and intravenous catheter; all these factors can contribute to the pro- thrombotic or hypercoagulable state, as defined by Vir- chow’s triad: stasis, hypercoagulability, and endothelial injury [7]. Low-molecular-weight heparin (LMWH), unfraction- ated heparin (UFH), and vitamin K antagonists (VKA) have been used to treat CAT. Previous phase III studies of patients with CAT have shown that the rate of recurrent thrombosis was lower with a six-month course of LMWH than with VKA, while the risk of bleeding was similar with both treatments [8, 9]. Therefore, LMWH has been recommended as an anticoagulant for the treatment of CAT in all major guidelines, including those of the Amer- ican Society of Clinical Oncology, National Comprehen- sive Cancer Network, and American Thoracic Society [10]. However, the efficacy of LMWH beyond six months remains unclear, and LMWH therapy is burdensome, as it requires daily subcutaneous injections [11]. In 2010, direct oral anticoagulants (DOACs) such as apixaban, rivaroxaban, and edoxaban emerged. Background data supporting the use of DOACs in patients with can- cer have become available. The comparison of an oral Factor Xa inhibitor with low molecular weight heparin in patients with cancer with venous thromboembolism (SELECT-D) study for rivaroxaban, the edoxaban for the treatment of cancer-associated venous thromboem- bolism (Hokusai VTE Cancer) study for edoxaban, and the apixaban for the treatment of venous thromboem- bolism associated with cancer (CARAVAGGIO) study for apixaban compared the efficacy of DOACs with that of LMWH, the first therapeutic choice for CAT treat- ment [11, 13, 14]. Patients with cancer are at an increased risk of throm- boembolic disease because of their hypercoagulable state [1]. Its incidence in patients with cancer is four to seven times higher than those without cancer [2], and it is reported that 5–20% of all patients with cancer exhibit thromboembolisms [3]. In a recent Danish population- based cohort study, twelve-month incidence in the can- cer cohort increased from 1.0% (95% CI (confidence interval): 0.9–1.2%) in 1997 to 3.4% (95% CI: 2.9–4.0%) in 2017, which was paralleled by the improved survival of patients and the increased use of computed tomography scans, chemotherapy, and targeted therapies [4].i [ , , ] In the Hokusai-VTE Cancer study, edoxaban was a non-inferior treatment compared to dalteparin for recur- rent VTE with major bleeding (12.8% vs. 13.5%, respec- tively) [11]. However, during six months of the study period, a significantly higher rate of major bleeding (6.9% vs. 4.0%) was observed with both treatments. Major bleeding episodes were mainly caused by upper gastroin- testinal bleeding in patients with gastrointestinal cancer. In the SELECT-D study, major bleeding cumulative inci- dence at six months was 6% in the rivaroxaban group and 4% in the dalteparin group (HR, 1.83; 95% CI: 0.68–4.96) [13]. Although DOACs appear to be acceptable alterna- tives to LMWH for the treatment of CAT, several factors need to be considered to tailor anticoagulation manage- ment strategies for patients with active cancer [15]. Drug- drug interactions are important factors to be considered as systemic cancer-related therapies may interfere with DOACs. Potent inhibitors or inducers of P-glycoprotein and cytochrome p4503A4 (CYP3A4) are known to influ- ence the metabolism of DOACs and potentially alter their efficacy and/or safety profiles [16]. However, the extent to which these drugs influence the blood concentrations of DOACs is unknown. Platinum and taxane agents are generally used for the initial treatment of gynecological cancers. Abstract 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. Oride et al. Thrombosis Journal (2023) 21:57 Oride et al. Thrombosis Journal (2023) 21:57 Page 2 of 11 Page 2 of 11 Participants p Participants included patients who were ≥ 20 and < 85 years-of-age, histologically diagnosed with ovar- ian or advanced uterine corpus cancer, and had VTE or pulmonary embolism (PE) which was confirmed by hematological tests (D-dimer) and imaging studies (lower-limb venous ultrasound and/or contrast-enhanced computed tomography). Patients with cervical and vulvar cancers were excluded from this study because of the dif- ferent treatment strategies used, such as radiotherapy and concurrent chemoradiotherapy. After the primary surgeries were performed and the presence of malig- nancy was histologically confirmed, written informed consent was obtained before the initial postoperative adjuvant chemotherapy. As remaining tumors may affect the hypercoagulability of patients, those who under- went suboptimal surgeries were excluded. Patients who were expected to survive for at least six months from the time of consent were included in the study. Patients with a performance status > 2, impaired renal function (creatinine clearance less than 30 mL/min), abnormal liver function, weighing < 40 kg, with active bleeding or a high risk of bleeding, uncontrolled hypertension, history of hypersensitivity to DOACs, pregnancy or lactation, use of antiplatelet agents, history of venous thrombosis, complications of acute bacterial endocarditis, or history or treatment of atrial fibrillation were excluded from the study. Blood sampling Th ll h The overall schematic of the blood sampling schedule is shown in Fig. 1. The trough concentration (Ctrough) is defined as the concentration of a drug immediately before the next dose is administered and shows how a drug accumulates over time, providing valuable infor- mation about drug disposition [20]. On the first day of chemotherapy, blood was collected from patients after breakfast and before edoxaban administration (time point 1; Ctrough day1). Single oral doses of edoxaban result in peak plasma concentrations within 1.0–2.0 h of administration [19]; therefore, additional blood sam- pling was performed 2 h after the administration of edoxaban under physician supervision and was defined as maximum drug concentration (Cmax) day1 (time point 2). Thereafter, chemotherapy was initiated as described above. The third and fourth sets of blood sampling were performed the following day and defined as Ctrough day2 (time point 3) and Cmax day2 (time point 4), respec- tively. The fifth and sixth sets of blood sampling were performed on the day after the last cycle of chemother- apy to assess the long-term effects of chemotherapy and were defined as Ctrough day2 (time point 5) and Cmax day2* (time point 6), respectively. These blood samples and blood coagulation tests were used to analyze the plasma concentrations of edoxaban, prothrombin time- international normalized ratio (PT-INR), activated par- tial thromboplastin time (APTT), and active factor Xa concentrations. Background A ran- domized Phase III trial comparing each DOAC with VKA for the treatment of venous thromboembolism (VTE) showed that DOACs are not inferior to VKA in terms of treatment efficacy and recurrence rate; VTE treatment has shifted from using VKAs to DOACs owing to their efficacy, standardized dosing, reduced monitoring, less frequent follow-up, and fewer inter- actions with food or drugs [12]. More recently, clinical In this study, we explore the safety of orally admin- istrating edoxaban in patients with gynecological can- cers undergoing chemotherapy, including paclitaxel, by evaluating the percentage change in the plasma con- centration of edoxaban during treatment. Oride et al. Thrombosis Journal (2023) 21:57 Page 3 of 11 Page 3 of 11 following criteria: moderate renal impairment (creati- nine clearance, 30–50 mL/min), body weight ≤ 60 kg, or concomitant use of potent P-glycoprotein inhibitors (such as erythromycin, cyclosporine, dronedarone, qui- nidine, or ketoconazole). Patients with ovarian cancer were scheduled to receive six cycles of carboplatin (area under the curve (AUC) = 5) with paclitaxel (175 mg/m2) with or without bevacizumab (15 mg/kg), while those with　uterine corpus cancer were scheduled to receive six cycles of carboplatin (AUC = 4), paclitaxel (150 mg/ m2), and epirubicin (50 mg/m2) [18]. A pharmacokinetic study of edoxaban revealed that a steady-state blood con- centration can be achieved after three days of treatment [19]; therefore, the scheduled postoperative chemothera- pies were initiated more than three days after the admin- istration of edoxaban. Blood samples were collected six times during chemotherapy. During the chemotherapy, all patients admitted the hospital. Edoxaban adherence was strictly checked by the pharmacologist in the ward during the admission. During the outpatient follow-up, participants presented self-reports regarding the adher- ence of their medication at every visit, and edoxaban adherence was strictly checked by attending physicians. Trial design and interventionsh The clinical trial assessing the safety of edoxaban with concomitant chemotherapy in patients with gynecologi- cal cancer-associated thrombosis (EGCAT study) was a single-arm, comparative, open-label, uncontrolled treat- ment intervention study. This was a specified clinical trial under the Clinical Research Act in Japan and was spon- sored by the Daiichi Sankyo Co., Ltd., (Tokyo, Japan). This study was conducted in accordance with the prin- ciples of the Declaration of Helsinki and the local regu- lations. The study protocol was reviewed and approved by the Osaka University Clinical Research Review Com- mittee (CRB5180007). A summary of this clinical study was registered and published as jRCTs051190024 on 2019/06/03 in Janpan Registry of Clinical Trials (https:// jrct.niph.go.jp/en-latest-detail/jRCTs051190024). After obtaining written informed consent followed the primary surgeries, patients were orally adminis- tered an edoxaban (30 or 60 mg) (Daiichi Sankyo Co., Ltd., Tokyo, Japan) tablet daily after breakfast based on their weight and renal function. The edoxaban dosage was reduced to 30 mg in patients who met any of the Page 4 of 11 Oride et al. Thrombosis Journal (2023) 21:57 g. 1 The overall schema of the trial and the schedule of blood sampling. A Trial design and intervention of this study. B The blood sampling hedule. Time point 1: Ctrough day1 (first day of chemotherapy before administrating edoxaban), time point 2: Cmax day1 (first day of emotherapy two hours after administrating edoxaban), time point 3: Ctrough day2 (day after chemotherapy initiation before administrating oxaban), time point 4: Cmax day2 (day after chemotherapy initiation two hours after administrating edoxaban), time point 5: Ctrough day2* (day er the final chemotherapy session before administrating edoxaban), time point 6: Cmax day2* (day after the final chemotherapy session two hours er administrating edoxaban) Fig. 1 The overall schema of the trial and the schedule of blood sampling. A Trial design and intervention of this study. B The blood sampling schedule. Time point 1: Ctrough day1 (first day of chemotherapy before administrating edoxaban), time point 2: Cmax day1 (first day of chemotherapy two hours after administrating edoxaban), time point 3: Ctrough day2 (day after chemotherapy initiation before administrating edoxaban), time point 4: Cmax day2 (day after chemotherapy initiation two hours after administrating edoxaban), time point 5: Ctrough day2* (day after the final chemotherapy session before administrating edoxaban), time point 6: Cmax day2* (day after the final chemotherapy session two hours after administrating edoxaban) Fig. Trial design and interventionsh 1 The overall schema of the trial and the schedule of blood sampling. A Trial design and intervention of this study. B The blood sampling schedule. Time point 1: Ctrough day1 (first day of chemotherapy before administrating edoxaban), time point 2: Cmax day1 (first day of chemotherapy two hours after administrating edoxaban), time point 3: Ctrough day2 (day after chemotherapy initiation before administrating edoxaban), time point 4: Cmax day2 (day after chemotherapy initiation two hours after administrating edoxaban), time point 5: Ctrough day2* (day after the final chemotherapy session before administrating edoxaban), time point 6: Cmax day2* (day after the final chemotherapy session two hours after administrating edoxaban) Plasma concentration of edoxaban The PT-INR and APTT were analyzed as part of general blood coagulation tests. The PT-INR was obtained by multiplying the calculated prothrombin time (PT) with the international sensitivity index. PT was measured using Thromborel® S (Siemens, Munich, Germany) containing human placenta-derived thromboplastin and calcium, which initiate the coagulation reaction. APTT was measured using a Thrombocheck APTT- SLA (Sysmex, Kobe, Japan) containing synthetic phos- pholipids and calcium chloride. Both parameters were measured using an automated blood coagulation ana- lyzer CS5100 (Sysmex, Kobe, Japan). Blood samples were collected by venous puncture and stored with heparin. Samples were immediately cen- trifuged at 2500 g for 10 min at 4 °C, and thereafter plasma was stored at -80 °C. Edoxaban concentrations were determined by Shin Nippon Biomedical Labora- tories (Tokyo, Japan). Ultra-high performance liquid chromatography (HPLC) with tandem mass spectrom- etry (API 4000; AB Sciex Pte. Ltd., Tokyo, Japan) and an HPLC analytical column (CAPCELL PAK C18 MGII; 2.0 mm i.d. × 150 mm, 3 µm; OSAKA SODA Co., Ltd., Osaka, Japan) were used for the concentration analysis as previously reported [21]. The quantification range was 1–500 ng/ml. Page 5 of 11 Page 5 of 11 Oride et al. Thrombosis Journal (2023) 21:57 Oride et al. Thrombosis Journal (2023) 21:57 The concentration of active factor Xa was meas- ured using a SensoLyte® Rh110 Factor Xa Assay Kit (#AS-72207; AnaSpec, Fremont, CA, USA), follow- ing the manufacturer’s instructions. The fluorescence intensity was measured using a microplate reader (SpectraMax iD3; Molecular Devices, San Jose, CA) to determine the concentration of Xa. The quantitative range was 0.0320–2.00 µmol/L. Statistical analysish The number of patients required for this study was cal- culated to be 14 to achieve a Type I error of 5% or less and a power of 80% or more in a one-tailed test. There- fore, considering cases of discontinuation during the study, the number of planned study participants was set at > 15. The lower limit (upper limit is ∞) of the 95% CI corresponding to a one-sided t-test of percentage change rate in edoxaban trough concentrations before and after chemotherapy administration is considered significant if it is greater than 0%. Each parameter, including the AUC values, PT-INR, APTT, and active factor Xa concentration between time points, was sta- tistically analyzed using the Wilcoxon signed-rank test or Friedman test, and P-values were adjusted using the Bonferroni method for multiple comparisons. Continu- ous variables are expressed as mean ± standard devia- tion (SD). Statistical significance was set at P < 0.05. All statistical analyses were performed using R version 4.2.2 [25]. Endpoint measurements h d The primary endpoint of this study was the percent- age change in plasma edoxaban trough concentra- tions before and after chemotherapy initiation, which indicated the effect of chemotherapy on the excretion capacity of edoxaban. The secondary endpoints included determining the percentage change in plasma edoxaban trough con- centrations before chemotherapy initiations and after a series of chemotherapy cycles, which indicates the effect of multiple doses of chemotherapy on excre- tion capacity. The area under the plasma drug con- centration–time curve (AUC) is a definite integral of the concentration of a drug in the blood plasma and reflects the actual exposure of the body to drugs after administration [22]. In this study, we calculated the AUC values of edoxaban before and two hours after administration using the linear trapezoidal method, which indicated the absorption capacity of edoxaban. The following formula was used to calculate AUC values: Characteristics of participants p p Patients with ovarian or uterine corpus cancer diag- nosed with VTE and/or PE received anticoagulant therapy, including UFH or DOACs, preoperatively. After surgery, the patients were asked to participate in this study, and the 16 patients who provided writ- ten informed consent were included. The participants’ characteristics are listed in Table 1. The median age of the patients was 57 (interquartile range; 55–68) years, and the mean body mass index was 22.1 (interquartile range; 19.7–24.5). Among these, 12 patients had ovar- ian cancer and 4 had uterine corpus cancer. Of the 12 patients with ovarian cancer, 1 had serous carcinoma, 4 had endometrioid carcinoma, 6 had clear cell car- cinoma, and 1 had mixed carcinoma (endometroid/ serous). Of the four uterine corpus cancers, three were endometrioid carcinomas, and one was a carci- nosarcoma. Eight patients were diagnosed at an early stage (stages I–II), while the remaining patients were diagnosed at advanced stages (stages III–IV). Twelve patients had VTE only and four had PE with VTE. Among those 4 cases, one case with ovarian cancer was found to have multiple cerebral infarctions and diag- nosed as Trousseau’s syndrome. Based on the body weight of the patients, three patients received 60 mg of edoxaban, and the remaining patients received 30 mg. None of the patients had a history of thrombosis or thrombotic predisposition. All patients strictly adhered to the edoxaban regimen prescribed during the study period. AUC = (Ctrough + Cmax) ∗1/2 : ∗2h(ng ∗h/ml) 2B); this was contrary to expectations, and indicated that the trough concen- trations of edoxaban, which represents excretion capac- ity, were not significantly increased by chemotherapy and paclitaxel administration. In eight patients whose blood samples were collected at time points 1, 3, and 5, the trough concentration of edoxaban changed from 21.6 ± 13.3 to 14.3 ± 7.8 ng/ml (Fig. 2C), and the mean percent change in edoxaban concentration was -22.0% (95% CI: -52.7–∞; P = 0.89) with a SD of 45.9% (Fig. 2D). This showed that the trough concentrations of edoxa- ban were not significantly affected by multiple cycles of chemotherapy. AUC values that reflected the actual body exposure to edoxaban two hours after administration were cal- culated on day1 (before chemotherapy initiation), day2 (after chemotherapy initiation), and day2(after the final chemotherapy session) from eight patients whose blood samples were collected at all time points. The average AUC values on day1, day2, and day2 were 238.2 ± 115.6, 277.1 ± 133.5, and 229.8 ± 118.2 ngh/ml, respectively. These AUC values were not significantly affected by multiple cycles of chemotherapy (P = 0.325) (Fig. 3A). Among the two patients who received 60 mg of edoxa- ban, the average AUC values on day1, day2, and day2 were 330.0 ± 257.3, 501.7 ± 160.2, and 418.0 ± 90.9 ngh/ ml, respectively (P = 0.607) (Fig. 3B). Among six patients who received 30 mg of edoxaban, the average AUC values on day1, day2, and day2 were 184.8 ± 101.6, 235.7 ± 62.9, and 176.2 ± 49.9 ngh/ml, respectively (P = 0.513) (Fig. 3C). The AUC values revealed that actual body exposure to edoxaban was not altered by chemotherapy at either of the edoxaban dosages. IQR Interquartile range, BMI Body mass index, VTE Venous thromboembolism, DVT Deep vein thrombosis, PE Pulmonary embolism IQR Interquartile range, BMI Body mass index, VTE Venous thromboembolism, DVT Deep vein thrombosis, PE Pulmonary embolism Percentage change of trough concentrations of edoxaban during chemotherapies with paclitaxelh The primary endpoint of this study was to determine whether the trough concentration of edoxaban was affected by the administration of chemotherapeu- tic drugs, including paclitaxel. The mean and SD of the plasma concentrations of edoxaban at each time point are shown in Fig. 2A. Among the 16 patients enrolled, plasma samples were collected after chemotherapy initia- tion in 15 patients and after the last chemotherapy cycle in eight patients. The mean values of Ctrough day1 (time point 1), Cmax day1 (time point 2), Ctrough day2 (time point 3), Cmax day2 (time point 4), Ctrough day2 (time point 5), and Cmax day2* (time point 6) were 17.7 ± 10.9, 216.6 ± 119.9, 20.0 ± 15.6, 257.1 ± 123.6, 14.3 ± 7.8, and 222.4 ± 119.6 ng/ml, respectively. In the 12 patients with ovarian cancer, the mean values of Ctrough day1 (time point 1) and Ctrough day2 (time point 3) were 14.9 ± 6.2 and 15.2 ± 7.4 ng/ml, respectively. In the four patients with uterine corpus cancer, the mean values of Ctrough day1 (time point 1) and Ctrough day2 (time point 3) AUC = (Ctrough + Cmax) ∗1/2 : ∗2h(ng ∗h/ml) AUC = (Ctrough + Cmax) ∗1/2 : ∗2h(ng ∗h/ml) Percentage changes of AUC values, active fac- tor Xa concentration, PT-INR, and APTT before and after chemotherapy were also calculated as secondary endpoints. Any adverse events such as bleeding and thrombotic recurrence were recorded up to six months after ini- tiating edoxaban administration. Bleeding events were defined as a combination of major or clinically relevant non-major bleeding events. Major bleeding events included a decrease in hemoglobin of 2 g/dL or more, requiring transfusion of two or more units of blood, occurring at a critical site, or contributing to death [23]. Clinically relevant non-major bleeding events did not meet the criteria for major bleeding but were asso- ciated with the need for medical intervention, such as contact with a physician or discontinuation of drug administration [24]. Recurrent thrombosis included cases identified by imaging studies with or without symptoms, and cases which resulted in death due to thrombus. Oride et al. Thrombosis Journal (2023) 21:57 Page 6 of 11 Page 6 of 11 Table 1 Characteristics of the trial population at baseline (n = 16) IQR Interquartile range, BMI Body mass index, VTE Venous thromboembolism, DVT Deep vein thrombosis, PE Pulmonary embolism Characteristics Median (IQR) Age (years) 57 (55–68) Mean (IQR) BMI 22.1 (19.7–24.5) No. (%) Type of cancer Ovary 12 (75%) Uterus corpus 4 (25%) Histology Ovary Uterus corpus Serous carcinoma 1 (6.3%) 0 (0%) Endometrioid carcinoma 4 (25%) 3 (18.8%) Clear cell carcinoma 6 (37.5%) 0 (0%) Carcinosarcoma 0 (0%) 1 (6.3%) Mixed carcinoma 1 (6.3%) 0 (0%) Stage of cancer Ovary Uterus corpus I 7 (43.8%) 1 (6.3%) II 1 (6.3%) 0 (0%) III 4 (25%) 2 (12.5%) IV 0 (0%) 1 (6.3%) Location of VTE DVT 12 (75%) PE + DVT 4 (25%) Edoxaban administration 60 mg 3 (19%) 30 mg 13 (81%) History of previous thrombosis or thrombotic predisposition-no 0 were 26.8 ± 16.6 and 33.1 ± 25.2 ng/ml, respectively. The mean trough concentrations were not significantly dif- ferent between different tumor types (Table 2), indicat- ing that different chemotherapy regimens did not alter the trough concentrations. The overall trough concen- tration of edoxaban changed from 17.7 ± 10.9 ng/ml to 20.0 ± 15.6 ng/ml after the initiation of chemotherapy with paclitaxel (n = 15), and the mean percent change in edoxaban concentration was 14.5% (95% CI: -6.4–∞; P = 0.12) with an SD of 45.9% (Fig. Percentage changes of active factor Xa concentration, PT‑INR, and APTT during chemotherapy with paclitaxelh The plasma concentrations of active factor Xa during the study are shown in Fig. 4A. The active factor Xa concen- trations before (time point 1) and two hours after edoxa- ban administration (time point 2) were 0.102 ± 0.080 and 0.124 ± 0.095 μM (n = 16), respectively. One day after chemotherapy initiation, the active factor Xa concentra- tions before (time point 3) and two hours after edoxa- ban administration (time point 4) were 0.113 ± 0.102 and Oride et al. Thrombosis Journal (2023) 21:57 Page 7 of 11 Fig. 2 The percentage change in trough concentrations of edoxaban during chemotherapy with paclitaxel. A Concentrations of plasma edoxaban at each time point. Dots and error bars represent mean ± SD. B Percentage change in trough concentration of edoxaban from time points 1 to 3 (n = 15). Dots and error bars represent mean ± 95% CI. C Trough concentrations of plasma edoxaban at time points 1, 3, and 5 (n = 8). Dots and error bars represent mean ± SD. D Percentage change in trough concentrations of edoxaban from time points 1 to 5 (n = 8). Dots and error bars represent mean ± 95% CI. CI: confidence interval; inf: infinity Fig. 2 The percentage change in trough concentrations of edoxaban during chemotherapy with paclitaxel. A Concentrations of plasma edoxaban at each time point. Dots and error bars represent mean ± SD. B Percentage change in trough concentration of edoxaban from time points 1 to 3 (n = 15). Dots and error bars represent mean ± 95% CI. C Trough concentrations of plasma edoxaban at time points 1, 3, and 5 (n = 8). Dots and error bars represent mean ± SD. D Percentage change in trough concentrations of edoxaban from time points 1 to 5 (n = 8). Dots and error bars represent mean ± 95% CI. these values were not significantly altered during the study period (P = 0.359) (Fig. 4B).h Percentage changes of active factor Xa concentration, PT‑INR, and APTT during chemotherapy with paclitaxelh CI: confidence interval; inf: infinity Table 2 Concentration of plasma edoxaban (ng/ml) during the study All values are expressed as mean ± SD Time point All patients (n = 16) Cancer type Edoxaban dosage Ovarian (n = 12) Uterus corpus (n = 4) 60 mg (n = 3) 30 mg (n = 13) 1 17.7 ± 10.9 14.9 ± 6.2 26.8 ± 16.6 15.6 ± 6.0 26.3 ± 22.4 2 216.6 ± 119.9 211.8 ± 93.2 229.9 ± 194.4 188.8 ± 78.2 330.4 ± 206.6 3 20.0 ± 15.6 15.2 ± 7.4 33.1 ± 25.2 16.0 ± 7.2 36.1 ± 30.4 4 257.1 ± 123.6 237.4 ± 82.5 311.3 ± 208.0 207.9 ± 68.8 453.7 ± 92.0 5 14.3 ± 7.8 12.8 ± 4.3 16.8 ± 12.7 11.9 ± 4.4 21.5 ± 13.9 6 222.4 ± 119.6 156.4 ± 53.4 332.3 ± 123.8 164.4 ± 51.6 396.5 ± 77.1 Table 2 Concentration of plasma edoxaban (ng/ml) during the study All values are expressed as mean ± SD these values were not significantly altered during the study period (P = 0.359) (Fig. 4B).h 0.145 ± 0.116 μM (n = 15), respectively. One day after the final chemotherapy session, the active factor Xa concen- trations before (time point 5) and two hours after edoxa- ban administration (time point 6) were 0.133 ± 0.173 and 0.104 ± 0.08 μM (n = 8), respectively. In eight patients whose blood samples were collected at all time points, The PT-INR values during the study period are shown in Fig. 4C. The PT-INR values before (time point 1) and two hours after edoxaban administration (time point 2) were 1.05 ± 0.05 and 1.47 ± 0.27 (n = 16), respectively. The PT-INR values during the study period are shown in Fig. 4C. The PT-INR values before (time point 1) and two hours after edoxaban administration (time point 2) were 1.05 ± 0.05 and 1.47 ± 0.27 (n = 16), respectively. Oride et al. Thrombosis Journal (2023) 21:57 Page 8 of 11 Fig. 3 The AUC values of edoxaban administrated at day1 (before chemotherapy initiation), day2 (after chemotherapy initiation), and day2* (after the final chemotherapy session) (n = 8). A All cases. B Cases who received 60 mg of edoxaban (n = 2). C Cases who received 30 mg of edoxaban (n = 6). Discussion Th The treatment of VTE in patients with active cancer remains challenging owing to complications such as an increased risk of bleeding and potential drug-drug interactions with chemotherapy. Although DOACs have significantly fewer drug-drug interactions than VKAs, drugs that strongly affect the CYP3A4 enzyme and/ or P-glycoprotein can alter the plasma concentration of DOACs and lead to clinically significant alterations in their anticoagulant effects [17]. The safety and efficacy data of edoxaban is comparable to that of dalteparin in the Hokusai-VTE Cancer study in patients with non- gastrointestinal cancers; this suggests that the drug-drug interactions between DOACs and anticancer agents are clinically manageable [26]. However, concrete safety data regarding the use of DOACs in cancer patients receiving chemotherapies indispensable for the cure are still lack- ing. This study is the first to monitor the plasma concen- tration of edoxaban in patients with ovarian and uterine corpus cancer who were receiving chemotherapy with paclitaxel, and to evaluate the percent change in trough concentrations and AUC values of edoxaban before and after treatment. The trough concentrations and AUC values of edoxaban were shown not to be significantly altered by multiple cycles of chemotherapy, indicating Fig. 3 The AUC values of edoxaban administrated at day1 (before chemotherapy initiation), day2 (after chemotherapy initiation), and day2* (after the final chemotherapy session) (n = 8). A All cases. B Cases who received 60 mg of edoxaban (n = 2). C Cases who received 30 mg of edoxaban (n = 6). Dots and error bars represent mean ± SD One day after chemotherapy initiation, the PT-INR val- ues before (time point 3) and two hours after edoxa- ban administration (time point 4) were 1.07 ± 0.24 and 1.30 ± 0.15 (n = 15), respectively. One day after the final chemotherapy session, the PT-INR values before (time point 5) and two hours after edoxaban administration (time point 6) were 1.05 ± 0.06 and 1.29 ± 0.18 (n = 8), respectively. Overall, the PT-INR values increased two hours after edoxaban administration (P = 0.036) and returned to a similar level the following day. In eight patients whose blood samples were collected at all time points, the PT-INR values were not affected by chemo- therapy (Fig. Percentage changes of active factor Xa concentration, PT‑INR, and APTT during chemotherapy with paclitaxelh Dots and error bars represent mean ± SD after chemotherapy initiation, the APTT values before (time point 3) and two hours after edoxaban adminis- tration (time point 4) were 27.7 ± 3.3 and 31.1 ± 3.7 s (n = 15), respectively. One day after the final chemother- apy session, the APTT values before (time point 5) and two hours after edoxaban administration (time point 6) were 26.8 ± 3.3 and 30.1 ± 4.7 s (n = 8), respectively. Over- all, the APTT values were slightly extended two hours after edoxaban administration (P = 0.035) and returned to a similar level the following day. The APTT values for the various time points were slightly shortened compared with that of time points 1 and 5 (P = 0.041); however, there were no other differences among the time points (Fig. 4F). Safety profiles No adverse events, including bleeding or recurrent thrombosis, occurred within six months of study. Out- side of the set observation period, there was one case of hemorrhage and one case of recurrent thrombosis. One patient presented with intermittent epistaxis that quickly resolved by applying pressure; therefore, oral edoxaban was not discontinued. In one patient with a recurrent thrombus, a thrombus in the lower inferior vena cava was observed nine months after the administration of edoxaban, and anticoagulation therapy was changed from edoxaban to heparin. Discussion Th 4D).h One day after chemotherapy initiation, the PT-INR val- ues before (time point 3) and two hours after edoxa- ban administration (time point 4) were 1.07 ± 0.24 and 1.30 ± 0.15 (n = 15), respectively. One day after the final chemotherapy session, the PT-INR values before (time point 5) and two hours after edoxaban administration (time point 6) were 1.05 ± 0.06 and 1.29 ± 0.18 (n = 8), respectively. Overall, the PT-INR values increased two hours after edoxaban administration (P = 0.036) and returned to a similar level the following day. In eight patients whose blood samples were collected at all time points, the PT-INR values were not affected by chemo- therapy (Fig. 4D).h The APTT values obtained during the study are shown in Fig. 4E. The APTT values before (time point 1) and two hours after edoxaban administration (time point 2) were 29.5 ± 3.0 and 34.4 ± 4.2 s (n = 16), respectively.. One day Oride et al. Thrombosis Journal (2023) 21:57 Page 9 of 11 Fig. 4 Percentage changes in active factor Xa concentration, PT-INR, and APTT values. A Active factor Xa concentrations at all time points. B Active factor Xa concentrations at time points 1, 3, and 5 (n = 8). C PT-INR values at all time points. D PT-INR values at time points 1, 3, and 5 (n = 8). E APTT values at all time points. F APTT values at time points 1, 3, and 5 (n = 8). Dots and error bars represent mean ± SD Fig. 4 Percentage changes in active factor Xa concentration, PT-INR, and APTT values. A Active factor Xa concentrations at all time points. B Active factor Xa concentrations at time points 1, 3, and 5 (n = 8). C PT-INR values at all time points. D PT-INR values at time points 1, 3, and 5 (n = 8). E APTT values at all time points. F APTT values at time points 1, 3, and 5 (n = 8). Dots and error bars represent mean ± SD that edoxaban is an acceptable option for the treatment of CAT in patients with gynecological cancer receiving chemotherapy, including paclitaxel. monoclonal antibodies, including bevacizumab, have not been reported to have significant inhibitory or inducing effects on CYP3A4 or P-glycoproteins [17]. Discussion Th The paclitaxel and carboplatin regime, with or without bevacizumab, is the most frequently used regimen for the treatment of ovarian and uterine corpus cancers; therefore, the use of paclitaxel might affect the plasma concentration of DOACs, which may interfere its treatment effect. Previ- ously studies on the efficacy of DOACs in treating CAT have not focused on gynecological malignancies; this is most likely due to the few cases included in these stud- ies. In the Hokusai-VTE Cancer study, only 19 and 15 cases of ovarian and uterine corpus cancers, respectively, were included in the edoxaban group, whereas 33 and Many chemotherapeutic drugs induce or inhibit the activity of CYP3A4, P-glycoprotein, or both. These include anthracyclines such as doxorubicin; antimy- cotic agents such as vincristine and paclitaxel; topoi- somerase inhibitors such as topotecan and etoposide; alkylating agents such as cyclophosphamide; tyrosine kinase inhibitors such as imatinib, lenvatinib, and suni- tinib; immune-modulating agents such as cyclosporine and tacrolimus; and hormonal agents such as tamox- ifen and anastrozole [27]. In contrast, platinum-based agents, including carboplatin; intercalating agents; and Oride et al. Thrombosis Journal (2023) 21:57 Page 10 of 11 Oride et al. Thrombosis Journal (2023) 21:57 treatment. These findings suggest that edoxaban is safe for the treatment of CAT during the treatment of gynecologi- cal cancers using chemotherapy. 22 cases were included in the dalteparin group [11]. In that study, only 40 cases in the edoxaban group and 47 cases in the dalteparin group received taxanes during the observation period [11]. Therefore, the safety and efficacy of edoxaban in patients with gynecological cancer receiv- ing chemotherapy requires investigation. Furthermore, although several phase III studies have shown the com- parable safety and efficacy of DOACs in patients with cancer, none have validated the plasma concentration of each DOAC during chemotherapy. Herein, we measured the specific changes in plasma edoxaban concentrations before and after chemotherapy. Author details 1D f 1 Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2‑15, Yamada‑Oka, Suita City, Osaka, Japan. 2 Graduate School of Public Health, Shizuoka Graduate University of Public Health, Shizuoka, Japan. Received: 12 March 2023 Accepted: 3 May 2023 Received: 12 March 2023 Accepted: 3 May 2023 Received: 12 March 2023 Accepted: 3 May 2023 Conclusion Patients with CAT and ovarian or uterine corpus cancer who were receiving edoxaban orally showed no significant increase in the trough concentration of edoxaban while undergoing chemotherapy, including paclitaxel, for cancer Availability of data and materials The datasets used and/or analyzed in the study are available upon reasonable request from the corresponding author. Authors’ contributions KS and AS designed this study. KS, YK, TT, MK, KH, and EK recruited participants. EN designed and performed the statistical analyses. AS and TO analyzed and organized the data. AS, TO, and KS interpreted the data. TO and KS drafted and revised the manuscript. All authors agree to be accountable for all aspects of the work. TK supervised the study. All authors agree to be accountable for all aspects of the study and ensure that questions related to the accuracy or integrity of any part of the study are appropriately investigated and resolved. The author(s) read and approved the final manuscript. The AUC values, PT-INR, APTT, and plasma concen- tration of active factor Xa were evaluated as secondary endpoints. The PT-INR and APTT values were altered two hours after edoxaban administration, as previously reported [29], and returned to similar levels one day after administration. Throughout time points 1, 3, and 5, no significant changes in AUC values, PT-INR values, or plasma concentration of active factor Xa were observed. Although APTT values were slightly shortened from time points 1 to 5 (P = 0.041), these results suggest that concomitant chemotherapy with paclitaxel and edoxaban did not induce hypercoagulability in patients.h Discussion Th Abbreviations CI Confidence interval CAT Cancer-associated thrombosis LMWH Low-molecular-weight heparin UFH Unfractionated heparin VKA Vitamin K antagonist DOAC Direct oral anticoagulant VTE Venous thromboembolism PE Pulmonary embolism AUC Area under the curve APTT Activated partial thromboplastin time PT-INR Prothrombin time-international normalized ratio SD Standard deviation In this study, percentage changes in the trough con- centrations of edoxaban before and after chemotherapy were set as the primary endpoints as the bleeding risk in patients receiving edoxaban orally was reported to corre- late more strongly with its plasma trough concentration than with AUC or Cmax values [28]. Although the number of participants was small due to the high cost of measur- ing the edoxaban concentrations, we successfully showed that the percentage change in trough concentration was not significantly increased the day following chemother- apy (14.5%; 95% CI: -6.4–∞; P = 0.12). Acknowledgements We thank Moe Matsui for the secretarial assistance. Ethics approval and consent to participate This study was conducted in accordance with the principles of the Declara- tion of Helsinki and the local regulations. The study protocol was reviewed and approved by the Osaka University Clinical Research Review Committee (CRB5180007). A summary of this clinical study was registered and published on 2019/06/03 as jRCTs051190024 in the database (https://jrct.niph.go.jp/en- latest-detail/jRCTs051190024). This study has several limitations. First, this was a sin- gle-institution study with a small sample size. Second, no reference values are available for percentage change in plasma edoxaban concentration exist owing to the lack previous studies on this topic. Third, it was neces- sary to evaluate the percentage change in plasma con- centration from only six blood-sampling time points as this was invasive for participants and the cost of meas- uring plasma edoxaban and active factor Xa concentra- tion levels were high. A greater number of patients with more blood sampling is desirable to confirm the safety of edoxaban during chemotherapy. Funding g This study was funded by Daiichi Sankyo Co., Ltd. (Tokyo, Japan). Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. References 1. Abdol Razak NB, Jones G, Bhandari M, Berndt MC, Metharom P. Cancer- associated thrombosis: An overview of mechanisms, risk factors, and Page 11 of 11 Page 11 of 11 Oride et al. Thrombosis Journal (2023) 21:57 treatment. 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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: ? Publisher’s Note 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: 17. Short NJ, Connors JM. New oral anticoagulants and the cancer patient. Oncologist. 2014;19:82–93. https://doi.org/10.1634/theoncologist. 2013-0239. 18. Egawa-Takata T, Ueda Y, Kuragaki C, Miyake T, Miyatake T, Fujita M, et al. Chemotherapy for endometrial carcinoma (GOGO-EM1 study): TEC (pacli- taxel, epirubicin, and carboplatin) is an effective remission-induction and adjuvant therapy. Cancer Chemother Pharmacol. 2011;68:1603–10. https://doi.org/10.1007/s00280-011-1638-4. 19. Parasrampuria DA, Truitt KE. Pharmacokinetics and pharmacodynamics of edoxaban, a non-vitamin K antagonist oral anticoagulant that inhibits clotting factor Xa. Clin Pharmacokinet. 2016;55:641–55. https://doi.org/ 10.1007/s40262-015-0342-7. 19. Parasrampuria DA, Truitt KE. Pharmacokinetics and pharmacodynamics of edoxaban, a non-vitamin K antagonist oral anticoagulant that inhibits clotting factor Xa. Clin Pharmacokinet. 2016;55:641–55. https://doi.org/ 10.1007/s40262-015-0342-7.
W4386845565.txt	https://pubs.rsc.org/en/content/articlepdf/2023/md/d3md90035e	en		Contents list	RSC medicinal chemistry	2,023	cc-by	1,772	RSC Medicinal Chemistry View Article Online View Journal \| View Issue Open Access Article. Published on 19 September 2023. Downloaded on 5/18/2024 2:48:29 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. rsc.li/medchem The Royal Society of Chemistry is the world's leading chemistry community. Through our high impact journals and publications we connect the world with the chemical sciences and invest the profits back into the chemistry community. IN THIS ISSUE ISSN 2632–8682 CODEN RMCSCX 14(9) 1583–1828 (2023) Cover See Marcelo E. Tolmasky et al., pp. 1591–1602. Image reproduced by permission of Marcelo E. Tolmasky and Prem Chapagain from RSC Med. Chem., 2023, 14, 1591. Inside cover See Zdeněk Musil, Radim Hrdina et al., pp. 1662–1666. 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US copyright law is applicable to users in the USA. Registered Charity No. 207890. View Article Online REVIEWS 1629 Research progress of anticancer drugs targeting CDK12 Open Access Article. Published on 19 September 2023. Downloaded on 5/18/2024 2:48:29 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Zhijia Yan, Yongli Du,* Haibin Zhang, Yong Zheng, Huiting Lv, Ning Dong and Fang He 1645 Druggable targets for the immunopathy of Alzheimer's disease Donald F. 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Enhancing allosteric inhibition of dihydrodipicolinate synthase through the design and synthesis of novel dimeric compounds Rebecca M. Christoff, Mohammad Al Bayer, Tatiana P. Soares da Costa, Matthew A. Perugini and Belinda M. Abbott* 1704 Proline pre-conditioning of Jurkat cells improves recovery after cryopreservation Alex Murray, Peter Kilbride and Matthew I. Gibson* 1712 Discovery of benzoxazole–thiazolidinone hybrids as promising antibacterial agents against Staphylococcus aureus and Enterococcus species Vijay Sai Krishna Cheerala, Abdul Akhir, Deepanshi Saxena, Rahul Maitra, Sidharth Chopra* and Sundaresan Chittor Neelakantan* 1722 De novo design of a stapled peptide targeting SARSCoV-2 spike protein receptor-binding domain Ravindra Thakkar,* Dilip K. Agarwal, Chathuranga B. Ranaweera, Susumu Ishiguro, Martin Conda-Sheridan, Natasha N. Gaudreault, Juergen A. Richt, Masaaki Tamura and Jeffrey Comer 1588 \| RSC Med. Chem., 2023, 14, 1585–1590 This journal is © The Royal Society of Chemistry 2023 View Article Online RESEARCH ARTICLES Open Access Article. Published on 19 September 2023. Downloaded on 5/18/2024 2:48:29 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 1734 Discovery and computational studies of piperidine/ piperazine-based compounds endowed with sigma receptor affinity Laura De Luca, Lisa Lombardo, Salvatore Mirabile, Agostino Marrazzo, Maria Dichiara, Giuseppe Cosentino, Emanuele Amata and Rosaria Gitto* 1743 Dual FLT3/haspin kinase inhibitor based on 3Hpyrazolo[4,3-f]quinoline scaffold with activities against acute myeloid leukemia Allison L. Kempen, Nickolas R. Brauer and Herman O. Sintim* 1755 Discovery of rigid biphenyl Plasmodium falciparum DHFR inhibitors using a fragment linking strategy Marie Hoarau,* Patpanat Sermmai, Thaveechai Varatthan, Ratthiya Thiabma, Tararat Jantra, Roonglawan Rattanajak, Danoo Vitsupakorn, Jarunee Vanichtanankul, Siriporn Saepua, Yongyuth Yuthavong, Chawanee Thongpanchang and Sumalee Kamchonwongpaisan 1767 First-in-class small molecule inhibitors of ICOS/ ICOSL interaction as a novel class of immunomodulators Somaya A. Abdel-Rahman, Katarzyna Świderek and Moustafa T. Gabr* This journal is © The Royal Society of Chemistry 2023 RSC Med. Chem., 2023, 14, 1585–1590 \| 1589 View Article Online RESEARCH ARTICLES 1778 Open Access Article. Published on 19 September 2023. Downloaded on 5/18/2024 2:48:29 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Hybrid molecules of protoflavones and spirooxindole derivatives with selective cytotoxicity against triplenegative breast cancer cells Gábor Girst, Elizabeth A. Lopes, Lídia M. Gonçalves, Margarida Espadinha, Norbert Kúsz, Hui-Chun Wang, Maria M. M. Santos* and Attila Hunyadi* 1787 Design, synthesis, and biological evaluation of novel pyrimidin-2-amine derivatives as potent PLK4 inhibitors Yanli Xue, Shuyi Mu, Pengkun Sun, Yin Sun, Nian Liu, Yu Sun, Lin Wang, Dongmei Zhao* and Maosheng Cheng 1803 Chloroacetamide fragment library screening identifies new scaffolds for covalent inhibition of the TEAD·YAP1 interaction Khuchtumur Bum-Erdene, Mona K. Ghozayel, Mark J. Zhang, Giovanni Gonzalez-Gutierrez and Samy O. Meroueh* 1817 Synthesis and antibacterial activity of 2-benzylidene-3-oxobutanamide derivatives against resistant pathogens Ankur Sood and Venkitasamy Kesavan* 1590 \| RSC Med. Chem., 2023, 14, 1585–1590 This journal is © The Royal Society of Chemistry 2023
https://openalex.org/W2889689154	https://europepmc.org/articles/pmc6135910?pdf=render	English	null	Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria	Frontiers in microbiology	2,018	cc-by	7,806	Edited by: Edited by: Peter Mullany, University College London, United Kingdom Reviewed by: Baltasar Mayo, Consejo Superior de Investigaciones Cientíﬁcas (CSIC), Spain Victor Krylov, I. I. Mechnikov Research Institute of Vaccines and Sera (RAS), Russia J. Tony Pembroke, University of Limerick, Ireland Correspondence: Dongchang Sun sundch@zjut.edu.cn Reviewed by: Baltasar Mayo, Consejo Superior de Investigaciones Cientíﬁcas (CSIC), Spain Victor Krylov, I. I. Mechnikov Research Institute of Vaccines and Sera (RAS), Russia J. Tony Pembroke, U i i f Li i k I l d MINI REVIEW published: 06 September 2018 doi: 10.3389/fmicb.2018.02154 MINI REVIEW Keywords: antibiotic resistance gene, multidrug resistance, DNA transfer, natural transformation, conjugation, Escherichia coli Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria Dongchang Sun College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China Horizontal gene transfer (HGT) plays an important role in bacterial evolution. It is well accepted that DNA is pulled/pushed into recipient cells by conserved membrane- associated DNA transport systems, which allow the entry of only single-stranded DNA (ssDNA). However, recent studies have uncovered a new type of natural bacterial transformation in which double-stranded DNA (dsDNA) is taken up into the cytoplasm, thus complementing the existing methods of DNA transfer among bacteria. Regulated by the stationary-phase regulators RpoS and cAMP receptor protein (CRP), Escherichia coli establishes competence for natural transformation with dsDNA, which occurs in agar plates. To pass across the outer membrane, a putative channel, which may compete for the substrate with the porin OmpA, may mediate the transfer of exogenous dsDNA into the cell. To pass across the inner membrane, dsDNA may be bound to the periplasmic protein YdcS, which delivers it into the inner membrane channel formed by YdcV. The discovery of cell-to-cell contact-dependent plasmid transformation implies the presence of additional mechanism(s) of transformation. This review will summarize the current knowledge about mechanisms of HGT with an emphasis on recent progresses regarding non-canonical mechanisms of natural transformation. Fully understanding the mechanisms of HGT will provide a foundation for monitoring and controlling multidrug resistance. INTRODUCTION Specialty section: This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology Received: 23 March 2018 Accepted: 22 August 2018 Published: 06 September 2018 Citation: Sun D (2018) Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria. Front. Microbiol. 9:2154. doi: 10.3389/fmicb.2018.02154 Specialty section: This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology Received: 23 March 2018 Accepted: 22 August 2018 Published: 06 September 2018 Specialty section: This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology Horizontal gene transfer (HGT) drives the evolution of bacteria. Transfer of antibiotic resistance genes (ARGs) plays an important role in the development of multidrug resistance (MDR) in bacteria (Forsberg et al., 2012). There are three “classical” methods of DNA transfer in nature: bacterial conjugation, natural transformation, and transduction (von Wintersdorﬀet al., 2016). Via HGT, exogenous DNA can be transferred from one bacterium to another even if they are only distantly related (Chen et al., 2005; Burton and Dubnau, 2010). With the accumulation of genes involved in diﬀerent resistance mechanisms from the exogenous DNA, bacteria are able to acquire MDR rapidly. For example, Acinetobacter and Enterobacter strains carrying the NDM-1 plasmid or mcr-1 plasmid, which contain a group of resistance genes, can tolerate even last-resort antibiotics (Yong et al., 2009; Wang and Sun, 2015; Liu et al., 2016; Shen et al., 2016; Zheng et al., 2017). Understanding the mechanisms of DNA transfer in bacteria would provide new strategies to help address the ongoing challenge of multi-drug-resistant bacteria in the future. Received: 23 March 2018 Accepted: 22 August 2018 Published: 06 September 2018 Citation: Sun D (2018) Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria. Front. Microbiol. 9:2154. doi: 10.3389/fmicb.2018.02154 September 2018 \| Volume 9 \| Article 2154 1 Frontiers in Microbiology \| www.frontiersin.org DNA Transport Systems Sun Natural bacterial transformation and conjugation have been found in bacteria and archaea. Two diﬀerent membrane protein complexes consisting of conserved proteins are responsible for pulling in and pushing out DNA during natural bacterial transformation and conjugation, respectively (Chen and Dubnau, 2004; Claverys et al., 2009; Burton and Dubnau, 2010; Johnston et al., 2014; Cabezon et al., 2015; Ilangovan et al., 2015, 2017). These DNA transport systems deliver single-stranded DNA (ssDNA) either from the donor cell (for conjugation) or into the recipient cell (for transformation) (Chen and Dubnau, 2004; Claverys et al., 2009; Burton and Dubnau, 2010; Johnston et al., 2014; Cabezon et al., 2015; Ilangovan et al., 2015, 2017). Recent studies have revealed two new types of DNA transfer in Escherichia coli. One of these methods has been shown to be independent of the conserved proteins for the transport of ssDNA during natural transformation or bacterial conjugation. Instead, double-stranded DNA (dsDNA) is taken up into the cytoplasm and internalized by E. coli cells on solid agar plates (Sun et al., 2006, 2009, 2013; Sun, 2011, 2016; Zhang et al., 2012). The other method of DNA transfer is dependent on cell- to-cell contact and DNA transfer occurs within a colony on agar plates (Maeda et al., 2004, 2006; Etchuuya et al., 2011; Sobue et al., 2011; Kurono et al., 2012; Matsuda et al., 2012; Matsumoto et al., 2016). DNA transfer via this method is sensitive to DNase I, indicating that DNA that is transported into the recipient cell is naked (rather than protein-protected). Although DNA transfer occurs on agar plates via both of the above two transformation methods, no evidence shows similarities between DNA transfer mechanisms of the two methods. In the former transformation method, DNA transfer occurs in the absence of donor cells. Whereas, in the latter transformation method, donor cells are required for DNA transfer in the colony. During bacterial conjugation, physical contact between the donor and the recipient cells is required. Nonetheless, the cell-to-cell contact- dependent plasmid transformation is diﬀerent from conjugation in that DNA transfer is not mediated by mobile elements (Maeda et al., 2004; Kurono et al., 2012; Matsuda et al., 2012). Crossing the OM For G−bacteria, a sophisticated protein complex is assembled in the OM, where the complex binds exogenous DNA and drags it into the periplasm (Figure 1). The assembly and FIGURE 1 \| Classical DNA uptake during natural transformation. Exogenous DNA is pulled into the cytoplasm by the extension and retraction of pseudopili, as a consequence of the assembly and disassembly of pseudopilin multimers (PilA). This is followed by the transfer of dsDNA across the OM protein PilQ/HofQ (for G−bacteria only). The DNA receptor (ComEA) mediates the transfer of one strand of DNA across the IM channel formed by ComEC with the assistance of the ATPase ComFA, accompanied by degradation of the other strand of DNA. The incoming ssDNA is protected by DprA which conveys it to RecA for homologous recombination in the cytoplasm. Citation: In this review, we will ﬁrst discuss the mechanisms of classical natural bacterial transformation and conjugation. Then, the non- canonical DNA transfer on agar plates will be described in detail, with an emphasis on how DNA is pulled into cells. Burton and Dubnau, 2010; Johnston et al., 2014; Cabezon et al., 2015; Ilangovan et al., 2015, 2017; Veening and Blokesch, 2017). Although conditions for competence induction vary widely among bacterial species (Aas et al., 2002; Berka et al., 2002; Claverys et al., 2006; Veening and Blokesch, 2017), proteins involved in DNA uptake are highly conserved even among distantly related bacteria (Johnston et al., 2014), except for Helicobacter pylori, which uses a conjugation-like system for DNA uptake during natural transformation (Smeets and Kusters, 2002). Here, the conserved DNA uptake system in bacteria is described. G−bacteria have an outer membrane (OM), whereas G+ bacteria have not. During natural transformation, G−bacteria need to pull DNA across both the OM and the inner membrane (IM), whereas G+ bacteria need to overcome the barrier of the peptidoglycan layer, which is much thicker and denser, and needs to be weakened before translocation of DNA across the IM. In this process, dsDNA is pulled across the OM in G−bacteria and ssDNA is pulled across the IM in both G+ and G−bacteria. The general mechanism underlying DNA transfer during natural transformation is summarized in Figure 1. Crossing the IM Bound by ComEA in the periplasm, exogenous DNA is translocated across the IM via a pore formed by ComEC, also named Rec2 in some G−bacteria (e.g., H. inﬂuenzae), a widely conserved IM protein for translocation of DNA across the IM (Barouki and Smith, 1985; Berge et al., 2002; Draskovic and Dubnau, 2005; Sinha et al., 2012; Baker et al., 2016; Salzer et al., 2016). It has been proposed that ComEC acts as both a translocase and nuclease during DNA translocation (Chen and Dubnau, 2004; Claverys et al., 2009; Burton and Dubnau, 2010; Johnston et al., 2014; Veening and Blokesch, 2017). In this process, one strand of the dsDNA is translocated into the cytoplasm, simultaneously the degradation of the other strand occurs (Barouki and Smith, 1985; Berge et al., 2002; Baker et al., 2016). In silico analysis of ComEC indicates that the β-lactamase- like domain at the C-terminus may function as a nuclease and Domain of Unknown function 4131 at the N-terminus has a DNA binding domain (Baker et al., 2016). It is possible that the dsDNA that is bound to the N-terminus of ComEC is divided into two strands of ssDNA and one of them is degraded by the nuclease at the C-terminus. g Protein-coated conjugative ssDNA is transported across the IM, periplasm and OM through a membrane channel formed by a group of proteins encoded by the conjugative DNA molecule (Goessweiner-Mohr et al., 2013; Cabezon et al., 2015; Ilangovan et al., 2015). Inside the channel, the conjugative pilus (formed by pilins) is responsible for pushing ssDNA out of membrane. The mechanism of DNA transfer via conjugation has been best exempliﬁed by the Vir system. To export DNA out of the donor cell, a conjugative plasmid encodes a complicated membrane protein complex. During conjugation, a plasmid- or ICE-encoded relaxase creates a nick in one strand of the conjugative DNA at the oriT site, followed by ssDNA translocation across the channel formed by components of the T4SS and replication of the remaining strand, either independently from or in concert with conjugation (Ilangovan et al., 2017). During translocation across cell membranes, three ATPases (VirD4, VirB4, and VirB11) provide the energy for DNA transport (Chen et al., 2005; Cabezon et al., 2015; Ilangovan et al., 2015). In natural transformation and conjugation, diﬀerent types of pili participate in the movement of DNA. PULL DNA IN DURING NATURAL TRANSFORMATION Natural transformation was discovered in Streptococcus pneumoniae in 1928 (Griﬃth, 1928). Induced by a heptadecapeptide pheromone, naturally transformable bacteria show a special physiological state termed “competence”, during which they are capable of pulling in exogenous DNA (Berka et al., 2002; Ogura et al., 2002). The mechanism of DNA transfer during natural transformation is well conserved among Gram-positive (G+) (e.g., Bacillus subtilis and S. pneumoniae) and Gram-negative (G−) bacterial species (e.g., Neisseria gonorrhoeae, Haemophilus inﬂuenzae, and Vibrio cholerae), as well as archaea (Chen and Dubnau, 2004; Claverys et al., 2009; FIGURE 1 \| Classical DNA uptake during natural transformation. Exogenous DNA is pulled into the cytoplasm by the extension and retraction of pseudopili, as a consequence of the assembly and disassembly of pseudopilin multimers (PilA). This is followed by the transfer of dsDNA across the OM protein PilQ/HofQ (for G−bacteria only). The DNA receptor (ComEA) mediates the transfer of one strand of DNA across the IM channel formed by ComEC with the assistance of the ATPase ComFA, accompanied by degradation of the other strand of DNA. The incoming ssDNA is protected by DprA which conveys it to RecA for homologous recombination in the cytoplasm. September 2018 \| Volume 9 \| Article 2154 Frontiers in Microbiology \| www.frontiersin.org 2 Sun DNA Transport Systems Crossing the IM Competence pili or pseudopili mediate the transfer of dsDNA across the membrane during natural transformation, whereas conjugative pili mediates the transfer of ssDNA across the membrane during conjugation (Cabezon et al., 2015; Ilangovan et al., 2015). In both cases, the assembly/disassembly of pili drives the movement of transferring DNA. It remains unclear how conjugative DNA is further transported in the recipient cell. The channel for DNA translocation is assumed to be formed by two ComEC monomers with seven transmembrane segments (Draskovic and Dubnau, 2005). Given that over-expression of ComEC is toxic to the cell, the structure of ComEC remains unresolved (Draskovic and Dubnau, 2005). The driving force for translocation of ssDNA across the IM may be provided by an ATPase (ComFA), which is also widely conserved in bacteria (Londono-Vallejo and Dubnau, 1994; Takeno et al., 2011; Chilton et al., 2017; Diallo et al., 2017). After translocation of ssDNA, DprA, and RecA bind ssDNA and catalyze the formation of joint DNA molecules for homologous recombination (Mortier- Barriere et al., 2007; Dwivedi et al., 2013; Yadav et al., 2013, 2014; Duﬃn and Barber, 2016; Diallo et al., 2017; Hovland et al., 2017; Le et al., 2017). In this way, the incoming foreign ssDNA displaces one strand of the chromosomal dsDNA (Mortier-Barriere et al., 2007), followed by being converted to homogeneous dsDNA through DNA replication. THE TRANSFER OF DNA DURING BACTERIAL CONJUGATION disassembly of a type IV pilus causes a ﬁber-like pseudopilus to be extruded out of and hauled back into the pore-forming OM proteins (Chen and Dubnau, 2004). The pore that accommodates exogenous DNA is 6–6.5 nm in diameter, and is formed by PilQ, a secretin that is 15 nm wide and 34 nm long with ﬁve rings and an extraordinary stable “cone” and “cup” structures (Chen and Dubnau, 2004). The pore cavity is large enough to accommodate dsDNA (∼2.4 nm) (Collins et al., 2001; Assalkhou et al., 2007; Burkhardt et al., 2011). Accompanied by the extension and retraction of type IV pili, DNA is transported across the OM through the pore (Laurenceau et al., 2013; Salzer et al., 2014, 2016; Leong et al., 2017). Between the OM and IM (the periplasm), the incoming DNA is bound by the substrate- binding protein ComEA, which prevents DNA from slipping by means of a “Brownian Ratcheting” mechanism (Inamine and Dubnau, 1995; Provvedi and Dubnau, 1999; Berge et al., 2002; Takeno et al., 2012; Seitz et al., 2014; Salzer et al., 2016). Bacterial conjugation was ﬁrst discovered in E. coli (Lederberg and Tatum, 1946). Relying on cell-to-cell contact, DNA can be pushed out of a donor cell and transported into a recipient cell during bacterial conjugation. A group of modular mobile genetic elements, known as integrative and conjugative elements (ICEs) or conjugative transposons (Franke and Clewell, 1981), has been found in many bacterial genomes (Wozniak and Waldor, 2010; Bi et al., 2012; Cury et al., 2017). ICEs can transfer from one bacterium to another, facilitating the spread of ARGs in environment (Wozniak and Waldor, 2010; Bi et al., 2012; Cury et al., 2017). The transfer of conjugative DNA across the membrane of the donor bacterium relies on a large membrane- associated protein complex, that belongs to the type IV secretion system (T4SS) (Goessweiner-Mohr et al., 2013; Cabezon et al., 2015; Ilangovan et al., 2015; Figure 2). Components of the T4SS for conjugation are encoded by genes of either self-replicable conjugative plasmids or ICEs in the chromosomal DNA of the donor bacterium (Cabezon et al., 2015; Ilangovan et al., 2015; Johnson and Grossman, 2015). The mechanism of DNA transfer is summarized in Figure 2. THE UPTAKE OF dsDNA IN E. coli E. coli has long been thought not to be naturally transformable. In this century, the natural transformation of E. coli has been observed initially on nutrient-deﬁcient agar plates and later September 2018 \| Volume 9 \| Article 2154 Frontiers in Microbiology \| www.frontiersin.org 3 DNA Transport Systems Sun FIGURE 3 \| A new route for dsDNA transfer in Escherichia coli. Via an unidentiﬁed channel, exogenous DNA transfers across the OM. The pore-forming protein OmpA can compete for DNA with the unidentiﬁed channel. To pass across the IM, the incoming DNA binds the substrate-binding protein YdcS and is translocated from the periplasm to the cytoplasm via an IM channel formed by YdcV. It remains unclear whether a plasmid enters E. coli as intact circular or linear dsDNA. FIGURE 3 \| A new route for dsDNA transfer in Escherichia coli. Via an unidentiﬁed channel, exogenous DNA transfers across the OM. The pore-forming protein OmpA can compete for DNA with the unidentiﬁed channel. To pass across the IM, the incoming DNA binds the substrate-binding protein YdcS and is translocated from the periplasm to the cytoplasm via an IM channel formed by YdcV. It remains unclear whether a plasmid enters E. coli as intact circular or linear dsDNA. FIGURE 3 \| A new route for dsDNA transfer in Escherichia coli. Via an unidentiﬁed channel, exogenous DNA transfers across the OM. The pore-forming protein OmpA can compete for DNA with the unidentiﬁed channel. To pass across the IM, the incoming DNA binds the substrate-binding protein YdcS and is translocated from the periplasm to the cytoplasm via an IM channel formed by YdcV. It remains unclear whether a plasmid enters E. coli as intact circular or linear dsDNA. E. coli cells is regulated by the transcriptional regulator RpoS and the cyclic AMP (cAMP) – cAMP receptor protein (CRP), and these cells can acquire exogenous DNA exclusively on agar plates (Zhang et al., 2012; Guo et al., 2015). The functions of RpoS or the cAMP-CRP complex in the chemical transformation of E. coli have not been found. E. coli cells is regulated by the transcriptional regulator RpoS and the cyclic AMP (cAMP) – cAMP receptor protein (CRP), and these cells can acquire exogenous DNA exclusively on agar plates (Zhang et al., 2012; Guo et al., 2015). The functions of RpoS or the cAMP-CRP complex in the chemical transformation of E. coli have not been found. THE UPTAKE OF dsDNA IN E. coli However, the stimulating eﬀect of agar on transformation is not due to an increase in Ca2+, Mg2+, or Mn2+ concentration (Sun et al., 2009). It remains unclear whether osmotic pressure and/or any other biological/physical factor(s) contribute to the increase of transformation on plates with a high concentration of agar. Second, an OM protein, OmpA, plays opposite roles in natural and chemical transformation of E. coli: it promotes chemical transformation but suppresses natural transformation (Sun et al., 2013). Third, exponentially growing E. coli cells are often employed for preparing chemically competent cells with the highest eﬃciency, and chemical transformation occurs in a liquid, whereas the natural transformation of stationary-phase on nutrient-rich agar plates (Tsen et al., 2002; Sun et al., 2006). Although natural plasmid transformation of E. coli shows single-hit kinetics, implying that dsDNA may enter the cell (Sun et al., 2009), there are basic diﬀerences between natural and chemical transformation. First, natural transformation is promoted by an increased concentration of agar, whereas chemical transformation relies on high concentrations of divalent ions (i.e., Ca2+, Mg2+, or Mn2+) (Sun et al., 2009). However, the stimulating eﬀect of agar on transformation is not due to an increase in Ca2+, Mg2+, or Mn2+ concentration (Sun et al., 2009). It remains unclear whether osmotic pressure and/or any other biological/physical factor(s) contribute to the increase of transformation on plates with a high concentration of agar. Second, an OM protein, OmpA, plays opposite roles in natural and chemical transformation of E. coli: it promotes chemical transformation but suppresses natural transformation (Sun et al., 2013). Third, exponentially growing E. coli cells are often employed for preparing chemically competent cells with the highest eﬃciency, and chemical transformation occurs in a liquid, whereas the natural transformation of stationary-phase THE UPTAKE OF dsDNA IN E. coli FIGURE 2 \| DNA transfer during bacterial conjugation. Conjugative DNA is processed into ssDNA by a relaxase (R) in the cytoplasm of the donor bacterium. To further transport ssDNA, a group of membrane and periplasmic proteins are assembled together to form a large complex, which can be subdivided into four distinct parts: (1) the pilus is formed by the assembly of pilins (VirB2) with adhesins (VirB5) at the distal end; (2) the OM component, which consists of VirB7, VirB9, and the C-terminus of VirB10; (3) the periplasmic component which consists of VirB8, VirB10, and VirB6; and (4) the IM component, formed of VirB3, VirB6, VirB8, and VirB10. Additionally, three hexameric ATPases (VirB4, VirB11, and VirD4) are attached to the IM to provide energy during DNA transfer. Natural transformation of E. coli also diﬀers from that of other naturally transformable bacteria in that the conserved DNA uptake machinery is not required for the uptake of exogenous dsDNA in E. coli (Sun et al., 2009). Some components of the conserved DNA uptake machinery are believed to function in using DNA as a nutrient in E. coli (Finkel and Kolter, 2001; Palchevskiy and Finkel, 2006). Nevertheless, attempts to conﬁrm these observations in three independent laboratories have not succeeded (Sun, 2011; Johnston et al., 2014). DNA, which has been thought to serve as the sole carbon source could not account for cell growth, implying that other nutrient sources should be present in the culture (Sun, 2011; Johnston et al., 2014). It is possible that degraded DNA in the minimal culture serves as a source of building blocks for the synthesis of new DNA in bacteria. During the natural transformation of E. coli, new ABC transporter proteins have been shown to participate in DNA transfer (Sun et al., 2009; Sun, 2016). These transporters are diﬀerent from the known classical DNA uptake proteins that mediate natural bacterial transformation. The mechanism of this new type of DNA transfer is proposed in Figure 3. on nutrient-rich agar plates (Tsen et al., 2002; Sun et al., 2006). Although natural plasmid transformation of E. coli shows single-hit kinetics, implying that dsDNA may enter the cell (Sun et al., 2009), there are basic diﬀerences between natural and chemical transformation. First, natural transformation is promoted by an increased concentration of agar, whereas chemical transformation relies on high concentrations of divalent ions (i.e., Ca2+, Mg2+, or Mn2+) (Sun et al., 2009). Frontiers in Microbiology \| www.frontiersin.org Crossing the OM According to the Transporter Classiﬁcation Database (TCDB1), ydcS and ydcV are predicted to encode proteins for binding a substrate in the periplasm and for translocation of the substrate across IM (Saier et al., 2014). YdcS has been shown as a PHB synthase in the periplasm (Dai and Reusch, 2008). The mutant lacking ydcT (a putative ATPase encoding gene) in the same operon as ydcS and ydcV, is naturally transformed with slightly but obviously reduced frequency (less than 50%) (Sun, 2016), indicating that this gene is also involved in natural plasmid transformation of E. coli. Nonetheless, with respect to signiﬁcantly reduced transformation frequency in the ydcV and the ydcS mutants, ydcT seems to have only a minor eﬀect on DNA transport. It is likely that additional energy source is required for eﬃcient transport of dsDNA across the IM. Crossing the OM Escherichia coli has a complete set of genes that potentially encode components of the classical DNA uptake machinery. These genes are homologous to the conserved DNA uptake genes in other naturally transformable bacteria. Comparative genomic analysis predicts that, in E. coli, putative DNA uptake genes hofQ and gspD September 2018 \| Volume 9 \| Article 2154 Frontiers in Microbiology \| www.frontiersin.org DNA Transport Systems Sun may encode proteins forming a channel for transporting DNA across the OM, and ppdD may encode pilins for the assembly of the competence pili or pseudopili which pull exogenous DNA in (Finkel and Kolter, 2001; Claverys and Martin, 2003; Sun et al., 2009). However, inactivation of hofQ, gspD, or ppdD does not aﬀect natural transformation with dsDNA, suggesting that the conserved DNA uptake machinery for translocation of ssDNA does not mediate dsDNA transfer across the OM in E. coli (Sun et al., 2009). To identify the OM pore used for DNA transport during natural transformation of E. coli, the pore-forming protein OmpA was evaluated, considering that OmpA performs functions in bacteriophage infection and bacterial conjugation. Inactivation of ompA increases natural transformation by 7- to 60-fold while decreasing chemical transformation by ∼10-fold, suggesting that OmpA blocks DNA transfer during natural transformation but promotes DNA transfer in artiﬁcial transformation (Sun et al., 2013). OmpA is unlikely to form an open gate under natural conditions, but can be switched to the open state with the molecular force of electrostatic interaction (i.e., salt-bridge), that drives structural transition of a protein under diﬀerent conditions (Hong et al., 2006). The closed and open states of the gate are dependent on the formation of salt bridges of Arg138-Glu52 and Lys82-Glu128, respectively (Hong et al., 2006). During natural transformation of E. coli, DNA may pass across an unidentiﬁed channel that competes with OmpA for transforming DNA. The putative channel may be consisted of OM components (i.e., OM protein and pili/psedopili) of a DNA transport system and pulls DNA into the cell on the LB-agar plate. In the default “gate-closed” state, OmpA traps the transforming DNA, making it unable to reach the right channel for completing transformation. By contrast, during chemical transformation or electroporation, a high concentration of Ca2+ or an electric current helps open the gate, allowing DNA to pass across the channel formed by OmpA (Sun et al., 2013). Crossing the IM Based on a membrane topology study, the conserved IM protein called ComEC is predicted to mediate the translocation of ssDNA during classical natural transformation (Chen and Dubnau, 2004; Claverys et al., 2009; Johnston et al., 2014). However, inactivation of the ComEC homolog YcaI in E. coli does not aﬀect natural plasmid transformation of E. coli, suggesting that DNA is translocated into the cytoplasm via a diﬀerent route (Sun et al., 2009). The single-hit kinetics in natural plasmid transformation of E. coli suggest that the establishment of a plasmid in the cytoplasm is mediated not by the annealing of partially overlapping opposite ssDNA derived from two independent plasmid monomers, but by a new route, i.e., the transfer of dsDNA across the IM of bacteria (Sun et al., 2009). Screening of RpoS-targeted transformation-related genes has identiﬁed ydcS and ydcV, which are located in the same ABC-transporter operon (Sun, 2016). Inactivation of ydcS and ydcV reduces natural transformation 6.7- and 9.5-fold, respectively (Sun, 2016). Chemical transformation is also reduced by Inactivation of ydcS, whereas the chemical transformation in a ydcV mutant is not reduced as compared to its wild-type counterpart (Sun, 2016). CELL-TO-CELL TRANSFER OF NON-CONJUGATIVE PLASMIDS Cell-to-cell contact is often required for bacterial conjugation. Of note, plasmid transformation of E. coli in colonies proceeds on the surface of agar plates and cell-to-cell contact is required for the transfer of plasmids that do not carry conjugative functions (Maeda et al., 2004, 2006). Cell-to-cell contact- dependent plasmid transformation occurs not only within the same genus but also across genera (Wang et al., 2007). In some naturally transformable G−bacteria (e.g., H. inﬂuenzae and N. gonorrhoeae), a short DNA sequence (named DUS) can increase DNA uptake (Claverys and Martin, 2003; Wang and Sun, 2015; Zheng et al., 2017). During cell-to-cell contact- dependent plasmid transformation of E. coli, an 88 bp DNA sequence promotes DNA transfer (Sobue et al., 2011). Screening of the Keio collection (Baba et al., 2006), a comprehensive library of E. coli knock-out mutants defective in non-essential genes, has identiﬁed rodZ, whose product regulates the rod-shape of the cell, as an essential gene for cell-to-cell contact-dependent transformation (Kurono et al., 2012). Because the homologs of DNA uptake genes (e.g., ycaI) have not been revealed to be essential genes, conventional DNA uptake machinery is not likely to be involved in cell-to-cell contact-dependent plasmid transformation of E. coli. Recently, cell-to-cell contact-dependent transformation is discovered in B. subtilis (Zhang et al., 2018). However, inactivation of the competence regulator ComK, that controls the expression of conserved DNA uptake genes, abolishes cell-to-cell DNA transfer (Zhang et al., 2018), indicating that mechanisms of the cell-to-cell DNA transfer in E. coli and B. subtilis are basically diﬀerent. 1http://www.tcdb.org September 2018 \| Volume 9 \| Article 2154 Frontiers in Microbiology \| www.frontiersin.org REFERENCES Claverys, J. P., Prudhomme, M., and Martin, B. (2006). Induction of competence regulons as a general response to stress in gram-positive bacteria. Annu. Rev. Microbiol. 60, 451–475. doi: 10.1146/annurev.micro.60.080805.142139 Aas, F. E., Wolfgang, M., Frye, S., Dunham, S., Lovold, C., and Koomey, M. (2002). Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression. Mol. Microbiol. 46, 749–760. doi: 10.1046/j.1365-2958.2002.03193.x Collins, R. F., Davidsen, L., Derrick, J. P., Ford, R. C., and Tonjum, T. 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https://openalex.org/W2470199523	https://www.frontiersin.org/articles/10.3389/fgene.2016.00117/pdf	English	null	The Genetic Architecture of Barley Plant Stature	Frontiers in genetics	2,016	cc-by	12,268	Edited by: Daniel Pinero, Universidad Nacional Autónoma de México, Mexico Edited by: Daniel Pinero, Universidad Nacional Autónoma de México, Mexico Reviewed by: Songnian Hu, Beijing Institute of Genomics-Chinese Academy of Sciences, China Caiguo Zhang, Universidad Nacional Autónoma de México, Mexico Reviewed by: Songnian Hu, Beijing Institute of Genomics-Chinese Academy of Sciences, China Caiguo Zhang, Universidad Nacional Autónoma de México, Mexico Correspondence: Ahmad M. Alqudah alqudah@ipk-gatersleben.de; Thorsten Schnurbusch thor@ipk-gatersleben.de The Genetic Architecture of Barley Plant Stature Ahmad M. Alqudah 1, Ravi Koppolu 1, Gizaw M. Wolde 1, Andreas Graner 2 and Thorsten Schnurbusch 1* Ahmad M. Alqudah 1, Ravi Koppolu 1, Gizaw M. Wolde 1, Andreas Graner 2 and Thorsten Schnurbusch 1 1 HEISENBERG-Research Group Plant Architecture, Leibniz-Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany, 2 Research Group Genome Diversity, Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany Plant stature in temperate cereals is predominantly controlled by tillering and plant height as complex agronomic traits, representing important determinants of grain yield. This study was designed to reveal the genetic basis of tillering at ﬁve developmental stages and plant height at harvest in 218 worldwide spring barley (Hordeum vulgare L.) accessions under greenhouse conditions. The accessions were structured based on row-type classes [two- vs. six-rowed] and photoperiod response [photoperiod-sensitive (Ppd-H1) vs. reduced photoperiod sensitivity (ppd-H1)]. Phenotypic analyses of both factors revealed profound between group effects on tiller development. To further verify the row-type effect on the studied traits, Six-rowed spike 1 (vrs1) mutants and their two-rowed progenitors were examined for tiller number per plant and plant height. Here, wild-type (Vrs1) plants were signiﬁcantly taller and had more tillers than mutants suggesting a negative pleiotropic effect of this row-type locus on both traits. Our genome-wide association scans further revealed highly signiﬁcant associations, thereby establishing a link between the genetic control of row-type, heading time, tillering, and plant height. We further show that associations for tillering and plant height are co-localized with chromosomal segments harboring known plant stature-related phytohormone and sugar-related genes. This work demonstrates the feasibility of the GWAS approach for identifying putative candidate genes for improving plant architecture. Keywords: barley, tillering, plant height, vrs1, Ppd-H1, GWAS INTRODUCTION Specialty section: This article was submitted to Plant Genetics and Genomics, a section of the journal Frontiers in Genetics Specialty section: This article was submitted to Plant Genetics and Genomics, a section of the journal Frontiers in Genetics Tillering is one of the key components for improving grain yield in temperate cereals, such as wheat (Triticum aestivum L.) and barley (Sreenivasulu and Schnurbusch, 2012; Kebrom et al., 2013; Hussien et al., 2014). Cereals are able to maximize grain yield through increased tillering (Evers and Vos, 2013) and increasing the number of fertile tillers (bearing fertile spikes) was proposed as one of the most important components for grain yield in wheat and barley (Sreenivasulu and Schnurbusch, 2012; Xie et al., 2016). Variation in tillering was attributed to genetic variation between barley genotypes in tiller production (Abeledo et al., 2004; Alqudah and Schnurbusch, 2014), or genetic variation in pre-anthesis phase duration in a bi-parental population (Borras et al., 2009), or partially to the environmental inﬂuence (Abeledo et al., 2004; Borras et al., 2009). Previous studies on tillering mainly focused on the ﬁnal tiller number at harvest but a few studies focused on tillering at diﬀerent developmental stages, such as Borras et al. (2009); however, until now no study has documented the natural variation of tillering at pre-anthesis stages. Received: 05 April 2016 Accepted: 07 June 2016 Published: 24 June 2016 ORIGINAL RESEARCH published: 24 June 2016 doi: 10.3389/fgene.2016.00117 Citation: Alqudah AM, Koppolu R, Wolde GM, Graner A and Schnurbusch T (2016) The Genetic Architecture of Barley Plant Stature. Front. Genet. 7:117. doi: 10.3389/fgene.2016.00117 June 2016 \| Volume 7 \| Article 117 Frontiers in Genetics \| www.frontiersin.org 1 The Genetic Architecture of Barley Plant Stature Alqudah et al. The generation of additional side-tillers (i.e., tillering) requires tiller bud formation and outgrowth, which is a complex developmental process under the control of genetic factors, environment, and phytohormone action (Kebrom et al., 2013). In barley, bud outgrowth into tillers (side-branches) happens sequentially after the three leaf stage (Kirby and Appleyard, 1987). The number of developing tiller buds or bud outgrowth is inﬂuenced by growing conditions such as light and water (Doust, 2007; Evers and Vos, 2013; Kebrom et al., 2013). Moreover, bud outgrowth in monocots and dicots is regulated by a complex and conserved pathway of phytohormones and their interactions including auxin, strigolactones [(SL; as suppressors)], cytokinins (as promoters by reducing auxin; Kebrom et al., 2013) and other hormones like brassinosteroids, abscisic acid (ABA), ethylene, and gibberellins (GAs). The role of phytohormones in bud outgrowth is well-reviewed (Evers and Vos, 2013; Kebrom et al., 2013) while Evers and Vos (2013) describe a mathematical model for tillering in cereals. So far, most of the phytohormonal knowledge in barley and wheat tillering regulation are based on results extrapolated from other grass species, such as rice and maize; however, hormonal pathways regulating bud outgrowth are not yet fully understood. many QTL for barley plant height overlapping with previously mapped QTL and known genes. However, natural variation in plant height is still insuﬃcient to understand the importance of this trait with respect to other agronomical traits. Thus, tools like GWAS analyses using high density genetic maps based upon diﬀerent population structures are key to increase our knowledge concerning genetic factors controlling plant height. g So far several barley tillering mutant loci were identiﬁed, including uniculme4 (cul4, Tavakol et al., 2015), many noded dwarf6/densinodosum6 (mnd6/den6, Dabbert et al., 2010), uniculme2 (cul2), intermedium spike-m (int-m) intermedium spike-b (int-b; Babb and Muehlbauer, 2003), granum-a (gra-a; Dabbert et al., 2010), and absent lower laterals (als; Dabbert et al., 2009), which also aﬀect other barley plant architectural traits. In addition to sdw1, other plant height mutants are available, including sdw2-4 and short culm 1(hcm; Borner et al., 1999; Franckowiak et al., 2005). Citation: Functional interaction studies of these mutants showed pleiotropic or epistatic eﬀects between plant height and tiller development such as gra-a (Dabbert et al., 2010). Therefore, studying these traits in diverse barley collection can potentially explain the interconnection between these traits. Recently, several studies highlighted the importance of sugars as a key component of plant stature regulations, for instance Evers (2015). The regulatory role of sugars during branching might be through regulating physiological mechanisms involving hormonal genes (Barbier et al., 2015). In any case, the role of sugars in shoot branching is not well-understood, and therefore, further genetic investigations on the role of sugars in shoot branching are required to reveal the underlying regulatory network of shoot branching in cereals. In barley, Vrs1 is the major gene controlling the row-type of the spike (Komatsuda et al., 2007). In its functional form, Vrs1 produces the two-rowed spike phenotype; while mutations in Vrs1 result in the six-rowed spike phenotype. In our previous study we found substantial diﬀerences between two- and six-row barleys in terms of tiller number under various growth conditions with high heritability values (Alqudah and Schnurbusch, 2014). Very recently, Liller et al. (2015) similarly found that the allelic status at vrs1 pleiotropically aﬀected tiller number. Furthermore, PHOTOPERIOD RESPONSE LOCUS 1 (Ppd-H1) is the key regulator of heading time in barley (Turner et al., 2005). Karsai et al. (1999) studied the eﬀect of Ppd-H1 on agronomical traits including tillering and plant height in a bi-parental barley mapping population. To the best of our knowledge, no research was performed to identify the natural variation of tillering and plant height based on row-type classes and allelic status at Ppd-H1 in barley. Thus, this study was designed to detect QTL underlying natural variation of tiller number per plant at diﬀerent pre-anthesis stages and plant height at harvest based upon diﬀerences in row-type and photoperiod response by phenotyping a worldwide spring barley collection under controlled greenhouse (GH) conditions. The GWAS analysis using a 9k gene-based single nucleotide polymorphisms (SNPs) chip (Comadran et al., 2012) provided an unprecedented genetic resolution for the studied traits. The strategy of phenotyping the plants at pre-anthesis stages emphasized that present genetic variation of tillering could be genetically dissected. In this study, development stage-speciﬁc QTL i.e., QTL that have not been reported before were detected for tillering and plant height. Frontiers in Genetics \| www.frontiersin.org The Collection and Population Structure A collection of 218 spring barley worldwide accessions was used in this study that includes 125 two- and 93 six-rowed accessions (Pasam et al., 2012 and Table S1). Moreover, the collection was divided into two groups based on allelic variation at the Ppd-H1 locus (SNP22, G/T, Turner et al. (2005) and Sharma et al., in preparation), 95 photoperiod-sensitive (Ppd-H1) and 123 accessions carrying the reduced photoperiod sensitivity (ppd-H1) allele (Alqudah et al., 2014). The collection was structured using 6355 polymorphic SNPs. The collection includes 149 cultivars, 57 landraces and 18 breeding lines previously described by Haseneyer et al. (2010). Genome-Wide Association Study (GWAS) Analysis Analysis GWAS of groups was performed using their corresponding genotype and phenotype datasets. A mixed linear model (MLM) using GenStat 16 (Genstat, 2014) was used to calculate associations between estimated phenotypic traits (BLUEs) and each single marker. Association analysis in MLM was performed using single trait association analysis with Eigen-analysis as correction of population structure and controlling false positive associations (Genstat, 2014). For detecting signiﬁcant associations, we considered a threshold P-value of 0.01 (i.e., –log10 P ≥2) in all traits. A multiple test, i.e., the false discovery rate (FDR), was calculated using GenStat 16 (Genstat, 2014) to determine the signiﬁcance level of the SNP P-value at <0.05 to exclude false-positive associations (Storey and Tibshirani, 2003). Through this conservative method, we tightly set the signiﬁcance level of the SNP P-value providing highly signiﬁcant associations (−log10 P ≥FDR). FDR approach is strictly used to validate the associations in complex traits such as heading date (Alqudah et al., 2014; Pauli et al., 2014). Allele eﬀects were estimated relative to the performance of cultivar “Mansholt zweizeilig” for six-rowed and Ppd-H1 groups and cultivar “Isaria” for two-rowed and ppd-H1 groups. We used SNP markers that passed the FDR threshold to determine highly associated QTL within conﬁdence interval ±5 cM. The interval ±5 cM was found as an average linkage disequilibrium in this population (Pasam et al., 2012), so we used it as a conﬁdence interval to determine highly associated QTL. Known tillering and plant height genes (bold and italicized) were genetically anchored and located according to the Barke × Morex RILs (POPSEQ) sequence contigs using IPK barley BLAST server, Gatersleben (http://webblast.ipk-gatersleben.de/barley/). More information about these genes, their genbank accession numbers, barley high conﬁdence probability gene, and their genetic positions are shown in Table S3. Citation: Apart from this, several putative orthologous barley genes (characterized for tillering and plant height in other species) were genetically mapped onto barley chromosomes based on SNP marker associations obtained from our GWAS study. Understanding the mechanisms of tillering may help to better understand and modify crop architecture in order to achieve better yield (Sreenivasulu and Schnurbusch, 2012). Several genes regulating tiller formation have already been identiﬁed and characterized such as TEOSINTE BRANCHED 1 (TB1) in maize, which is under the control of SL, inhibit bud outgrowth by regulating maize GRASSY TILLERS 1 (GT1; Kebrom et al., 2013). INTERMEDIUM-SPIKE C (Int-C; Ramsay et al., 2011) in barley, which is an ortholog of TB1; whereas barley SIX-ROWED SPIKE 1 (Vrs1) is a homolog of GT1 (Whipple et al., 2011). Even though, these genes inhibit lateral growth (branching), they do so in a diﬀerent developmental context (Kebrom et al., 2013). Decreasing barley plant height was the main strategy for improving grain yield and harvest index through reduced lodging (Bezant et al., 1996), using SEMI-DWARF 1 (sdw1 or denso) in Europe (EU) and East Asia (EA; Hellewell et al., 2000). Wang et al. (2010) found that dwarﬁng genes in barley have negative impact on spike agronomical traits such as spike length and grain density. The relationship between plant height and heading date was documented by Lin et al. (1995), where three alleles at the sdw1 locus were associated with delay in heading (Hellewell et al., 2000) and some other alleles are day-length sensitive (Wang et al., 2010). Recently, Wang et al. (2014) found a new plant height QTL that positively aﬀects barley agronomic traits and grain yield. Through Genome-Wide Association Studies (GWAS), Pasam et al. (2012) and Pauli et al. (2014) detected June 2016 \| Volume 7 \| Article 117 Frontiers in Genetics \| www.frontiersin.org 2 The Genetic Architecture of Barley Plant Stature Alqudah et al. Genotyping Genotyping of this collection was performed using a genome- wide high-density 9K SNPs chip from IlluminaTM that assayed 7842 SNPs (Comadran et al., 2012). The markers that passed minor allele frequency (MAF) ≥0.05 were used in association analysis (6355 SNPs, Table S2). Finally, we used 4323, 4320, 4228, and 4050 SNPs for GWAS analysis of two-rowed, six-rowed, Ppd-H1, and ppd-H1groups, respectively. On average about 4200 SNPs per accession were scored and around 210 accessions per marker were used in analysis. We used genetic marker positions anchored by physical map positions SNPs markers generated based on Barke × Morex RILs POPSEQ population (Mascher et al., 2013). MATERIALS AND METHODS The vrs1 mutants of Barke, Bonus, and Foma were used in this study to collect tillering data at two developmental stages (Z37, ﬂag leaf just visible and heading time, Z55). Barke mutant (8408- 1) was described by Gottwald et al. (2009); whereas vrs1 mutants from Bonus (hex-v.03) and Foma (Int-d.12) were described by Komatsuda et al. (2007). Phenotypic data of 218 accessions were analyzed by REML (Residual Maximum Likelihood) and BLUEs (Best Linear Unbiased Estimates) to estimate each accession’s phenotypic mean, which in turn were used in the association analysis (SAS, 2006). Fisher’s least signiﬁcant diﬀerence (LSD) was used to compare between groups (i.e., two- vs. six-rowed and photoperiod-sensitive vs. reduced photoperiod sensitivity) and to compare between genotypes with mutants at the probability level P ≤0.05. Broad-sense heritability for traits in each group was calculated across growing times as the ratio between the genetic variance and the phenotypic variance which includes genotypic by growing times (environment) interaction variance and error variance components using PROC VARCOMP (SAS, 2006). Frontiers in Genetics \| www.frontiersin.org RESULTS to very high in all groups (Table 2), indicating that they are predominantly genetically controlled. to very high in all groups (Table 2), indicating that they are predominantly genetically controlled. Correlation Analysis between Thermal Time of Developmental Stages and Studied Traits Using 6355 polymorphic SNPs markers from 9k array, the collection of 218 worldwide spring barley accessions was separable into two subpopulations: (i) based on row-type classes (two- and six-rowed phenotypes; Figure S1A), and (ii) based on alleles for photoperiod response to long day conditions [photoperiod-sensitive, Ppd-H1, and one speciﬁc reduced photoperiod sensitivity allele, ppd-H1; SNP22, G/T, Turner et al. (2005) Figure S1B]. Correlation analysis between studied traits and thermal time at developmental stages was performed on the whole collection (Figure 3). Generally, correlation values were moderate (r ≈ 0.6∗∗) between total tiller number per plant and growing-degree days (GDD) at AP, TIP, and HD stages (Figure 3), while only low (r ≈0.4∗∗) at AE. The correlation values between total number of tillers at pre-anthesis developmental stages (e.g., at AP and at TIP) and GDD ranged between 0.30∗∗–0.65∗∗(Figure 3). There was no clear trend of correlations between productive tiller number at Hrv and GDD of pre-anthesis stages and total tiller number at these stages. In contrast, only weak correlations were obtained between non-productive tillers at Hrv and GDD of pre-anthesis stages and total tiller number at these stages (r ≈0.2∗; Figure 3). For plant height, there were no correlations between GDD and plant height at diﬀerent developmental stages (Figure 3). These ﬁndings suggest that longer phase duration may lead to more tillers during pre-anthesis phases and more non-productive tillering at Hrv. Natural Variation of Tillering The major loci for row-type (Vrs1) and heading time (Ppd- H1) appear to be the key genetic determinants aﬀecting tiller number in the whole collection (Figure S6A). These genes were consistently detectable during early pre-anthesis stages (AP, TIP, and HD). Therefore, GWAS was conducted for the four groups separately (two-rowed, six-rowed, Ppd-H1, ppd-H1). Phenotypic Variation of Tillering at Different Developmental Stages p g Signiﬁcant diﬀerences (P ≤0.05) in tiller number per plant were found between row-type classes and photoperiod response groups. Two-rowed barley had signiﬁcantly higher total number of tillers per plant compared to six-rowed at all developmental stages (Figure 1A). To further investigate the row-type eﬀect on tillering, analyses of vrs1 mutants and their progenitors were performed which showed that the total number of tillers per plant was signiﬁcantly higher in two-rowed progenitors (Table 1). For photoperiod response groups, we found that plants with reduced photoperiod sensitivity (ppd-H1) had signiﬁcantly more total tillers per plant at all developmental stages compared to photoperiod sensitive plants (Ppd-H1; Figure 1B). The variation within two-rowed and ppd-H1 was larger than in other groups (Figures 1A,B). At harvest stage, the number of productive and non-productive tillers were signiﬁcantly higher in two-rowed and ppd-H1 groups (Figures 1A,B). Based on the origin of accessions in each group, EU accessions had more tillers per plant at pre- anthesis stages and non-productive tillers at Hrv in case of two- rowed, six-rowed and Ppd-H1 groups; whereas the diﬀerence was not evident in the ppd-H-1 group (Figures S2A–D). In terms of biological status, we found that improved cultivars had signiﬁcantly higher number of total tillers per plant at pre- anthesis stages and non-productive tillers at Hrv (at P ≤0.05) than breeder’s lines and landraces likely because of selection (Figure S3). Phenotypic Data yp Seeds from each of the 218 spring barley accessions were grown for 10 days under controlled GH condition (LD condition, 16/8 h day/night and ∼20/16◦C day/night). Thirty seedlings of each accession were grown in 0.5-L pots (one plant per pot; 9-cm pot diameter and 9-cm height) in the GH. Previous tests by Alqudah and Schnurbusch (2014) showed that this pot size eﬀectively restricted excessive tillering and enabled to genetically evaluate single plant potential for tillering under GH conditions. Pots were randomized three times per week to reduce border and temperature-gradient eﬀects on plant growth and development. The phenotypic experiments were performed from September 2011 to April 2012 in eight consecutive batches due to limited GH space and feasibility of workload. The experiment had a completely randomized design with 30 plants per accession. Details about growth conditions, experimental setup and phenotyping for dissecting the pre-anthesis phase, i.e., developmental stages [awn primordium (AP, Z31–33); Tipping (TIP, Z49); heading (HD, Z55); anther extrusion (AE, Z65)]; can be found in Alqudah and Schnurbusch (2014) and Alqudah et al. (2014). The total tiller number per plant was recorded from three plants/accession (each plant was considered as a biological replicate) at each developmental stages (AP, TIP, HD, and AE), while at harvest (Hrv) total tillers from six biological replicates (plants) were grouped as productive (tiller, carrying spike) and non-productive tillers (tiller, without spike). Plant height data were collected from six biological replicates (plants) at Hrv as the distance between soil and the top of the plant without spike. June 2016 \| Volume 7 \| Article 117 3 The Genetic Architecture of Barley Plant Stature Alqudah et al. Frontiers in Genetics \| www.frontiersin.org QTL Detection for Tillering within Row-Type Groups GWAS analysis for 125 two- and 93 six-rowed accessions was performed to study the natural variation within each group. We detected in total 53 signiﬁcant marker-trait associations (≥FDR; Table 3) distributed across 15 chromosomal QTL regions (chromosomal region in red color). Only one six-rowed- speciﬁc QTL (5H 31.7–34.3 cM) was identiﬁed, while 14 QTL were two-rowed-speciﬁc (Figure 4). Plenty of natural genetic variation was found at pre-anthesis stages (AP, TIP, and HD; Figure 4). Phenotypic Variation of Plant Height Analysis of plant height at harvest did not show any signiﬁcant diﬀerence between row-type classes and between photoperiod groups (P ≤0.05; Figures 2A,B). However, analyses of vrs1 mutants and their progenitors found that wild-type plants were taller than mutants at HD stage (Z 55, Table 1). The geographical origins of the accessions showed signiﬁcant diﬀerences (at P ≤ 0.05) in plant height within photoperiod response groups (Figure S4). AM accessions were the tallest in the Ppd-H1 group; whereas these accessions were also shortest in the ppd-H1 group (Figures S4C,D). In our study, we did not ﬁnd any eﬀect of biological status (at P ≤0.05) on plant height (Figure S5). Through detailed association analysis, we found several interesting regions for tillering based on row-type classes. Six chromosomal regions have tillering eﬀects, such as on 1H, 61.5–66.3 cM; 2H, 6.6–7.4 cM; 3HL, 128–137.7 cM; 4H, 101– 102 cM; 5H, 31.7–34.1; and 6H, 16.9–24.6 cM (Figure 4) of which ﬁve regions are putatively novel QTL lacking known candidate genes. The chromosomal regions at 1H, 2H, and 4H strongly appeared at earlier stages (AP∗and TIP). We were unable to co- locate known genes for other signiﬁcant chromosomal regions on 5H (31.7–34.1 cM), where we hypothesize that the EARLY MATURITY 7 (eam7) locus could underlie the 6H 17 cM QTL (Alqudah et al., 2014). The phenotypic data of studied traits at diﬀerent developmental stages for 218 accessions are available in Table S4. Interestingly, broad-sense heritability values for the traits studied (tiller number and plant height) ranged from high June 2016 \| Volume 7 \| Article 117 4 Alqudah et al. The Genetic Architecture of Barley Plant Stature FIGURE 1 \| Boxplots of total tiller number per plant in both row-type classes (A) and photoperiod groups (B). The degree of signiﬁcance indicated as P, 0.05; P, 0.01; *P, 0.001. Signiﬁcant differences (p ≤0.05) were determined with a one-way ANOVA using LSD. Signiﬁcant differences between the groups were calculated for each developmental stage separately. Three biological replicates were used from each accession at each pre-anthesis developmental stage and six biological replicates were used for counting productive and non-productive tiller at harvest stage. (n = 125 and 93 for two- and six-rowed barleys, respectively; and n = 95 and 123 for photoperiod sensitive and reduced photoperiod sensitivity barley, respectively). Phenotypic Variation of Plant Height AP, awn primordium, Alqudah and Schnurbusch (2014); TIP, tipping, Z49; HD, heading, Z55; AE, anther extrusion, Z65; Hrv, Harvest, Zadoks et al. (1974). Developmental stages calculated based on thermal time ◦C × D−1 (GDD). FIGURE 1 \| Boxplots of total tiller number per plant in both row-type classes (A) and photoperiod groups (B). The degree of signiﬁcance indicated as P, 0.05; P, 0.01; P, 0.001. Signiﬁcant differences (p ≤0.05) were determined with a one-way ANOVA using LSD. Signiﬁcant differences between the groups were calculated for each developmental stage separately. Three biological replicates were used from each accession at each pre-anthesis developmental stage and six biological replicates were used for counting productive and non-productive tiller at harvest stage. (n = 125 and 93 for two- and six-rowed barleys, respectively; and n = 95 and 123 for photoperiod sensitive and reduced photoperiod sensitivity barley, respectively). AP, awn primordium, Alqudah and Schnurbusch (2014); TIP, tipping, Z49; HD, heading, Z55; AE, anther extrusion, Z65; Hrv, Harvest, Zadoks et al. (1974). Developmental stages calculated based on thermal time ◦C × D−1 (GDD). The highest signiﬁcant marker eﬀects were found for SNPs on 2H (19.9 cM), which co-localized with Ppd-H1 at all pre-anthesis stages in two-rowed barleys (Figure 4), whereby the Ppd-H1 group reduced the number of tillers per plant at AP, TIP and HD stages by −0.86, −1.32, and −0.68 tillers per plant, respectively. Another signiﬁcant region is co-localized with the position of Barley FLORICAULA/LEAFY (BFL, 2HL 107.3 cM) and SOLUBLE STARCH SYNTHASE (HvSSIIIb, 2HL 112.1 cM, Figure 4,), which appeared at TIP, reducing tillering by ∼−1.0 tiller per plant. Moreover, several chromosomal regions were precisely co-localized with genes in the centromeric region of 2H (∼58 cM) with signiﬁcant eﬀects at AP and TIP [(e.g., 2H 58 cM, CENTRORADIALIS (eps2/HvCEN/eam6), and SUGAR SIGNALLING IN BARLEY 2 (HvSUSIBA2); 2H 59.4 cM, HEADING DATE6 (HD6-2H); 2H 64.73 cM, CONSTANS4 (HvCO4)]. The centromeric region on 2H also includes very interesting tillering-related genes like GIBBERELLIN-INSENSITIVE DWARF2 (HvGID2); KNOTTED1-LIKE HOMEOBOX1 (HvKNOX1); and DWARF11 (HvD11); (Figure 4 and Table S3). However, the possibility of linkage is high in centromeric region, so we cannot be sure which gene(s) cause the phenotypic eﬀect. Phenotypic Variation of Plant Height Genotype Tiller per plant Plant height (cm) Z37† Z55 Z37 Z55 Barke 12.0a 17.3a 46.3a 75.6a 8408-1 mutant 6.3b 11.0b 49.6a 67.3b Bonus 14.3a 18.6a 43.3a 84.6a hex-v.3 7.3b 11.3b 41.3a 71.3b Foma 12.3a 20.3a 36.6b 55.3a int-d.12 4.3b 12.0b 50.0a 59.6a Wild type Vrs1 12.8a 18.7a 42.1a 71.7a Mutant vrs1 5.9b 11.4b 46.9a 66.1b †Z37, ﬂag leaf just visible; and Z55, heading time. Three biological replicates were used from each genotype at each developmental stage. different letters in each pair indicate there is signiﬁcant difference at P = 0.05 according to the LSD test. Other interesting associations were also found around the centromeric region of 4H (51 cM) including DORMANCY-ASSOCIATED1 (HvDRM1, 44.90 cM); HvCO16, HvPRR59, HvPhyB/HvPRR73 (51.1-51.4 cM); GDSL ESTERASE/LIPASE PROTEIN112, WILTED DWARF AND LETHAL1 (HvGELP112/HvWDL1, 51.4 cM; Figure 4), or on 5H (46.3–47.5 cM), which includes HvCMF13, ASPARAGINE SYNTHASE1 (HvAS1), HvD53, and BRITTLE CULM12/GIBBERELLIN-DEFICIENT DWARF1 (HvBC12/GGD1). Other interesting associations were also found around the centromeric region of 4H (51 cM) including DORMANCY-ASSOCIATED1 (HvDRM1, 44.90 cM); HvCO16, HvPRR59, HvPhyB/HvPRR73 (51.1-51.4 cM); GDSL ESTERASE/LIPASE PROTEIN112, WILTED DWARF AND LETHAL1 (HvGELP112/HvWDL1, 51.4 cM; Figure 4), or on 5H (46.3–47.5 cM), which includes HvCMF13, ASPARAGINE SYNTHASE1 (HvAS1), HvD53, and BRITTLE CULM12/GIBBERELLIN-DEFICIENT DWARF1 (HvBC12/GGD1). GWAS analysis in the ppd-H1 group expresses the importance of heading time genes on tillering. On 2H (40.8–52.8 cM) is an example about heading time genes, including HvCO18 and HvFT4, and the region on 5H (119.8–125.8 cM) covering Vrn- H1/Phy-C. These ﬁndings reinforce that some of the heading time genes may have a pleiotropic eﬀect on tillering at diﬀerent developmental stages. In this study we detected two interesting regions for non- productive tillering in six-rowed barleys on 7H (140.9 cM), which is close to BRASSINOSTEROID DEFICIENT DWARF2/ DIMINUTO, DWARF1 (HvBRD2/HvDIM/HvDWF1, 140.6 cM), and on 6H (9.1 cM). Findings in this section conﬁrmed that there is plenty of variation in tillering especially at early developmental stages. Several associations are co-located with regions being associated with putative candidate genes while few appear to be novel. By association analysis, we found three overlapping, seemingly sugar-related QTL aﬀecting tiller number. Here, chromosome 3H (57.1–62.5 cM includes HvHXK9 and HvHXK6) showed a major eﬀect in the Ppd-H1 group [(TIP∗∗, and AE∗(Figure 5) thereby promoting tillering by +1.7 and +1 tillers, respectively)]; while it also increased productive tillering at Hrv in the ppd- H1 group. Phenotypic Variation of Plant Height Besides these associations, we found other associations close to putative heading time genes on 6H [(49.22 cM, CCT MOTIF FAMILY3 (HvCMF3); 52.62 cM, HvCO7; 54.2 cM, CYTOCHROME P450 (HvCYP734A7); 55.38 cM, ARABIDOPSIS PSEUDO-RESPONSE REGULATOR1/ TIMING OF CAB EXPRESSION1 (HvPRR1/HvTOC1); 59.06 cM, CRYPTOCHROMES1b (HvCry1b); 67.91 cM, HvCO14; 68.20 cM, HvCO2; and 69.2, HvCO11, Figure 4 and Table S3)]. June 2016 \| Volume 7 \| Article 117 Frontiers in Genetics \| www.frontiersin.org 5 The Genetic Architecture of Barley Plant Stature Alqudah et al. TABLE 1 \| Total tiller number per plant and plant height in Barke, Bonus, and Foma (Vrs1) and their induced mutants Barke mutant (8408-1), hex-v.3, and int-d.12 (vrs1), respectively, at two developmental stages. TABLE 1 \| Total tiller number per plant and plant height in Barke, Bonus, and Foma (Vrs1) and their induced mutants Barke mutant (8408-1), hex-v.3, and int-d.12 (vrs1), respectively, at two developmental stages. in the Ppd-H1 group; whereas the eﬀect was negative (reduced tillering) for the ppd-H1 allele. We detected one strong group- speciﬁc association for non-productive tillering (ppd-H1/Non- P∗), including three markers on 2H at 29.4 cM, but failed to ﬁnd known candidate gene close to this QTL. On 4H, we found one signiﬁcantly associated region at 43.5–45.7 cM [i.e., TWISTED DWARF 1/TUBULIN ALPHA-2 (HvTID1/HvTUA2) and HvDRM1], which is important for productive tillering in both groups speciﬁcally for increasing productive tiller number. We found several signiﬁcant chromosomal regions without known candidate genes. For instance, group-speciﬁc (Ppd-H1) associations on 1H (95.9–96.9 and 103.8–106.2 cM) and 2H at 6.5–8.9 cM and 73.7–83.8 cM included the major row-type gene Vrs1, which inﬂuenced productive tiller number at Hrv (Figure 5). The associated region on chromosome 4H at 54.0– 54.3 cM is without candidate gene and important for productive tiller number in Ppd-H1 and non-productive tillering in the ppd- H1 group. In addition, we detected putatively novel associations on 5H (143.7–146.1 cM) and 6H (28.3–28.9 cM), which appear important for tillering at diﬀerent developmental stages. These results clearly demonstrate that using photoperiod responses as a basis for dividing our population is worthwhile to better understand the natural genetic variation of tillering in this germplasm panel. TABLE 1 \| Total tiller number per plant and plant height in Barke, Bonus, and Foma (Vrs1) and their induced mutants Barke mutant (8408-1), hex-v.3, and int-d.12 (vrs1), respectively, at two developmental stages. Phenotypic Variation of Plant Height The signiﬁcant chromosomal region on 4H (81.2– 91.3 cM) includes the SUCROSE TRANSPORTER 1 (HvSUT1) gene and had an impact on productive tiller number within Ppd-H1 and non-productive tillering in ppd-H1. The third QTL (i.e., for productive tillering; in the Ppd-H1 group) is located on 5HS close to HvSUT2. These observations may hint toward the importance of sugar-related genes on tillering in cereals. QTL Detection for Tillering within Photoperiod Response Groups †AP, awn primordium, Alqudah and Schnurbusch (2014); TIP, tipping, Z49; HD, heading, Z55; AE, anther extrusion, Z65; Hrv, Harvest, Zadoks et al. (1974). H2: broad-sense heritability for each group overall growing times based on accessions mean. n = 125 and 93 for two- and six-rowed barleys, respectively. n = 95 and 123 for barleys with photoperiod-sensitive and reduced photoperiod sensitivity, respectively. Developmental stages calculated based on thermal time ◦C × D-1 (GDD). ASSOCIATED1 (HvLHY/HvCCA1) and similarly on 3H (44.3– 46.2 cM; HvD2, HvHXK7, HvGI, HvGA3ox2/HvD18). The importance of the latter QTL is that it is group-speciﬁc for productive tillering in the Ppd-H1 group. Finally, the QTL on 6H (87.6–95 cM) includes ﬁve associated markers close to CONSTITUTIVELY PHOTOMORPHOGENIC1 (HvCOP1, 88.6 cM), and TREHALOSE-6-PHOSPHATE SYNTHASE2 (HvTPS2). GWAS results stratiﬁed according to photoperiod response show that tillering is complex and that genetic variation at late-developmental stages is important to understand the genetic factors controlling the formation of productive and non-productive tillers. 10 signiﬁcant chromosomal regions (Figure 6) with a total of 26 signiﬁcant marker-trait associations displaying signiﬁcance (≥FDR). Looking at the genetic variation within groups no marker-trait association was detected for the photoperiod- sensitive group (Ppd-H1). ASSOCIATED1 (HvLHY/HvCCA1) and similarly on 3H (44.3– 46.2 cM; HvD2, HvHXK7, HvGI, HvGA3ox2/HvD18). The importance of the latter QTL is that it is group-speciﬁc for productive tillering in the Ppd-H1 group. Finally, the QTL on 6H (87.6–95 cM) includes ﬁve associated markers close to CONSTITUTIVELY PHOTOMORPHOGENIC1 (HvCOP1, 88.6 cM), and TREHALOSE-6-PHOSPHATE SYNTHASE2 (HvTPS2). GWAS results stratiﬁed according to photoperiod response show that tillering is complex and that genetic variation at late-developmental stages is important to understand the genetic factors controlling the formation of productive and non-productive tillers. g p Six signiﬁcant regions belong to the ppd-H1 group of which one is without known candidate genes on 1H (10.9–13.4 cM; Figure 6); interestingly all of these six QTL are also closely co- localized with plant height QTL in two- and six-rowed accessions. Two regions very precisely co-localized with putative heading time genes (e.g., on 1H 47.5–48 cM (HvCMF10); and on 7H (41.9–42.3 cM) close to HvCO8. One signiﬁcant chromosomal region is a putatively sugar-related QTL on 3H (54.5–59.6 cM) including HvHXK9. While two other interesting associations were found around the centromeric region on 4H and 6H including several associated candidate genes for plant height and heading time. QTL Detection for Tillering within Photoperiod Response Groups H2: broad-sense heritability for each group overall growing times based on accessions mean. n = 125 and 93 for two- and six-rowed barleys, respectively. n = 95 and 123 for barleys with photoperiod-sensitive and reduced photoperiod sensitivity, respectively. Developmental stages calculated based on thermal time ◦C × D-1 (GDD). FIGURE 2 \| Boxplots of plant height (cm) in both row-type classes (A) and photoperiod response groups (B). Signiﬁcant differences (p ≤0.05) were determined with a one-way ANOVA using LSD. Signiﬁcant differences between the groups were calculated harvest stage. Six biological replicates were used from each accession at harvest stage. (n = 125 and 93 for two- and six-rowed barleys, respectively; and n = 95 and 123 for photoperiod sensitive and reduced photoperiod sensitive barley, respectively). TABLE 2 \| Estimation of broad-sense heritability (H2) for tiller number per plant at different developmental stages and plant height at harvest. FIGURE 2 \| Boxplots of plant height (cm) in both row-type classes (A) and photoperiod response groups (B). Signiﬁcant differences (p ≤0.05) were determined with a one-way ANOVA using LSD. Signiﬁcant differences between the groups were calculated harvest stage. Six biological replicates were used from each accession at harvest stage. (n = 125 and 93 for two- and six-rowed barleys, respectively; and n = 95 and 123 for photoperiod sensitive and reduced photoperiod sensitive barley, respectively). TABLE 2 \| Estimation of broad-sense heritability (H2) for tiller number per plant at different developmental stages and plant height at harvest. Group Tiller per plant Plant height AP† TIP HD AE Hrv Hrv Productive Non-productive Two-rowed 0.80 0.92 0.94 0.96 0.92 0.88 0.92 Six-rowed 0.70 0.94 0.90 0.85 0.92 0.90 0.95 Ppd-H1 0.78 0.90 0.93 0.87 0.90 0.85 0.93 ppd-H1 0.76 0.91 0.92 0.91 0.91 0.87 0.92 †AP, awn primordium, Alqudah and Schnurbusch (2014); TIP, tipping, Z49; HD, heading, Z55; AE, anther extrusion, Z65; Hrv, Harvest, Zadoks et al. (1974). H2: broad-sense heritability for each group overall growing times based on accessions mean. n = 125 and 93 for two- and six-rowed barleys, respectively. n = 95 and 123 for barleys with photoperiod-sensitive and reduced photoperiod sensitivity, respectively. Developmental stages calculated based on thermal time ◦C × D-1 (GDD). ad-sense heritability (H2) for tiller number per plant at different developmental stages and plant height at harvest. QTL Detection for Tillering within Photoperiod Response Groups p p p GWAS analysis in both photoperiod response groups, i.e., Ppd- H1 (95 accessions) and ppd-H1 (123 accessions), identiﬁed 51 marker-trait associations (≥FDR) distributed across 17 chromosomal regions (Figure 5). Most of the associated markers were detected from AE to harvest and 10 QTL appeared to be stage-speciﬁc at Hrv for productive and non-productive tillering (Figure 5). p g g g Four strong associations included genes related to plant stature, sugar and heading time. On 5H (43.7–50.0 cM; HvCO3, TREHALOSE-6-PHOSPHATE SYNTHASE1 (HvTPS1), BRASSINOSTEROID C-23 HYDROXYLASE (HvCPD), NARROW LEAF AND DWARF1/ TERMINAL FLOWER1 (HvND1, TFL1), HvAS1, HvD53, and HvBC12/GGD1), all of these genes are located in the centromeric region, and hence, it is not clear which gene(s) cause the eﬀect. The same conclusion can be drawn for the region on 7H (64–71 cM; HvCO12/HvCO13/H; HvCO1, WEALTHY FARMERS PANICLE1/ IDEAL PLANT ARCHITECTURE1/SQUAMOSA PROMOTER BINDING PROTEIN-LIKE14 (HvWFP1/HvIPA1/HvSPL14); LATE ELONGATED HYPOCOTYL/CIRCADIAN CLOCK One major association was on 1H (43.1–55.7 cM centromeric region), which included 22 associated markers (≥FDR) and the GA INSENSITIVE DWARF 1 (HvGID1), HEXOKINASE 1 (HvHXK1), SOLUBLE STARCH SYNTHASE (HvSSIIIa), HEXOSE TRANSPORTATION 1, 2/ SUGAR TRANSPORTER (HvSTP1,2/HvSuT4) genes (Figure 5). This region has conﬂicting eﬀects on tiller number, i.e., a positive eﬀect (enhanced tillering) June 2016 \| Volume 7 \| Article 117 Frontiers in Genetics \| www.frontiersin.org 6 The Genetic Architecture of Barley Plant Stature Alqudah et al. FIGURE 2 \| Boxplots of plant height (cm) in both row-type classes (A) and photoperiod response groups (B). Signiﬁcant differences (p ≤0.05) were determined with a one-way ANOVA using LSD. Signiﬁcant differences between the groups were calculated harvest stage. Six biological replicates were used from each accession at harvest stage. (n = 125 and 93 for two- and six-rowed barleys, respectively; and n = 95 and 123 for photoperiod sensitive and reduced photoperiod sensitive barley, respectively). TABLE 2 \| Estimation of broad-sense heritability (H2) for tiller number per plant at different developmental stages and plant height at harvest. Group Tiller per plant Plant height AP† TIP HD AE Hrv Hrv Productive Non-productive Two-rowed 0.80 0.92 0.94 0.96 0.92 0.88 0.92 Six-rowed 0.70 0.94 0.90 0.85 0.92 0.90 0.95 Ppd-H1 0.78 0.90 0.93 0.87 0.90 0.85 0.93 ppd-H1 0.76 0.91 0.92 0.91 0.91 0.87 0.92 †AP, awn primordium, Alqudah and Schnurbusch (2014); TIP, tipping, Z49; HD, heading, Z55; AE, anther extrusion, Z65; Hrv, Harvest, Zadoks et al. (1974). Genetic Variation of Plant Height There is no clear eﬀect of vrs1 and Ppd-H1 on the plant height (Figure S6B), however, we used them to structure the population. GWAS analysis of the entire population detected June 2016 \| Volume 7 \| Article 117 Frontiers in Genetics \| www.frontiersin.org 7 The Genetic Architecture of Barley Plant Stature Alqudah et al. FIGURE 3 \| Correlation matrix for the studied traits with growing-degree days (GGD). The degree of signiﬁcance indicated as P, 0.05; P, 0.01; P, 0.001. AP, awn primordium, Alqudah and Schnurbusch (2014); TIP, tipping, Z49; HD, heading, Z55; AE, anther extrusion, Z65; Hrv, Harvest, Zadoks et al. (1974). Developmental stages calculated based on thermal time ◦C × D−1 (GDD). TABLE 3 \| False Discovery Rate (FDR) threshold (P = 0 05) for tiller number per plant at each developmental stage and plant height at harvest in group of FIGURE 3 \| Correlation matrix for the studied traits with growing degree days (GGD) The degree of signiﬁcance indicated as P 0 05; P 0 01; P 0 001 FIGURE 3 \| Correlation matrix for the studied traits with growing-degree days (GGD). The degree of signiﬁcance indicated as P, 0.05; P, 0.01; *P, 0.001. AP, awn primordium, Alqudah and Schnurbusch (2014); TIP, tipping, Z49; HD, heading, Z55; AE, anther extrusion, Z65; Hrv, Harvest, Zadoks et al. (1974). Developmental stages calculated based on thermal time ◦C × D−1 (GDD). TABLE 3 \| False Discovery Rate (FDR) threshold (P = 0.05) for tiller number per plant at each developmental stage and plant height at harvest in group of barley accessions. Group Tiller number per plant Plant height AP† TIP HD AE Hrv Hrv Productive Non-productive Two-rowed 3.44 (52) 3.46 (58) 3.44 (15) 3.52 (24) 3.70 (0) 4.28 (7) 3.49 (5) Six-rowed 3.45 (1) 3.32 (0) 3.21 (0) 3.48 (8) 3.72 (0) 4.30 (3) 3.42 (17) Ppd-H1 5.12 (2) 4.98 (4) 4.61 (3) 5.05 (2) 3.60 (72) 2.97 (0) 3.60 (1) ppd-H1 4.51 (2) 4.75 (6) 3.71 (0) 5.21 (1) 4.06 (8) 3.10 (38) 3.41 (17) Whole population 3.32 (6) SNPs exceeding FDR threshold are considered as highly signiﬁcant SNPs. Number of SNPs exceeding FDR threshold are shown in brackets. †AP, awn primordium, (Alqudah and Schnurbusch, 2014; Alqudah et al., 2014); TIP, tipping, Z49; HD, heading, Z55; AE, anther extrusion, Z65; Hrv, Harvest, (Zadoks et al., 1974; Alqudah et al., 2014). 6.4 cM) and two were located in signiﬁcant chromosomal regions. Genetic Variation of Plant Height The two-rowed-speciﬁc QTL on 1H (10.9–13.4 cM) is shared with a ppd-H1-speciﬁc QTL, while the second QTL Four marker-trait associations were detected in the subpopulation of two-rowed barley; two of which were single-marker-trait associations (Figure 6; 5H 85.6 and 6H June 2016 \| Volume 7 \| Article 117 Frontiers in Genetics \| www.frontiersin.org 8 Alqudah et al. The Genetic Architecture of Barley Plant Stature 4 \| Genetically anchored position of highly associated QTL for tiller number per plant at all barley developmental stages based on spike row-type (two-rowed/six-rowed) groups using 9K kers. Bold and italicized gene names indicate genetically anchored positions of known heading time and plant stature genes in the Barke x Morex RILs. Associated chromosomal regions are highlighted with olors according to developmental stages. Red chromosomal areas indicate the range of signiﬁcantly associated QTL (within conﬁdence interval ±5 cM) which are exceeding FDR level of each developmental nchored position of highly associated QTL for tiller number per plant at all barley developmental stages based on spike row-type (two-rowed/six-rowed) groups using 9K licized gene names indicate genetically anchored positions of known heading time and plant stature genes in the Barke x Morex RILs. Associated chromosomal regions are highlighted with developmental stages. Red chromosomal areas indicate the range of signiﬁcantly associated QTL (within conﬁdence interval ±5 cM) which are exceeding FDR level of each developmental FIGURE 4 \| Genetically anchored position of highly associated QTL for tiller number per plant at all barley developmental stages based on spike row-type (two-rowed/six-rowed) groups using 9K SNP markers. Bold and italicized gene names indicate genetically anchored positions of known heading time and plant stature genes in the Barke x Morex RILs. Associated chromosomal regions are highlighted with different colors according to developmental stages. Red chromosomal areas indicate the range of signiﬁcantly associated QTL (within conﬁdence interval ±5 cM) which are exceeding FDR level of each developmental stage. FIGURE 4 \| Genetically anchored position of highly associated QTL for tiller number per plant at all barley developmental stages based on spike row-type (two-rowed/six-rowed) groups using 9 SNP markers. Bold and italicized gene names indicate genetically anchored positions of known heading time and plant stature genes in the Barke x Morex RILs. Associated chromosomal regions are highlighted wi different colors according to developmental stages. Genetic Variation of Plant Height Red chromosomal areas indicate the range of signiﬁcantly associated QTL (within conﬁdence interval ±5 cM) which are exceeding FDR level of each development stage. plant at all barley developmental stages based on spike row-type (two-rowed/six-rowed) groups usin eading time and plant stature genes in the Barke x Morex RILs. Associated chromosomal regions are highlighted signiﬁcantly associated QTL (within conﬁdence interval ±5 cM) which are exceeding FDR level of each developm FIGURE 4 \| Genetically anchored position of S FIGURE 4 \| Genetically anchored position SNP markers Bold and italicized gene name June 2016 \| Volume 7 \| Article 117 9 Frontiers in Genetics \| www.frontiersin.org Alqudah et al. The Genetic Architecture of Barley Plant Stature FIGURE 5 \| Genetically anchored position of highly associated QTL for tiller number per plant at all barley developmental stages based on photoperiod responses [photoperiod-sensitive (Ppd-H1)/reduced photoperiod sensitivity (ppd-H1)] groups using 9K SNP markers. Bold and italicized gene names indicate genetically anchored positions of known heading time and plant stature genes in the Barke × Morex RILs. Associated chromosomal regions are highlighted with different colors according to developmental stages. Red chromosomal areas indicate the range of signiﬁcantly associated QTL (within conﬁdence interval ±5 cM) which are exceeding FDR level of each developmental stage. 5 \| Genetically anchored position of highly associated QTL for tiller number per plant at all barley developmental stages based on photoperiod responses [photoperiod-sensitive )/reduced photoperiod sensitivity (ppd-H1)] groups using 9K SNP markers. Bold and italicized gene names indicate genetically anchored positions of known heading time and plant stature genes in e × Morex RILs. Associated chromosomal regions are highlighted with different colors according to developmental stages. Red chromosomal areas indicate the range of signiﬁcantly associated QTL (within ce interval ±5 cM) which are exceeding FDR level of each developmental stage. nchored position of highly associated QTL for tiller number per plant at all barley developmental stages based on photoperiod responses [photoperiod-sensitive period sensitivity (ppd-H1)] groups using 9K SNP markers. Bold and italicized gene names indicate genetically anchored positions of known heading time and plant stature genes in ssociated chromosomal regions are highlighted with different colors according to developmental stages. Red chromosomal areas indicate the range of signiﬁcantly associated QTL (within which are exceeding FDR level of each developmental stage. Genetic Variation of Plant Height FIGURE 5 \| Genetically anchored position of highly associated QTL for tiller number per plant at all barley developmental stages based on photoperiod responses [photoperiod-sensitiv (Ppd-H1)/reduced photoperiod sensitivity (ppd-H1)] groups using 9K SNP markers. Bold and italicized gene names indicate genetically anchored positions of known heading time and plant stature the Barke × Morex RILs. Associated chromosomal regions are highlighted with different colors according to developmental stages. Red chromosomal areas indicate the range of signiﬁcantly associated QT conﬁdence interval ±5 cM) which are exceeding FDR level of each developmental stage. FIGURE 5 \| Genetically anchored position of highly associated QTL for tiller number per plant at all barley developmental stages based on photoperiod responses [photoperiod-sen (Ppd-H1)/reduced photoperiod sensitivity (ppd-H1)] groups using 9K SNP markers. Bold and italicized gene names indicate genetically anchored positions of known heading time and plant st the Barke × Morex RILs. Associated chromosomal regions are highlighted with different colors according to developmental stages. Red chromosomal areas indicate the range of signiﬁcantly associat conﬁdence interval ±5 cM) which are exceeding FDR level of each developmental stage. IGURE 5 \| Genetically anchored position of highly a Ppd-H1)/reduced photoperiod sensitivity (ppd-H1)] he Barke × Morex RILs. Associated chromosomal region onﬁdence interval ±5 cM) which are exceeding FDR leve June 2016 \| Volume 7 \| Article 117 Frontiers in Genetics \| www.frontiersin.org 10 Alqudah et al. The Genetic Architecture of Barley Plant Stature nchored position of highly associated QTL for plant height at harvest stage based on spike row-type (two-rowed/six-rowed) and photoperiod responses Ppd-H1)/reduced photoperiod sensitivity (ppd-H1)] groups using 9K SNP markers. Bold and italicized gene names indicate genetically anchored positions of known heading time he Barke × Morex RILs. Associated chromosomal regions are highlighted with different colors according to group. Red chromosomal areas indicate the range of signiﬁcantly associated l ±5 M) hi h di FDR l l f h FIGURE 6 \| Genetically anchored position of highly associated QTL for plant height at harvest stage based on spike row-type (two-rowed/six-rowed) and photoperiod responses [photoperiod-sensitive (Ppd-H1)/reduced photoperiod sensitivity (ppd-H1)] groups using 9K SNP markers. Bold and italicized gene names indicate genetically anchored positions of known heading time and plant stature genes in the Barke × Morex RILs. Associated chromosomal regions are highlighted with different colors according to group. The Signiﬁcance of the Experimental Approach Analysis of tillering at diﬀerent pre-anthesis developmental stages provided an unprecedented overview on the natural variation of tiller outgrowth in our worldwide barley collection. This approach appears to be helpful to better understand genetic factors controlling tillering in cereals. Previous tillering studies in barley were mainly conducted under ﬁeld conditions to associate the ﬁnal number of tillers at harvest with yield or developmental stages like HD (Borras et al., 2009; Alqudah and Schnurbusch, 2014). High broad-sense heritability values for plant stature- related traits were obtained in comparison with previous studies (Rasmusson, 1987; Borras et al., 2009; Pauli et al., 2014), most likely because of accurate phenotyping following a single-plant phenotyping strategy at diﬀerent developmental stages under controlled GH conditions (Alqudah and Schnurbusch, 2014, 2015; Alqudah et al., 2014). Therefore, the power of the current GWAS to detect associated loci was increased compared with previous ﬁeld studies of the same germplasm (e.g., Pasam et al., 2012), demonstrating that GH conditions are appropriate for studying plant stature-related traits. Analysis of tillering at diﬀerent pre-anthesis developmental stages provided an unprecedented overview on the natural variation of tiller outgrowth in our worldwide barley collection. Accessions with delayed heading time (those carrying the ppd-H1 allele) showed a more complex genetic constitution for tillering, possibly due to longer pre-anthesis phase durations and more non-productive tillering than early heading accessions (Ppd-H1). This observation may also reinforce our previous ﬁndings that the pre-anthesis period is critical for tiller development and any tiller developed after heading might not develop productive spikes. Spike row-type classes in barley (two- and six-rowed) were found as one of the major determinants of population structure in most barley GWAS analyses (Pasam et al., 2012; Pauli et al., 2014); however, we subdivided our population based on Ppd- H1 alleles and row-type classes and hence were able to detect a rich source of genetic variation for plant stature traits. The solidity of the found marker-trait associations (FDR) approach, e.g., also used by Pauli et al. (2014), in combination with the latest version of the barley physical map enabled us to clearly locate genetic marker positions and associate detected QTL with Results obtained for hormone-related QTL aﬀecting tillering showed associations especially at Hrv. For instance, expression of DRM1-like in wheat is known to be associated with tiller bud dormancy in a tiller inhibition (tin) mutant (Kebrom et al., 2012). DISCUSSION The two-rowed group exhibited a more complex genetic make-up for tillering than six-rowed types. The eﬀect of the row- type gene Vrs1 on tillering at early developmental stages became evident after studying vrs1 mutants. Here, wild-type plants had signiﬁcantly more tillers than mutants, which is in agreement with recently published results obtained by Liller et al. (2015). It is known that Vrs1 determines spike row-type (Komatsuda et al., 2007). Due to its known role as a negative regulator of lateral spikelet fertility in the spike, one might assume that wild- type Vrs1 also negatively aﬀects tillering; but this was evidentially not the case in our wild-type/mutant analyses and GWAS panel. In fact, lateral spikelet abortion of the spike, but increased tiller number in two-rowed types, is most likely explainable as a negative pleiotropic eﬀect of Vrs1, whereby grain setting potential is compensated through tillering; or vice-versa for six-rowed types. QTL for Tiller Number per Plant In six-rowed barley accessions, six marker-trait associations were detected. Two were single-marker-trait associations (Figure 6; 3H 1.0 cM and 5H 95.5 cM); while from the remaining four QTL, one lacks any candidate genes (putatively novel QTL) on 5H (21.3–23.6 cM). The QTL on 3H (54.5–59.6 cM) includes HvHXK9 and was shared (ppd-H1 group). The strongest association for plant height was located on 4H between 59.6–59.8 cM∗, which is co-located with HvD4, and supported by 10 markers reducing plant height in six-rowed accessions by 7 cm. Another interesting six-rowed speciﬁc association is located in the centromeric region on 7H (71.2–73.6 cM). QTL for Tiller Number per Plant Several putatively novel candidate regions were associated with tillering at diﬀerent developmental stages based on row-type and/or photoperiod response groups. For instance, in the present study we detected putatively novel QTL without known candidates for productive tiller number in the Ppd-H1 group. The QTL on 1H (95.9–96.9 cM), 2H (6.5–8.9 cM) and 5H (143.7–146.1 cM) showed that there may be an opportunity to genetically optimize yield through increased productive tillering. Interestingly, the QTL on 2H at 6.5–8.9 cM is close to HvBRD, known as an important regulator of barley plant stature traits (Dockter et al., 2014), suggesting that this gene could be a putative candidate for controlling productive tiller number, too. Most of the putatively novel QTL appeared at earlier developmental stages in two-rowed barley, which are mostly carrying the ppd-H1 allele, showing delayed development and thus may produce more tillers. Functional analysis of these novel genomic regions will help to expand our knowledge about tillering in cereals. GWAS analysis for plant height using diﬀerent population structures revealed an association with sugar-related and heading time genes on plant height. Nonetheless, we found putatively novel QTL regions, which certainly require further validation work. Frontiers in Genetics \| www.frontiersin.org Genetic Variation of Plant Height Red chromosomal areas indicate the range of signiﬁcantly associated QTL (within conﬁdence interval ±5 cM) which are exceeding FDR level of each group. e (two-rowed/six-rowed) and photoperiod responses gene names indicate genetically anchored positions of known heading oup. Red chromosomal areas indicate the range of signiﬁcantly associa FIGURE 6 \| Genetically anchored position of highly associated QTL for plant height at harvest stage based on spike row-type (two-rowed/six-rowed) and photoperiod responses [photoperiod-sensitive (Ppd-H1)/reduced photoperiod sensitivity (ppd-H1)] groups using 9K SNP markers. Bold and italicized gene names indicate genetically anchored positions of known hea and plant stature genes in the Barke × Morex RILs. Associated chromosomal regions are highlighted with different colors according to group. Red chromosomal areas indicate the range of signiﬁcantly as QTL (within conﬁdence interval ±5 cM) which are exceeding FDR level of each group. arvest stage based on spike row-type g 9K SNP markers. Bold and italicized ge ted with different colors according to grou [photoperiod-sensitive (Ppd-H1)/reduced pho and plant stature genes in the Barke × Morex RIL icle 1 GU 6 \| Ge e ca y a c o ed pos o [photoperiod-sensitive (Ppd-H1)/reduced and plant stature genes in the Barke × Morex FIGURE 6 \| Genetically anchored position [ h t i d iti (P d H1)/ d d June 2016 \| Volume 7 \| Article 117 11 Frontiers in Genetics \| www.frontiersin.org The Genetic Architecture of Barley Plant Stature Alqudah et al. located at the end of 2H (146.4–147.5 cM) is overlapping with associations from the whole collection (i.e., ALL; Figure 6). These QTL appeared to be novel without known candidate genes associated with them, indicating that this study is able to reveal potentially new plant height QTL in two-rowed barley. candidate gene(s). This approach has the potential to create novel, hypothesis-driven research questions but similarly may provide a glimpse into ontogenetic traits, which are associated with speciﬁc gene classes, families, hormones, or metabolic pathways. QTL for Plant Height In this study, we found three putatively novel plant height QTL (1H, 10.9–13.4; 2H, 146.4–147.5; 5H, 21.3–23.6) which were not reported in previous GWAS analyses such as Pasam et al. (2012) and Pauli et al. (2014) or bi-parental mapping studies (Wang et al., 2014) conducted under ﬁeld conditions. In-depth genetic analyses of these important QTL are worthwhile targets to improve lodging resistance and subsequently yield. g Marker-trait associations explored the importance of putative heading time genes particularly those carrying CCT [CO, CO- LIKE, TIMING OF CAB1 (TOC1)] domain and B-box domains (CO-like genes) in the natural variation of tillering. These ﬁndings imply that CO-like genes might also be involved in tillering; however, more genetic analyses are required to elucidate their role and expand our current knowledge about these genes. Notably, the region around BFL (2H, 107.3 cM) was strongly associated with several SNPs in the two-rowed group, suggesting that this region has an important role in tillering in addition to regulating phase duration (Alqudah et al., 2014). Here, signiﬁcant eﬀects were found for markers co-locating with BFL thereby reducing tiller number by one tiller per plant. Further characterization of this gene is necessary to evaluate its importance in barley plant stature. In our germplasm panel, natural genetic variation for plant height was genetically less complex than for tillering, most likely due to the low variation in plant height as was reported by Pasam et al. (2012). The vrs1 mutant analysis suggests that Vrs1 also regulates plant height in addition to lateral spikelet/ﬂoret development and tillering. Associations close to HvD4 predominantly appeared in six-rowed accessions. This gene is known to impact plant height in rice, where mutants show mild semi dwarﬁsm due to defects in brassinosteroid biosynthesis (Sakamoto et al., 2006). Thus, variation for plant height in our collection could be attributed to brassinosteroid deﬁciency. Another interesting association was found in the chromosomal region that includes COP1 (6H, 88.6 cM) especially when Ppd-H1 alleles were less active (i.e., ppd-H1, more tillers). Arabidopsis COP1 regulates photomorphogenesis in seedlings and it also has pleiotropic phenotypes during late developmental stages (Nakagawa and Komeda, 2004). HvCOP1 appears to be a late heading time gene (Alqudah et al., 2014) which likely promotes tillering in the late heading ppd-H1 group. Taken together, allelic variation around HvCOP1 appears as the ﬁrst report for temperate cereals that this gene aﬀects tillering possibly through controlling vegetative-to-generative phase-transition. The Signiﬁcance of the Experimental Approach The HvDRM1 region (4H, 44.9 cM) appeared in six-rowed and photoperiod sensitive groups, suggesting that allelic variation at this chromosomal region is crucial for producing less but mainly productive tillers. Similar conclusions can be drawn for the June 2016 \| Volume 7 \| Article 117 12 The Genetic Architecture of Barley Plant Stature Alqudah et al. chromosomal region around HvTID1/HvTUA2 (4H, 39.8 cM), which is known to control plant stature through changing the number of cells in the shoot apical meristem in rice (Sunohara et al., 2009). In contrast to the HvDRM1 region, associations close to BRASSINOSTEROID DEFICIENT DWARF2/ DIMINUTO, DWARF1 (HvBRD2/HvDIM/HvDWF1; i.e., 7H, 140.6 cM) lead to produce non-productive tillers possibly due to trade-oﬀs with other plant stature traits. These results indicate potential loci controlling tiller number that can be utilized for future breeding programs. chromosomal region around HvTID1/HvTUA2 (4H, 39.8 cM), which is known to control plant stature through changing the number of cells in the shoot apical meristem in rice (Sunohara et al., 2009). In contrast to the HvDRM1 region, associations close to BRASSINOSTEROID DEFICIENT DWARF2/ DIMINUTO, DWARF1 (HvBRD2/HvDIM/HvDWF1; i.e., 7H, 140.6 cM) lead to produce non-productive tillers possibly due to trade-oﬀs with other plant stature traits. These results indicate potential loci controlling tiller number that can be utilized for future breeding programs. Two strong associations were found in the centromeric regions of 5 and 7H with tight linkage to hormone and heading time genes in photoperiod response groups; due to the uncertainty of marker orders in these regions, drawing ﬁnal conclusions require more genetic evidences. Interestingly, we found that improved cultivars produced more tillers likely as an output of breeding programs. This feature appeared in many EU cultivars, which are mostly two-rowed, possess the late ppd-H1 allele and thus produce more tillers that are non-productive. Manipulating tiller number genetically by decreasing non-productive tillering and/or increasing productive tiller number will be a challenge for breeders to maximize yield. Using QTL analysis in wheat, Xie et al. (2016) proposed that large genetic variation in tillering is advantageous to select for higher tillering capacity and survival thereby producing more fertile tillers that then may contribute to higher grain yield. Considering all of our ﬁndings from tillering, one can conclude that natural variation of tillering is under a complex genetic regulation. Our ﬁndings reinforce that pleiotropic gene actions do exist for tiller number, for example in case of Vrs1. The Signiﬁcance of the Experimental Approach Here, we set out to obtain a broad overview of the genetic factors that inﬂuence tillering in barley while follow-up work in other cereals will gain value-added information in this context. p g Obtained association signals show for the ﬁrst time a genetic association for a potential role of sugar-related genes in tillering of barley. In accordance with recent ﬁndings in sorghum, sugar is one of the major key regulators of axillary bud outgrowth (Kebrom and Mullet, 2015). Three putatively sugar-related QTL were found to be associated with HEXOKINASE and SUCROSE TRANSPORTER genes reinforcing the hypothesis about the importance of sugars in tillering. Hexokinases were characterized in rice as being important for sugar phosphorylation, sugar sensing, and signaling (Cho et al., 2006). Recently, it was shown that sucrose plays a key role during shoot branching in wheat and pea (Kebrom et al., 2012; Mason et al., 2014). Moreover, the expression of sucrose-inducible genes was down regulated in dormant buds of the tin mutant of wheat (Kebrom et al., 2012). In summary, our association analysis suggests that there is a tight linkage between sugar-related genes and productive tiller number that predominantly appears in accessions carrying Ppd-H1 alleles. Future studies should investigate the mechanisms of how sugar-related genes inﬂuence tillering and plant height. Frontiers in Genetics \| www.frontiersin.org June 2016 \| Volume 7 \| Article 117 REFERENCES appearance and tillering dynamics in a barley population. Field Crop Res. 113, 95–104. doi: 10.1016/j.fcr.2009.03.012 appearance and tillering dynamics in a barley population. Field Crop Res. 113, 95–104. doi: 10.1016/j.fcr.2009.03.012 Abeledo, L. G., Calderini, D. F., and Slafer, G. A. (2004). Leaf appearance, tillering and their coordination in old and modern barleys from Argentina. 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Genet. 44, 1388–1392. doi: 10.1038/ng.2447 Alqudah, A. M., and Schnurbusch, T. (2015). Barley leaf area and leaf growth rates are maximized during the pre-anthesis phase. Agronomy 5, 107–129. doi: 10.3390/agronomy5020107 Alqudah, A. M., Sharma, R., Pasam, R. K., Graner, A., Kilian, B., and Schnurbusch, T. (2014). Genetic dissection of photoperiod response based on GWAS of pre-anthesis phase duration in spring barley. PLoS ONE 9:e113120. doi: 10.1371/journal.pone.0113120 Dabbert, T., Okagaki, R. J., Cho, S., Boddu, J., and Muehlbauer, G. J. (2009). The genetics of barley low-tillering mutants: absent lower laterals (als). Theor. Appl. Genet. 118, 1351–1360. doi: 10.1007/s00122-009-0985-6 Dabbert, T., Okagaki, R. J., Cho, S., Heinen, S., Boddu, J., and Muehlbauer, G. J. (2010). The genetics of barley low-tillering mutants: low number of tillers-1 (lnt1). Theor. Appl. Genet. 121, 705–715. doi: 10.1007/s00122-010- 1342-5 Babb, S., and Muehlbauer, G. J. (2003). Genetic and morphological characterization of the barley uniculm2 (cul2) mutant. Theor. Appl. Genet. 106, 846–857. doi: 10.1007/s00122-002-1104-0 Barbier, F. F., Lunn, J. E., and Beveridge, C. A. (2015). Ready, steady, go! A sugar hit starts the race to shoot branching. Curr. Opin. Plant Biol. 25, 39–45. doi: 10.1016/j.pbi.2015.04.004 Dockter, C., Gruszka, D., Braumann, I., Druka, A., Druka, I., Franckowiak, J., et al. (2014). CONCLUSION The authors would like to thank Nils Stein for providing seeds of the Vrs1 mutant (8408-1) and Martin Mascher for sharing unpublished physical map data. Special thanks go to Annett Beyer for excellent technical assistance. We also thank IPK gardeners for help during this work. This study was ﬁnancially supported by the German Research Council (DFG), grant number SCHN 768/4-1, the German Federal Ministry of Education and Research (BMBF) GABI-FUTURE Start Program, grant number 0315071, and IPK core funding to TS. In the context of plant architecture, we found substantial diﬀerences for tillering and plant height in our barley worldwide collection. The analysis once more demonstrated the power of the GWAS approach for identifying putative candidate genes and improving plant architecture. Several physically anchored and co-locating chromosomal segments harboring known plant stature-related phytohormone metabolism and signaling genes in addition to sugar-related genes were identiﬁed. Based on GWAS results, a link between the genetic control of row-type, heading time, tillering, and plant height in barley was established. Our ﬁndings suggest that considering sugar-related genes seems very promising for future barley plant architecture works. Further investigation to conﬁrm these associations, i.e., further functional SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fgene. 2016.00117 AUTHOR CONTRIBUTIONS Conceived the project: TS. Designed and performed the experiments: AA, RK, GW, TS. Analyzed the data: AA, TS. Contributed reagents/materials/analysis tools: AG. Wrote the paper: AA, TS with contributions from all co-authors. QTL for Plant Height Interestingly, our GWAS analysis suggests that sugar-related genes are involved in regulating plant height. For instance, associations at HvHXK9 (3H, 59.3 cM) and allelic variation around this gene appear as the ﬁrst report for temperate cereals that sugar-related genes are possibly also important for plant height. Clearly, further molecular and genetic investigations are required in order to reveal the role of sugars in plant height. Putative heading time genes, such as HvCMF10 and HvCO8, were closely associated with plant height. An eﬀect of heading time genes like Ppd-H1 and Flt-2L on plant height was already reported in previous studies (Karsai et al., 1999; Chen et al., 2009). Interestingly, the centromeric region around June 2016 \| Volume 7 \| Article 117 Frontiers in Genetics \| www.frontiersin.org 13 The Genetic Architecture of Barley Plant Stature Alqudah et al. OsWFP1/OsIPA1/OsSPL14 (7H, 70.5 cM) was associated with plant height in six-rowed barley; while this gene regulates plant architecture, including plant height in rice (Jiao et al., 2010; Miura et al., 2010), we cannot exclude the eﬀect of other closely linked genes, such as HvLHY and HvCCA1 (7H, 70.8 cM). Similar conclusion can be postulated for genes in the centromeric regions of 4 and 6H. Thus, further genetic and functional analyses of these regions may reveal the importance of these genes in barley plant height research. 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https://openalex.org/W4381929540	https://www.nature.com/articles/s41598-023-37513-2.pdf	English	null	Risk factors for insulin resistance related to polycystic ovarian syndrome in Iranian population	Scientific reports	2,023	cc-by	8,399	Risk factors for insulin resistance related to polycystic ovarian syndrome in Iranian population Asieh Mansour 1,5, Maryam Mirahmad 1,5, Mohammad Reza Mohajeri‑Tehrani 1, Mahdieh Jamalizadeh 2, Sedigheh Hosseinimousa 3, Fatemeh Rashidi 4, Pooria Asili 1 & OPEN Polycystic ovary syndrome (PCOS) has significant metabolic sequelae linked to insulin resistance. This study aimed to compare clinical, metabolic, and hormonal characteristics of PCOS women with and without insulin resistance. The second aim was to compare the clinico-biochemical profiles of the various PCOS phenotypes. In this cross-sectional secondary analysis, we combined the baseline data from two separate randomized controlled trials (RCTs) in women diagnosed with PCOS. PCOS patients were categorized into the four Rotterdam PCOS phenotypes according to the presence of at least two criteria of oligomenorrhea/anovulation (O), hyperandrogenism (H), and polycystic ovary morphology (P): O–H–P, H–P, O–H, and O–P. Participants were categorized into two groups according to the homeostasis model assessment index of insulin resistance (HOMA-IR) levels: < 3.46, and ≥ 3.46. The correlation between the HOMA-IR and biometric, clinical, and biochemical variables was assessed in normal weight (BMI < 25) and overweight/obese (BMI ≥ 25) PCOS women. Then, the association between PCOS phenotypes and insulin resistance was investigated using logistic regression analysis. A total of 125 PCOS patients aged 18–40 years were included in the present study. Based on our results, the HOMA-IR index was positively correlated with diastolic blood pressure, free androgen index, and triglycerides levels; and negatively correlated with sex hormone-binding globulin in overweight/obese PCOS women. In addition, the HOMA-IR index was found to be positively correlated with alanine transaminase and negatively correlated with diastolic blood pressure in normal weight PCOS women. Moreover, individuals with O–H–P phenotype (odds ratio [OR] 2.52, 95% confidence interval [CI] 1.02–6.24) had about two-fold increased risk of insulin resistance. In conclusion, the full-blown PCOS (O–H–P) phenotype has an increased risk of insulin resistance. Accordingly, phenotype division may help physicians to predict adverse metabolic outcomes. Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrine disorders in females, affecting 10–15% of reproductive-age women worldwide1. Heterogeneous by nature, PCOS involves a variety of signs and symptoms of ovulatory dysfunction and androgen excess. There is a close connection between PCOS and insulin resistance, abdominal adiposity, hyperinsulinemia, and metabolic complications of these conditions2. The most common metabolic feature of PCOS, insulin resistance and compensatory hyperinsulinemia, affects about 35–80% of PCOS women3. A combination of chronic anovulation and high androgen levels correlates with insulin resistance and a higher risk of cardiovascular complications like dyslipidemia, impaired glucose tolerance, diabetes mellitus, and metabolic syndrome4. www.nature.com/scientificreports www.nature.com/scientificreports Methods Study design and subjects. In this cross-sectional secondary analysis, we combined the baseline data from two separate randomized controlled trials (RCTs) in women diagnosed with PCOS10,11. The two studies were randomized, prospective, placebo-controlled double-blind trials performed in Tehran, Iran, from August 2017 to May 2019. RCTs aimed to evaluate the efficacy of natural compounds (Resveratrol/Oligopin) on the hormonal and metabolic features of women diagnosed with PCOS. The protocols of both studies are avail- able at the Iranian Registry of Clinical Trials (irct.ir) with the identifier numbers IRCT20140406017139N3 and IRCT2017061917139N2. In order to compare the hormonal parameters in insulin-resistant and non-insulin- resistant groups, the total sample size was estimated at 128 patients, using GPower software (version 3.1.9.4) with the alpha of 0.05, power of 80% (β = 0.2) and medium effect size of 0.5. Inclusion criteria. Participants were included if they were diagnosed with PCOS and aged 18–40 years. PCOS was diagnosed using Rotterdam criteria12, by the presence of at least two of the following three conditions: (a) oligomenorrhea (menstrual cycles > 35 days)/amenorrhea (no menses for 3–6 months or longer), (b) clinical (acne, hirsutism and/or androgenic hair loss) and/or biochemical hyperandrogenism (defined by total testoster- one > 70 ng/dl), (c) polycystic ovarian morphology (PCOM) on the ultrasound exam (presence of ≥ 12 follicles measuring 2–9 mm and/or ovarian volume ≥ 10 ml in at least one ovary). Based on the presence of the three criteria participants were divided into the following groups: O–H–P (i.e. presence of all three criteria including oligomenorrhea/amenorrhea (O), hyperandrogenism (H), and PCOM (P), H–P, O–H, and O–P. Exclusion criteria. Exclusion criteria were as follows: Breastfeeding, pregnancy, use of medications known to affect metabolism and/or ovarian function (e.g., metformin or oral contraceptives) for at least one month before the screening, history of diabetes, acromegaly, Cushing’s disease, or any condition that mimics features of PCOS (e.g., non-classical congenital adrenal hyperplasia, hyperprolactinemia, or thyroid disorders). Measurements and assays. At the baseline visit, included participants underwent a physical examination and height, as well as systolic and diastolic blood pressure were measured. Hypertension was defined as systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg and/or using anti-hypertensive drugs13. In addition, the waist circumference was measured halfway between the lowest rib and the iliac crest. Body com- position and weight were assessed using the body impedance analyzer (BIA) (Tanita, Japan). Methods Body mass index (BMI) was calculated based on the following formula: BMI = weight (kg)/height (m2). Participants were divided into two groups according to their BMI (normal weight [BMI < 25]; overweight/obese [BMI ≥ 25]). Acne score was assessed in four grades, as previously described elsewhere14. The score of hirsutism was evaluated using the Ferriman-Gallwey score system15. y y Trans-abdominal ultrasonography was performed by experienced radiologists. Ovarian volume and a total number of antral follicles (2–9 mm in diameter) were measured using a 3 to 5.5 MHz curvilinear probe (acuson s2000, Siemens Medical Solutions, USA).t Blood samples were collected after overnight fasting and stored at − 20 °C until the analysis. Sex hormone binding globulin (SHBG) was determined by enzyme-linked immunosorbent assay (ELISA) kits (Demeditec, Germany) with the inter-assay coefficient of variation (CV) of 5.2%. The free androgen index (FAI) was deter- mined by the equation FAI = total testosterone (ng/mL) × 3.47/SHBG (nmol/L)16. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, dehydroepiandrosterone (DHEA), C-peptide, and fasting insu- lin levels were measured by the ELISA kits (Monobind Inc. Lake Forest, California, USA) with the inter-assay CVs of 4.9%, 4.8%, 5.1%, 2.9%, and 3.8%, respectively. Concentrations of the fasting blood sugar (FBS), serum triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL), high-sensitivity C-reactive protein (hs-CRP), alanine transaminase (ALT), aspartate transaminase (AST), and creatinine (Cr) were determined by an auto-analyzer (Cobas c 311, Roche Diagnostics, Risch-Rotkreuz, Switzerland). Hemoglobin A1c (HbA1c) concentration was measured using a high-performance liquid chromatography analyzer (Tosoh, Tokyo, Japan). Friedewald formula was used to calculate low-density lipoprotein cholesterol (LDL) levels. The insulin resist- ance was estimated by homeostasis model assessment index of insulin resistance (HOMA-IR) with the following formula: HOMA-IR = FBS (mg/dL) × fasting insulin (μIu/mL)/40517. The HOMA-IR cut-off value of 3.46 was used to determine insulin resistance in PCOS patients18. Statistical analysis. Statistical analysis was conducted using the SPSS software (SPSS Inc., Chicago, IL, USA). Chi-squared test was employed to analyze trends in categorical variables. The Shapiro–Wilk test was employed to assess the normality of continuous variables. Nonparametric tests (Mann–Whitney U and Kruskal– Wallis tests) were used if the statistical assumptions of normality were violated. For normally distributed varia- bles, independent sample t-test and one-way analysis of variance (ANOVA) were used for comparisons between means as appropriate. Risk factors for insulin resistance related to polycystic ovarian syndrome in Iranian population Asieh Mansour 1,5, Maryam Mirahmad 1,5, Mohammad Reza Mohajeri‑Tehrani 1, Mahdieh Jamalizadeh 2, Sedigheh Hosseinimousa 3, Fatemeh Rashidi 4, Pooria Asili 1 & OPEN According to the Rotterdam criteria, the diagnosis of PCOS requires the presence of at least two of the following three features: ovulatory dysfunction, clinical and/ or biochemical hyperandrogenism, and polycystic ovarian morphology5. Based on combinations of these three classic manifestations, PCOS women can be categorized into four main phenotypes. , g p yp Nevertheless, the question as to whether metabolic features are the same amongst all phenotypes of PCOS has yet to be addressed. Several studies have suggested that PCOS phenotypes involving oligomenorrhea or 1Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran. 2Endocrinology and Metabolic Research Center, Institute of Basic and Clinic Physiology Science and Department of Internal Medicine, Kerman University of Medical Science, Kerman, Iran. 3Department of Obstetrics and Gynecology, Infertility Unit, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran. 4School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 5These authors contributed equally: Asieh Mansour and Maryam Mirahmad. email: mahmood.sajadi@gmail.com \| https://doi.org/10.1038/s41598-023-37513-2 Scientific Reports \| (2023) 13:10269 Scientific Reports \| (2023) 13:10269 www.nature.com/scientificreports/ hyperandrogenemia portend a higher risk of metabolic disorders6,7, while some studies reported that the risk of metabolic complications does not differ among the various phenotypes, and is only related to obesity8,9. hyperandrogenemia portend a higher risk of metabolic disorders6,7, while some studies reported that the risk of metabolic complications does not differ among the various phenotypes, and is only related to obesity8,9. Firstly, this study aimed to make a comparison between PCOS patients with and without insulin resistance, f Firstly, this study aimed to make a comparison between PCOS patients with and without insulin resista and then compare four different phenotypes of PCOS in terms of clinical and biochemical parameters. Results One hundred twenty-five women were included in the present analysis. Table 1 depicts the demographic, clini- cal, and biochemical characteristics of PCOS patients, who were divided into two distinct groups: (i) 96 (76.8%) participants with normal HOMA-IR values (< 3.46) and (ii) 29 (23.2%) participants with abnormal HOMA-IR values (≥ 3.46). The study participants were young reproductive-aged women with a median age of 28 years and a mean BMI of 27.15 ± 5.90 (kg/m2). The proportion of participants who reported hair loss was significantly higher in PCOS women with normal HOMA-IR compared with those with abnormal HOMA-IR (p = 0.033). PCOS women with abnormal HOMA-IR index were found to have significantly higher mean levels of BMI (p < 0.001), and anthropometric parameters including body fat percent (p < 0.001), fat mass (p < 0.001), fat-free mass (FFM) (p = 0.024), and waist circumference (p < 0.001) compared with those with normal HOMA-IR index. www.nature.com/scientificreports/ and O–H–P variables to estimate the odds of insulin resistance by different PCOS phenotypes. A p-value less than 0.05 was considered to be significant. and O–H–P variables to estimate the odds of insulin resistance by different PCOS phenotypes. A p-value less than 0.05 was considered to be significant. Ethical considerations. The study was conducted in accordance with the Declaration of Helsinki and its subsequent revisions, and was approved by the ethics committee of the Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1399.1058). Informed consent was obtained from all study participants. Results Variables Total (n = 125) HOMA-IR < 3.46 (n = 96) HOMA-IR ≥ 3.46 (n = 29) p-value Age (years)a 28.00 (22.00–32.00) 27.50 (22.00–32.00) 29.00 (23.50–34.50) 0.256 Irregular menstruation, yesb 104.00 (83.20) 78.00 (81.30) 26.00 (89.70) 0.289 Hair loss, yesb 88.00 (70.40) 63.00 (65.60) 25 (86.20) 0.033 Acne scorea 2.00 (0–2.00) 2.00 (0–2.00) 2.00 (0–2.00) 0.769 Hirsutism scorec 10.69 ± 6.12 10.92 ± 5.84 9.96 ± 7.030 0.466 BMI (kg/m2)c 27.15 ± 5.90 25.96 ± 5.080 30.99 ± 6.80 < 0.001 Fat percent (%)a 35.70 (30.20–41.60) 34.55 (29.42–39.22) 41.9 (35.50–48.15) < 0.001 Fat mass (kg)a 24.20 (18.20–32.10) 22.05 (17.32–28.77) 33.30 (24.20–40.95) < 0.001 FFM (kg)a 42.80 (40.30–46.70) 42.20 (39.50–46.12) 44.20 (41.80–47.45) 0.024 Waist circumference (cm)a 91.00 (82.87–100.00) 89 (81–96.12) 100.5 (94.25–108.00) < 0.001 Hypertension, yesb 15 (12.00) 9 (9.40) 6 (20.70) 0.112 FSH (mIU/mL)a 5.60 (4.20–6.80) 5.65 (4.12–6.60) 5.60 (4.20–7.40) 0.490 LH (mIU/mL)a 9.40 (4.40–17.15) 9.00 (4.02–16.45) 12.80 (4.65–18.05) 0.265 LH/FSH ratioa 1.82 (0.92–3.22) 1.75 (0.82–3.07) 2.10 (1.20–3.34) 0.357 Testosterone (ng/mL)a 0.40 (0.30–0.60) 0.45 (0.32–0.60) 0.40 (0.30–0.60) 0.534 FAIa 3.37 (1.86–6.88) 3.04 (1.73–6.45) 5.98 (2.35–8.21) 0.047 DHEA (ng/dL)a 152.40 (96.60–205.50) 153.70 (91.27–214.35) 149.00 (124.70–195.00) 0.625 SHBG (nmol/L)a 41.00 (23.95–82.00) 48.50 (28.05–90.85) 27.00 (21.05–41.60) 0.003 hs-CRP (mg/L)a 1.20 (0.50–2.90) 1.05 (0.42–2.17) 2.70 (0.95–6.45) 0.005 Fasting insulin (uIU/mL)a 12.20 (9.15–15.80) 11.30 (8.70–13.07) 18.70 (16.75–21.85) < 0.001 C-peptide (ng/mL)a 1.00 (0.75–1.60) 1.00 (0.70–1.20) 1.60 (1.45–1.95) < 0.001 FBS (mg/dL)a 84.00 (80.50–92.00) 83.00 (79.25–88.00) 94.00 (86.5–102.00) < 0.001 HbA1c (%)a 5.30 (5.10–5.50) 5.30 (5.00–5.50) 5.40 (5.15–50.50) 0.148 AST (U/L)a 18.00 (15.50–20.00) 18.00 (15.25–20.75) 18.00 (15.50–19.00) 0.970 ALT (U/L)a 10.00 (8.00–14.00) 9.50 (8.00–13.75) 12.00 (9.50–14.00) 0.064 Total cholesterol (mg/dL)a 171.00 (149.00–193.5) 167.5 (149.00–191.75) 180 (150.50–200.50) 0.1499 HDL- cholesterol (mg/dL)a 42.00 (34.00–48.00) 41 (34.00–49.50) 43.00 (36.00–47.00) 0.801 LDL-cholesterol (mg/dL)a 97.00 (82.00–114.00) 96 (81.00–110.75) 100.00 (83.50–122.00) 0.142 Triglycerides (mg/dL)a 106.00 (72.50–145.50) 100.50 (69.00–130.25) 141.00 (93.00–196.00) 0.003 Cr (mg/dL)c 0.80 ± 0.12 0.80 ± 0.12 0.80 ± 0.11 0.888 PCOM, yesa 104 (83.20) 79 (82.30) 25 (86.20) 0.621 Table 1. Demographic, biochemical, and anthropometric characteristics of patients and comparison between the two groups according to HOMA-IR cutoff value of 3.46. Significant values are in bold. Data are presented as median (IQR), mean ± SD or number (%). a Mann–Whitney U test. b Chi-squared test. c Independent sample t test. Methods In case of significant differences in the ANOVA or Kruskal–Wallis test, the Bonferroni post hoc test was used for pairwise comparison.h p p p The correlation of HOMA-IR with clinical and biochemical variables was investigated using Spearman cor- relation. Finally, the data were analyzed using the backward stepwise selection model with age, H–P, O–H, O–P, Scientific Reports \| (2023) 13:10269 \| https://doi.org/10.1038/s41598-023-37513-2 www.nature.com/scientificreports/ Table 1. Demographic, biochemical, and anthropometric characteristics of patients and comparison between the two groups according to HOMA-IR cutoff value of 3.46. Significant values are in bold. Data are presented as median (IQR), mean ± SD or number (%). a Mann–Whitney U test. b Chi-squared test. c Independent sample t test. HOMA-IR homeostasis model assessment insulin resistance, BMI body mass index, FFM fat-free mass, FSH follicle-stimulating hormone, LH luteinizing hormone, FAI free androgen index, DHEA Dehydroepiandrosterone, SHBG Sex hormone-binding globulin, hs-CRP high-sensitivity C-reactive protein, FBS fasting blood sugar, HbA1c hemoglobin A1c, AST aspartate aminotransferase, ALT alanine aminotransferase, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Cr creatinine, PCOM polycystic ovarian morphology. www.nature.com/scientificreports/ Furthermore, the median hs-CRP, fasting insulin, C-peptide, FBS, and triglyceride levels were significantly higher in participants with abnormal HOMA-IR values. There was also an association between FAI and SHBG levels with HOMA-IR index. Participants with abnormal HOMA-IR were shown to have higher levels of FAI (p = 0.047) and lower levels of SHBG (p = 0.003) compared with PCOS women with normal HOMA-IR index.h p p p The correlations of HOMA-IR with continuous variables are demonstrated in Table 2. HOMA-IR index levels were positively correlated with the BMI (r = 0.400, p < 0.001), and anthropometric measures including fat percentage (r = 0.437, p < 0.001), fat mass (r = 0.437, p < 0.001), FFM (r = 0.270, p = 0.002), and waist circumfer- ence (r = 0.418, p < 0.001) in PCOS women. However, no significant correlation was found between HOMA-IR index levels and age, acne score, hirsutism score, or systolic/diastolic blood pressure in study subjects. Among biochemical variables, we found that the HOMA-IR index was positively correlated with FAI (r = 0.286, p = 0.001), hs-CRP (r = 0.238, p = 0.008), triglycerides (r = 0.332, p < 0.001) and ALT (r = 0.260, p = 0.003); and was negatively correlated with SHBG (r = − 0.312, p < 0.001) in study participants. It is noteworthy that, despite predictable biochemical variables like fasting insulin, C-peptide and FBS, the HbA1c was not correlated with the HOMA-IR index. Despite the limited number of participants, we performed another correlation analysis stratified by BMI (Table 3), as the HOMA-IR was significantly correlated with BMI and anthropometric indices. gi y p Analysis of data from normal weight participants revealed that HOMA-IR index levels were positively cor- related with the fasting insulin (r = 0.975, p < 0.001), FBS (r = 0.376, p = 0.008), and ALT (r = 0.290, p = 0.046); it was also negatively correlated with diastolic blood pressure (r = − 0.357, p = 0.013). g y p p In overweight/obese participants, the HOMA-IR index was positively correlated with fasting insulin (r = 0.958, p < 0.001), FBS (r = 0.499, p < 0.001), C-peptide (r = 0.551, p < 0.001), FAI (r = 0.303, p = 0.007), triglycerides (r = 0.330, p = 0.003) and diastolic blood pressure (r = 0.239, p = 0.038); and was negatively correlated with SHBG (r = − 0.372, p = 0.001). www.nature.com/scientificreports/ p Clinical and laboratory parameters have been compared between various PCOS phenotype groups (Table 4). Among a total of 125 PCOS participants, the O–H–P phenotype (56%) was the most prevalent, followed by Table 2. Correlations of HOMA-IR with clinical and biochemical variables. Significant values are in bold. HOMA-IR homeostasis model assessment insulin resistance, BMI body mass index, FSH follicle-stimulating hormone, LH luteinizing hormone, FAI free androgen index, DHEA Dehydroepiandrosterone, SHBG Sex hormone-binding globulin, hs-CRP high-sensitivity C-reactive protein, FBS fasting blood sugar, HbA1c hemoglobin A1c, AST aspartate aminotransferase, ALT alanine aminotransferase, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Cr creatinine. Variables r coefficient p-value Age (years) 0.045 0.619 Acne score 0.037 0.681 Hirsutism score − 0.034 0.707 BMI (kg/m2) 0.400 < 0.001 Fat percent (%) 0.437 < 0.001 Fat mass (kg) 0.437 < 0.001 FFM (kg) 0.270 0.002 Waist circumference (cm) 0.418 < 0.001 Systolic blood pressure (mmHg) 0.165 0.068 Diastolic blood pressure (mmHg) 0.145 0.108 FSH (mIU/mL) 0.043 0.632 LH (mIU/mL) 0.063 0.482 LH/FSH ratio 0.057 0.530 Testosterone (ng/mL) 0.051 0.569 FAI 0.286 0.001 DHEA (ng/dL) − 0.018 0.841 SHBG (nmol/L) − 0.312 < 0.001 hs-CRP (mg/L) 0.238 0.008 Fasting insulin (uIU/mL) 0.970 < 0.001 C-peptide (ng/mL) 0.512 < 0.001 FBS (mg/dL) 0.451 < 0.001 HbA1c (%) 0.130 0.153 AST (U/L) 0.125 0.166 ALT (U/L) 0.260 0.003 Total cholesterol (mg/dL) 0.125 0.165 HDL-cholesterol (mg/dL) − 0.055 0.546 LDL-cholesterol (mg/dL) 0.114 0.206 Triglycerides (mg/dL) 0.332 < 0.001 Cr (mg/dL) − 0.037 0.680 Variables r coefficient p-value Age (years) 0.045 0.619 Acne score 0.037 0.681 Hirsutism score − 0.034 0.707 BMI (kg/m2) 0.400 < 0.001 Fat percent (%) 0.437 < 0.001 Fat mass (kg) 0.437 < 0.001 FFM (kg) 0.270 0.002 Waist circumference (cm) 0.418 < 0.001 Systolic blood pressure (mmHg) 0.165 0.068 Diastolic blood pressure (mmHg) 0.145 0.108 FSH (mIU/mL) 0.043 0.632 LH (mIU/mL) 0.063 0.482 LH/FSH ratio 0.057 0.530 Testosterone (ng/mL) 0.051 0.569 FAI 0.286 0.001 DHEA (ng/dL) − 0.018 0.841 SHBG (nmol/L) − 0.312 < 0.001 hs-CRP (mg/L) 0.238 0.008 Fasting insulin (uIU/mL) 0.970 < 0.001 C-peptide (ng/mL) 0.512 < 0.001 FBS (mg/dL) 0.451 < 0.001 HbA1c (%) 0.130 0.153 AST (U/L) 0.125 0.166 ALT (U/L) 0.260 0.003 Total cholesterol (mg/dL) 0.125 0.165 HDL-cholesterol (mg/dL) − 0.055 0.546 LDL-cholesterol (mg/dL) 0.114 0.206 Triglycerides (mg/dL) 0.332 < 0.001 Cr (mg/dL) − 0.037 0.680 Table 2. Correlations of HOMA-IR with clinical and biochemical variables. Significant values are in bold. Results HOMA-IR homeostasis model assessment insulin resistance, BMI body mass index, FFM fat-free mass, FSH follicle-stimulating hormone, LH luteinizing hormone, FAI free androgen index, DHEA Dehydroepiandrosterone, SHBG Sex hormone-binding globulin, hs-CRP high-sensitivity C-reactive protein, FBS fasting blood sugar, HbA1c hemoglobin A1c, AST aspartate aminotransferase, ALT alanine aminotransferase, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Cr creatinine, PCOM polycystic ovarian morphology. Table 1. Demographic, biochemical, and anthropometric characteristics of patients and comparison between the two groups according to HOMA-IR cutoff value of 3.46. Significant values are in bold. Data are presented as median (IQR), mean ± SD or number (%). a Mann–Whitney U test. b Chi-squared test. c Independent sample t test. HOMA-IR homeostasis model assessment insulin resistance, BMI body mass index, FFM fat-free mass, FSH follicle-stimulating hormone, LH luteinizing hormone, FAI free androgen index, DHEA Dehydroepiandrosterone, SHBG Sex hormone-binding globulin, hs-CRP high-sensitivity C-reactive protein, FBS fasting blood sugar, HbA1c hemoglobin A1c, AST aspartate aminotransferase, ALT alanine aminotransferase, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Cr creatinine, PCOM polycystic ovarian morphology. https://doi.org/10.1038/s41598-023-37513-2 Scientific Reports \| (2023) 13:10269 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ HOMA-IR homeostasis model assessment insulin resistance, BMI body mass index, FSH follicle-stimulating hormone, LH luteinizing hormone, FAI free androgen index, DHEA Dehydroepiandrosterone, SHBG Sex hormone-binding globulin, hs-CRP high-sensitivity C-reactive protein, FBS fasting blood sugar, HbA1c hemoglobin A1c, AST aspartate aminotransferase, ALT alanine aminotransferase, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Cr creatinine. Table 2. Correlations of HOMA-IR with clinical and biochemical variables. Significant values are in bold. HOMA-IR homeostasis model assessment insulin resistance, BMI body mass index, FSH follicle-stimulating hormone, LH luteinizing hormone, FAI free androgen index, DHEA Dehydroepiandrosterone, SHBG Sex hormone-binding globulin, hs-CRP high-sensitivity C-reactive protein, FBS fasting blood sugar, HbA1c hemoglobin A1c, AST aspartate aminotransferase, ALT alanine aminotransferase, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Cr creatinine. https://doi.org/10.1038/s41598-023-37513-2 Scientific Reports \| (2023) 13:10269 \| www.nature.com/scientificreports/ Table 3. Correlations of HOMA-IR with clinical and biochemical variables categorized based on the BMI. Significant values are in bold. HOMA-IR homeostasis model assessment insulin resistance, BMI body mass index, FSH follicle-stimulating hormone, LH luteinizing hormone, FAI free androgen index, DHEA Dehydroepiandrosterone, SHBG Sex hormone-binding globulin, hs-CRP high-sensitivity C-reactive protein, FBS fasting blood sugar, HbA1c hemoglobin A1c, AST aspartate aminotransferase, ALT alanine aminotransferase, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Cr creatinine. www.nature.com/scientificreports/ HOMA-IR homeostasis model assessment insulin resistance, BMI body mass index, FSH follicle-stimulating hormone, LH luteinizing hormone, FAI free androgen index, DHEA Dehydroepiandrosterone, SHBG Sex hormone-binding globulin, hs-CRP high-sensitivity C-reactive protein, FBS fasting blood sugar, HbA1c hemoglobin A1c, AST aspartate aminotransferase, ALT alanine aminotransferase, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Cr creatinine. O–H (16.8%) and H–P (16.8%), as well as O–P (10.4%) phenotypes. No difference between the four groups was recorded with respect to demographic, clinical, and biochemical characteristics, except for FBS levels (p = 0.038), which was the highest among O–H–P phenotype. In addition, in the Bonferroni post hoc test, no significant difference was found between each two groups. O–H (16.8%) and H–P (16.8%), as well as O–P (10.4%) phenotypes. No difference between the four groups was recorded with respect to demographic, clinical, and biochemical characteristics, except for FBS levels (p = 0.038), which was the highest among O–H–P phenotype. In addition, in the Bonferroni post hoc test, no significant difference was found between each two groups. f g p In logistic regression analysis using insulin resistance (HOMA-IR ≥ 3.46) as a dependent variable, phenotype O–H–P (odds ratio [OR] = 2.52, 95% confidence interval [CI] 1.02–6.24) was shown to be associated with about two-fold increased risk of insulin resistance (p < 0.05) as shown in Table 5. www.nature.com/scientificreports/ Variables HOMA-IR Normal weight (BMI < 25) (n = 48) Overweight/obese (BMI ≥ 25) (n = 77) Rs coefficient p-value Rs coefficient p-value Age (years) − 0.065 0.663 -0.035 0.764 Acne score 0.027 0.856 0.084 0.466 Hirsutism score 0.058 0.696 -0.013 0.911 Systolic blood pressure (mmHg) − 0.101 0.493 0.130 0.263 Diastolic blood pressure (mmHg) − 0.357 0.013 0.239 0.038 FSH (mIU/mL) 0.108 0.464 0.009 0.936 LH (mIU/mL) 0.069 0.641 0.091 0.431 LH/FSH ratio 0.056 0.706 0.081 0.483 Testosterone (ng/mL) 0.102 0.488 0.001 0.991 FAI 0.150 0.314 0.303 0.007 DHEA (ng/dL) 0.050 0.738 0.037 0.747 SHBG (nmol/L) − 0.120 0.418 − 0.372 0.001 hs-CRP (mg/L) 0.217 0.139 0.159 0.168 Fasting insulin (uIU/mL) 0.975 < 0.001 0.958 < 0.001 C-peptide (ng/mL) 0.271 0.063 0.551 < 0.001 FBS (mg/dL) 0.376 0.008 0.499 < 0.001 HbA1c (%) 0.085 0.567 0.141 0.228 AST (U/L) 0.215 0.142 0.010 0.929 ALT (U/L) 0.290 0.046 0.173 0.132 Total cholesterol (mg/dL) − 0.079 0.591 0.199 0.083 HDL-cholesterol (mg/dL) − 0.188 0.200 0.114 0.323 LDL-cholesterol (mg/dL) − 0.107 0.470 0.181 0.116 Triglycerides (mg/dL) 0.264 0.070 0.330 0.003 Cr (mg/dL) 0.046 0.755 − 0.079 0.493 Variables HOMA-IR Normal weight (BMI < 25) (n = 48) Overweight/obese (BMI ≥ 25) (n = 77) Rs coefficient p-value Rs coefficient p-value Age (years) − 0.065 0.663 -0.035 0.764 Acne score 0.027 0.856 0.084 0.466 Hirsutism score 0.058 0.696 -0.013 0.911 Systolic blood pressure (mmHg) − 0.101 0.493 0.130 0.263 Diastolic blood pressure (mmHg) − 0.357 0.013 0.239 0.038 FSH (mIU/mL) 0.108 0.464 0.009 0.936 LH (mIU/mL) 0.069 0.641 0.091 0.431 LH/FSH ratio 0.056 0.706 0.081 0.483 Testosterone (ng/mL) 0.102 0.488 0.001 0.991 FAI 0.150 0.314 0.303 0.007 DHEA (ng/dL) 0.050 0.738 0.037 0.747 SHBG (nmol/L) − 0.120 0.418 − 0.372 0.001 hs-CRP (mg/L) 0.217 0.139 0.159 0.168 Fasting insulin (uIU/mL) 0.975 < 0.001 0.958 < 0.001 C-peptide (ng/mL) 0.271 0.063 0.551 < 0.001 FBS (mg/dL) 0.376 0.008 0.499 < 0.001 HbA1c (%) 0.085 0.567 0.141 0.228 AST (U/L) 0.215 0.142 0.010 0.929 ALT (U/L) 0.290 0.046 0.173 0.132 Total cholesterol (mg/dL) − 0.079 0.591 0.199 0.083 HDL-cholesterol (mg/dL) − 0.188 0.200 0.114 0.323 LDL-cholesterol (mg/dL) − 0.107 0.470 0.181 0.116 Triglycerides (mg/dL) 0.264 0.070 0.330 0.003 Cr (mg/dL) 0.046 0.755 − 0.079 0.493 Table 3. Correlations of HOMA-IR with clinical and biochemical variables categorized based on the BMI. Significant values are in bold. Discussion In this cross-sectional analysis, abnormalities of anthropometric parameters, higher triglyceride levels, hs-CRP, ALT, and FAI as well as lower SHBG were observed among insulin-resistant women with PCOS. g We noticed higher hs-CRP levels in participants with abnormal HOMA-IR values. In recent years, increas- ing attention has been paid to the significance of inflammation in PCOS. Interleukin-18, as a potent proin- flammatory biomarker, was shown to be related to indices of adiposity and insulin resistance in PCOS19. In response to hyperglycemia, mononuclear cells produce tumor necrosis factor-α (TNF-α), which can exacerbate the metabolic abnormalities of PCOS20. Adipose tissue-resident macrophages release interleukin-6, which is contributed to insulin resistance in PCOS21. A systematic review and meta-analysis of 63 studies found that women with PCOS had significantly higher circulating CRP levels than controls (standardized mean difference 1.26, 95% CI 0.99–1.53). However, a high heterogeneity among studies was found22. The inflammatory state in PCOS is thought to interact with hyperinsulinemia, insulin resistance, and obesity23. Preliminary data indicate that application of insulin-sensitizing agent therapy may decrease the inflammatory state in PCOS24,25. Serum hs-CRP was shown to be positively correlated with serum insulin, insulin resistance, fat mass, and body weight in women with PCOS26,27. Our analyses suggest that the inflammation is due to PCOS-related insulin resistance rather than PCOS itself; but further evidence is required to support the mechanisms of inflammation and the role of insulin in PCOS. Discussion H hyperandrogenism, O oligomenorrhea, P polycystic ovarian morphology, BMI body mass index, FFM fat-free mass, FSH follicle-stimulating hormone, LH luteinizing hormone, hs-CRP high-sensitivity C-reactive protein, HOMA-IR homeostasis model assessment insulin resistance, FBS fasting blood sugar, HbA1c hemoglobin A1c, AST aspartate aminotransferase, ALT alanine aminotransferase, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Cr creatinine. Table 5. Association between PCOS phenotypes with insulin resistance (HOMA-IR ≥ 3.46) in logistic regression analysis (sample size n = 125). Significant values are in bold. H hyperandrogenism, O oligomenorrhea, P polycystic ovarian morphology, OR odds ratio, CI confidence interval. a Variables included into the multivariate model were age, H–P, O–H, O–P, and O–H–P using the backward selection approach. Variablesa OR 95%CI p-value H–P 2.08 0.72–5.98 0.175 O–H 2.84 0.91–8.88 0.073 O–P 1.80 0.70–4.65 0.22 O–H–P 2.52 1.02–6.24 0.046 Variablesa OR 95%CI p-value H–P 2.08 0.72–5.98 0.175 O–H 2.84 0.91–8.88 0.073 O–P 1.80 0.70–4.65 0.22 O–H–P 2.52 1.02–6.24 0.046 Table 5. Association between PCOS phenotypes with insulin resistance (HOMA-IR ≥ 3.46) in logistic regression analysis (sample size n = 125). Significant values are in bold. H hyperandrogenism, O oligomenorrhea, P polycystic ovarian morphology, OR odds ratio, CI confidence interval. a Variables included into the multivariate model were age, H–P, O–H, O–P, and O–H–P using the backward selection approach. Table 5. Association between PCOS phenotypes with insulin resistance (HOMA-IR ≥ 3.46) in logistic regression analysis (sample size n = 125). Significant values are in bold. H hyperandrogenism, O oligomenorrhea, P polycystic ovarian morphology, OR odds ratio, CI confidence interval. a Variables included into the multivariate model were age, H–P, O–H, O–P, and O–H–P using the backward selection approach. Our study also found an association between FAI and SHBG levels with the HOMA-IR index. Previous studies suggested that low SHBG levels in the general population are a predictor of increased risk of devel- oping type 2 diabetes mellitus28, hypertension29, and metabolic syndrome30. We also found a weak negative correlation between HOMA-IR and SHBG in PCOS overweight/obese women. A moderate correlation was observed in another study in PCOS women (r = − 0.557, p < 0.001)31. Compensatory hyperinsulinemia as a result of insulin resistance can suppress the synthesis of SHBG in the liver32. In addition, the relationship between low serum SHBG and impaired glucose metabolism can be explained by the hypothesis that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is regulated by SHBG. Discussion https://doi.org/10.1038/s41598-023-37513-2 Scientific Reports \| (2023) 13:10269 \| www.nature.com/scientificreports/ Variables H–P (n = 21) O–H (n = 21) O–P (n = 13) O–H–P (n = 70) p-value Age (years)a 28.00 (25.00–34.00) 26.00 (20.50–32.00) 30.00 (23.00–34.00) 28.00 (21.75–32.00) 0.438 BMI (kg/m2)b 26.05 ± 4.64 24.69 ± 4.69 26.52 ± 5.97 28.34 ± 6.34 0.061 Fat percent (%)a 33.4 (30.75–37.20) 35.30 (29.10–39.35) 30.40 (27.20–42.75) 37.50 (32.35–42.60) 0.104 Fat mass (kg)a 22.00 (18.20–25.60) 22.10 (15.85–28.90) 19.40 (14.97–32.70) 27.70 (19.75–34.30) 0.114 FFM (kg)a 44.40 (40.00–47.30) 41.1 (39.10–44.35) 42.20 (40.17–44.17) 43.20 (40.75–47.40) 0.181 Waist circumference (cm)a 88.00 (78.00–98.25) 89.00 (78.00–97.50) 91.75 (89.00–96.50) 94.00 (84.50–103.00) 0.215 Systolic blood pressure (mmHg)b 103.81 ± 16.42 108.09 ± 12.50 101.92 ± 11.28 108.29 ± 14.96 0.358 Diastolic blood pressure (mmHg)b 68.09 ± 14.62 75.00 ± 10.25 70.77 ± 8.62 74.93 ± 10.27 0.064 FSH (mIU/mL)a 6.20 (4.25–7.35) 4.80 (2.75–5.90) 5.70 (4.20–7.15) 5.70 (4.27–6.62) 0.307 LH (mIU/mL)a 8.40 (4.20–14.60) 4.60 (2.10–14.95) 9.10 (3.25–17.15) 11.00 (6.40–19.62) 0.083 LH/FSH ratioa 1.68 (0.92–2.32) 1.00 (0.58–2.45) 1.75 (0.66–2.80) 2.06 (1.14–3.52) 0.173 hs-CRP (mg/L)a 1.30 (0.40–1.75) 0.90 (0.20–4.00) 0.70 (0.30–2.50) 1.50 (0.67–3.22) 0.147 HOMA-IRb 2.55 (1.78–3.03) 2.39 (1.51–3.28) 2.75 (2.34–2.89) 2.65 (1.89–3.65) 0.438 Fasting insulin (uIU/mL)a 12.30 (8.90–15.15) 11.70 (7.20–16.05) 12.10 (11.15–13.50) 12.25 (9.40–16.45) 0.794 C-peptide (ng/mL)a 0.90 (0.60–1.55) 0.55 (1.00–1.20) 1.00 (0.65–1.25) 1.10 (0.80–1.60) 0.169 FBS (mg/dL)a, c 83.00 (76.50–89.00) 82.00 (77.50–89.00) 85.00 (83.00–94.50) 86.00 (81.00–95.00) 0.038 HbA1c (%)a 4.92 (5.40–5.57) 5.30 (5.00–5.45) 5.20 (4.92–5.37) 5.30 (5.20–5.50) 0.394 AST (U/L)a 17.00 (16.00–20.50) 17.00 (15.00–19.00) 18.00 (16.00–20.00) 17.50 (15.00–21.00) 0.903 ALT (U/L)a 11.00 (9.50–14.50) 9.00 (7.00–14.00) 11.00 (7.00–13.50) 10.00 (8.00–13.25) 0.586 Total cholesterol (mg/dL)a 177.00 (161.50–191.00) 165.00 (142.50–200.00) 166.00 (131.50–189.00) 171.00 (149.00–194.00) 0.777 HDL-cholesterol (mg/dL)a 40.00 (34.50–48.50) 40.00 (32.50–49.50) 42.00 (35.00–43.50) 44 (34.00–50.00) 0.619 LDL-cholesterol (mg/dL)a 100.00 (89.00–109.50) 86.00 (75.50–122.50) 96.00 (76.00–114.5) 96.50 (82.00–115.00) 0.654 Triglycerides (mg/dL)a 88.00 (69.00–138.50) 99.00 (67.00–141.50) 104.00 (62.50–156.50) 110.00 (75.50–148.00) 0.591 Cr (mg/dL)b 0.80 ± 0.13 0.81 ± 0.14 0.81 ± 0.15 0.79 ± 0.10 0.867 Table 4. Distribution of clinical and biochemical features among PCOS phenotypes. Significant values are in bold. Data are presented as median (IQR) or mean ± SD. a Kruskal–Wallis. b One-way analysis of variance (ANOVA). c The differences between each two groups was not significant (p > 0.05) using Bonferroni post hoc test. www.nature.com/scientificreports/ correlations between FAI or SHBG and HOMA-IR in overweight/obese PCOS women were not evident in normal weight PCOS women in our study. We found a positive correlation between HOMA-IR and diastolic blood pressure in obese/overweight PCOS women, which is consistent with a previous study that indicated higher diastolic blood pressure in obese hyperin- sulinemic PCOS women compared to lean hyperinsulinemic ones34. Higher diastolic pressure was also reported among obese PCOS women compared with non-obese subjects35–37. Additionally, we found an opposite weak correlation between HOMA-IR and diastolic blood pressure in normal weight PCOS women. It might be a potential source of uncertainty in our study and it is difficult to extrapolate our results to other PCOS women. fi Our analyses also showed a weak positive correlation between HOMA-IR and ALT levels in normal weight participants. It is to be noted that we did not screen participants regarding non-alcoholic fatty liver disease (NAFLD). Nevertheless, elevated liver enzymes, as a surrogate marker of NAFLD, are common in women with PCOS38,39. A recent meta-analysis reported that higher values of HOMA-IR, FAI, ALT, and triglycerides, as well as obesity were all associated with significantly higher risk-adjusted odds of NAFLD in PCOS women40. It was also shown that the HOMA-IR indexes in the PCOS group complicated with NAFLD were higher than those in the control group complicated with NAFLD41.h There is evidence of high ALT levels in lean PCOS patients42. Our study suggests that ALT levels in PCOS may be linked to insulin resistance, irrespective of obesity. The possible explanation is that in young lean insulin- sensitive subjects, energy is mainly stored in the liver and muscle glycogen. However, in lean insulin-resistant subjects, energy is commonly diverted to the liver triglyceride synthesis, causing hepatic steatosis and NAFLD in PCOS43. Nevertheless, more evidence is required to confirm such associations. We also made a comparison among different PCOS phenotypes. We found no difference among the four phenotypes of PCOS regarding demographic, clinical, and biochemical characteristics, except for FBS levels. FBS levels were highest among PCOS women with O–H–P phenotype. Conflicting results are reported on the blood sugar status in the PCOS phenotypes from different ethnic groups. Contrary to the results of our study, several researchers did not show differences in blood glucose levels between PCOS phenotypes44–46, while there is also evidence of higher FBS in O–H–P phenotype47–49. Conclusion In conclusion, the results of this study revealed that PCOS women diagnosed with insulin resistance based on HOMA-IR should be monitored in regard to visceral obesity, blood pressure, liver enzymes, and hypertriglyceri- demia. Moreover, phenotype division may help physicians to predict adverse metabolic outcomes. The results of this study suggest that full-blown PCOS (O–H–P) women have an increased risk of insulin resistance and may need routine screening for metabolic disturbances. www.nature.com/scientificreports/ It remains to be elucidated whether blood sugar status differs among PCOS phenotypes. Th f h bl h d d h p yp gf g p yp The association of HOMA-IR in PCOS phenotypes is variable across the studies. Some previous studies have investigated the risk of insulin resistance in the various PCOS phenotypes and showed that HOMA-IR does not differ among the four PCOS phenotypes50,51, while the results of some other studies indicated a higher risk of insulin resistance in the phenotype O–H–P52–55. In this study, we showed a higher risk of insulin resistance in the classic PCOS phenotype. This disagreement may be due to different study methodologies, defining criteria for insulin resistance, cut-off values for HOMA-IR, or PCOS classification criteria.hi fi This study has its strengths; a comprehensive comparison of clinical and biochemical profiles of PCOS women with/without insulin resistance and also among different PCOS phenotypes was provided. Phenotypic categoriza- tion of PCOS women can help to predict risk of insulin resistance. To mention the limitations, the current study was restricted to Iranian women, and further research is required with a larger sample size involving patients of different ethnicities. Another limitation of the current study is the use of HOMA-IR cut-off for diagnosis of insulin resistance. However, the euglycemic hyperinsulinemic clamp is the gold standard method for direct measurement of insulin resistance. Data availabilityh y The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Received: 15 March 2023; Accepted: 22 June 2023 Discussion Activation of upstream PI3K may cause phosphorylation of Mammalian target of rapamycin (mTOR), and consecutive insulin resistance32. Noteworthy, another study indicated that the association between glycemic parameters and SHBG depends on BMI, and SHBG is not reflective of insulin resistance in PCOS women33. 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https://openalex.org/W4390422207	https://ieeexplore.ieee.org/ielx7/6287639/6514899/10376174.pdf	English	null	Deepfake Detection: Analysing Model Generalisation Across Architectures, Datasets and Pre-Training Paradigms	IEEE access	2,024	cc-by	23,246	This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 0 Date of publication xxxx 00, 0000, date of current version xxxx 00, 0000. Date of publication xxxx 00, 0000, date of current version xxxx 00, 0000. Digital Object Identifier 10.0000/ACCESS.20xx.DOI ABSTRACT As deepfake technology gains traction, the need for reliable detection systems is crucial. Recent research has introduced various deep learning-based detection systems, yet they exhibit limitations in generalizing effec- tively across diverse data distributions that differ from the training data. Our study focuses on understanding the generalization challenges by exploring specific aspects such as deep learning model architecture, pre-training strategy and datasets. Through a comprehensive comparative analysis, we evaluate multiple supervised and self-supervised deep learning models for deepfake detection. Specifically, we evaluate eight supervised deep learning architectures and two transformer-based models pre-trained using self-supervised strategies (DINO, CLIP) on four different deepfake detection benchmarks (FakeAVCeleb, CelebDF-V2, DFDC and FaceForensics++). Our analysis includes intra-dataset and inter-dataset evaluations, examining the best performing models, generalisation capabilities and impact of augmentations. We also investigate the trade-off between model size, efficiency and performance. Our main goal is to provide insights into the effectiveness of different deep learning architectures (transformers, CNNs), training strategies (supervised, self-supervised) and deepfake detection benchmarks. Through our extensive analysis, we established that Transformer models outperform CNN models in deepfake detection. Also, we show that FaceForensics++ and DFDC datasets equip models with comparably better generalisation capabilities, as compared to FakeAVCeleb and CelebDF-V2 datasets. Our analysis also show that image augmentations can be helpful in achieving better performance, at least for the Transformer models. INDEX TERMS deepfakes; image classification; convolutional neural networks; transformers; video processing. INDEX TERMS deepfakes; image classification; convolutional neural networks; transformers; video processing. Deepfake Detection: Analysing Model Generalisation Across Architectures, Datasets and Pre-Training Paradigms SOHAIL AHMED KHAN1 and DUC-TIEN DANG-NGUYEN1 (Member, IEEE) 1MediaFutures, Department of Information Science and Media Studies, University of Bergen, Norway Corresponding authors: Sohail Ahmed Khan and Duc-Tien Dang-Nguyen (e-mail: {sohail.khan, ductien.dangnguyen}@uib.no 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 with limited technical knowledge, facilitating the production of remarkably convincing deepfakes that closely resemble genuine content. D EEPFAKES, or deepfake media, are digital media that have been generated or modified using deep learning algorithms [1]. They have gained notoriety in recent years due to their potential to manipulate and deceive by producing fraudulent and deceptive media content. While deepfakes can serve innocent or even entertaining purposes, they also harbor substantial dangers when harnessed for malicious intentions, like crafting convincing fraudulent media to sway public opinion, manipulate electoral outcomes, or incite vi- olence [2], [3], [4]. Also, given the prevalence of pow- erful and budget-friendly computing resources along with the widespread accessibility of paid, as well as open-source software, the creation of deepfakes has become increasingly straightforward [5]. This accessibility extends to individuals D The research community has been actively proposing novel AI-based automated deepfake detection models, trying to address these issues posed by deepfake media [6], [7], [8], [9], [10], [11]. However, a significant issue associated with current deepfake detection models is their lack of generalisa- tion capability [1], [7], [12]. This means that these detection systems work very well when dealing with deepfakes that come from the same data distribution as they were trained on. However, they struggle to perform well when exposed to deepfakes generated using different methods than the ones used for training. Previous research efforts have introduced a multitude of 1 1 VOLUME 4, 2016 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 carefully designed deep learning models for deepfake de- tection, accompanied by novel techniques for training (e.g., augmentations, multi-modal training setup, diverse set of training features etc) and evaluating these models on well- known deepfake datasets. I. INTRODUCTION However, given the vast volume of research publications, it has become increasingly chal- lenging to discern which kind of architectures yield optimal results and which datasets are most effective in facilitating robust model performance, thus enhancing generalisation to unseen data. In light of these considerations, we contend that a comprehensive analysis that unites a diverse range of deep learning architectures, trained and assessed across multiple prominent deepfake datasets in a unified manner, is imperative to gain a deeper understanding of this issue. We also think that such a comparative analysis has the potential to uncover valuable insights for identifying the most suitable architecture and dataset(s) to enhance the effectiveness of deepfake detection. Consequently, we believe that this anal- ysis can contribute significantly to addressing the current challenge of model generalisation in the realm of deepfake detection. well-known self-supervised learning strategies: DINO [24] and CLIP [25]. We choose to ViT in this case since it is shown to achieve better performance as compared to CNN architecture, i.e., ResNet [24]. To study these models and find out how good the self-supervised features are, we use self- supervised ViT-Base models (DINO and CLIP) as feature extractors and train a classification head on top of them. It is important to note that we only train the classification head and freeze the weights of the feature extractors to avoid backpropagating gradients through them. In summary, our study aims to provide insights into various aspects, including: (1) identifying the most effective mod- els for detecting deepfakes among those being tested, (2) pinpointing the model with the highest ability to adapt to new and unseen data, (3) assessing the difficulty of different datasets for model training, (4) determining the dataset that best facilitates generalisation to unseen data, (5) evaluating the performance of self-supervised training strategies and (6) examining the impact of augmentations on enhancing model performance. This next parts of this paper is organised as follows. In Section II we present a brief literature review on the topic of deepfake detection. Section III presents the proposed frame- work. In Section IV we present the results and discussion of our findings and finally Section V concludes this study by summarising our analysis and presents future research direction. In this study, we carry out a comprehensive comparative analysis of several widely recognised deep architectures for image and video recognition, aiming to assess their efficacy in detecting deepfakes. 1models trained and evaluated on the same dataset 2models trained on one dataset and evaluated on another dataset I. INTRODUCTION Our primary goal is to determine which among these models achieves superior performance on unseen, out-of-distribution data, i.e., exhibit impressive generalisation capability. The models selected for our study comprise of both Convolutional Neural Networks (CNNs) and Transformer models. The rationale behind incorporating transformer models is rooted in their recent notable achieve- ments across a spectrum of computer vision tasks such as im- age classification [13], [14], [15], object detection [16], [17], image segmentation [18], video classification, multi-modal learning [19], [20], 3D analysis [21], [22] and beyond [23]. II. LITERATURE REVIEW II. LITERATURE REVIEW Since recently quite a large number of research studies fo- cused on deepfake media detection have been proposed. Most studies employ CNN models trained on large amounts of data in order to detect deepfake media. The proposed studies also employ different strategies e.g., novel augmentation tech- niques [29], hybrid models [9], [30], biological features [31], multi-modal features [6], [9], temporal features along with spatial information [9], [10], [32], recurrent networks, trans- former models [8], [9] etc to detect deepfake images/videos while trying to increase the models’ generalisation capabili- ties. Below we present some well-known, as well as some of the recently proposed deepfake detection studies. We chose to review studies in this section that share similarities with ours, focusing on common aspects such as the selection of detection models and the datasets used to train and evaluate the proposed models. For our analysis we train all participating models on four deepfake detection datasets and evaluate them in both intra- dataset 1 and inter-dataset 2 configurations (see Figure 1). Ad- ditionally, we evaluate the difficulty level of each benchmark and investigate whether a more challenging benchmark leads to better generalisation performance on unseen data. To this end, we train participating models on all four datasets twice: first, without any image augmentations and then with various image augmentations to find out if they improve models’ performance. Since recently, transformer models trained using self- supervised methods have exhibited their capability to pro- duce robust visual features [24], [25], [26]. Subsequently, models trained through self-supervised methods have been shown to achieve excellent performance on new tasks, often without the need for additional training or with minimal train- ing [18], [24], [25], [27], [28]. Owing to this, we also analyse Vision Transformer (ViT) architecture pre-trained using two This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ A. CNN BASED DETECTION MODELS Through extensive experiments authors establish the effectiveness of M2TR and show their model outperforms SOTA Deepfake detection models by acceptable margins. Ciftci et al. [31], shifted away from traditional image features and proposed to employ biological signals (i.e., photoplethysmography or PPG signals which detects subtle alterations in color and motion within RGB videos) to train their models. The proposed model was comprised of a CNN, as well as a SVM. The CNN and SVM models made their individual predictions on the provided features sets, which were then fused together in order to get a final classification score. The proposed deepfake detection scheme achieved promising results when tested using both intra-dataset as well as inter-dataset configurations on multiple different deepfake detection benchmarks including, CelebDF [40], FaceForen- sics [41] and FaceForensics++ [33] datasets. Coccomini et al., in [30] propose a video deepfake de- tection model based on a hybrid transformer architecture. Authors used an EfficientNet-B0 as feature extractor. The extracted features were then used to train two different types of Vision Transformer models in their study, e.g., (1) Efficient ViT and (2) Convolutional Cross ViT. Through experimen- tation, authors established that the model comprising of EfficientNet-B0 feature extractor and Convolutional Cross ViT achieved the best performance scores as compared to other models that they tested. [ ] [ ] In study [6], Zhu et al. introduced a deepfake detection framework that leveraged 3D face decomposition features for detecting deepfakes. The authors demonstrated that the fusion of 3D identity texture and direct light features no- tably enhanced the detection performance, simultaneously promoting the model’s generalisation ability when assessed across different datasets. The training of the detection model involved both a cropped facial image and its corresponding 3D attributes. Authors employed XceptionNet [35] for fea- ture extraction. The study also provides an extensive analysis of various feature fusion strategies. The proposed model was trained on the FaceForensics++ [33] benchmark and subse- quently evaluated on three datasets: (1) FaceForensics++, (2) Google Deepfake Detection Dataset [42] and (3) DFDC [43] dataset. The reported evaluation statistics showed promising results across all three datasets, highlighting the model’s robust generalisation capability in comparison to previously proposed deepfake detection methods. Zhao et al., [10] propose an Interpretable Spatial-Temporal Video Transformer (ISTVT) for deepfake detection was pro- posed. A. CNN BASED DETECTION MODELS The authors conducted compre- hensive evaluations of their models across prominent deep- fake detection benchmarks, including FaceForensics++ [33], DFDC [43] and Google DFD [42]. Their models showed im- pressive performance across all these datasets, underscoring their efficacy in deepfake detection. In study [9], Khan et al. introduced the utilisation of trans- former architecture for the purpose of deepfake detection, presenting two novel models: (1) Image Transformer and (2) Video Transformer. Both models were trained using 3D face features [44] in addition to standard cropped face images. The integration of 3D face features aimed to swiftly ob- tain accurately aligned facial details, enhancing the learning process. The combination of these aligned features with conventional cropped face data contributed to the acquisition of pertinent facial details. To harness temporal information within videos, authors modified the standard Vision Trans- former (ViT) [13] to accommodate multiple successive face frames. Notably, the proposed model exhibited incremen- tal learning capabilities, accommodating new data without forgetting prior knowledge. The authors conducted compre- hensive evaluations of their models across prominent deep- fake detection benchmarks, including FaceForensics++ [33], DFDC [43] and Google DFD [42]. Their models showed im- pressive performance across all these datasets, underscoring their efficacy in deepfake detection. In [32] Sabir et al. proposed a deepfake detection system by focusing on the temporal information present in video streams to exploit temporal discrepancies across multiple frames. In order to analyse temporal data authors employed a recurrent convolutional architecture [36], [37] comprising of a CNN for feature extraction and a BiDirectional Recurrent Neural Network (BiDir RNN) to analyse temporal infor- mation present in videos. Specifically, authors studied two different CNN architectures, ResNet [38] and DenseNet [39] for feature extraction. The authors also employed a carefully crafted pre-processing regime to preprocess facial frames before inputting them into the models. The models were evaluated using the renowned FaceForensics++ deepfake de- tection benchmark [33], showing excellent results in an intra- dataset evaluation regime. Authors do not carry out a cross- dataset analysis in their study. i Wang et al., [8] introduced a Multi-modal Multi-scale TRansformer (M2TR) model, which processes patches of multiple sizes to identify local abnormalities in a given image at multiple different spatial levels. M2TR also utilises the fre- quency domain information along with RGB information us- ing a sophisticated cross-modality information fusion block to detect forgery related artifacts in a better way. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ A. CNN BASED DETECTION MODELS A. CNN BASED DETECTION MODELS In 2019, Rossler et al. released FaceForensics++, a dataset for deepfake detection [33]. The dataset, containing over 1.8 million manipulated images, was made publicly avail- able. Using the dataset authors also conducted an extensive analysis of several data-driven forgery detection methods. The methods included traditional machine learning models (SVMs) trained on handcrafted features, as well as contem- porary deep learning architectures including MesoNet [34] and XceptionNet [35]. By conducting a thorough analysis, 2 2 VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 B. TRANSFORMER BASED DETECTION MODELS authors discovered that a vanilla deep CNN model, Xcep- tionNet [35], outperforms other participating models signif- icantly in the context of detection task on compressed low quality data. Authors additionally demonstrated, via experi- ments and surveys, that the data-driven models outperform humans in detecting deepfakes. Nevertheless, the paper lacks a cross-dataset analysis of the models, which could have been beneficial in understanding the generalisation performance across diverse and unseen domains. In study [9], Khan et al. introduced the utilisation of trans- former architecture for the purpose of deepfake detection, presenting two novel models: (1) Image Transformer and (2) Video Transformer. Both models were trained using 3D face features [44] in addition to standard cropped face images. The integration of 3D face features aimed to swiftly ob- tain accurately aligned facial details, enhancing the learning process. The combination of these aligned features with conventional cropped face data contributed to the acquisition of pertinent facial details. To harness temporal information within videos, authors modified the standard Vision Trans- former (ViT) [13] to accommodate multiple successive face frames. Notably, the proposed model exhibited incremen- tal learning capabilities, accommodating new data without forgetting prior knowledge. A. CNN BASED DETECTION MODELS The proposed model incorporates a novel decomposed spatio-temporal self-attention as well as a self-subtract mech- anism to learn forgery related spatial artifacts and temporal inconsistencies. ISTVT can be also visualise the discrimi- native regions for both spatial and temporal dimensions by using the relevance propagation algorithm [10]. Extensive ex- periments on large-scale datasets were conducted, showing a strong performance of ISTVT both in intra-dataset and inter- dataset deepfake detection establishing the effectiveness and robustness of proposed model. Through this literature review it becomes apparent that the research community actively employs deep learning mod- els along with other techniques to try develop robust and VOLUME 4, 2016 3 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 FIGURE 1. The proposed framework. The process involves several steps, starting with the extraction and cropping of face frames from videos, followed by augmentation, normalisation and resizing. The pre-trained models are then used as feature extractors, with a new classification head (linear layer) added on top for supervised models. During training, the weights of both the feature extractor and the classification head are updated for supervised models, while only the newly added classification head is updated for self-supervised models. The models are evaluated through both intra-dataset and inter-dataset evaluations to test their performance and generalisation capabilities. For image models, the input data is a single cropped face image, while for video models, it is a tensor containing eight consecutive cropped face images from a given video. FIGURE 1. The proposed framework. The process involves several steps, starting with the extraction and cropping of face frames from videos, followed by augmentation, normalisation and resizing. The pre-trained models are then used as feature extractors, with a new classification head (linear layer) added on top for supervised models. During training, the weights of both the feature extractor and the classification head are updated for supervised models, while only the newly added classification head is updated for self-supervised models. The models are evaluated through both intra-dataset and inter-dataset evaluations to test their performance and generalisation capabilities. A. CNN BASED DETECTION MODELS For image models, the input data is a single cropped face image, while for video models, it is a tensor containing eight consecutive cropped face images from a given video. efficient deepfake detectors. However, while carefully read- ing the research studies it also becomes noticeable that the models perform poorly on unseen, out-of-distribution data. In addition to this, there is a lack of comparative studies which aim to identify which specific family of deep learning architectures is better for the task of deepfake detection. Furthermore, it’s not easy to determine without thorough experimentation that which of the well-known datasets offer improved generalisation potential to the models, i.e., allow models to better handle new and unseen data. quality of the representations they produce since they were initially trained through self-supervised training strategies. Consequently, for these models we only update weights of the newly added classification head while maintaining the frozen weights of the feature extractor backbone. This strategy enables us to directly compare the self-supervised feature representations with those obtained from supervised models. Since we deal DINO and CLIP as feature extractors, we follow the guidelines provided in their respective code repositories to extract features. For DINO, we extract features from the last four encoder blocks, as this configuration yielded optimal results. On the other hand, for CLIP, we exclusively extract features from the last encoder block. We then feed these features into the classification head. To address this, we study some of the most frequently used architectures (EfficentNets, XceptionNet, Vision Transform- ers) in the literature of deepfake detection in this research. We also employ widely known datasets for experimentation and try to find out the datasets offering best generalisation capabilities to the models. We also analyse some of the understudied approaches for deepfake detection i.e., we train and evaluate the performance of self-supervised models on deepfake detection and compare their performance with that of the supervised models. For intra-dataset evaluation we evaluate models on the same dataset (test set) it was trained on, e.g., model trained on dataset D1 is evaluated on the test set of D1. The primary ob- jective of intra-dataset evaluation is to discern which model achieves the highest performance score as compared to other participating models on each of the dataset. Moreover, this evaluation will offer insights into which dataset presents the greatest learning challenge for the models and which dataset is comparatively easier to learn. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ III. THE PROPOSED FRAMEWORKl The workflow followed in this study for training and evaluat- ing the models is illustrated in Figure 1. On top we show the training pipeline where we start by extracting and cropping faces from videos. The cropped face frames are then aug- mented, normalised and resized before being fed to the model for training. We load pre-trained models as feature extractors, i.e., we remove the last layer from the loaded models and add a new classification head (linear layer) on top. For supervised models, during training we update weights of both feature extractor as well as the classification head. In the context of inter-dataset evaluation, we evaluate models that were trained on one dataset across the remaining three datasets. For instance, a model trained on dataset D1 is tested on D2, D3 and D4 datasets. The objective of inter- dataset evaluation is two-fold: first, to investigate the models’ ability to generalise across datasets and second, to understand how effectively the training dataset empowers models to generalise well on unseen data. i For self-supervised models, our objective is to assess the The input data for training and evaluating image models 4 VOLUME 4, 2016 VOLUME 4, 2016 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 0 is a single face cropped image ([3, 224, 224]), whereas, input data for training and evaluating video models is a tensor con- taining 8 consecutive face cropped images ([8, 3, 224, 224]) from any given video. as validation set. For testing the models we use 4000 face frames randomly selected from 3500 (3200 fake and 300 real) videos. CelebDF-V2 [40] contains 5639 fake and 590 real videos. The real videos are collected from YouTube and contain interview videos of 59 celebrities having diverse ethnic back- grounds, genders, age groups. CelebDF-V2 dataset com- prises of fake videos generated using Encoder-Decoder mod- els. A. DATASETS Face2Face and NeuralTextures subsets are generated by a different process called, face re-enactment. In contrast to face swapping, face re-enactment swaps the faces of source and target, however, keeps the original iden- tity of the target face. TABLE 1. The amount of real/fake images used to train, validate and test our image models. Train/Test Data Dataset Train Validation Test Real Fake Real Fake Real Fake FakeAVCeleb [45] 47,808 47,808 5,360 5,360 2,000 2,000 CelebDF-V2 [40] 50,000 50,000 10,000 10,000 1,000 1,000 DFDC [43] 50,000 50,000 10,000 10,000 2,000 2,000 FaceForensics++ [33] 50,000 50,000 10,000 10,000 2,000 2,000 Deepfake Detection Challenge (DFDC) dataset [43] comprises of around 128k videos, out of which, around 104k are fake. Similar to the FaceForensics++, the DFDC also comprises of videos generated using more than one face manipulation algorithms. Five different methods were employed to generate fake videos, namely, (1) Deepfake Autoencoder [43], (2) MM/NN [50], (3) NTH [51], (4) FSGAN [52] and (5) StyleGAN [53]. In addition to these, a random selection of videos also underwent a simple sharpening post-processing operation which increases the videos’ perceptual quality. Unlike FaceForensics++ dataset, the DFDC dataset also contains videos having undergone audio-swapping. However, in this study we do not use audio features to train and evaluate our models. III. THE PROPOSED FRAMEWORKl Post-processing operations are also employed to circum- vent color mismatch, temporal flickering and inaccurate face masks. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ A. DATASETS In this study we train and evaluate several differ- ent deep learning models on four deepfake detection datasets/benchmarks: FakeAVCeleb [45], CelebDF-V2 [40], DFDC [43] and FaceForensics++ [33]. All of the four datasets comprise of real and fake videos, where fake videos are generated using different deepfake generation methods. In upcoming sections, we present a brief description of these datasets. FakeAVCeleb [45] is the most recently proposed deep- fake detection dataset. FakeAVCeleb dataset contains 19500 fake and 500 real videos. This dataset also contains au- dio modality and manipulates audio as well as video con- tent to generated deepfake videos. For video manipulation, FaceSwap [54] and FSGAN [52] alogrithms are used. For audio manipulation, a real-time voice cloning tool called SV2TTS [55] and Wav2Lip [56] are used. The dataset is divided into 4 subsets, i.e., (1) FakeVideo/FakeAudio, (2) RealVideo/RealAudio, (3) FakeVideo/RealAudio and (4) Re- alVideo/FakeAudio. FaceForensics++ [33] is one of the most widely studied deepfake detection benchmarks. FaceForensics++ comprises of 1000 real video sequences (mostly from YouTube) of mostly frontal faces and without any occlusions. These real videos were then manipulated using four different face ma- nipulation methods: (1) FaceSwap [46], (2) Deepfakes [47], (3) Face2Face [48] and (4) NeuralTextures [49] to have four subsets. Each subset comprises of 1000 videos each. In total, the dataset contains 5000 videos, i.e., 1000 real and 4000 fake videos. FaceForensics++ offers 3 different qualities of data, (1) Raw, (2) High-Quality and (3) Low-Quality. In our study, we experimented the high-quality videos. In this study, we only employ 2 of the mentioned subsets to train our models, i.e., (1) FakeVideo/FakeAudio and (2) RealVideo/RealAudio. FaceSwap and Deepfakes subset contains videos generated using what is called, face swapping. As the name suggests, face of the target person is replaced with the face of source person and results in transferring the identity of the source person onto the target. Face2Face and NeuralTextures subsets are generated by a different process called, face re-enactment. In contrast to face swapping, face re-enactment swaps the faces of source and target, however, keeps the original iden- tity of the target face. FaceSwap and Deepfakes subset contains videos generated using what is called, face swapping. As the name suggests, face of the target person is replaced with the face of source person and results in transferring the identity of the source person onto the target. B. DATASET PREPARATION The data preparation process was notably time-consuming due to two main factors: firstly, the datasets being substantial in size and secondly, some selected datasets lacking clear dataset preparation guidelines. For instance, FakeAVCeleb does not provide predefined train/validation/test splits. Con- sequently, we had to manually develop a strategy to effec- tively partition the dataset into train, validation and test sets. Ensuring that a single identity didn’t appear in multiple splits added another layer of complexity to this task. Since DFDC dataset is huge as compared to other par- ticipating datasets, we only use a subset of the full dataset to train and evaluate our models i.e., to keep the number of training, validation and test data nearly similar. For training we use roughly around 19500 (around 16500 fake and 3100 real) randomly selected videos from which we get 100k face cropped images (50k real and 50k fake). We use 20k images Additionally, all the datasets exhibit an imbalance, with a significantly higher number of "fake" videos compared to "real" ones. To address this, we took steps to ensure that the resulting datasets of cropped face images are balanced by extracting faces from videos. Our efforts aimed to include at least one frame from every video selected for training and evaluation. You can refer to Table 1 for detailed information 5 VOLUME 4, 2016 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 regarding the number of face frames used for training, vali- dation and model evaluation from each dataset. are trained and evaluated on images independently, dealing with each image on its own. This differs from video-based deepfake detection, where models are trained and tested on consecutive video frames to capture temporal discrepancies between frames along with spatial cues within each frame. 3https://imgaug.readthedocs.io/en/latest/ B. DATASET PREPARATION The data provided in Table 1 clearly illustrates that the training and validation sets for FakeAVCeleb contain a rel- atively smaller number of frames. This discrepancy can be attributed to the dataset containing a limited count of real videos (only 500), while the number of fake videos is sub- stantially larger (19500). As the video clips are of shorter duration, this translates to 47,808 frames being extracted from the chosen 300 real videos for the training set and 5360 frames from 100 videos for the validation set. Despite this slight variance in the size of the training and validation sets, we assume that it has a minimal impact on the models’ per- formance. This assumption is supported by our observations from training and evaluating the models using even fewer frames (approximately 25k real and 25k fake frames), which resulted in no significant deviations in the final test scores. Below, we provide a brief introduction to the image models employed in this study. • Xception [35] is a convolutional neural network (CNN) architecture that builds upon the Google’s Inception CNN architecture [60]. It distinguishes itself by using depth-wise separable convolutions in place of conventional Inception modules. Unlike standard convolutions applied across all N channels at once, depth-wise convolutions operate se- quentially on individual image channels. This characteristic reduces Xception’s trainable parameters compared to other prominent deep CNN models. Despite this reduction, Xcep- tion’s performance remains on par with models having more parameters, as evidenced on the ImageNet benchmark [57]. Furthermore, its smaller parameter count enhances resistance to overfitting on unseen data and decreases computational load, making it an efficient choice. Figure 2A illustrates the concept of depth-wise convolution, the fundamental building block of Xception. Xception not only demonstrates excel- lence on the ImageNet benchmark but also boasts significant achievements in previous deepfake detection studies [6], [33], [43]. Based on its proven track record in this domain, we include Xception for analysis in this study. • Xception [35] is a convolutional neural network (CNN) architecture that builds upon the Google’s Inception CNN architecture [60]. It distinguishes itself by using depth-wise separable convolutions in place of conventional Inception modules. Unlike standard convolutions applied across all N channels at once, depth-wise convolutions operate se- quentially on individual image channels. This characteristic reduces Xception’s trainable parameters compared to other prominent deep CNN models. D. MODELS We opt to explore six supervised image recognition models, equally divided into three CNNs and three transformer-based models. Furthermore, we assess two variations of trans- former models trained via self-supervised methods, namely (1) DINO [24] and (2) CLIP [25]. In addition to the image classification models, our study encompasses the training and evaluation of two distinct video classification models: (1) ResNet-3D [58], a CNN model for video classification and (2) TimeSformer [59], a transformer model tailored for video classification. We choose models based on their performance on the ImageNet benchmark [57], their parameter count and, in the case of certain models like Xception [35] and Efficient- Net [43] their established performance in deepfake detection, as reported by some of the previous studies [33], [43]. • EfficientNet-B7 [65] belongs to the EfficientNet family of CNN architectures. In their paper, the authors propose a scaling technique that uniformly adjusts depth, width and resolution using a compound coefficient. The central concept revolves around systematically scaling the model’s archi- tecture and parameters to achieve better efficiency. Unlike the conventional approach of arbitrarily scaling individual dimensions, the proposed strategy employs a consistent set of scaling coefficients across all dimensions. Consequently, the C. PREPROCESSING AND AUGMENTATIONS We adopt two distinct approaches to train our models in this study. Initially, we train models without applying any image augmentations. Subsequently, we train the models using a range of randomly chosen image augmentations, such as horizontal flips, affine transformations and random cut- out augmentations. All the cropped face images are then normalised according to the same method used for pre- training models on the ImageNet [57]. For implementing the augmentations, we utilise the imgaug3 library. p y y • Res2Net-101 [61] is a CNN architecture which is built upon the widely adopted ResNet architecture [38]. Res2Net introduces a new building block named the "Res2Net Block," which replaces the conventional bottleneck residual blocks utilised in ResNet models. By operating at a granular level, the Res2Net architecture captures multi-scale features and extends the receptive field range for every network layer. As a result, the network becomes more potent and efficient, leading to enhanced performance across diverse computer vi- sion tasks, including image classification, segmentation and object detection [61]. The innovative Res2Net block can be seamlessly integrated into other leading-edge backbone CNN models, such as ResNet [38], DLA [62], BigLittleNet [63] and ResNeXt [64]. We visualise the Res2Net block in Fig- ure 2B. In this study, we employ Res2Net-101 to explore whether multi-scale CNN features contribute to improved deepfake detection performance. Additionally, we investigate whether these enhancements extend to cross-dataset perfor- mance, gauging the model’s generalisation capability.i • Res2Net-101 [61] is a CNN architecture which is built upon the widely adopted ResNet architecture [38]. Res2Net introduces a new building block named the "Res2Net Block," which replaces the conventional bottleneck residual blocks utilised in ResNet models. By operating at a granular level, the Res2Net architecture captures multi-scale features and extends the receptive field range for every network layer. As a result, the network becomes more potent and efficient, leading to enhanced performance across diverse computer vi- sion tasks, including image classification, segmentation and object detection [61]. The innovative Res2Net block can be seamlessly integrated into other leading-edge backbone CNN models, such as ResNet [38], DLA [62], BigLittleNet [63] and ResNeXt [64]. We visualise the Res2Net block in Fig- ure 2B. In this study, we employ Res2Net-101 to explore whether multi-scale CNN features contribute to improved deepfake detection performance. Additionally, we investigate whether these enhancements extend to cross-dataset perfor- mance, gauging the model’s generalisation capability.ii B. DATASET PREPARATION Despite this reduction, Xcep- tion’s performance remains on par with models having more parameters, as evidenced on the ImageNet benchmark [57]. Furthermore, its smaller parameter count enhances resistance to overfitting on unseen data and decreases computational load, making it an efficient choice. Figure 2A illustrates the concept of depth-wise convolution, the fundamental building block of Xception. Xception not only demonstrates excel- lence on the ImageNet benchmark but also boasts significant achievements in previous deepfake detection studies [6], [33], [43]. Based on its proven track record in this domain, we include Xception for analysis in this study. ii In addition, the test set for CelebDF-V2 contains fewer frames for the same underlying reason – the test set of the dataset includes only 50 real and 50 fake videos. In response, we meticulously extracted a total of 2000 frames from this set of 100 test videos for the purpose of evaluation. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 1) Image Models Deepfake detection is typically treated as an image clas- sification problem. In this context, deep learning models 6 6 VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 ccepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 0 RE 2. Visual representation of the models used for analysis in this study. Due to space limitations, only basic, key concepts for each model are illustrated d of the whole model. For optimal understanding of the essential components of each model, we recommend viewing this figure in color and at a higher fication. n of the models used for analysis in this study. Due to space limitations, only basic, key concepts for each model are illustrated optimal understanding of the essential components of each model, we recommend viewing this figure in color and at a higher FIGURE 2. Visual representation of the models used for analysis in this study. Due to space limitations, only basic, key concepts for each model are illustrated instead of the whole model. For optimal understanding of the essential components of each model, we recommend viewing this figure in color and at a higher magnification. • Vision Transformer (ViT-Base) [66] belongs to the fam- ily of transformer models which were initially designed for natural language processing tasks. In the realm of computer vision, the Vision Transformer (ViT) emerged as a pioneering transformer-based architecture designed specifically for im- age classification tasks [13]. ViT harnesses the power of self- attention mechanisms to processes visual data. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 1) Image Models In our study, we use the ViT-Base as feature extractor and add a classifica- tion head on top. We only train the added classification head on participating deepfake detection datasets, while freezing the weights of the ViT-Base feature extractor. p g p p g y • Swin Transformer (Swin-Base) [14] is a class of Vision Transformer models. Swin Transformer architecture comes with a hierarchical structure, utilising a shifted windows approach for computing image representations. The shifted windowing strategy enhances efficiency by confining self- attention computation to non-overlapping local windows, while still enabling cross-window connections. This hierar- chical design offers flexibility for modeling at different scales and maintains linear computational complexity concerning image size. Swin Transformers achieve competitive perfor- mance, comparable to other state-of-the-art image classifica- tion models like EfficientNets [14], [65] and even outperform Vision Transformers and ResNets [13], [38]. Not only limited to image classification, Swin Transformers also excel in tasks such as image segmentation and object detection [14]. Fig- ure 2G provides an illustration of the window generation and attention calculation process in Swin Transformers. Because of the excellent performance Swin Transformer achieve on ImageNet, we use it for the task of deepfake detection and try to study how it performs as compared to other participating models. g • Contrastive Language-Image Pre-Training (CLIP) [25] is a neural network that has been trained on a diverse set of (image, text) pairs in a self-supervised contrastive manner. It has the ability to infer the most suitable text excerpt for a given image using natural language, without explicit supervision for this task. It exhibits zero-shot capabilities similar to the ones exhibited by GPT-2/GPT-3 [67], [68]. In CLIP’s original research paper, authors show that it achieves performance scores equivalent to the original ResNet50 [38] CNN model when evaluated on ImageNet [57] in a "zero- shot" fashion, i.e., even though CLIP does not use any of the 1.28 million labelled examples from the original dataset it achieves comparable performance as a ResNet50 model trained on ImageNet in a supervised manner. CLIP is il- lustrated in Figure 2H. For more details on CLIP, we refer readers to [25]. We employ a ViT-Base model trained using CLIP as a feature extractor for our study. Similar to DINO, we add a classification head on top of ViT-Base trained using CLIP. 1) Image Models Swin Transformer architecture comes with a hierarchical structure, utilising a shifted windows approach for computing image representations. The shifted windowing strategy enhances efficiency by confining self- attention computation to non-overlapping local windows, while still enabling cross-window connections. This hierar- chical design offers flexibility for modeling at different scales and maintains linear computational complexity concerning image size. Swin Transformers achieve competitive perfor- mance, comparable to other state-of-the-art image classifica- tion models like EfficientNets [14], [65] and even outperform Vision Transformers and ResNets [13], [38]. Not only limited to image classification, Swin Transformers also excel in tasks such as image segmentation and object detection [14]. Fig- ure 2G provides an illustration of the window generation and attention calculation process in Swin Transformers. Because of the excellent performance Swin Transformer achieve on ImageNet, we use it for the task of deepfake detection and try to study how it performs as compared to other participating models. A classification token is introduced at the outset of this input, which is subsequently processed by the transformer encoder—a mechanism reminiscent of the encoders in text- oriented transformer models. This approach empowers the model to better capture the context and relationships between different parts of the image. As a result, the network ef- fectively captures contextual nuances and interrelationships across distinct segments of the image, achieving performance comparable to state-of-the-art CNN models on the Ima- geNet dataset, especially when trained on giant datasets like ImageNet-21k or JFT-300M. The ViT architecture is visually depicted in Figure 2E. In our analysis, we undertake the training and evaluation of the base version of ViT-Base model for the deepfake detection task and subsequently compare its performance against other models participating in the study. emerge from the ViT model. Authors make the following observations in their study, i.e., (1) self-supervised ViT fea- tures (DINO) incorporate explicit visual information within an image, useful for computer vision tasks such as semantic segmentation, which does not come along as evidently with supervised ViTs and also not with CNNs; (2) self-supervised ViT features are also shown to achieve excellent performance when tested as k-NN classifiers, attaining 78.3% top-1 on ImageNet with a ViT-small architecture. For more details about this strategy, we would like to point readers towards the original paper [24]. The DINO training strategy is shown in Figure 2I. Inspired from these findings, we also employ ViT- Base [13] architecture trained using DINO [24]. 1) Image Models For our analysis, we only train the classification head and keep the CLIP ViT-Base features frozen i.e., we do not update its weights during training. • Multiscale Vision Transformer (MViT-V2-Base) [15] is another class of ViT models. Unlike traditional ViTs, the MViTs have multiple stages that vary in both channel capac- ity and resolution. These stages create a hierarchical pyramid of features, where initial shallow layers focus on capturing low-level visual information with high spatial resolution, while deeper layers extract complex, high-dimensional fea- tures at a coarser spatial resolution. This approach allows the network to capture the context and relationships between different parts of the image in a better way, which results in improved performance on a broad range of computer vision tasks including image classification, image segmen- tation [15]. A broad overview of the architecture of MViT is shown in Figure 2C. Since MViTs are relatively new and achieve excellent performance on different vision tasks, we employ these in our study to analyse how well they perform on the task of deepfake detection. 2) Video Models This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 1) Image Models Its method- ology employs a deceptively simple yet impactful strategy: the division of images into smaller patches, which are then fed into a transformer model as a unified entity. These patches are enriched with positional embeddings, enabling them to retain their spatial context within the original image. architecture offers a family of seven models spanning various scales [65]. Impressively, EfficientNet achieves top-notch performance across several image classification benchmarks, while maintaining computational efficiency that surpasses other architectures like ResNet and Inception [65]. In a manner similar to Xception, a specific variant of EfficientNet, namely EfficientNet-B7, has also demonstrated remarkable prowess in deepfake detection tasks. Notably, the triumphant solution of the Google-sponsored Deepfake Detection Chal- lenge (DFDC) was built upon the strengths of EfficientNet- B7 models [43]. Given this notable track record, our research aims to delve into the potential of this model in our study. 7 VOLUME 4, 2016 VOLUME 4, 2016 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 A classification token is introduced at the outset of this input, which is subsequently processed by the transformer encoder—a mechanism reminiscent of the encoders in text- oriented transformer models. This approach empowers the model to better capture the context and relationships between different parts of the image. As a result, the network ef- fectively captures contextual nuances and interrelationships across distinct segments of the image, achieving performance comparable to state-of-the-art CNN models on the Ima- geNet dataset, especially when trained on giant datasets like ImageNet-21k or JFT-300M. The ViT architecture is visually depicted in Figure 2E. In our analysis, we undertake the training and evaluation of the base version of ViT-Base model for the deepfake detection task and subsequently compare its performance against other models participating in the study. • Swin Transformer (Swin-Base) [14] is a class of Vision Transformer models. 2) Video Models We examined two distinct video classification models in this paper: (1) ResNet-3D [58], a CNN-based video classifier and (2) TimeSformer [59], a transformer-based video clas- sification model. Our investigation encompasses assessing the performance of both these models in both intra-dataset and inter-dataset contexts across four renowned deepfake detection benchmarks. Our decision to include video-based models alongside image-based detection models stems from our curiosity about the potential impact of temporal informa- tion present in videos for the deepfake detection task. • ResNet-3D [58] is based on the same principles as the original ResNet architecture [38], but they are specifically designed to work with 3D data, such as videos and volumetric medical images. These models use 3D convolutions, instead of 2D layers, for feature extraction. In addition to that, ResNet-3D models generally use a large number of layers, • ResNet-3D [58] is based on the same principles as the original ResNet architecture [38], but they are specifically designed to work with 3D data, such as videos and volumetric medical images. These models use 3D convolutions, instead of 2D layers, for feature extraction. In addition to that, ResNet-3D models generally use a large number of layers, • DINO [24] is a self-supervised training method, which is interpreted as self-DIstillation with NO labels. Authors train ViT using DINO and show interesting properties which • DINO [24] is a self-supervised training method, which is interpreted as self-DIstillation with NO labels. Authors train ViT using DINO and show interesting properties which 8 8 VOLUME 4, 2016 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication Citation information: DOI 10 1109/ACCESS 2023 3348450 0 which allows them to learn complex and abstract features in the data. ResNet-3D models have been utilised for a variety of computer vision tasks, including video classification, ac- tion recognition and medical image segmentation [58], [69]. 3) Accuracy Accuracy is another prominent classification metric. Accu- racy score is basically the measure of correct predictions made by a model in relation to all the predictions made by the model. Accuracy does not indicate how well a model has made a certain classification, as was the case with LogLoss and AUC. Accuracy score can be obtained by dividing the number of correct predictions by total predictions. 2) Area Under the Curve (AUC) 2) Area Under the Curve (AUC) AUC is another widely known metric used to evaluate classi- fication models. AUC basically refers to calculating the entire two-dimensional area under the Receiver Operating Curve (ROC). AUC gives hints about how well a model has made a certain prediction. Quite understandably, the higher the area falling under the ROC, i.e., AUC, the better the performance of the model at discriminating between "real" and "fake" samples in our case. Most of the recently proposed deepfake detection studies employ AUC as the evaluation metric to study the performance of their models. Note that the ROC curve is created by varying the thresh- old used to make predictions from 0 to 1, so the AUC provides a summary of the model’s performance across all possible thresholds. 2) Video Models For reference, we illustrate both 2D and 3D convolutions in Figure 2F. We choose to employ ResNet-3D model for our study because, (1) it is widely studied in regards of video recognition and (2) pre-trained models are easily available. We chose ResNet-3D model pre-trained on 8 frames per video to experiment in this study. L = −1 N N X i=1 [yi log(pi) + (1 −yi) log(1 −pi)] (1) (1) Where L is the LogLoss, N is the total number of samples in the dataset, yi is the true label of the i-th sample, pi is the predicted probability for the i-th sample. It is worth noting that Logloss is a widely used evaluation metric in machine learning competitions such as Kaggle competitions, as it gives a general idea of how good the predictions of the model are. We use LogLoss as one of the evaluation metrics in this study as other previously proposed deepfake detection research studies often use it as their evaluation metric and thus would allow us to compare our results with them. • TimeSformer [59] is a video recognition model based on the transformer architecture. TimeSformer utilises self- attention over space and time, instead of traditional convo- lutional layers, or the spatial attention as employed by ViT for image recognition. The TimeSformer model modifies the transformer architecture, generally used for image recogni- tion, by directly learning the spatio-temporal features from a sequence of frame-level patches. This is accomplished by ex- tending the self-attention mechanism from the image space to the 3D space-time volume. Similar to the Vision Transformer (ViT) model, the TimeSformer employs linear mapping and positional embeddings to interpret ordering of the resulting sequence of features. In TimeSformer paper [59], authors experimented with different self-attention techniques. Out of different techniques, the "divided attention" technique which calculates temporal and spatial attention separately within each block, was found to perform better than other self-attention calculation techniques and thus we choose to analyse the same architecture in this study. Divided space- time attention is illustrated in Figure 2D. We opt to evaluate TimeSformer on the task of deepfake detection and compare it with convolutional video classification network, ResNet- 3D. We also chose 8 frame per video version of the TimeS- former model, same as the ResNet-3D model we described above. E. EVALUATION METRICS In order to analyse the performance of our models in a com- prehensive way, we employ multiple widely used classifica- tion metrics, e.g., (1) LogLoss, (2) AUC and (3) Accuracy. Below we briefly introduce the chosen evaluation metrics. Accuracy = TP + TN TP + FP + TN + FN (2) (2) Where TP is the number of true positives, TN refers to the number of true negatives, FP refers to the number of false positives and FN refers to the number of false negatives. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 1) LogLoss LogLoss, also known as logarithmic loss or cross-entropy loss, is used to measure the classification performance of machine/deep learning models. LogLoss calculates the dis- similarity between the predicted probability score with the true label (0, 1 in case of binary classification). The LogLoss score is computed as the negative logarithm of the likelihood of the true labels given a set of predicted probabilities. The range of the LogLoss function is from 0 to infinity, with 0 representing the ideal outcome and higher values represent- ing worse outcomes. It is worth noting that accuracy is the proportion of cor- rectly classified samples out of the total number of samples. It is a common evaluation metric used in binary classification tasks, however, it can be misleading in cases where the classes (real, fake) are imbalanced, or if the cost associated with the false positives and false negatives is different. In such cases, other evaluation metrics like F1 score, precision, recall, or AUC may provide a more accurate evaluation of It is worth noting that accuracy is the proportion of cor- rectly classified samples out of the total number of samples.i VOLUME 4, 2016 9 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 the classification model’s performance. In our study however, since we have balanced number of samples both for real and fake classes, we can use accuracy as one of the evaluation metric. 4) Efficiency Comparison 4) Efficiency Comparison To gain a comprehensive understanding of models’ perfor- mance in deepfake detection, we conduct an in-depth analysis using three distinct classification performance metrics out- lined in earlier sections. Additionally, we provide efficiency metrics (see Table 2) for each model to offer insights into the trade-off between a model’s effectiveness in detecting deepfakes and its efficiency in real-world deployment. This analysis highlights the financial implications of deploying de- tection models on cloud services, emphasising the trade-off between efficiency and detection performance. For example, while models like Xception or ViT demonstrate high effi- ciency (on GPU), the forthcoming sections show that slower, more heavy models often outperform faster, lighter models in deepfake detection. For visual depiction of these efficiency scores, please see Figure 12 and 13 in the Appendix. In addition to the supervised models, our investigation includes two vision transformer (ViT-Base) models that have been pre-trained using the self-supervised techniques DINO [24] and CLIP [25], as previously outlined in Sec- tion III. We then compare these two self-supervised models against a supervised Vision Transformer (ViT) [13]. It’s important to note that all three models - DINO, CLIP and the supervised ViT - are all ViT-Base models. By training a classification head on top of these three models, our goal is to discern whether self-supervised features offer superior representations in comparison to supervised features. We employ fvcore4 library to compute GFLOPs for our models. While various libraries exist for GFLOPs measure- ment, it’s crucial to acknowledge that results may exhibit slight variations. To determine CPU and GPU inference times, we execute inference on 300 random images and then calculate the av- erage time spent on each image in milliseconds. For GPU, a warm-up phase precedes inference, involving the processing of 10 images to ensure optimal GPU performance before the actual inference on 300 images commences. Our machine is equipped with an RTX 3080 GPU, Ryzen 5800X CPU and 32GB RAM. 4https://github.com/facebookresearch/fvcore 5https://pytorch.org/ 6https://scikit-learn.org/stable/auto_examples/model_selection/plot_det.html 7https://github.com/huggingface/pytorch-image-models 8https://imgaug.readthedocs.io/en/latest/ F. IMPLEMENTATION DETAILS We use PyTorch5 framework to facilitate the training and testing of our models. In our training approach, we employ a batch size of 16 for image models and 4 for video models. The learning rate remains constant at 3×10−3 for both image and video models. Our chosen loss function is CrossEn- tropyLoss and we utilise Stochastic Gradient Descent (SGD) as the optimiser for model training. Our models undergo training for a span of 5 epochs, with final selection of the model having lowest validation loss for subsequent testing and evaluation purposes. For the evaluation stage, we use Scikit-Learn library [70]. We use Scikit-Learn to calculate and report LogLoss, AUC, Accuracy scores, as well as ROC and DET6 (Detection Error Tradeoff) curves [70]. TABLE 2. This table presents a detailed account of efficiency metrics of all the participating supervised models, including, the parameter count, inference times both on CPU and GPU and the number of floating point operations per second (FLOPs). Model Efficiency Model Parameters CPU GPU FLOPs Xception 21 million 49.28ms 7.65ms 4.6G Res2Net-101 43 million 110.23ms 31.81ms 8.2G EfficientNet-B7 64 million 148.77ms 37.37ms 5.4G ViT 86 million 239.18ms 6.18ms 16.9G Swin-Base 87 million 254.03ms 27.31ms 15.5G MViT-V2-Base 51 million 238.65ms 43.79ms 10.2G ResNet-3D 32 million 392.04ms 10.07ms 41.92G TimeSformer 121 million 2498.54ms 36.56ms 196.1G Model Efficiency To facilitate our model implementations and leverage pre- trained weights, we heavily rely on the PyTorch Image Mod- els7 repository by Ross Wightman. Additionally, we adapt certain code snippets from [24] to train linear classification heads on top of self-supervised feature extractors like DINO and CLIP. We augment images for training using the imgaug8 library. IV. RESULTS We conducted extensive experimentation and evaluation on six image recognition models and two video classification models, which we specifically trained for deepfake detec- tion. These evaluations are conducted across four different datasets, as outlined in Section III.The analysis includes evaluating all models under both intra-dataset conditions (trained and evaluated on the same dataset) and inter-dataset conditions (trained on one dataset and evaluated on other datasets, excluding the training dataset). Subsequent sections present the performance outcomes of all participating models within both intra-dataset and inter-dataset contexts. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ B. CELEBDF-V2 Table 4 presents the performance of supervised models when trained and evaluated on CelebDF-V2 [40] dataset. Same as it was the case with FakeAVCeleb dataset, almost all of the participating models achieve excellent scores i.e., more than 97% accuracy and more than 99% AUC score, while having a very small LogLoss. We can thus infer that the models quite comfortably learnt to discriminate between real/fake samples of the CelebDF-V2 dataset, similar to FakeAVCeleb dataset. Table 11 in Appendix reports results achieved by all the models when trained on FakeAVCeleb and evaluated on the remaining three datasets. When we look at the numbers in Table 11, it is apparent that almost all of the models perform poorly on all the other datasets. We can see that in terms of accuracy scores, the models are making random guesses. LogLoss and AUC scores are also not remarkably good in inter-dataset evaluation. To gauge the extent to which this dataset aids models in acquiring robust features for enhanced generalisation, we carry out extensive inter-dataset evaluation involving all par- ticipating models trained on CelebDF-V2. The outcomes of this evaluation are presented in Table 13 in the Appendix. Surprisingly similar to the observations from models trained on the FakeAVCeleb dataset and assessed on other datasets, the models trained on CelebDF-V2 and subjected to inter- dataset evaluation also display suboptimal performance. This outcome could possibly be attributed to CelebDF-V2 not being particularly challenging for the models to differentiate, as they almost flawlessly categorise every real/fake sample. Nonetheless, this dominance in classification also renders the models less adept at handling unseen data, as evidenced by the performance metrics detailed in Table 13 in the Appendix. For self-supervised models, the intra-dataset evaluation scores are not as high as those achieved by the supervised models, however, they are still not bad. This is understand- able as these models aren’t trained in an end-to-end manner, rather only the classification heads are trained on frozen features, as previously mentioned. On this dataset, DINO outperforms the other two models, i.e., CLIP and supervised ViT, with a significant margin as indicated in Table 8. In an inter-dataset evaluation setting, self-supervised mod- els provide intriguing insights. Notably, DINO, trained on the FakeAVCeleb dataset and evaluated on CelebDF-V2 and FaceForensics++ datasets, demonstrates comparable results to supervised image models. A. FAKEAVCELEB FakeAVCeleb [45] is a newly released deepfake detection dataset containing four different categories of videos as given in section III-A earlier. Since we focus only on visual deepfakes in this study, we do not use the audio data (real 10 VOLUME 4, 2016 VOLUME 4, 2016 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 his article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 TABLE 3. Intra-dataset performance comparison of image models. The table below presents scores achieved by image models when trained and evaluated on FakeAVCeleb [45] dataset. Best results are highlighted in yellow. FakeAVCeleb Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 0.0047 100.00% 99.93% 0.0040 100.00% 99.85% Res2Net-101 0.0008 100.00% 99.98% 0.0037 100.00% 99.93% EfficientNet-B7 0.0132 100.00% 99.63% 0.0047 100.00% 99.83% ViT 0.2073 99.29% 94.60% 0.3768 98.78% 92.43% Swin-Base 0.0033 100.00% 99.88% 0.0058 100.00% 99.83% MViT-V2-Base 0.0008 100.00% 100.00% 0.0023 100.00% 99.95% ResNet-3D 0.0041 100.00% 100.00% 0.0066 100.00% 100.00% TimeSformer 0.0796 99.96% 97.50% 0.1238 99.94% 97.00% TABLE 3. Intra-dataset performance comparison of image models. The table below presents scores achieved by image models when trained and evaluated on FakeAVCeleb [45] dataset. Best results are highlighted in yellow. FakeAVCeleb Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 0.0047 100.00% 99.93% 0.0040 100.00% 99.85% Res2Net-101 0.0008 100.00% 99.98% 0.0037 100.00% 99.93% EfficientNet-B7 0.0132 100.00% 99.63% 0.0047 100.00% 99.83% ViT 0.2073 99.29% 94.60% 0.3768 98.78% 92.43% Swin-Base 0.0033 100.00% 99.88% 0.0058 100.00% 99.83% MViT-V2-Base 0.0008 100.00% 100.00% 0.0023 100.00% 99.95% ResNet-3D 0.0041 100.00% 100.00% 0.0066 100.00% 100.00% TimeSformer 0.0796 99.96% 97.50% 0.1238 99.94% 97.00% and fake) for training and evaluating the models. Thus out of the four subsets of FakeAVCeleb dataset, we only use two for our experiments i.e., (1) FakeVideo/FakeAudio, (2) RealVideo/RealAudio. A. FAKEAVCELEB From the results given in Tables 3, 8, 10, 11 and 12 we can infer that FakeAVCeleb dataset is not challenging enough for the models to learn and is fairly easy to distinguish between fake and real samples for both supervised and self-supervised models. In addition to that, this dataset does not enhance the models’ ability to learn robust distinguishing features between real and fake faces, or in other words, it lacks at integrating the generalisation capability into the models, as is apparent from Tables 10, 11 and 12 in Appendix. We present scores of intra-dataset evaluation in Table 3 showing that all models perform pretty well in distinguishing between fake and real faces. From Table 3, we can see that all of the participating models achieved almost 99% AUC and very low LogLoss score when tested in an intra-dataset configuration. The numbers in 3 suggest that FakeAVCeleb dataset is relatively easy and thus the models can accurately distinguish between real and fake samples. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ B. CELEBDF-V2 It’s worth highlighting that DINO achieves this performance while only training the classification head, in contrast to supervised models that undergo full training. Also, the results suggest that training more complex models on easier datasets do not yield good performance scores when tested on out-of-distribution data (overfitting). The evidence of CelebDF-V2 being less challenging to learn is further substantiated by the outcomes obtained from the self-supervised models, as illustrated in Table 8. The numbers clearly demonstrate that even when training merely a classification head on feature extractors that remain frozen, VOLUME 4, 2016 VOLUME 4, 2016 11 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 TABLE 4. Intra-dataset comparison of image models. The table below presents scores achieved by image models when trained and evaluated on CelebDF-V2 [40] dataset. TABLE 4. Intra-dataset comparison of image models. The table below presents scores achieved by image models when trained and evaluated on CelebDF-V2 [40] d t t TABLE 4. Intra-dataset comparison of image models. The table below presents scores achieved by image models when trained and evaluated on CelebDF-V2 [40] d CelebDF Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 0.0712 99.73% 97.00% 0.0367 99.95% 98.55% Res2Net-101 0.0237 100.00% 98.95% 0.0185 99.99% 99.45% EfficientNet-B7 0.0433 99.95% 98.40% 0.0340 99.98% 98.75% ViT 0.0336 99.96% 98.60% 0.0350 99.95% 98.60% Swin-Base 0.0340 99.94% 98.80% 0.0202 99.97% 99.40% MViT-V2-Base 0.0075 100.00% 99.70% 0.0096 100.00% 99.70% ResNet-3D 0.0748 99.68% 97.00% 0.1525 98.68% 95.00% TimeSformer 0.0309 100.00% 98.00% 0.0220 99.96% 99.00% in Table 8. This reaffirms the notion that accurately distin- guishing between fake and real faces in the FaceForensics++ dataset poses a formidable task. This prompts us to question whether a more demanding dataset corresponds to enhanced generalisation capabilities. models still manage to achieve commendable results. B. CELEBDF-V2 For inter-dataset evaluation, self-supervised models trained on CelebDF-V2 and tested on the other datasets yield outcomes akin to those of supervised models, but in some cases, e.g., for DFDC self-supervised models show a considerable performance drop. For additional details, kindly consult Ta- bles 13 and 14 in the Appendix. Consequently, we move forward with evaluating all super- vised models trained on the FaceForensics++ dataset using an inter-dataset evaluation framework. The insights from this evaluation are outlined in Table 15 within the Appendix. The models exhibit satisfactory performance even when con- fronted with data originating from previously unseen do- mains. Noteworthy is the improved ability of models trained on the FaceForensics++ dataset and assessed on diverse datasets to generalise effectively. This contrasts with mod- els trained on the FakeAVCeleb and CelebDF-V2 datasets, which tend to exhibit comparatively poor generalisation ca- pabilities. To illustrate, the assessment of MViT trained on FaceForensics++ and evaluated on the FakeAVCeleb dataset yields an accuracy exceeding 80% and an AUC score ex- ceeding 90%. Furthermore, not only on the FakeAVCeleb This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ C. FACEFORENSICS++ The table below presents scores achieved by image models when trained and evaluated on DFDC [43] d t t DFDC Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 0.5613 88.75% 77.63% 0.5120 91.68% 80.65% Res2Net-101 0.5570 90.64% 79.98% 0.5691 91.78% 83.45% EfficientNet-B7 0.5542 89.97% 79.30% 0.4263 93.30% 84.15% ViT 0.4696 91.89% 81.08% 0.5709 89.44% 78.35% Swin-Base 0.5602 90.89% 82.60% 0.6650 87.77% 79.05% MViT-V2-Base 0.6079 88.41% 78.90% 0.5491 90.65% 82.40% ResNet-3D 0.5865 85.64% 75.75% 0.6739 84.69% 73.50% TimeSformer 0.4870 91.18% 83.25% 0.6176 92.30% 81.75% FIGURE 3. Performance (accuracy) comparison of participating models on all datasets. The reported scores result in an intra-dataset evaluation. Results in this figure are obtained by evaluating each model separately on each dataset and averaging the resulting scores. In addition to this, the figure presents the performance of each model trained with and without the augmentations, along with their parameter count. dataset, we can also see encouraging performance from all models trained on this dataset and evaluated on others. The results in Tables 15 and 16 in Appendix support the statement that more challenging datasets mean better generalisation capability. But we have to further re-enforce this statement after evaluating the models trained using DFDC [43] dataset in the upcoming section. C. FACEFORENSICS++ The performance metrics for all supervised models when trained and evaluated on the FaceForensics++ [33] dataset are presented in Table 5. These results are noticeably less favor- able compared to those achieved with the previous datasets, FakeAVCeleb and CelebDF-V2. Few models managed to exceed 95% accuracy and LogLoss scores are also less im- pressive in comparison. The metrics imply that this dataset presents a relatively intricate challenge for the models to dif- ferentiate between real and fake samples. The self-supervised models also encounter difficulties in achieving good scores on the FaceForensics++ dataset, as evident from the numbers TABLE 5. Intra-dataset comparison of image models. The table below presents scores achieved by image models when trained and evaluated on FaceForensics++ [33] dataset. FaceForensics++ Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 0.2342 96.96% 91.05% 0.2957 95.85% 89.03% Res2Net-101 0.2165 97.87% 93.48% 0.3213 97.30% 91.85% EfficientNet-B7 0.3111 96.92% 90.33% 0.3737 94.02% 86.95% ViT 0.2445 97.27% 92.18% 0.3571 94.04% 85.15% Swin-Base 0.1573 98.58% 94.90% 0.2191 97.60% 92.18% MViT-V2-Base 0.1828 98.34% 94.10% 0.1918 97.63% 93.00% ResNet-3D 0.3224 96.42% 90.36% 0.3085 96.19% 91.07% TimeSformer 0.2807 97.10% 90.00% 0.2451 96.76% 90.71% son of image models. The table below presents scores achieved by image models when trained and evaluated on TABLE 5. Intra-dataset comparison of image models. The table below presents scores achieved by image models when trained and evaluated on FaceForensics++ [33] dataset. 12 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 TABLE 6. Intra-dataset comparison of image models. The table below presents scores achieved by image models when trained and evaluated on DFDC [43] dataset. TABLE 6. Intra-dataset comparison of image models. D. DFDC DFDC is one of the biggest and widely adopted deepfake detection benchmarks. We present intra-dataset evaluation scores of our models trained and evaluated on DFDC in Table 6. Res2Net-101 turned out to be the best model in this evaluation, managing to achieve more than 84% accuracy score, 93% AUC score on the DFDC dataset. Self-supervised models also achieve relatively low scores when trained and evaluated on DFDC, as apparent from Table 8. This estab- lishes that DFDC is comparably more challenging dataset out of all the four datasets in this study. FIGURE 3. Performance (accuracy) comparison of participating models on all datasets. The reported scores result in an intra-dataset evaluation. Results in this figure are obtained by evaluating each model separately on each dataset and averaging the resulting scores. In addition to this, the figure presents the performance of each model trained with and without the augmentations, along with their parameter count. In Table 17 inside the Appendix section we present inter- dataset evaluation scores achieved by the supervised models trained on DFDC dataset. It is evident from the numbers that the models trained using DFDC dataset still achieve accept- able performance on unseen data, as compared to the scores achieved by the models which were trained on FakeAVCeleb and CelebDF-V2. Also, by looking at the results now, we can affirm the statement that models trained using more challeng- ing datasets seem to achieve better results. This finding is evident from Tables 10, 15, 16, 17 and 18. achieve accuracy levels ranging from approximately 92% to 94%. Notably, the figure underscores that image augmenta- tions do not always yield significant performance gains. For instance, XceptionNet, Res2Net-101, MViT-V2-Base and EfficientNet-B7 display superior performance when trained without image augmentations, as compared to their coun- terparts trained with augmentations. Nonetheless, the diver- gence in accuracy scores between models trained with and without image augmentations is generally modest, except in the case of ViT. Specifically, the ViT trained with image augmentations achieves an accuracy of 91.62%, whereas the ViT trained without augmentations records an accuracy of 88.63%. In addition to this, Figure 3 highlights that transformer models consistently perform better when trained This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME 4, 2016 1) Supervised Models Performance Comparison of Supervised Models on All Datasets Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 0.2179 96.36% 91.40% 0.2121 96.87% 92.02% Res2Net-101 0.1395 97.12% 93.10% 0.2282 97.27% 93.67% EfficientNet-B7 0.2305 96.71% 91.92% 0.2097 96.83% 92.42% ViT 0.2388 97.10% 91.62% 0.3350 95.55% 88.63% Swin-Base 0.1494 97.35% 94.05% 0.2275 96.34% 92.63% MViT-V2-Base 0.1998 96.68% 93.16% 0.1882 97.07% 93.76% Resnet-3D 0.2470 95.44% 90.77% 0.1620 94.89% 89.89% TimeSformer 0.2196 97.06% 92.18% 0.2521 97.24% 92.12% VOLUME 4, 2016 For more information, see https://creativecommons.org/licenses/by/4.0/ Furthermore, it’s worth noting that the transformer models (Swin-Base and MViT-V2-Base, TimeSformer) demonstrate superior performance compared to their CNN counterparts. Interestingly, the Res2Net-101 model also achieves remark- able numbers in the intra-dataset evaluation context, despite having roughly half the number of parameters (43 million pa- rameters) compared to the top-performing Swin-Base model (87 million parameters). Figure 3 and Table 7 collectively indicate a valuable observation: models equipped with multi- scale feature processing capabilities, such as Res2Net, MViT- V2 and Swin Transformer, exhibit the best performance among all the models. TABLE 7. This table compares the performance of all the participating (supervised) models. We present scores after averaging the scores (LogLoss, AUC, Accuracy) achieved by each model when evaluated in an intra-dataset setting. Performance Comparison of Supervised Models on All Datasets Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 0.2179 96.36% 91.40% 0.2121 96.87% 92.02% Res2Net-101 0.1395 97.12% 93.10% 0.2282 97.27% 93.67% EfficientNet-B7 0.2305 96.71% 91.92% 0.2097 96.83% 92.42% ViT 0.2388 97.10% 91.62% 0.3350 95.55% 88.63% Swin-Base 0.1494 97.35% 94.05% 0.2275 96.34% 92.63% MViT-V2-Base 0.1998 96.68% 93.16% 0.1882 97.07% 93.76% Resnet-3D 0.2470 95.44% 90.77% 0.1620 94.89% 89.89% TimeSformer 0.2196 97.06% 92.18% 0.2521 97.24% 92.12% VOLUME 4, 2016 For more information, see https://creativecommons.org/licenses/by/4.0/ TABLE 7. This table compares the performance of all the participating (supervised) models. We present scores after averaging the scores (LogLoss, AUC, Accuracy) achieved by each model when evaluated in an intra-dataset setting. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 1) Supervised Models In Figure 3, we illustrate a comparison of all participating supervised models based on their attained accuracy scores in an intra-dataset evaluation context. The visualisation clearly indicates that there exists minimal performance difference among the models. Across the majority of cases, the models 13 VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 FIGURE 4. TSNE visualisations of the participating detection models. We chose the best performing models on all datasets (with/without image augmentations). FIGURE 4. TSNE visualisations of the participating detection models. We chose the best performing models on all datasets (with/without image augmentations). of the participating detection models. We chose the best performing models on all datasets (with/without image augmentations) FIGURE 4. TSNE visualisations of the participating detection models. We chose the best performing models on all datasets (w using augmentations. Additionally, video models also exhibit better performance when trained using image augmentations. An important insight is that the best-performing model, Swin-Base, attains its peak accuracy when trained with image augmentations, further advocating for the incorporation of augmentations in training protocols. reduced performance levels in inter-dataset evaluation com- pared to intra-dataset evaluation. This discrepancy is reason- able since detection models tend to experience performance degradation when confronted with data originating from unseen distributions. However, the Figure 7 in Appendix TABLE 7. This table compares the performance of all the participating (supervised) models. We present scores after averaging the scores (LogLoss, AUC, Accuracy) achieved by each model when evaluated in an intra-dataset setting. 1) Supervised Models Performance Comparison of Self-Supervised Models on Individual Datasets Model With Augs No Augs Dataset LogLoss AUC ACC LogLoss AUC ACC Supervised 0.4105 90.19% 82.50% 0.3727 91.77% 85.50% FakeAVCeleb Dino 0.1444 99.00% 95.33% 0.0801 99.64% 97.25% CLIP 0.4369 89.88% 81.20% 0.3715 93.37% 84.55% Supervised 0.2941 95.52% 88.05% 0.2237 97.18% 91.80% CelebDF-V2 Dino 0.3655 97.31% 90.90% 0.3930 97.10% 88.90% CLIP 0.3750 91.43% 82.80% 0.3399 94.73% 85.40% Supervised 0.5182 83.11% 74.95% 0.4971 85.47% 77.43% FaceForensics++ Dino 1.1758 88.67% 80.60% 1.1186 89.48% 81.85% CLIP 0.5019 82.75% 74.15% 0.5093 85.16% 75.80% Supervised 0.5836 79.19% 68.65% 0.5829 80.93% 72.63% DFDC Dino 2.2839 80.72% 72.38% 1.5812 83.03% 74.15% CLIP 0.5601 79.08% 71.75% 0.5196 83.12% 75.00% g pp Performance Comparison of Self-Supervised Models on Individual Datasets Model With Augs No Augs Dataset LogLoss AUC ACC LogLoss AUC ACC Supervised 0.4105 90.19% 82.50% 0.3727 91.77% 85.50% FakeAVCeleb Dino 0.1444 99.00% 95.33% 0.0801 99.64% 97.25% CLIP 0.4369 89.88% 81.20% 0.3715 93.37% 84.55% Supervised 0.2941 95.52% 88.05% 0.2237 97.18% 91.80% CelebDF-V2 Dino 0.3655 97.31% 90.90% 0.3930 97.10% 88.90% CLIP 0.3750 91.43% 82.80% 0.3399 94.73% 85.40% Supervised 0.5182 83.11% 74.95% 0.4971 85.47% 77.43% FaceForensics++ Dino 1.1758 88.67% 80.60% 1.1186 89.48% 81.85% CLIP 0.5019 82.75% 74.15% 0.5093 85.16% 75.80% Supervised 0.5836 79.19% 68.65% 0.5829 80.93% 72.63% DFDC Dino 2.2839 80.72% 72.38% 1.5812 83.03% 74.15% CLIP 0.5601 79.08% 71.75% 0.5196 83.12% 75.00% Performance Comparison of Self-Supervised Models on Individual Datasets Model With Augs No Augs Dataset LogLoss AUC ACC LogLoss AUC ACC Supervised 0.4105 90.19% 82.50% 0.3727 91.77% 85.50% FakeAVCeleb Dino 0.1444 99.00% 95.33% 0.0801 99.64% 97.25% CLIP 0.4369 89.88% 81.20% 0.3715 93.37% 84.55% Supervised 0.2941 95.52% 88.05% 0.2237 97.18% 91.80% CelebDF-V2 Dino 0.3655 97.31% 90.90% 0.3930 97.10% 88.90% CLIP 0.3750 91.43% 82.80% 0.3399 94.73% 85.40% Supervised 0.5182 83.11% 74.95% 0.4971 85.47% 77.43% FaceForensics++ Dino 1.1758 88.67% 80.60% 1.1186 89.48% 81.85% CLIP 0.5019 82.75% 74.15% 0.5093 85.16% 75.80% Supervised 0.5836 79.19% 68.65% 0.5829 80.93% 72.63% DFDC Dino 2.2839 80.72% 72.38% 1.5812 83.03% 74.15% CLIP 0.5601 79.08% 71.75% 0.5196 83.12% 75.00% Activation Mapping (Grad-CAM)9 [72]. Figure 6 in appendix section presents Grad-CAMs of the supervised image models on all datasets. It is interesting to observe that all models, to varying degrees, concentrate on different facial regions when making predictions. reports a useful finding: across all datasets, as compared to CNN models the transformers consistently emerge as the top- performing models. 9https://github.com/jacobgil/pytorch-grad-cam This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 1) Supervised Models Performance Comparison of Supervised Models on All Datasets Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 0.2179 96.36% 91.40% 0.2121 96.87% 92.02% Res2Net-101 0.1395 97.12% 93.10% 0.2282 97.27% 93.67% EfficientNet-B7 0.2305 96.71% 91.92% 0.2097 96.83% 92.42% ViT 0.2388 97.10% 91.62% 0.3350 95.55% 88.63% Swin-Base 0.1494 97.35% 94.05% 0.2275 96.34% 92.63% MViT-V2-Base 0.1998 96.68% 93.16% 0.1882 97.07% 93.76% Resnet-3D 0.2470 95.44% 90.77% 0.1620 94.89% 89.89% TimeSformer 0.2196 97.06% 92.18% 0.2521 97.24% 92.12% VOLUME 4, 2016 Moving towards inter-dataset analysis, we present the out- comes attained by the supervised models when assessed in an inter-dataset context through Figure 7 in Appendix section. The figure showcases that the models exhibit noticeably 14 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 TABLE 8. This table compares the performance of all the participating (self-supervised) models when evaluated in an intra-dataset setting. The statistics of this table are illustrated in Figure 10 in Appendix. 8 s tab e co pa es t e pe o a ce o a t e pa t c pat g (se supe sed) ode s e e a uated a t a dataset sett g e stat st cs o t s illustrated in Figure 10 in Appendix. VOLUME 4, 2016 1) Supervised Models We refer readers to Table 10 in the Ap- pendix section to examine the inter-dataset scores achieved by models on each of the dataset. Furthermore, we provide the ROC, DET curves for the participating models assessed in an intra-dataset context, as illustrated in Figures 8 and 9 in the Appendix respectively. The corresponding AUC scores reinforce the notion that FakeAVCeleb and CelebDF-V2 datasets present less com- plexity to the models in comparison to FaceForensics++ and DFDC datasets. This underscores the idea that training the models on more challenging datasets, rather than easier ones, enhances their generalisation capabilities for deepfake detection. We also present the TSNE [71] plots of all the supervised models in Figure 4, to visually represent how the models separate real faces from the fake ones. Also, it gives us an idea about how the models group together faces coming from same datasets near to each other as compared to the faces coming from a different dataset. The TSNE plots also help us visualise which datasets are more challenging than the others. For example, if we look at the TSNE plots in Figure 4, we can see that the models tend to separate the easier datasets (FakeAVCeleb and CelebDF-V2) in a better way, as compared to how they separate the more challenging datasets (FaceForensics++ and DFDC). The scores (LogLoss, AUC, ACC) reported in Tables 7 and 9 for each model are calculated by averaging the individual scores achieved by that specific model on each dataset. For example, s1, s2, s3, s4 are scores that a model achieved on datasets d1, d2, d3 and d4. Another notable observation is that image models tend to perform the separation task more effectively compared to video models. This is expected, considering our earlier mention that video models typically require larger amounts of training data (we trained both image and video models on the same dataset in this study). As part of our future research, we aim to explore video models on larger datasets to further validate this hypothesis. Despite this, the t-SNE visualizations reveal an interesting insight: while the video model ResNet-3D may struggle to distinguish between real and fake faces within the same dataset, it excels at effectively separating data from different datasets. 3) The Outcome Answering the six questions that we posed at the beginning of this study in Section I: FIGURE 5. Performance (accuracy) comparison of two self-supervised ViT models and one supervised ViT. The reported scores result in an intra-dataset evaluation. Results in this figure are obtained by evaluating each model separately on each dataset and averaging the resulting scores. In addition to this, the figure presents the performance of each model trained with and without the augmentations. All of of the three models have the same amount of trainable parameters since they all are ViT-Base models and the only difference is the pre-training schemes used to train the models. • identifying the most effective models for detecting deep- fakes among those being tested - Ans: Models equipped with multi-scale feature representation capabilities, such as MViT-V2, Res2Net-101 and Swin Transformer (hierar- chical representations). • identifying the most effective models for detecting deep- fakes among those being tested - Ans: Models equipped with multi-scale feature representation capabilities, such as MViT-V2, Res2Net-101 and Swin Transformer (hierar- chical representations). • pinpointing the model with the highest ability to adapt to new and unseen data - Ans: Upon examining the tables in the Appendix section, it becomes evident that MViT-V2 consistently achieves superior performance scores com- pared to other models in the majority of cases. Further- more, these tables also highlight that Transformer models generally outperform CNN models in most scenarios.i ing both supervised and self-supervised approaches and then evaluated on four prominent deepfake detection datasets. Our extensive experiments revealed that models adept at processing multi-scale features, such as Res2Net-101, MViT- V2 and Swin Transformer, consistently outperformed others in intra-dataset comparisons. Notably, MViT-V2-Base and Res2Net-101 achieved superior performance with approx- imately half the parameters of the Swin-Base transformer model. Regarding generalisation across datasets, transformer models consistently outperformed CNN models, with Face- Forensics++ [33] and DFDC [43] enhancing generalisation capabilities. • assessing the difficulty of different datasets for model training - Ans: DFDC and FaceForensics++ datasets pose greater challenges for the models to learn in comparison to CelebDF-V2 and FakeAVCeleb datasets. • determining the dataset that best facilitates generalisation to unseen data - Ans: Table 10 in the Appendix confirms that the FaceForensics++ dataset promotes strong gener- alisation of models to unseen data, with the DFDC dataset ranking second in this regard. 2) Self-Supervised Models In Figure 5 we show a similar comparison involving self- supervised models. It is clear that DINO outperforms the other two models. A careful examination of the outcomes in Tables 8 and 9 enables us to deduce that self-supervised features, particularly DINO, yield superior feature represen- tations in comparison to CLIP and supervised. To strengthen this finding further, we illustrate the ROC and DET curves in Figures 10 and 11 in the Appendix respectively. In addition to that, for a better diagnosis of the models we also visualise the predictions using Gradient-weighted Class 15 VOLUME 4, 2016 VOLUME 4, 2016 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 FIGURE 5. Performance (accuracy) comparison of two self-supervised ViT models and one supervised ViT. The reported scores result in an intra-dataset evaluation. Results in this figure are obtained by evaluating each model separately on each dataset and averaging the resulting scores. In addition to this, the figure presents the performance of each model trained with and without the augmentations. All of of the three models have the same amount of trainable parameters since they all are ViT-Base models and the only difference is the pre-training schemes used to train the models. TABLE 9. This table compares the performance of the self-supervised models. We present scores after averaging the scores (LogLoss, AUC, Accuracy) achieved by each model on the four datasets, when evaluated in an intra-dataset setting. In this table, Supervised refer to ViT-Base model pre-trained using supervised training scheme. DINO refers to ViT-Base model pre-trained using self-supervised scheme proposed in [24] and CLIP refers to ViT-Base pre-trained using self-supervised scheme prposed in [25]. All of these ViT-Base models are used as feature extractors, where we only train a classification head on top of each of the feature extractor and freeze the weights of feature extractors. Performance Comparison of Self-Supervised Models on All Datasets Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 0.4516 87.00% 78.54% 0.4191 88.84% 81.59% Dino 0.9924 91.43% 84.80% 0.7932 92.31% 85.54% CLIP 0.4685 85.78% 77.48% 0.4351 89.09% 80.19% 3) The Outcome 3) The Outcome Our investigation into models pre-trained using self- supervised strategies showed that the ViT-Base model, pre- trained using DINO [24], outperformed both supervised ViT- Base and self-supervised CLIP [25] ViT-Base models. Addi- tionally, our findings indicate that the selected image aug- mentations lead to improved performance for Transformer models, while offering comparably less notable benefits for CNN models. • evaluating the performance of self-supervised training strategies - Ans: From Tables 8 and 9, it is evident that DINO [24] outperforms the other two competing strate- gies in intra-dataset evaluation across all datasets. • examining the impact of augmentations on enhancing model performance - Ans: Within the scope of this study, the augmentations that we have employed have a min- imal effect on models’ performance i.e., in some cases, augmentations help models achieve better performance, while in other cases, they don’t. VI. 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He is a PC member in a number of conferences in the fields of lifelogging, multimedia forensics and pattern recognition. He is co-organiser of over 50 special sessions, workshops and research challenges from ACM MM, ACM ICMR, NTCIR, ImageClef and MediaEval during the last 10 years. He is also the General Chair of MMM 2023, TPC Chair of MMM 2022 and ACM ICMR 2024. DUC TIEN DANG NGUYEN is an associate professor of computer science at the Department of Information Science and Media Studies, Uni- versity of Bergen. His main area of expertise is on multimedia forensics, lifelogging, multimedia retrieval and computer vision. He is the author and co-author of more than a hundred peer-reviewed and widely cited research papers. He is a PC member in a number of conferences in the fields of lifelogging, multimedia forensics and pattern recognition. He is co-organiser of over 50 special sessions, workshops and research challenges from ACM MM, ACM ICMR, NTCIR, ImageClef and MediaEval during the last 10 years. He is also the General Chair of MMM 2023, TPC Chair of MMM 2022 and ACM ICMR 2024. [60] C. Szegedy, W. Liu, Y. Jia, P. Sermanet, S. E. Reed, D. Anguelov, D. Erhan, V. Vanhoucke, and A. Rabinovich, “Going deeper with convolutions,” in Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 2014, pp. 1–9. [61] S. Gao, M.-M. Cheng, K. Zhao, X. Zhang, M.-H. Yang, and P. H. S. Torr, “Res2Net: A new multi-scale backbone architecture,” IEEE Transactions on Pattern Analysis and Machine Intelligence, vol. 43, pp. 652–662, 2021. [62] F. Yu, D. Wang, and T. Darrell, “Deep layer aggregation,” in Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recogni- tion, 2017, pp. 2403–2412. [63] C.-F. Chen, Q. Fan, N. Mallinar, T. Sercu, and R. Feris, “Big-little net: An efficient multi-scale feature representation for visual and speech recognition,” arXiv preprint arXiv:1807.03848, 2018. [64] S. Xie, R. [73] OpenAI, “Introducing ChatGPT,” https://openai.com/blog/chatgpt, 2021, [Online; accessed 08-August-2023]. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ REFERENCES B. Girshick, P. Dollár, Z. Tu, and K. He, “Aggregated residual transformations for deep neural networks,” in Proceedings of the IEEE 18 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 VOLUME 4, 2016 VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ ccepted for publication in IEEE Access. This is the author's version which has not been fully edited and This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 his article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication Citation information: DOI 10 1109/ACCESS 2023 3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 0 APPENDIX FIGURE 6. CBAM visualisations of the supervised image models. PPENDIX FIGURE 6. CBAM visualisations of the supervised image models. FIGURE 6. CBAM visualisations of the supervised image models. VOLUME 4, 2016 19 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 FIGURE 7. Performance (accuracy) comparison of participating models evaluated using inter-dataset scheme. Results in this figure are obtained by, (1) evaluating each model trained on one dataset on each of the remaining datasets and (2) averaging the achieved scores, i.e., add the 3 accuracy scores and divide by 3. FIGURE 7. Performance (accuracy) comparison of participating models evaluated using inter-dataset scheme. Results in this figure are obtained by, (1) evaluating each model trained on one dataset on each of the remaining datasets and (2) averaging the achieved scores, i.e., add the 3 accuracy scores and divide by 3. VOLUME 4, 2016 20 VOLUME 4, 2016 VOLUME 4, 2016 VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 LE 10. This table compares the performance of all the participating (supervised) models evaluated in an inter-dataset setting. Results in this table are obtained ) evaluating each model trained on one dataset on each of the remaining datasets and (2) averaging the achieved scores, i.e., add the 3 accuracy scores and e by 3. Figure 7 illustrate the statistics of this table. I t D t t E l ti This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 TABLE 10. This table compares the performance of all the participating (supervised) models evaluated in an inter-dataset setting. Results in this table are obtained by, (1) evaluating each model trained on one dataset on each of the remaining datasets and (2) averaging the achieved scores, i.e., add the 3 accuracy scores and divide by 3. Figure 7 illustrate the statistics of this table. VOLUME 4, 2016 VOLUME 4, 2016 Inter-Dataset Evaluation Training Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 7.4484 57.69% 52.43% 6.8728 55.41% 51.99% FakeAVCeleb Res2Net-101 8.5574 58.77% 51.98% 7.0666 60.64% 52.18% EfficientNet-B7 8.5664 62.32% 53.42% 10.7718 60.05% 52.49% ViT 6.7348 61.01% 51.60% 9.1672 58.45% 51.38% Swin-Base 5.1077 62.54% 53.32% 4.3274 64.88% 54.78% MViT-V2-Base 4.7564 58.78% 51.73% 4.2891 59.38% 52.11% ResNet-3D 4.4308 57.61% 52.99% 3.8206 60.09% 51.99% TimeSformer 4.7334 61.55% 50.76% 4.7759 63.95% 53.89% Xception 3.9439 65.06% 52.25% 4.8776 66.40% 52.22% CelebDF-V2 Res2Net-101 5.4266 65.90% 51.97% 5.6891 66.21% 52.21% EfficientNet-B7 5.9514 66.99% 53.62% 8.9668 67.13% 53.14% ViT 5.4921 68.52% 52.71% 8.9981 66.36% 52.21% Swin-Base 5.6007 70.06% 52.88% 4.8405 70.56% 53.60% MViT-V2-Base 4.8723 70.71% 53.02% 4.6419 67.20% 53.09% ResNet-3D 6.8365 61.57% 51.30% 5.0504 64.52% 51.76% TimeSformer 4.5629 69.04% 54.50% 4.9391 69.43% 54.89% Xception 1.0701 69.92% 60.73% 1.1262 67.78% 59.62% FaceForensics++ Res2Net-101 1.0165 73.46% 64.09% 1.2360 73.61% 66.44% EfficientNet-B7 0.8792 79.51% 68.71% 1.0068 69.80% 63.52% ViT 0.7899 78.45% 68.32% 0.8301 73.24% 65.87% Swin-Base 0.8517 77.94% 66.21% 0.8482 78.03% 65.46% MViT-V2-Base 0.8407 79.75% 70.72% 0.7292 75.85% 67.67% ResNet-3D 1.0639 74.47% 67.00% 1.3331 66.61% 59.50% TimeSformer 1.0665 75.59% 68.67% 0.8492 77.03% 68.33% Xception 1.2959 63.62% 59.15% 1.6780 64.18% 58.18% DFDC Res2Net-101 2.0224 67.80% 62.58% 1.7396 69.50% 62.85% EfficientNet-B7 1.0388 71.32% 65.28% 1.2764 72.28% 66.56% ViT 1.2198 70.45% 63.86% 1.2498 64.71% 60.65% Swin-Base 1.2423 73.49% 67.72% 1.3802 69.49% 64.55% MViT-V2-Base 1.2329 72.37% 64.38% 1.2254 72.68% 66.98% ResNet-3D 1.1354 66.69% 61.45% 1.1354 66.27% 62.00% TimeSformer 1.1421 70.66% 66.60% 1.6584 71.77% 65.00% VOLUME 4, 2016 21 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 TABLE 11. Inter-dataset evaluation scores of models trained on FakeAVCeleb [45] dataset and evaluated on the remaining three datasets. TABLE 11. Inter-dataset evaluation scores of models trained on FakeAVCeleb [45] dataset and evaluated on the remaining three datasets. TABLE 11. VOLUME 4, 2016 Inter-dataset evaluation scores of models trained on FakeAVCeleb [45] dataset and evaluated on the remaining thr Training Dataset: FakeAVCeleb Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 3.1366 50.52% 56.10% 3.9659 44.63% 54.25% CelebDF-V2 Res2Net-101 3.9007 57.61% 54.60% 3.2127 56.73% 54.50% EfficientNet-B7 5.7925 59.31% 54.50% 12.3786 54.30% 51.65% ViT 4.7035 56.51% 51.60% 7.0900 52.18% 46.65% Swin-Base 3.5148 61.13% 57.70% 2.8360 62.54% 61.45% MViT-V2-Base 4.5526 65.37% 54.00% 3.6492 58.18% 55.05% ResNet-3D 2.4752 53.88% 51.00% 1.7475 57.36% 49.00% TimeSformer 3.9086 51.60% 48.00% 3.2374 58.52% 55.00% Xception 10.3539 62.90% 50.38% 8.9711 63.06% 50.33% FaceForensics++ Res2Net-101 11.6456 59.23% 50.18% 9.7934 58.54% 50.53% EfficientNet-B7 10.5412 63.80% 52.45% 10.4825 63.13% 52.05% ViT 9.3036 61.70% 51.10% 12.3768 58.44% 50.93% Swin-Base 6.1675 62.97% 50.70% 5.5166 64.87% 51.23% MViT-V2-Base 4.8833 56.51% 50.65% 4.7116 63.40% 50.85% ResNet-3D 6.7343 51.30% 50.71% 5.9796 53.99% 50.71% TimeSformer 6.0010 62.61% 51.79% 6.1889 62.60% 51.43% Xception 8.8546 59.65% 50.80% 7.6813 58.53% 51.40% DFDC Res2Net-101 10.1260 59.48% 51.15% 8.1937 66.66% 51.50% EfficientNet-B7 9.3656 63.86% 53.30% 9.4543 62.71% 53.78% ViT 6.1972 64.81% 52.10% 8.0348 64.71% 56.58% Swin-Base 5.6410 63.51% 51.55% 4.6297 67.25% 51.65% MViT-V2-Base 4.8333 54.46% 50.55% 4.5065 56.55% 50.43% ResNet-3D 4.0828 67.65% 57.25% 3.7347 68.91% 56.25% TimeSformer 4.2907 70.43% 52.50% 4.9015 70.74% 55.25% TABLE 12. Inter-dataset evaluation scores of self-supervised models fine-tuned on FakeAVCeleb [45] dataset and evaluated ion scores of self-supervised models fine-tuned on FakeAVCeleb [45] dataset and evaluated on the remaining three datasets. 12. Inter-dataset evaluation scores of self-supervised models fine-tuned on FakeAVCeleb [45] dataset and evaluated on the remaining three datasets. Training Dataset: FakeAVCeleb Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 1.1051 60.65% 58.50% 1.2554 58.82% 55.75% CelebDF-V2 Dino 2.7798 64.07% 60.65% 2.7178 63.39% 60.50% CLIP 1.8561 50.22% 50.50% 3.2978 52.29% 50.15% Supervised 2.2969 62.99% 53.48% 2.2189 64.15% 55.08% FaceForensics++ Dino 7.5589 61.59% 57.73% 8.4257 62.74% 52.05% CLIP 1.1427 58.52% 55.30% 1.5146 60.47% 56.83% Supervised 1.8896 65.60% 56.05% 1.8893 68.37% 57.70% DFDC Dino 8.2808 59.25% 54.10% 9.0520 62.46% 52.40% CLIP 1.1260 64.49% 59.45% 2.0150 65.06% 56.98% VOLUME 4, 2016 Thi k i li d d C ti C Att ib ti 4 0 Li F i f ti htt // ti /li /b /4 0/ LE 12. Inter-dataset evaluation scores of self-supervised models fine-tuned on FakeAVCeleb [45] dataset and evaluated on the remaining three datasets. VOLUME 4, 2016 Training Dataset: FakeAVCeleb Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 1.1051 60.65% 58.50% 1.2554 58.82% 55.75% CelebDF-V2 Dino 2.7798 64.07% 60.65% 2.7178 63.39% 60.50% CLIP 1.8561 50.22% 50.50% 3.2978 52.29% 50.15% Supervised 2.2969 62.99% 53.48% 2.2189 64.15% 55.08% FaceForensics++ Dino 7.5589 61.59% 57.73% 8.4257 62.74% 52.05% CLIP 1.1427 58.52% 55.30% 1.5146 60.47% 56.83% Supervised 1.8896 65.60% 56.05% 1.8893 68.37% 57.70% DFDC Dino 8.2808 59.25% 54.10% 9.0520 62.46% 52.40% CLIP 1.1260 64.49% 59.45% 2.0150 65.06% 56.98% VOLUME 4, 2016 TABLE 12. Inter-dataset evaluation scores of self-supervised models fine-tuned on FakeAVCeleb [45] dataset and evaluated on the remaining three datasets. Training Dataset: FakeAVCeleb Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 1.1051 60.65% 58.50% 1.2554 58.82% 55.75% CelebDF-V2 Dino 2.7798 64.07% 60.65% 2.7178 63.39% 60.50% CLIP 1.8561 50.22% 50.50% 3.2978 52.29% 50.15% Supervised 2.2969 62.99% 53.48% 2.2189 64.15% 55.08% FaceForensics++ Dino 7.5589 61.59% 57.73% 8.4257 62.74% 52.05% CLIP 1.1427 58.52% 55.30% 1.5146 60.47% 56.83% Supervised 1.8896 65.60% 56.05% 1.8893 68.37% 57.70% DFDC Dino 8.2808 59.25% 54.10% 9.0520 62.46% 52.40% CLIP 1.1260 64.49% 59.45% 2.0150 65.06% 56.98% 2 VOLUME 4 2016 22 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 0 TABLE 13. Inter-dataset evaluation scores of models trained on CelebDF-V2 [40] dataset and evaluated on the remaining three datasets. TABLE 13. Inter-dataset evaluation scores of models trained on CelebDF-V2 [40] dataset and evaluated on the remaining three datasets. VOLUME 4, 2016 Training Dataset: CelebDF-V2 Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 4.7313 65.82% 51.68% 5.1136 67.77% 51.78% FakeAVCeleb Res2Net-101 5.7429 69.08% 52.30% 4.3332 71.01% 52.83% EfficientNet-B7 7.4940 63.86% 52.05% 9.6846 65.93% 51.45% ViT 5.0347 69.00% 53.08% 9.6735 61.89% 52.18% Swin-Base 6.0084 67.63% 52.20% 4.8922 68.28% 52.70% MViT-V2-Base 5.1980 72.43% 52.75% 5.3953 61.24% 51.55% ResNet-3D 6.0756 62.79% 50.50% 4.9703 61.58% 50.50% TimeSformer 4.8465 69.73% 53.00% 5.8829 68.77% 54.00% Xception 4.2473 63.26% 53.53% 5.6357 63.68% 53.58% FaceForensics++ Res2Net-101 6.3947 64.79% 53.33% 6.9000 63.59% 52.90% EfficientNet-B7 6.3164 65.07% 54.80% 8.9065 66.31% 53.98% ViT 6.1010 68.14% 53.53% 9.9676 65.50% 53.50% Swin-Base 6.0278 70.13% 54.23% 5.5408 68.45% 54.30% MViT-V2-Base 5.2175 70.01% 53.15% 4.6160 67.88% 54.08% ResNet-3D 7.1877 60.00% 52.14% 5.6544 66.01% 54.29% TimeSformer 4.8228 68.84% 57.50% 5.0219 67.55% 56.43% Xception 2.8532 66.11% 51.55% 3.8835 67.74% 51.30% DFDC Res2Net-101 4.1424 63.83% 50.28% 5.8342 64.01% 50.90% EfficientNet-B7 4.0438 72.05% 54.00% 8.3092 69.17% 54.00% ViT 5.3405 68.41% 51.53% 7.3534 71.69% 50.95% Swin-Base 4.7659 72.42% 52.20% 4.0886 74.95% 53.80% MViT-V2-Base 4.2014 69.69% 53.15% 3.9144 72.48% 53.65% ResNet-3D 7.2461 61.91% 51.25% 4.5265 65.97% 50.50% TimeSformer 4.0195 68.56% 53.00% 3.9124 71.98% 54.25% TABLE 14. Inter-dataset evaluation scores of self-supervised models fine-tuned on CelebDF-V2 [40] dataset and evaluated on the remaining three datasets. Training Dataset: CelebDF-V2 Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 1.8680 65.64% 53.70% 1.7462 66.18% 56.58% FakeAVCeleb Dino 8.5606 68.16% 57.35% 10.6620 66.48% 53.10% CLIP 2.0280 61.23% 52.60% 2.2300 60.48% 53.28% Supervised 1.9176 63.57% 54.63% 2.0464 64.77% 55.63% FaceForensics++ Dino 9.3992 64.71% 56.60% 9.2731 66.18% 56.25% CLIP 1.5848 60.23% 53.58% 1.5040 66.12% 58.88% Supervised 3.0170 53.42% 49.65% 3.3809 51.49% 50.20% DFDC Dino 13.8247 52.52% 50.60% 10.2818 54.76% 52.60% CLIP 2.2078 59.35% 50.80% 2.6224 58.78% 51.02% VOLUME 4, 2016 23 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ TABLE 14. Inter-dataset evaluation scores of self-supervised models fine-tuned on CelebDF-V2 [40] dataset and evaluated on the remaining three datasets. VOLUME 4, 2016 Training Dataset: CelebDF-V2 Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 1.8680 65.64% 53.70% 1.7462 66.18% 56.58% FakeAVCeleb Dino 8.5606 68.16% 57.35% 10.6620 66.48% 53.10% CLIP 2.0280 61.23% 52.60% 2.2300 60.48% 53.28% Supervised 1.9176 63.57% 54.63% 2.0464 64.77% 55.63% FaceForensics++ Dino 9.3992 64.71% 56.60% 9.2731 66.18% 56.25% CLIP 1.5848 60.23% 53.58% 1.5040 66.12% 58.88% Supervised 3.0170 53.42% 49.65% 3.3809 51.49% 50.20% DFDC Dino 13.8247 52.52% 50.60% 10.2818 54.76% 52.60% CLIP 2.2078 59.35% 50.80% 2.6224 58.78% 51.02% VOLUME 4, 2016 23 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ TABLE 14. Inter-dataset evaluation scores of self-supervised models fine-tuned on CelebDF-V2 [40] dataset and evaluated o ion scores of self-supervised models fine-tuned on CelebDF-V2 [40] dataset and evaluated on the remaining three datasets. 23 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 TABLE 15. Inter-dataset evaluation scores of models trained on FaceForensics++ [33] dataset and evaluated on the remaining three datasets. TABLE 15. Inter-dataset evaluation scores of models trained on FaceForensics++ [33] dataset and evaluated on the remaining three datasets. TABLE 15. VOLUME 4, 2016 Inter-dataset evaluation scores of models trained on FaceForensics++ [33] dataset and evaluated on the remaining Training Dataset: FaceForensics++ Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 0.8691 79.62% 65.88% 0.7795 76.14% 66.93% FakeAVCeleb Res2Net-101 0.7693 83.01% 71.28% 0.6527 85.48% 76.83% EfficientNet-B7 0.5782 89.59% 77.05% 0.7375 77.88% 70.08% ViT 0.6648 83.05% 70.65% 0.7419 76.40% 69.23% Swin-Base 0.5880 87.72% 72.95% 0.6373 89.10% 71.15% MViT-V2-Base 0.3654 92.96% 84.65% 0.4047 90.25% 81.90% ResNet-3D 0.7903 83.55% 68.00% 1.1338 73.34% 62.50% TimeSformer 0.9135 79.33% 75.00% 0.7900 76.65% 70.50% Xception 1.0426 65.92% 61.60% 1.2566 62.39% 58.65% CelebDF-V2 Res2Net-101 1.0751 67.85% 62.40% 1.4218 65.46% 59.80% EfficientNet-B7 0.7759 78.46% 69.95% 1.0103 67.24% 61.25% ViT 0.5915 82.44% 74.10% 0.8504 75.11% 65.40% Swin-Base 0.7136 74.58% 67.05% 0.7879 70.94% 63.75% MViT-V2-Base 0.9791 76.66% 65.35% 0.7912 68.69% 62.70% ResNet-3D 1.1992 66.12% 65.00% 1.5866 59.44% 55.00% TimeSformer 1.1745 73.68% 63.00% 0.7446 80.40% 71.00% Xception 1.2988 64.22% 54.70% 1.3424 64.81% 53.28% DFDC Res2Net-101 1.2052 69.51% 58.60% 1.6336 69.89% 62.70% EfficientNet-B7 1.2835 70.49% 59.13% 1.2726 64.29% 59.23% ViT 1.1135 69.87% 60.20% 0.8981 68.20% 62.98% Swin-Base 1.2534 71.53% 58.63% 1.1194 74.04% 61.48% MViT-V2-Base 1.1775 69.63% 62.15% 0.9917 68.61% 58.40% ResNet-3D 1.2023 73.75% 68.00% 1.2788 67.04% 61.00% TimeSformer 1.1116 73.77% 68.00% 1.0129 74.04% 63.50% TABLE 16. Inter-dataset evaluation scores of self-supervised models fine-tuned on FaceForensics++ [33] dataset and evalua scores of self-supervised models fine-tuned on FaceForensics++ [33] dataset and evaluated on the remaining three datasets. nter-dataset evaluation scores of self-supervised models fine-tuned on FaceForensics++ [33] dataset and evaluated on the remaining three datasets. Training Dataset: FaceForensics++ Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 0.7400 69.21% 64.70% 0.7968 69.54% 64.35% FakeAVCeleb Dino 3.4485 62.66% 60.32% 3.6146 64.73% 61.45% CLIP 0.8256 65.37% 59.63% 0.9682 66.54% 59.35% Supervised 0.6256 74.47% 66.70% 0.6993 71.45% 66.00% CelebDF-V2 Dino 3.0070 68.39% 59.50% 3.4239 65.94% 60.10% CLIP 0.6627 68.77% 61.45% 0.6678 73.97% 65.45% Supervised 1.1463 61.54% 58.38% 1.1353 66.51% 61.90% DFDC Dino 7.5253 58.14% 54.35% 5.9902 61.83% 57.73% CLIP 0.6647 71.49% 66.60% 0.8275 67.59% 62.85% VOLUME 4, 2016 Thi k i li d d C ti C Att ib ti 4 0 Li F i f ti htt // ti /li /b /4 0/ TABLE 16. Inter-dataset evaluation scores of self-supervised models fine-tuned on FaceForensics++ [33] dataset and evaluated on the remaining three datasets. VOLUME 4, 2016 Training Dataset: FaceForensics++ Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 0.7400 69.21% 64.70% 0.7968 69.54% 64.35% FakeAVCeleb Dino 3.4485 62.66% 60.32% 3.6146 64.73% 61.45% CLIP 0.8256 65.37% 59.63% 0.9682 66.54% 59.35% Supervised 0.6256 74.47% 66.70% 0.6993 71.45% 66.00% CelebDF-V2 Dino 3.0070 68.39% 59.50% 3.4239 65.94% 60.10% CLIP 0.6627 68.77% 61.45% 0.6678 73.97% 65.45% Supervised 1.1463 61.54% 58.38% 1.1353 66.51% 61.90% DFDC Dino 7.5253 58.14% 54.35% 5.9902 61.83% 57.73% CLIP 0.6647 71.49% 66.60% 0.8275 67.59% 62.85% 24 VOLUME 4, 2016 ABLE 16. Inter-dataset evaluation scores of self-supervised models fine-tuned on FaceForensics++ [33] dataset and evaluated on the remaining three datasets. Training Dataset: FaceForensics++ Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 0.7400 69.21% 64.70% 0.7968 69.54% 64.35% FakeAVCeleb Dino 3.4485 62.66% 60.32% 3.6146 64.73% 61.45% CLIP 0.8256 65.37% 59.63% 0.9682 66.54% 59.35% Supervised 0.6256 74.47% 66.70% 0.6993 71.45% 66.00% CelebDF-V2 Dino 3.0070 68.39% 59.50% 3.4239 65.94% 60.10% CLIP 0.6627 68.77% 61.45% 0.6678 73.97% 65.45% Supervised 1.1463 61.54% 58.38% 1.1353 66.51% 61.90% DFDC Dino 7.5253 58.14% 54.35% 5.9902 61.83% 57.73% CLIP 0.6647 71.49% 66.60% 0.8275 67.59% 62.85% 4 VOLUME 4, 2016 24 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 0 BLE 17. Inter-dataset evaluation scores of models trained on DFDC [43] dataset and evaluated on the remaining three datasets TABLE 17. Inter-dataset evaluation scores of models trained on DFDC [43] dataset and evaluated on the remaining three datasets. TABLE 17. Inter-dataset evaluation scores of models trained on DFDC [43] dataset and evaluated on the remaining three datasets. VOLUME 4, 2016 Training Dataset: DFDC Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Xception 1.4046 58.38% 55.25% 1.8346 60.31% 53.63% FakeAVCeleb Res2Net-101 2.0891 59.23% 56.33% 1.6953 59.77% 55.43% EfficientNet-B7 1.0800 65.31% 61.63% 1.0920 71.87% 65.40% ViT 1.2515 59.31% 56.00% 1.1361 60.12% 57.43% Swin-Base 1.2053 67.81% 62.90% 1.2668 63.48% 60.25% MViT-V2-Base 1.2121 63.46% 60.05% 1.2139 65.75% 61.30% ResNet-3D 1.1114 63.19% 54.50% 1.2748 62.02% 56.50% TimeSformer 1.1582 65.34% 62.00% 1.6968 67.80% 61.00% Xception 1.1784 67.90% 61.25% 1.7465 64.95% 58.20% CelebDF-V2 Res2Net-101 1.2293 83.01% 74.95% 1.1859 83.57% 72.35% EfficientNet-B7 0.8278 79.82% 70.15% 1.2972 74.27% 68.45% ViT 0.7301 85.62% 76.45% 0.9351 73.81% 67.25% Swin-Base 0.8411 84.36% 76.60% 1.1246 80.34% 73.20% MViT-V2-Base 0.8548 87.83% 71.55% 0.7711 84.75% 76.75% ResNet-3D 0.7638 79.60% 72.00% 0.7806 77.88% 72.00% TimeSformer 1.0558 76.48% 71.00% 1.3635 79.60% 74.00% Xception 1.3048 64.59% 60.95% 1.4530 67.29% 62.70% FaceForensics++ Res2Net-101 2.7490 61.15% 56.48% 2.3375 65.15% 60.78% EfficientNet-B7 1.2085 68.82% 64.08% 1.4401 70.71% 65.83% ViT 1.6779 66.43% 59.13% 1.6781 60.19% 57.28% Swin-Base 1.6806 68.32% 63.65% 1.7493 64.66% 60.20% MViT-V2-Base 1.6317 65.82% 61.55% 1.6911 67.54% 62.88% ResNet-3D 1.5308 57.27% 57.86% 1.3507 58.91% 57.50% TimeSformer 1.2122 70.17% 66.79% 1.9148 67.90% 60.00% TABLE 18. Inter-dataset evaluation scores of self-supervised models fine-tuned on DFDC [43] dataset and evaluated on the remaining three datasets. Training Dataset: DFDC Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 1.2860 57.72% 52.25% 0.9593 60.67% 57.58% FakeAVCeleb Dino 3.5511 60.45% 56.70% 3.8135 61.42% 59.20% CLIP 1.1978 55.09% 52.33% 1.1680 55.04% 54.80% Supervised 0.8549 72.57% 65.20% 0.8149 69.37% 65.95% CelebDF-V2 Dino 2.8856 69.28% 63.50% 3.4654 62.35% 57.50% CLIP 0.7905 66.77% 60.65% 0.6538 76.81% 71.40% Supervised 0.9295 64.05% 59.15% 0.8214 67.33% 62.10% FaceForensics++ Dino 3.3117 63.87% 59.28% 2.9844 66.65% 61.95% CLIP 0.7500 66.29% 61.90% 0.8216 68.50% 63.08% VOLUME 4, 2016 25 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ TABLE 18. Inter-dataset evaluation scores of self-supervised models fine-tuned on DFDC [43] dataset and evaluated on the aluation scores of self-supervised models fine-tuned on DFDC [43] dataset and evaluated on the remaining three datasets. TABLE 18. Inter-dataset evaluation scores of self-supervised models fine-tuned on DFDC [43] dataset and evaluated on the remaining three datasets. VOLUME 4, 2016 Training Dataset: DFDC Evaluation Dataset Model With Augs No Augs LogLoss AUC ACC LogLoss AUC ACC Supervised 1.2860 57.72% 52.25% 0.9593 60.67% 57.58% FakeAVCeleb Dino 3.5511 60.45% 56.70% 3.8135 61.42% 59.20% CLIP 1.1978 55.09% 52.33% 1.1680 55.04% 54.80% Supervised 0.8549 72.57% 65.20% 0.8149 69.37% 65.95% CelebDF-V2 Dino 2.8856 69.28% 63.50% 3.4654 62.35% 57.50% CLIP 0.7905 66.77% 60.65% 0.6538 76.81% 71.40% Supervised 0.9295 64.05% 59.15% 0.8214 67.33% 62.10% FaceForensics++ Dino 3.3117 63.87% 59.28% 2.9844 66.65% 61.95% CLIP 0.7500 66.29% 61.90% 0.8216 68.50% 63.08% VOLUME 4, 2016 2 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 25 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 FIGURE 8. ROC curves of each of the model when evaluated on each of the 4 different participating datasets in an intra-dataset evaluation setting. FIGURE 8. ROC curves of each of the model when evaluated on each of the 4 different participating datasets in an intra-dataset evaluation setting. FIGURE 9. DET curves of each of the model when evaluated on each of the 4 different participating datasets in an intra-dataset evaluation setting. FIGURE 9. DET curves of each of the model when evaluated on each of the 4 different participating datasets in an intra-da FIGURE 9. DET curves of each of the model when evaluated on each of the 4 different participating datasets in an intra-dataset evaluation setting. 26 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. VOLUME 4, 2016 For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 0 FIGURE 12. This bar chart highlights the efficiency of the supervised models in terms of inference time on both GPU and CPU devices. It reveals that CNN models outperform transformer models, taking nearly half the time for processing a single image frame on CPU. On GPU, the figure illustrates that all models achieve inference in less than 45 milliseconds at most. ViT and Xception models are the fastest among other models on GPU inference speeds, taking less than 10 milliseconds to process a single frame. FIGURE 12. This bar chart highlights the efficiency of the supervised models in terms of inference time on both GPU and CPU devices. It reveals that CNN models outperform transformer models, taking nearly half the time for processing a single image frame on CPU. On GPU, the figure illustrates that all models achieve inference in less than 45 milliseconds at most. ViT and Xception models are the fastest among other models on GPU inference speeds, taking less than 10 milliseconds to process a single frame. FIGURE 13. This figure illustrates the performance of supervised image models, showcasing both total parameters and the number of floating-point operations per second (GFLOPs). The results align with the preceding bar chart, emphasising the superior efficiency of CNN models, as compared to transformer models. It’s important to note that video models, although not depicted here, exhibit a significantly higher number of floating-point operations per second, acting as outliers in the figure and slightly affecting its visual coherence. VOLUME 4, 2016 This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 epted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication Citation information: DOI 10 1109/ACCESS 2023 3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 0 FIGURE 10. ROC curves of self-supervised models trained and evaluated on each dataset using the intra-dataset evaluation scheme. VOLUME 4, 2016 RE 10. ROC curves of self-supervised models trained and evaluated on each dataset using the intra-dataset evaluation scheme 27 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2023.3348450 FIGURE 11. DET curves of self-supervised models trained and evaluated on each dataset using the intra-dataset evaluation scheme. VOLUME 4, 20 FIGURE 11. DET curves of self-supervised models trained and evaluated on each dataset using the intra-dataset evaluation scheme. VOLUME 4, 2016 FIGURE 11. DET curves of self-supervised models trained and evaluated on each dataset using the intra-dataset evaluation scheme. VOLUME 4, 2016 FIGURE 11. DET curves of self-supervised models trained and evaluated on each dataset using the intra-dataset evaluation scheme. VOLUME 4, 2016 RE 11. DET curves of self-supervised models trained and evaluated on each dataset using the intra-dataset evaluation scheme 28 28 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. VOLUME 4, 2016 This disparity arises from the nature of video models processing more data at once, specifically 8 image frames, compared to image models that handle only one image at a time. FIGURE 13. This figure illustrates the performance of supervised image models, showcasing both total parameters and the number of floating-point operations per second (GFLOPs). The results align with the preceding bar chart, emphasising the superior efficiency of CNN models, as compared to transformer models. It’s important to note that video models, although not depicted here, exhibit a significantly higher number of floating-point operations per second, acting as outliers in the figure and slightly affecting its visual coherence. This disparity arises from the nature of video models processing more data at once, specifically 8 image frames, compared to image models that handle only one image at a time. 29 VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/
https://openalex.org/W4392454312	https://www.researchsquare.com/article/rs-4000622/latest.pdf	English	null	Recognition of High-Grade Invasiveness and Achievement of Gross Total Resection via an Endoscopic Endonasal Transsphenoidal Approach Promise Favorable Outcomes in Growth Hormone Pituitary Adenomas: 16 Years of Experience in a Single Institute	Research Square (Research Square)	2,024	cc-by	5,740	Recognition of High-Grade Invasiveness and Achievement of Gross Total Resection via an Endoscopic Endonasal Transsphenoidal Approach Promise Favorable Outcomes in Growth Hormone Pituitary Adenomas: 16 Years of Experience in a Single Institute Ting-Wei Chang Chang Gung Memorial Hospital at Linkou, Chang Gung University Chun-Chia Tseng Chang Gung Memorial Hospital at Linkou, Chang Gung University Yu-Chi Wang Chang Gung Memorial Hospital at Linkou, Chang Gung University Yin-Cheng Huang Chang Gung Memorial Hospital at Linkou, Chang Gung University Peng-Wei Hsu Chang Gung Memorial Hospital at Linkou, Chang Gung University Chi-Cheng Chuang Chang Gung Memorial Hospital at Linkou, Chang Gung University Cheng-Chi Lee Chang Gung Memorial Hospital at Linkou, Chang Gung University Ting-Wei Chang Chang Gung Memorial Hospital at Linkou, Chang Gung University Chun-Chia Tseng Chang Gung Memorial Hospital at Linkou, Chang Gung University Yu-Chi Wang Chang Gung Memorial Hospital at Linkou, Chang Gung University Yin-Cheng Huang Chang Gung Memorial Hospital at Linkou, Chang Gung University Peng-Wei Hsu Chang Gung Memorial Hospital at Linkou, Chang Gung University Chi-Cheng Chuang Chang Gung Memorial Hospital at Linkou, Chang Gung University Cheng-Chi Lee Chang Gung Memorial Hospital at Linkou, Chang Gung University Research Article Posted Date: March 5th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-4000622/v1 Page 1/15 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Page 2/15 Abstract Background: Growth hormone (GH)-secreting pituitary tumors produce systemic comorbidities, thus achieving gross total resection (GTR) and biochemical remission are imperative. The aim of this study was to identify predictors affecting resection status and biochemical remission. Methods: We retrospectively reviewed 54 GH adenoma patients receiving endoscopic endonasal transsphenoidal approach (EETSA). Medical records and magnetic resonance imaging were reviewed for tumor size, volume, resection status, invasion status, and Knosp and Hardy-Wilson grades. We also classified invasion status into high- and low-grade groups. Biochemical remission was defined as an insulin-like growth factor 1 value within sex- and age-adjusted reference or a random GH level < 1.0 ng/mL. Results: The degrees of horizontal and vertical invasion based on preoperative Knosp and Hardy-Wilson grade were highly associated with intraoperative resection status (p=0.015, 0.014 and 0.017 respectively), but micro- or macroadenoma, tumor size and volume were not. We also found more significant differences between resection status and higher-grade invasion (p=0.006, 0.006 and 0.001, respectively). Knosp, Hardy-Wilson grades and resection status were significantly associated with biochemical remission (p=0.0007, 0.0428, 0.0006 and 0.0012, respectively). Although there was no difference between micro- or macroadenoma, tumor size and volume were statistically significant associated with outcomes (p=0.024, 0.0032, respectively). Similarly, more significant differences between biochemical remission and higher-grade invasion were found (p=0.00005, 0.009 and 0.00001, respectively). Conclusions: EETSA remains the best treatment option for GH adenomas. Biochemical remission was highly associated with invasion status and the possibility of achieving GTR. Earlier diagnosis and more aggressive resection are key to favorable outcomes. Materials and Methods We retrospectively reviewed the medical records of patients with GH-secreting pituitary tumors at Linkou Chang Gung Memorial Hospital from October 2004 to December 2020. Data collection was approved by the Institutional Review Board (IRB) of Chang Gung Memorial Hospital. During this period, 54 patients received EETSA for pituitary tumor removal. Of these patients, 50 had sufficient medical records including pre- and postoperative magnetic resonance imaging (MRI), and data on GH and IGF-1 levels. Two patients did not have postoperative MRI scans, and two patients were lost to follow-up from our outpatient department. Pre- and postoperative MRI scans were reviewed by a neurosurgeon and neuroradiologist for general items including tumor size, tumor volume and resection status, and some specific characteristics including cavernous sinus invasion, parasellar invasion, suprasellar invasion, infrasellar invasion, Knosp classification, and Hardy-Wilson classification. We used Knosp classification for evaluating the degree of cavernous sinus invasion, and Hardy-Wilson classification for the degree of infrasellar, suprasellar and parasellar invasion [18, 19]. In addition, we also divided the tumors into two groups on the basis of the level of cavernous sinus invasion. A less invasive tumor was defined as Knosp grade < 2, and a more invasive tumor as Knosp grade 3 and 4 [12]. Tumors classified below Hardy-Wilson grade 2 were defined as less infrasellar or sphenoidal sinus invasion, and those with Hardy-Wilson grade 3 or higher were defined as more infrasellar or sphenoidal sinus invasion [6]. The level of suprasellar invasion was based on Hardy-Wilson grade 0 ~ C. Tumors classified below Hardy-Wilson grade C were considered less parasellar invasion, and grade D and E tumors were considered to have more parasellar invasion ability [6]. Tumor size was defined by measuring the maximal horizontal length of the tumor in MRI coronal view. Tumors were classified as microadenoma if the size was < 1 cm, and macroadenoma if the size was ≥ 1 cm. Tumor volume was calculated as “ABC/2”, where A represents the longest tumor length, B represents the longest perpendicular line to A, and C represents the tumor height. Biochemical remission was determined according to the current consensus guidelines as an IGF-1 value within sex- and age- adjusted reference or random GH level < 1.0 ng/mL [20, 21]. We used MedCalc version 19.7 (MedCalc Software Ltd., Ostend, Belgium) for data analysis. Introduction Acromegaly is a condition caused by the overproduction of growth hormone (GH) due to pituitary tumors, and the subsequent production of insulin-like growth factor 1 (IGF-1). The severe systemic comorbidities of acromegaly including cardiovascular disease, diabetes mellitus, and musculoskeletal disease make controlling acromegaly necessary. The extent of associated complications and mortality risk are related to the length of exposure to excess GH and IGF-1, and thus early diagnosis and treatment are imperative [1]. Achieving biochemical remission remains the most important goal when treating patients with GH- secreting pituitary tumors. Surgery is the first-line treatment modality, and an endoscopic endonasal transsphenoidal approach (EETSA) is the most appropriate [2, 3]. An important factor to predict the outcome after surgery is postoperative remission, and several predictors of postoperative biochemical remission have been identified, including preoperative human growth hormone and IGF-1 levels, tumor size, tumor volume, Hardy-Wilson classification, Knosp classification, sphenoid sinus invasion and prior surgery [3–9]. Most of these factors are related to the difficulty in achieving gross total resection (GTR) Page 3/15 Page 3/15 and biochemical remission [2, 4, 10–17]. However, very few studies have comprehensively discussed the factors influencing outcomes in patients with GH adenomas. Therefore, we retrospectively reviewed our 16-year experience in treating patients with GH adenomas who received EETSA as primary treatment. The aim of this study was to identify the pre- and postoperative factors affecting GTR and biochemical remission in our patients, and to compare our results with other studies. Materials and Methods Fisher’s exact test was used for comparing categorical variables, the chi-square test was used for comparing ordinal variables, and the Mann-Whitney test was used for comparing continuous variables. A p value ≤ 0.05 was considered to indicate a statistically significant difference. Discussion Acromegaly is a disease which manifests as progressive bone and cartilage growth as well as systemic complications including cardiovascular, metabolic, and respiratory issues. The first-line therapy for acromegaly is surgical resection, and surgical resection alone leads to disease control in approximately 90% of microadenoma cases and 40–60% of macroadenoma cases [3]. Postoperative biochemical remission is highly related to the preoperative condition and intraoperative resection status [3-5, 7, 22, 23]. The importance of biochemical remission cannot be overemphasized, because it ameliorates the systemic effects of elevated GH, IGF-1, and the resultant mortality/morbidity [24]. Results Page 4/15 Page 4/15 As shown in Table 1, the overall rate of GTR in the patients receiving surgery was As shown in Table 1, the overall rate of GTR in the patients receiving surgery was 78% (39 of 50 patients), and 27 patients (54%) achieved biochemical remission. We then compared the patients according to the status of resection (GTR or subtotal resection), and found no significant differences in tumor characteristics including micro- or macroadenoma, tumor size and tumor volume between the two groups (p > 0.99, p = 0.09 and 0.06 respectively) (Table 2). In addition, the possibility of GTR decreased with increasing Knosp grade (p = 0.015), and Hardy-Wilson grade was also significantly associated with GTR status (p = 0.014 and 0.017 respectively). We then further divided the patients into two groups according to the degree of invasiveness by Hardy-Wilson and Knosp grade, and found that the GTR rate for both classification systems reached statistical significance (p = 0.006, 0.006 and 0.001, respectively) (Table 2-1). In analysis of the patients with and without biochemical remission, the patients who achieved biochemical remission had a smaller tumor size (p = 0.024) and volume (p = 0.0032) (Table 3). We also found that the rate of achieving biochemical remission decreased with higher invasiveness according to Knosp and Hardy-Wilson grade (p = 0.0007, 0.0428 and 0.0006, respectively). However, there were no significant differences between the patients with or without supra-, intra- or infrasellar invasion (p = 0.57, 0.08 and 0.37, respectively). Biochemical remission was also related to resection status (p = 0.0012), and achieving GTR was also highly related to invasion status. The rate of biochemical remission also reached a statistical difference in these two classification systems. We further divided the patients into two groups by the degree of invasiveness in the different grading systems, and found that the rate of achieving biochemical remission was significantly associated with both grading systems (p = 0.00005, 0.009 and 0.00001, respectively) (Table 3-1). Factors influencing intraoperative resection status It is known that cells secreting GH reside in the inferior and lateral aspects of the pituitary gland. Consequently, tumors tend to grow in an inferior and lateral direction. Our results are similar to previous studies in which GTR was associated with achieving biochemical remission [4, 11, 12]. Therefore, achieving GTR is an important predictive factor for disease control. On the other hand, the most crucial determinant in resection is horizontal and vertical invasion. In this study, intraoperative resection status was highly associated with preoperative Knosp and Hardy-Wilson grades (p = 0.015, 0.014 and 0.017, respectively) (Table 2). These results show that the surgical difficulty increased when the tumor extended beyond the sellar region in a horizontal or vertical direction. However, our data showed that tumor size and volume were not significantly associated with achieving GTR (p = 0.09 and 0.06, respectively), in contrast to the results reported by Zeng et al [12]. The main reason for this difference may be due to the mean size of macroadenoma in our study of around 1.76 cm, which may indicate a lack of higher-grade invasiveness, and subsequently that the possibility of achieving GTR was similar to the microadenoma patients. This finding also strengthens the importance of making an earlier diagnosis in GH adenoma patients, as the smaller tumor size would increase the possibility of achieving GTR and subsequent biochemical remission. Furthermore, the p-value for tumor volume (p = 0.06) in the view of GTR was closer to 0.05 than tumor size (p = 0.09), which may suggest that more precise volumetric measurement is important when defining the mass effect of a tumor, especially in irregularly shaped or residual tumors [26, 27]. We also found stronger associations between higher Knosp and Hardy grades and a lower probability of achieving GTR (p = 0.006, 0.006, and 0.001, respectively) (Table 2-1). These results further highlight the importance of carefully interpretating preoperative MRI and the degree of peripheral invasion, especially in those with higher invasion classification. Classification of parasellar invasion Knosp grade and Hardy-Wilson classification based on preoperative MRI are used to determine cavernous sinus and parasellar invasion [8, 9, 18, 19, 25]. Detailed descriptions of Knosp and Hardy grades are shown in Figure 1 and 2. A higher grade of tumor invasion is associated with greater difficulty in achieving GTR, particularly pituitary functioning tumors in which biochemical remission is even more critical than non-functioning tumors. Page 5/15 Page 5/15 Association of tumor size and volume with resection status and biochemical remission Many studies have reported an association between tumor size and biochemical remission rate, and some studies have mentioned that patients who achieve remission have a smaller tumor size [3, 6, 11]. In addition, some studies have reported that patients with microadenoma have a higher remission rate [6, 12]. In our study, the patients with microadenoma (< 1 cm) did not have a significantly higher remission rate compared to those with macroadenoma. However, the patients who achieved remission did have a smaller tumor size (< 1.3 cm, p = 0.024), which is similar to the study by Samuel et al [3]. A possible explanation for this finding is that our patients had smaller macroadenomas (mean size 1.76±0.67 cm). As a result, we suggest that the crucial cut-off value of tumor size to achieve GTR should be ³ 1.3 cm, or the average width of the sellar region. Hence, the 1 cm definition for the classification of micro- and macroadenomas may not be necessary, especially in GH-secreting or other functioning tumors necessitating surgical resection. Furthermore, in tumors that did not invade the cavernous sinus or extend into the supra- or infrasellar region, the tumor size and volume were determining predictors; in contrast, in tumors that extended beyond the sellar region, the determining predictor of biochemical remission was the presence or absence of higher-grade invasion. Factors influencing biochemical remission Unlike intraoperative resection status, we found that tumor size and volume were positively correlated with postoperative biochemical remission (p = 0.024 and 0.0032, respectively) (Table 3), which is similar to the results of other studies [4, 5, 11, 22]. In addition, preoperative Knosp and Hardy-Wilson classification (p = 0.007, 0.0428, and 0.0006, respectively) and the resultant intraoperative resection status (p = 0.0012) were also significantly associated with postoperative biochemical remission as mentioned above. Interestingly, suprasellar, pure intrasellar and infrasellar invasion were not risk factors for biochemical remission (p = 0.57, 0.08 and 0.37, respectively). This result further supports that the grading of classification is far more important than the presence or absence of surrounding invasion status. The abovementioned suprasellar, pure intrasellar and infrasellar factors describe vertical tumor invasion status, which is not thought to be as critical as horizontal invasion. Although intrasellar tumors were easier to resect, the surgical techniques were similar to those with lower grade Knosp grade. In addition, for patients with infrasellar invasion toward the sphenoid floor or clivus, complete resection of tumors infiltrating into bone was challenging. In addition, GTR may be feasible for lower grade suprasellar invasion, but it remains challenging for higher grade suprasellar invasion, as demonstrated in Table 3-1. Furthermore, although tumor size and volume were predictive factors of biochemical remission, Page 6/15 Page 6/15 the lower preoperative GH and IGF-1 levels were not correlated with postoperative biochemical remission (p = 0.19 and 0.12, respectively). Tumors with a smaller size and volume are thought to harbor fewer secreting tumor cells; however, not every tumor cell produces an equivalent amount of GH and the resultant systemic effects. We found stronger associations between higher Knosp and Hardy-Wilson grades and a lower probability of postoperative biochemical remission (p = 0.00005, 0.009, and 0.00001, respectively) (Table 3-1). These results further support the importance of carefully interpretation ofing preoperative MRI and the degree of peripheral invasion, especially in those with higher invasion classification. Regardless of postoperative biochemical remission or intraoperative resection status, we found that the most pivotal predictive factor was preoperative grading of invasion status, especially in those with a higher grade. Careful interpretation of preoperative MRI and removing as much of the tumor as possible are the key factors associated with favorable outcomes. Association of tumor invasion grading with resection status and biochemical remission The Hardy-Wilson and Knosp grading systems are widely used to classify the level of tumor invasion, and they are also key indicators of whether GTR can be achieved. A higher level of tumor invasion is considered to impede complete surgical resection, and therefore biochemical remission. Although some studies have reported that a higher grade was associated with a lower rate of biochemical remission, the results have been inconsistent [2, 3, 7, 22]. Whether Hardy-Wilson grade can predict biochemical remission remains controversial. Mohammad et al reported that biochemical remission was independent of Hardy-Wilson grade [4], whereas Campbell et al and Yildirim et al found that remission was related to Hardy-Wilson grade [6, 22]. In addition, Shin et al and Yildirim et al found that Hardy-Wilson grade was a predictor of biochemical remission [3, 6]. In our study, we comprehensively reviewed all classifications, and found that the probability of achieving GTR and rate of biochemical remission decreased with higher Page 7/15 Page 7/15 Hardy-Wilson grade and. In addition, we also found that the rates of achieving GTR and biochemical remission were related to cavernous sinus invasion. Furthermore, when considering the level of suprasellar extension only, there was no statistical significance among Hardy-Wilson grade 0~C. This may imply that with mature endoscopic endonasal surgery, GTR can also be achieved even when the tumor extends into the suprasellar space. In addition, although we used an EETSA, which is via the infrasellar space, sphenoid sinus or clivus invasion may imply a higher degree of tumor invasion, and consequently greater difficulty in curing the disease [6]. However, cavernous sinus invasion is still associated with the greatest difficulty in achieving complete tumor removal and ideal hormone control because of the vital structures inside. Ethics Approval Data collection was approved by the Institutional Review Board (IRB) of Chang Gung Memorial Hospital. Data Availability Data collection was approved by the Institutional Review Board (IRB) of Chang Gung Memorial Hospital. Conclusions Pituitary GH adenomas exert systemic effects, and EETSA remains the best treatment for biochemical remission. GTR was associated with biochemical remission and was influenced by invasion status, especially in those with higher grade invasion. Biochemical remission was highly associated with invasion status and resection status rather than tumor size and volume. Earlier diagnosis and more aggressive resection regardless of the 1 cm definition of micro- and macroadenomas are the key factors associated with favorable outcomes. Funding This research was funded by a grant from Chang Gung Memorial Hospital (CMRPG1K0131, Cheng Chi Lee), and the funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no relevant financial or non-financial interests to disclose. The authors have no relevant financial or non-financial interests to disclose. Data Availability The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. Author Contribution Author Contribution Page 8/15 T.W.C. and C.C.T. wrote the main manuscript text, Y.C.H., P.W.H., C.C.C. and C.C.L. collected the data, T.W.C. and Y.C.W. did the analysis and prepared the tables and figures, C.C.C. and C.C.L. supervised the manuscript. T.W.C. and C.C.T. wrote the main manuscript text, Y.C.H., P.W.H., C.C.C. and C.C.L. collected the data, T.W.C. and Y.C.W. did the analysis and prepared the tables and figures, C.C.C. and C.C.L. supervised the manuscript. T.W.C. and C.C.T. wrote the main manuscript text, Y.C.H., P.W.H., C.C.C. and C.C.L. collected the data, T.W.C. and Y.C.W. did the analysis and prepared the tables and figures, C.C.C. and C.C.L. supervised the manuscript. References 1. Abreu A et al (2016) Challenges in the diagnosis and management of acromegaly: a focus on comorbidities. Pituitary 19(4):448–457 2. Araujo-Castro M et al (2021) Predictive model of surgical remission in acromegaly: age, presurgical GH levels and Knosp grade as the best predictors of surgical remission. J Endocrinol Invest 44(1):183–193 3. Shin SS et al (2013) Endoscopic endonasal approach for growth hormone secreting pituitary adenomas: outcomes in 53 patients using 2010 consensus criteria for remission. Pituitary 16(4):435–444 4. Taghvaei M et al (2018) Endoscopic Endonasal Approach to the Growth Hormone-Secreting Pituitary Adenomas: Endocrinologic Outcome in 68 Patients. World Neurosurg 117:e259–e268 5. Hofstetter CP et al (2010) Endoscopic endonasal transsphenoidal surgery for growth hormone- secreting pituitary adenomas. Neurosurg Focus 29(4):E6 5. Hofstetter CP et al (2010) Endoscopic endonasal transsphenoidal surgery for growth hormone- secreting pituitary adenomas. Neurosurg Focus 29(4):E6 6. Yildirim AE et al (2014) Endoscopic endonasal transsphenoidal treatment for acromegaly: 2010 consensus criteria for remission and predictors of outcomes. Turk Neurosurg 24(6):906–912 6. Yildirim AE et al (2014) Endoscopic endonasal transsphenoidal treatment for acromegaly: 2010 consensus criteria for remission and predictors of outcomes. Turk Neurosurg 24(6):906–912 7. Jane JA Jr. et al (2011) Endoscopic transsphenoidal surgery for acromegaly: remission using modern criteria, complications, and predictors of outcome. J Clin Endocrinol Metab 96(9):2732– 2740 7. Jane JA Jr. et al (2011) Endoscopic transsphenoidal surgery for acromegaly: remission using modern criteria, complications, and predictors of outcome. J Clin Endocrinol Metab 96(9):2732– 2740 8. Davies BM et al (2016) Assessing size of pituitary adenomas: a comparison of qualitative and quantitative methods on MR. Acta Neurochir (Wien) 158(4):677–683 8. Davies BM et al (2016) Assessing size of pituitary adenomas: a comparison of qualitative and quantitative methods on MR. Acta Neurochir (Wien) 158(4):677–683 9. Chatzellis E et al (2015) Aggressive pituitary tumors. Neuroendocrinology 101(2):87–104 9. Chatzellis E et al (2015) Aggressive pituitary tumors. Neuroendocrinology 101(2):87–104 9. Chatzellis E et al (2015) Aggressive pituitary tumors. Neuroendocrinol 10. Cardinal T et al (2021) Postoperative GH and Degree of Reduction in IGF-1 Predicts Postoperative Hormonal Remission in Acromegaly. Front Endocrinol (Lausanne) 12:743052 10. Cardinal T et al (2021) Postoperative GH and Degree of Reduction in IGF-1 Predicts Postoperative Hormonal Remission in Acromegaly. Front Endocrinol (Lausanne) 12:743052 11. Cardinal T et al (2020) Impact of tumor characteristics and pre- and postoperative hormone levels on hormonal remission following endoscopic transsphenoidal surgery in patients with acromegaly. Neurosurg Focus 48(6):E10 11. References Shirvani M, Motiei-Langroudi R (2014) Transsphenoidal surgery for growth hormone-secreting pituitary adenomas in 130 patients. World Neurosurg 81(1):125–130 24. Holdaway IM, Rajasoorya RC, Gamble GD (2004) Factors influencing mortality in acromegaly. J Clin Endocrinol Metab 89(2):667–674 24. Holdaway IM, Rajasoorya RC, Gamble GD (2004) Factors influencing mortality in acromegaly. J Clin Endocrinol Metab 89(2):667–674 25. Hardy J, Vezina JL (1976) Transsphenoidal neurosurgery of intracranial neoplasm. Adv Neurol 15:261–273 25. Hardy J, Vezina JL (1976) Transsphenoidal neurosurgery of intracranial neoplasm. Adv Neurol 15:261–273 26. Lee CC et al (2015) Prediction of Long-term Post-operative Testosterone Replacement Requirement Based on the Pre-operative Tumor Volume and Testosterone Level in Pituitary Macroadenoma. Sci Rep 5:16194 26. Lee CC et al (2015) Prediction of Long-term Post-operative Testosterone Replacement Requirement Based on the Pre-operative Tumor Volume and Testosterone Level in Pituitary Macroadenoma. Sci Rep 5:16194 27. Chuang CC et al (2017) Different Volumetric Measurement Methods for Pituitary Adenomas and Their Crucial Clinical Significance. Sci Rep 7:40792 27. Chuang CC et al (2017) Different Volumetric Measurement Methods for Pituitary Adenomas and Their Crucial Clinical Significance. Sci Rep 7:40792 References Cardinal T et al (2020) Impact of tumor characteristics and pre- and postoperative hormone levels on hormonal remission following endoscopic transsphenoidal surgery in patients with acromegaly. Neurosurg Focus 48(6):E10 12. Zheng Y, Wang CD, Mai Y, Zhu RK (2020) ZF, Surgical management of growth hormone-secreting pituitary adenomas: A retrospective analysis of 33 patients. Medicine, 2020;99:19(e19855) 12. Zheng Y, Wang CD, Mai Y, Zhu RK (2020) ZF, Surgical management of growth hormone-secreting pituitary adenomas: A retrospective analysis of 33 patients. Medicine, 2020;99:19(e19855) 13. Shen SC et al (2019) Long-Term Effects of Intracapsular Debulking and Adjuvant Somatostatin Analogs for Growth Hormone-Secreting Pituitary Macroadenoma: 10 Years of Experience in a Single Institute. World Neurosurg 126:e41–e47 14. Almeida JP et al (2018) Reoperation for growth hormone-secreting pituitary adenomas: report on an endonasal endoscopic series with a systematic review and meta-analysis of the literature. J Page 9/15 Page 9/15 Neurosurg 129(2):404–416 Neurosurg 129(2):404–416 15. Yano S et al (2017) Intraoperative Scoring System to Predict Postoperative Remission in Endoscopic Endonasal Transsphenoidal Surgery for Growth Hormone-Secreting Pituitary Adenomas. World Neurosurg 105:375–385 16. Briceno V et al (2017) Efficacy of transsphenoidal surgery in achieving biochemical cure of growth hormone-secreting pituitary adenomas among patients with cavernous sinus invasion: a systematic review and meta-analysis. Neurol Res 39(5):387–398 17. Babu H et al (2017) Long-Term Endocrine Outcomes Following Endoscopic Endonasal Transsphenoidal Surgery for Acromegaly and Associated Prognostic Factors. Neurosurgery 81(2):357–366 18. Knosp E et al (1993) Pituitary adenomas with invasion of the cavernous sinus space: a magnetic resonance imaging classification compared with surgical findings. Neurosurgery, 33(4): p. 610-7; discussion 617-8 19. Hardy J (1969) Transphenoidal microsurgery of the normal and pathological pituitary. Clin Neurosurg 16:185–217 20. Giustina A et al (2010) A consensus on criteria for cure of acromegaly. J Clin Endocrinol Metab 95(7):3141–3148 21. Giustina A et al (2023) Consensus on criteria for acromegaly diagnosis a 21. Giustina A et al (2023) Consensus on criteria for acromegaly diagnosis and remission. Pituitary 21. Giustina A et al (2023) Consensus on criteria for acromegaly diagnosis and remission. Pituitary 22. Campbell PG et al (2010) Outcomes after a purely endoscopic transsphenoidal resection of growth 22. Campbell PG et al (2010) Outcomes after a purely endoscopic transsphenoidal resection of growth hormone-secreting pituitary adenomas. Neurosurg Focus 29(4):E5 23. Shirvani M, Motiei-Langroudi R (2014) Transsphenoidal surgery for growth hormone-secreting pituitary adenomas in 130 patients. World Neurosurg 81(1):125–130 23. Tables Statistical Analysis of the Patients with or without Gross Total Resection According to the Degree of Invasiveness Page 12/15 Values Variable GTR (n = 39) STR (n = 11) P value Knosp Grade 0.006 0~2 32 4 3~4 7 7 Hardy-Wilson Grade 0.006 0~2 26 2 3~4 13 9 Hardy-Wilson Grade 0.001 0~C 32 7 D~E 3 8 GTR, gross total resection; STR, subtotal resection Table 3. Statistical Analysis of the Patients with or without Biochemical Remission Values Variable Remission (n = 27) Non-remission (n = 23) P value Demographics Male 13 (48.15%) 10 (43.48%) 0.78 Age (years) 44.3±12.8 44.6±15.6 0.88 Tumor Characteristics Macroadenoma 20 (74.07%) 20(86.96%) 0.78 Tumor Size (cm) 1.3±0.5 1.8±0.8 0.024 Tumor Volume (cm3) 1.4±1.5 3±2.9 0.0032 Suprasellar invasion 12 (44.44%) 13 (56.52%) 0.57 Pure intrasellar lesion 14 (51.85%) 6 (26.09%) 0.08 Infrasellar invasion 7 (25.93%) 9 (39.13%) 0.37 Knosp Grade 0.0007 0 3 0 1 14 4 2 9 6 3 1 11 4 0 2 Hardy-Wilson Grade 0.0428 1 7 2 2 13 6 3 4 10 4 3 5 Hardy-Wilson Grade 0.0006 0 14 4 A 7 1 B 3 3 Values Variable GTR (n = 39) STR (n = 11) P value Knosp Grade 0.006 0~2 32 4 3~4 7 7 Hardy-Wilson Grade 0.006 0~2 26 2 3~4 13 9 Hardy-Wilson Grade 0.001 0~C 32 7 D~E 3 8 R, gross total resection; STR, subtotal resection le 3. Statistical Analysis of the Patients with or without Biochemical Remission Values Variable Remission (n = 27) Non-remission (n = 23) P value Demographics Male 13 (48.15%) 10 (43.48%) 0.78 Age (years) 44.3±12.8 44.6±15.6 0.88 Tumor Characteristics Macroadenoma 20 (74.07%) 20(86.96%) 0.78 Tumor Size (cm) 1.3±0.5 1.8±0.8 0.024 Tumor Volume (cm3) 1.4±1.5 3±2.9 0.0032 Suprasellar invasion 12 (44.44%) 13 (56.52%) 0.57 Pure intrasellar lesion 14 (51.85%) 6 (26.09%) 0.08 Infrasellar invasion 7 (25.93%) 9 (39.13%) 0.37 Knosp Grade 0.0007 0 3 0 1 14 4 2 9 6 3 1 11 4 0 2 Hardy-Wilson Grade 0.0428 1 7 2 2 13 6 3 4 10 4 3 5 Hardy-Wilson Grade 0.0006 0 14 4 A 7 1 B 3 3 C 2 1 D 0 7 E 1 7 Gross Total Resection 26(96.3%) 13(56.52%) 0.0012 Preoperative hGH 14.3±15.1 32.4±37.7 0.19 Preoperative IGF-1 603.7±235.8 688.9±196.4 0.12 , gross total resection; STR, subtotal resection e 3. Tables Table 1. Characteristics of the Study Patients Page 10/15 Variable Value Demographics Male 23 (46%) Age (years) 44.4±14.2 Tumor Characteristics Macroadenoma 40 (80%) Tumor Size (cm) 1.6±0.7 Tumor Volume (cm3) 2.1±2.4 Knosp Grade 0 3 (6%) 1 18 (36%) 2 15 (30%) 3 12 (24%) 4 2 (4%) Hardy-Wilson Grade 1 9 (18%) 2 19 (38%) 3 14 (28%) 4 18 (36%) Hardy-Wilson Grade 0 18 (38%) A 8 (16%) B 6 (12%) C 3 (6%) D 7 (14%) E 8 (16%) Post-OP Characteristics Page 11/15 Variable Value Post-OP IGF-1 279±191.9 Biochemical Remission 27 (54%) Follow-up period (months) 80.7±46.9 Variable Value Post-OP IGF-1 279±191.9 Biochemical Remission 27 (54%) Follow-up period (months) 80.7±46.9 Values are given as frequency (percentage) for categorical variables or mean ± standard deviation for continuous variables. Values are given as frequency (percentage) for categorical variables or mean ± standard deviation for continuous variables. 2. Statistical Analysis of the Patients with or without Gross Total Resection Values Variable GTR (n = 39) STR (n = 11) P value Demographics Male 16(41.03%) 7(63.64%) 0.30 Age (years) 45.3±14.5 41.4±12.5 0.41 Tumor Characteristics Macroadenoma 31(79.49%) 9(81.82%) 1 Tumor Size (cm) 1.4±0.5 2.1±1.1 0.09 Tumor Volume (cm3) 1.7±1.8 3.5±3.6 0.06 Knosp Grade 0.015 0 3 0 1 17 1 2 12 3 3 7 5 4 0 2 Hardy-Wilson Grade 0.014 1 7 2 2 19 0 3 9 5 4 4 4 Hardy-Wilson Grade 0.017 0 16 2 A 8 0 B 5 1 C 3 0 D 4 3 E 3 5 Table 2. Statistical Analysis of the Patients with or without Gross GTR, gross total resection; STR, subtotal resection; hGH, human growth hormone; IGF- 1, insulin-like growth factor 1 GTR, gross total resection; STR, subtotal resection; hGH, human growth hormone; IGF- 1, insulin-like growth factor 1 Values are given as frequency (percentage) for categorical variables or mean ± standard deviation for continuous variables. Table 2-1. Statistical Analysis of the Patients with or without Gross Total Resection According to the Degree of Invasiveness g Values are given as frequency (percentage) for categorical variables or mean ± standard deviation for continuous variables. Table 2-1. Tables Statistical Analysis of the Patients with or without Biochemical Remission Values Variable Remission (n = 27) Non-remission (n = 23) P value Demographics Male 13 (48.15%) 10 (43.48%) 0.78 Age (years) 44.3±12.8 44.6±15.6 0.88 Tumor Characteristics Macroadenoma 20 (74.07%) 20(86.96%) 0.78 Tumor Size (cm) 1.3±0.5 1.8±0.8 0.024 Tumor Volume (cm3) 1.4±1.5 3±2.9 0.0032 Suprasellar invasion 12 (44.44%) 13 (56.52%) 0.57 Pure intrasellar lesion 14 (51.85%) 6 (26.09%) 0.08 Infrasellar invasion 7 (25.93%) 9 (39.13%) 0.37 Knosp Grade 0.0007 0 3 0 1 14 4 2 9 6 3 1 11 4 0 2 Hardy-Wilson Grade 0.0428 1 7 2 2 13 6 3 4 10 4 3 5 Hardy-Wilson Grade 0.0006 0 14 4 A 7 1 B 3 3 C 2 1 D 0 7 E 1 7 Gross Total Resection 26(96.3%) 13(56.52%) 0.0012 Preoperative hGH 14.3±15.1 32.4±37.7 0.19 Preoperative IGF-1 603.7±235.8 688.9±196.4 0.12 Page 13/15 hGH, human growth hormone; IGF-1, insulin-like growth factor 1 Values are given as frequency (percentage) for categorical variables or mean ± standard deviation for continuous variables. Values are given as frequency (percentage) for categorical variables or mean ± standard deviation for continuous variables. Values are given as frequency (percentage) for categorical variables or mean ± standard deviation for continuous variables. Table 3-1. Statistical Analysis of the Patients with or without Biochemical Remission According to the Degree of Invasiveness Values Variable Remission (n = 27) Non-remission (n =23) P value Knosp Grade 0.00005 0~2 26 10 3~4 1 13 Hardy-Wilson Grade 0.009 0~2 20 8 3~4 7 15 Hardy-Wilson Grade 0.00001 0~C 26 9 D~E 1 14 Figures Figure 1 Knosp grade. This system grades the parasellar extension of a tumor towards the cavernous sinus in relation to the intracavernous carotid artery [8, 18] Table 3-1. Statistical Analysis of the Patients with or without Biochemical Remission According to the Degree of Invasiveness Values Variable Remission (n = 27) Non-remission (n =23) P value Knosp Grade 0.00005 0~2 26 10 3~4 1 13 Hardy-Wilson Grade 0.009 0~2 20 8 3~4 7 15 Hardy-Wilson Grade 0.00001 0~C 26 9 D~E 1 14 Figures Figure 1 Knosp grade. This system grades the parasellar extension of a tumor towards the cavernous sinus in relation to the intracavernous carotid artery [8, 18] Figure 1 Knosp grade. This system grades the parasellar extension of a tumor towards the cavernous sinus in relation to the intracavernous carotid artery [8, 18] Knosp grade. This system grades the parasellar extension of a tumor towards the cavernous sinus in relation to the intracavernous carotid artery [8, 18] Page 14/15 Page 14/15 Figure 2 Hardy-Wilson classification of pituitary tumors. Upper panel shows the classification of downward sphenoid bone invasion (grade 0: intact with normal contour; grade I: intact with bulging floor; grade II: intact with enlarged fossa; grade III: localized sellar destruction; grade IV: diffuse sellar destruction). Only grade III and IV tumors are considered invasive or high grade. Lower panel describes classification of upward suprasellar extension [grade A: only intrasellar location; grade B: recess of the third ventricle; grade C: whole anterior third ventricle; grade D: intracranial extradural; grade E: extracranial extradural] [9] Figure 2 Hardy-Wilson classification of pituitary tumors. Upper panel shows the classification of downward sphenoid bone invasion (grade 0: intact with normal contour; grade I: intact with bulging floor; grade II: intact with enlarged fossa; grade III: localized sellar destruction; grade IV: diffuse sellar destruction). Only grade III and IV tumors are considered invasive or high grade. Lower panel describes classification of upward suprasellar extension [grade A: only intrasellar location; grade B: recess of the third ventricle; grade C: whole anterior third ventricle; grade D: intracranial extradural; grade E: extracranial extradural] [9] Hardy-Wilson classification of pituitary tumors. Upper panel shows the classification of downward sphenoid bone invasion (grade 0: intact with normal contour; grade I: intact with bulging floor; grade II: intact with enlarged fossa; grade III: localized sellar destruction; grade IV: diffuse sellar destruction). Only grade III and IV tumors are considered invasive or high grade. Lower panel describes classification of upward suprasellar extension [grade A: only intrasellar location; grade B: recess of the third ventricle; grade C: whole anterior third ventricle; grade D: intracranial extradural; grade E: extracranial extradural] [9] Hardy-Wilson classification of pituitary tumors. Upper panel shows the classification of downward sphenoid bone invasion (grade 0: intact with normal contour; grade I: intact with bulging floor; grade II: intact with enlarged fossa; grade III: localized sellar destruction; grade IV: diffuse sellar destruction). Only grade III and IV tumors are considered invasive or high grade. Lower panel describes classification of upward suprasellar extension [grade A: only intrasellar location; grade B: recess of the third ventricle; grade C: whole anterior third ventricle; grade D: intracranial extradural; grade E: extracranial extradural] [9] Page 15/15
https://openalex.org/W2005316064	https://research.bangor.ac.uk/portal/files/7156632/PDB33-00.pdf	English	null	Disciplined Improvisation: Characteristics of Inquiry in Mindfulness-Based Teaching	Mindfulness	2,014	cc-by	7,613	Dyfyniad o'r fersiwn a gyhoeddwyd / Citation for published version (APA): Crane, R. S., Stanley, S., Rooney, M., Bartley, P., Cooper, L., & Mardula, J. (2015). Disciplined Improvisation: Characteristics of inquiry in mindfulness-based teaching. Mindfulness, 6(5), 1104- 1114. https://doi.org/10.1007/s12671-014-0361-8 Hawliau Cyffredinol / General rights Hawliau Cyffredinol / General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit making activity or commercial gain study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? y • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Cyswllt i'r cyhoeddiad / Link to publication Dyfyniad o'r fersiwn a gyhoeddwyd / Citation for published version (APA): Crane, R. S., Stanley, S., Rooney, M., Bartley, P., Cooper, L., & Mardula, J. (2015). Disciplined Improvisation: Characteristics of inquiry in mindfulness-based teaching. Mindfulness, 6(5), 1104- 1114. https://doi.org/10.1007/s12671-014-0361-8 Hawliau Cyffredinol / General rights Disciplined Improvisation Crane, R.S.; Stanley, Steven; Rooney, Michael; Bartley, Patricia; Cooper, Lucinda; Mardula, Jody Mindfulness DOI: 10.1007/s12671-014-0361-8 Published: 01/10/2015 Publisher's PDF, also known as Version of record Cyswllt i'r cyhoeddiad / Link to publication Dyfyniad o'r fersiwn a gyhoeddwyd / Citation for published version (APA): Crane, R. S., Stanley, S., Rooney, M., Bartley, P., Cooper, L., & Mardula, J. (2015). Disciplined Improvisation: Characteristics of inquiry in mindfulness-based teaching. Mindfulness, 6(5), 1104- 1114. https://doi.org/10.1007/s12671-014-0361-8 PRIFYSGOL BANGOR / BANGOR UNIVERSITY PRIFYSGOL BANGOR / B DOI: 10.1007/s12671-014-0361-8 Publisher's PDF, also known as Version of record Cyswllt i'r cyhoeddiad / Link to publication Cyswllt i'r cyhoeddiad / Link to publication # The Author(s) 2014. This article is published with open access at Springerlink.com # The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Evidence for the effectiveness of mindfulness-based stress reduction (MBSR) and mindfulness-based cognitive therapy (MBCT) is rapidly growing as interest in this field expands. By contrast, there are few empirical analyses of the pedagogy of MBSR and MBCT. Development of the evidence base concerning the teaching of MBCT or MBSR would support the integrity of the approach in the context of rapid expansion. This paper describes an applied conversation anal- ysis (CA) of the characteristics of inquiry in the MBSR and MBCT teaching process. Audio-recordings of three 8-week MBCT and MBSR classes, with 24, 12, and 6 participants, were transcribed and systematically examined. The study fo- cused on the teacher-led interactive inquiry which takes place in each session after a guided meditation practice. The study describes and analyzes three practices within the inquiry pro- cess that can be identified in sequences of talk: turn-taking talk involving questions and reformulations; the development of participant skills in a particular way of describing experience; and talk that constructs intersubjective connection and affilia- tion within the group. CA enables fine-grained analysis of the interactional work of mindfulness-based inquiry. Inquiry is a process of disciplined improvisation which is both highly specific to the conditions of the moment it took place in and uses repeated and recognizable patterns of interaction. Disciplined Improvisation: Characteristics of Inquiry in Mindfulness-Based Teaching Rebecca S. Crane & Steven Stanley & Michael Rooney & Trish Bartley & Lucinda Cooper & Jody Mardula M. Rooney City and Hackney Primary Care Psychology Service, Homerton University Hospital Trust, London, UK S. Stanley School of Social Sciences, Cardiff University, Cardiff, UK Take down policy If b li th t th Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. 24. Oct. 2024 Mindfulness Mindfulness DOI 10.1007/s12671-014-0361-8 DOI 10.1007/s12671-014-0361-8 ORIGINAL PAPER Introduction There is an extensive empirical evidence base for the effec- tiveness of mindfulness-based stress reduction (MBSR) and mindfulness-based cognitive therapy (MBCT) (hereon abbre- viated to MB) approaches as an intervention in clinical set- tings, education, and the workplace (e.g., Fjorback et al. 2011; Piet and Hougaard 2011). However, there is a dearth of studies concerning the pedagogical processes involved in teaching MB courses. Leaders in the field have expressed concerns about the potential for a dilution of integrity of the approach, in part because of lack of in-depth understanding about MB teaching practice (Williams and Kabat-Zinn 2011). There is a descriptive and theoretical literature on the pedagogy of MBSR and MBCT (Crane 2009; Kabat-Zinn 2013; McCown et al. 2010; Santorelli 2000; Segal et al. 2012). This practitioner literature would be strengthened by empirical studies of the MB teaching process. Gaining an insight into key aspects of the pedagogy would play an important part in understanding how the approach achieves its affects and how teacher training can effectively support the development of competence. The only study to date which takes the teacher as the research object (van Aalderen et al. 2012) is a qualita- tive analysis of the role of the teacher in MBCT, in- volving interviews with course participants and teachers, a focus group of teachers, and an observational analysis of an MBCT course. Their findings offer support to the practitioner view that the teacher’s embodiment of mindfulness is a central way through which participant learning is facilitated. Keywords Conversation analysis . Mindfulness-based stress reduction . Mindfulness-based cognitive therapy . Inquiry . Pedagogy . Integrity R. S. Crane (): T. Bartley: L. Cooper: J. Mardula Centre for Mindfulness Research and Practice, School of Psychology, Bangor University, Bangor, UK e-mail: r.crane@bangor.ac.uk R. S. Crane (): T. Bartley: L. Cooper: J. Mardula Centre for Mindfulness Research and Practice, School of Psychology, Bangor University, Bangor, UK e-mail: r.crane@bangor.ac.uk Crane et al. (2012) developed a MB teaching competence framework and evaluated its psychometric properties (Crane et al. 2013). This work demonstrated that a group of experi- enced MB teachers can agree on a structured and consistent rubric for assessing MB teaching against criteria and that the tool can be used reliably by experienced MB trainers who are trained in its use. S. Introduction Stanley School of Social Sciences, Cardiff University, Cardiff, UK Mindfulness There is also a small but growing body of research examining mediators of change in MB courses. For example, the Kuyken et al. (2010a) research established that cultivation of both self- compassion and mindfulness plays an important role in protecting participants from future depression. This suggests that pedagogical processes that support the development of self-compassion are likely to be important in supporting positive participant outcome and are there- fore likely to be important areas of teacher competence. Trials are increasingly including analysis of mediator variables in their design (Huijbers et al. 2012; Kuyken et al. 2010b; Williams et al. 2010), so it is likely that studies of mechanisms of action will increase over the next few years and that this will feed the knowledge base on MB teaching. develop skills in. The results therefore have the potential to inform teacher training developments. Secondly, we were interested in piloting the CA method and therefore chose to focus the research on the interactional aspects of MB teaching. There is a well-developed practitioner literature which describes the process and pedagogical principles underpin- ning inquiry in MBCT and MBSR (Crane 2009; Felder et al. 2012; McCown et al. 2010; Santorelli 2000; Segal et al. 2012). An inquiry sequence is described in this literature as occurring in the following generic way. Following the meditation practice, the teacher begins a conversation by asking participants what they noticed during the practice. They do this to encourage reflection and explo- ration on their experience; work together through dialogue about these observations to find out what is being discovered; and link these observations and discoveries to the learning themes of the program. Inquiry aims to reveal and bring into conscious awareness automated and unrecognized habits and patterns of thinking and feeling and to make known some of the properties of thoughts and feelings. The manner of attend- ing to the experience, the teacher, and the relational process are all thus aiming to offer an embodiment of the attitudinal qualities of mindfulness. It is suggested that this supports participants to internalize a mindful way of relating to expe- rience which includes increased levels of self-compassion, reduced levels of cognitive reactivity, and the development of a capacity to allow, rather than problem solve, whatever experience is present in the moment. Introduction In order to maximize outcomes and ensure fidelity to appropriate teaching standards, it is important to build on this initial knowledge base and develop systematic investigations of the pedagogy of the teaching process by directly studying it. There are, however, some intriguing tensions in examining the practice of MB teaching. The outcome evidence base is located within the scientific/medical paradigm, and some writers on mindfulness teaching argue that many of its dimen- sions lie outside that paradigm (McCown et al. 2010). Even if we accept that MB teaching is amenable to empirical exami- nation, we still face methodological challenges. Many of these challenges would be familiar to researchers examining the process aspects of psychotherapy and education, and some are unique to a mindfulness context. The study aimed to discover how MB inquiry sequences are actually conducted in practice by skilled MB teachers in collaboration with their participants. We aimed to investigate the interactional practices employed by experienced MB teachers in order to investigate: How do MB teachers lead inquiry? How are sequences of inquiry organized? And are there recognizable patterns to the interactional practices? q A number of methodologies can be used to analyze the key features of the conversational practice of inquiry in MBCT and MBSR. Applied conversation analysis (CA; Hutchby and Wooffitt 2009) is a particularly suitable method because it provides a naturalistic, observational investigation of the pro- cess. Antaki (2011) offered the following working definition of CA: “the close examination of language in interaction. It answers the concrete questions: how do you and I bring off the business we transact with each other?” (p. 2). Conversation analysts study social life through analysis of social interaction, specifically, turn design and interactional organization are investigated to understand how meaning, social action, and context are created moment-by-moment. CA imposes a disci- pline on the researcher to interpret how participants display their understandings of what is happening within interaction. This contrasts with a typical MB practitioner perspective of focusing on the intention that might have been behind a teacher’s utterance. CA allows a close examination of what speakers are observably and hearably doing with their talk through an investigation of recurrent patterns or practices of interaction. Participants Audio-recordings of three MBSR/MBCT classes taught by different teachers who consistently score proficient/advanced on the MBI:TAC (Crane et al. 2012) were analyzed. All follow the usual MBSR/MBCT format of eight 2–2.5 h week- ly group sessions, with a daylong session of guided mindful- ness practice during week 6 of the course. See Table 1. Procedure Prior to commencement of the research, the study was reviewed and approved by the university’s research ethics and governance committee. The three teachers were The study focused on the practice of inquiry for two rea- sons. First, it is the aspect of MB teaching practice which trainees frequently identify as being the most challenging to Mindfulness Table 1 Demographic character- istics of intervention, teachers, and course participants Group Numbers of participants Gender Population Program Teacher Participants M F A 23 F 6 17 General public MBSR B 12 F 4 8 Trainee MBCT/MBSR teachers MBSR C 6 F 2 4 People with cancer MBCT Table 1 Demographic character- istics of intervention, teachers, and course participants Peräkylä (2004) suggested that the “backbone” of CA work involves “qualitative case-by-case analysis” (p. 299). We an- alyzed a small number of cases, illustrating a specific form of MB teacher–participant interaction. collaborators and co-applicants. Consent was required from all course participants prior to commencement of recording and ceased if any participant decided to withdraw from the research. Recording of the entire 8-week course took place where possible. Recordings were stored on a password- protected hard drive and kept in a locked safe. The reliability of the study is mainly ensured by examining naturally occurring interaction, rather than using researcher- prompted interactions (i.e., interviews) which then need to be generalized to everyday life. The data captures what actually happened in these MB courses. Data Analysis We aimed to adopt a stance of “unmotivated looking” as de- scribed by Psathas (1990) which is open to discovering interac- tional practices without being led by pre-formulated conceptu- alizations of what MB teaching should look like. To cultivate the capacity to simply listen with minimum intrusion of the concep- tual mind and pre-formulated ideas about the teaching, the researchers sat in mindfulness meditation before reviewing each recorded exemplar. A key discipline during the analysis was to notice when we had moved beyond observation and into sug- gesting a motivation or intention for an interactional practice. To ensure the validity of the analytic claims, we did the following. Firstly, the first three authors made transcriptions and analytic observations independently and then collectively transcribed and analyzed the data extracts we selected. The three teachers reviewed the findings in light of their practitioner expe- rience of the teaching process. Secondly, we adopted the standard CA practice of looking at how participants themselves display their understanding of what is happening in interaction, particu- larly the actions being performed in previous turns at talk. This is a crucial way of establishing validity because we could check our claims against the understandings displayed by participants. We started with 60 h of recorded material and then narrowed the focus to sections of teacher-led inquiry conduct- ed after the first guided meditation practice in sessions 1–7 (approximately 7 h of material). The three researchers repeat- edly listened to these inquiry sequences alongside reviewing first draft non-annotated transcripts, with the aim of identify- ing recurring conversational patterns. They then came togeth- er to collaboratively review recordings and transcripts. Sequences of interaction that offered examples of repeatedly observed interactional practices were selected and transcribed using a simplified CA transcription notation (Hutchby and Wooffitt 2009). The notation aims to capture all of the qual- ities present in actual speech, such as length of silences, overlapping speech acts, and qualitative features such as rising pitch, volume, added stress, and noises and utterances by speakers other than words (Table 2 details the transcribing conventions used). These extracts were then analyzed collab- oratively by the researchers to enable detailed investigation of the interactional patterning of sequences. Results Material from all three teachers was included evenly in the analytical process, but the extracts presented in the paper are teachers B and C. Extracts were chosen on the basis that they Table 2 CA transcription symbols used (simplified from Hutchby and Wooffitt 2009) [ Starting point of overlapping speech =word No break or gap between words or turns (3.0) Silence measured in seconds (.) Pause of less than 0.2 s word Emphasis °word° Especially quiet wo:rd Prolongation of sound WORD Words in capitals mark a section of speech noticeable louder than that surrounding it wo- Cut off .hhh Inhalation ↑↓ Shifts into especially high or low pitch In this study, we gathered examples of MB teacher–partic- ipant interaction which were available to us and built up a corpus of inquiry sequences. We have not used random sampling or statistical sampling of populations to ensure the generalizability and representativeness of our findings. Mindfulness 1. Turn-Taking Talk that Involves Questioning and Reformulations 1. Turn-Taking Talk that Involves Questioning and Reformulations represent repeatedly seen conversational patterns, were suc- cinct, and could make sense to a reader outside the overall context of the conversation that had preceded the extract. A turn-taking feature of generic pedagogical discourse contexts is a three-turn sequence in which the teacher asks a question (first turn), followed by the participant(s) an- swer (second turn), and then the turn routinely goes back to the teacher who gives a response (third turn) (Lee 2007). We found this characteristic three-turn sequence to be consistently employed by the MB teacher during inquiry sequences. Participants usually self-select to re- spond and shape the teacher’s third-turn response by choosing the timing and content of their second-turn contributions. A range of methods of teacher first- and third-turn responses were observed with a common theme of reformulating participants’ talk to satisfy the institu- tional aims of the MB course. There are a wide range of interactive patterns involved in inquiry. Here, we focus on three overarching practices which we observed repeatedly across the whole data set: turn-taking talk that involves questions and reformulations; talk that develops participants’ competence in a specific way of talking about experience; and talk that reinforces intersubjective connection and affiliation. The observed features of these three interactional practices are presented below and are illustrated through refer- ence to numbered lines on transcript examples. The interactional practices are interrelated so overlap is inevitable. Results In each of the three sections below, therefore, priority is given to highlighting the key features of the practice under consideration, but aspects of the other two are also mentioned (T = teacher; A, B, or C = group; P = participant numbered in the order of first to speak). Extract 1 (session 2) In extract 1, the teacher offers a complex first-turn part which contains a number of rephrased questions and instruc- tions for the participants (1–7). In questioning the participants, the teacher also does some instructing work: establishing what is expected and required of participants in their next turn. The teacher alternates between a permissive, tentative openness which allows whatever was “experienced” or “noticed” to be shared (“perhaps sharing,” “different things”) and a prescrip- tive instruction about what is required of them (“let’s start off just by … just … little snippets”). The teacher limits the type of preferred range of possible response options and specifical- ly directs the participants to talk about what they noticed in their experience as opposed to, for example, asking for feedback on the exercise or an evaluative question like “how well did you manage that?” When a participant offers a second turn that is an evalua- tive, comparative response (8), the teacher is quick to offer a third turn (10). She talks over the participant and rather than responding directly to the evaluative aspect of the talk (i.e., class vs. home) she subtly reformulates what the participant has said by redescribing the participants’ recent experience: the comparative “easier,” which might have been developed into a story, becomes an emphasized and stand-alone “ease of focus.” This reformulation does at least two further things. First, it is affiliative, in the sense that the teacher echoes the Mindfulness participants’ own words (easier) with a slight, but significant, modification (ease) that is actually a repair that redirects the participant’s offered focus, while at the same time doing description and acknowledgment rather than challenge or evaluation. Second, it successfully generalizes to, and in- cludes, the group (note the multiple-participant “yes” in lines 12 and 14). The teacher then broadens the reformulation to the whole by giving emphasis to “all” (13). This interactional pattern of widening the learning outcomes from one individ- ual’s experience to the group was frequently seen. cludes, the group (note the multiple-participant “yes” in lines 12 and 14). Results The teacher then broadens the reformulation to the whole by giving emphasis to “all” (13). This interactional pattern of widening the learning outcomes from one individ- ual’s experience to the group was frequently seen. Extract 2 (later in same session from which extract 1 was taken) Extract 2 (later in same session from which extract 1 was taken) Extract 2 also shows how third-turn responses are used to widen the learning to the whole group following a series of turn-taking exchanges with one participant. In the lead up to this exchange, the teacher and participant 3 have together recreated her memory of her experience of her mind repeat- edly being carried away with “bizarre thoughts.” In extract 2, the teacher and participant collaboratively construct the idea that the participant was not aware of her mind drifting to “bizarre thoughts” (see “thought” (104) repaired to “aware” (106, 113, 117)). example of the key theme of this stage of the program: “automatic” (111). She then extends this reformulation to others (“we are not aware” (113)), normalizes the experience (114), and communicates (through demonstrating her own curiosity) that this new awareness is a significant piece of learning (116–7). Note how the participant moves from “I” (104) to the less personal “you” (108), demonstrating her shifting sense that this is not a personal phenomenon. Thus, there is a co-constructed interactional build up to the learning point of universality, which the teacher directionally steers and participants collaborate in. The teacher opens the exchange with a first-turn question, prefaced by a demonstration of keen curiosity (“love to ask you”) in the learning theme that she is directing the conversa- tion toward (knowing that the mind wanders). In a series of closely overlapping turns (97–104), the participant responds to the teacher’s questions with recognition that she had “never really thought about it.” The teacher then introduces this as an In summary, the turn-taking process is a co-construction in which the teacher opens the dialogue, a series of turns take place during which participants’ memories of their experience are collaboratively re-constructed, and then the teacher gathers, expands, and reformulates the learning for the benefit of the whole group. This gathering process sometimes takes place Mindfulness of them emerge in participants’ accounts of their specific experience. after a turn-taking sequence with one participant and at other times after a series of turns with several participants. Results The teacher determines the end point for the series of turns of questions and answers. 2 The Development of Skills in a Particular Way of Describing Experience When the teacher offers third-turn reformulations of partic- ipant experience for the whole group, they sometimes keep their turn for an extended time. Thus, didactic teaching fol- lows on and is linked to themes that participants have already introduced. Learning themes are only introduced as examples Participants’ talk is shaped by the teacher toward the con- versational norms of a MB class. This can be seen in action within the talk in a number of ways. Extract 3 (which occurs in session 2 between extracts 1 and 2) Extract 3 (which occurs in session 2 between extracts 1 and 2) Extract 3 shows how the teacher directionally leads the focus of the conversation back to the sorts of areas of focus for a MB class—in this particular instance to a focus on specific experience in a specific practice. Following an exchange with a participant who is experiencing difficulty with mind wandering in the prac- tice, the teacher widens the exploration to the whole group (56–8). The question builds toward the theme of univer- sality through appeal to others’ experiences. However, participant 2 takes the interaction away from a specific exploration of experience within recent practice (65–70). The teacher interrupts and overlaps with the participant with an emphatic “Okay” (71) before bringing the focus back (71–6) with the highly directive “so we are going to stay with this practice” with its emphatic “this.” The teacher continues with a softer affiliative tone and an invitation to “just for now stay” (75). The teacher redirects the focus of discussion back to the recent practice. In summary, a range of interactional practices through which the teacher aims to train participants’ competence in describing their experience in ways consistent with mindful- ness practice were seen in the data: First, participants are learning to anchor their learning in specific direct experience rather than in generalized ideas about experience, and when participants become less specific, they are firmly redirected. Second, participants generally only speak about their own immediate experience, so they predominantly use the pronoun Mindfulness “I,” are redirected when they generalize beyond their own expe- rience, and are not generally given space to elaborate about their experience. Results The teacher draws out themes that are likely to be universal to everyone, so during the third-turn reformulations, they make a pronoun shift to “us” and “we” (see 113–7, extract 2) and speak about “the” mind rather than using the possessive “your” mind (see 113, extract 3). Generally, the teacher is the member of the group who is given space to generalize experi- ence in these ways. noticed mind wandering and expresses that she had never thought of it before (104–5)). The teacher is actively directing participants toward recognition that in this context the appar- ently ordinary becomes an important topic, and socially nor- mative responses are descriptive reports about noticing recent direct experience. Fifth, participants are being trained to speak in the language of “noticing” and “being aware of” experience (e.g., see 2, 6, 10, 14 in extract 1) as they retrospectively co-construct their experience using language. In other recordings, teachers would specifically reward participants’ noticing skills with the expres- sion “good noticing”, and noticing of a “small” experience is rewarded with an emphasized positive assessment. Third, participant talk that is a detailed, rich, non-analytic account of a specific and recent example of immediate experience that relates to key learning themes produces greater displays of interest from the teacher and to longer time sequences of turn- taking with the teacher. In extract 2, the teacher uses each turn with participant 3 as an invitation to display key learning themes to the whole group. By contrast, when participants are not offering contributions that fit, teachers either explicitly redirect the interac- tion or offer short emphaticminimal responsetokenswhich project for an end to the turn-taking (see the “okay” at line 71 in extract 3). 3 Talk that Constructs Intersubjective Connection and Affiliation As has been noted in Sections 1 and 2 above the MB teachers’ talk seems designed to produce a sense of affiliation and connection both between teacher and participant, and across the whole group (including the teacher), through a repeated practice of constructing a connection with the uni- versality of human experience (e.g., extract 3, lines 113–117). Fourth, teachers appear to be training participants to dis- play interest and curiosity in ordinary everyday experience and in mind patterns that might have previously been off radar (see 94–6 in extract 2 where participant 3 is asked if she has Extract 4 (session 4) experience and communicates affiliation through the soft, long “mmm” (3). There are regular, and often long, pauses Extract 4 shows teacher–participant affiliation being creat- ed through interaction. The teacher validates the participants’ Mindfulness “right,” “sure,” “yeh,” “mm”) often overlapping with par- ticipants’ talk. Here, in extract 4, the teacher’s “mm’s” overlap with the participant’s words and in this instance communicate affiliation and actively signal to the partic- ipant to continue because what she is saying is of interest and value. As expressed earlier, these minimal responses can also be used to close down and change topic (see the “okay” in 71, extract 3). “right,” “sure,” “yeh,” “mm”) often overlapping with par- ticipants’ talk. Here, in extract 4, the teacher’s “mm’s” overlap with the participant’s words and in this instance communicate affiliation and actively signal to the partic- ipant to continue because what she is saying is of interest and value. As expressed earlier, these minimal responses can also be used to close down and change topic (see the “okay” in 71, extract 3). (11, 13, 17, 19) which may demonstrate willingness of both the teacher and the participant to stay with her experience and that this is an okay place to be together. Notice also how teacher and participant match each other’s pace and tone (see the rising intonation of the teacher in 16 which follows the participant’s emphasis on “coming”). 3 Talk that Constructs Intersubjective Connection and Affiliation The teachers generally used many highly positive min- imal response tokens (Jefferson 1984) (words, such as Extract 5 (session 2) (picking up a short while after extract 1) Extract 5 (session 2) (picking up a short while after extract 1) In extract 5, the teacher offers an echo of tone and content of the phrase “very difficult,” performing a sense of alliance and affiliation with participant 3 as she shares her experience of challenge and communicates difficulty (though interpolated laughter). The participant is able to come in with a completely contrasting experience (28), implying that (al- though this is only session 2) an encouraging, invitational atmosphere has been established and is continually being reinforced through conversation. In other session recordings, the teachers explicitly encourage participants to share expe- riences that might “be the same as or different to” what has just been shared. teaching. This offers insights into the pedagogic relationship between the participants’ development of understanding and the interactional practices used. Further work is needed to investigate how these understandings relate to participant outcomes. It is clear that the contingencies of the social organiza- tion of the participatory learning process influence the shape and character of each moment. We suggest that these implicit but important social processes have a sig- nificant influence on participant learning. It is therefore important to recognize and account for them in teaching and in the training of teachers. CA offers a methodology for unpacking these taken-for-granted processes and re- vealing the practical enactment of the teachers’ pedagogi- cal work. Although the sequences of transcript tend to be between the teacher and one other participant, it is important to remember that every conversation happens in the presence of the whole group. The teachers frequently use strategies during a turn- taking sequence with one participant to ensure wider partici- pation, to encourage affiliation between everyone present, and to encourage recognition that the theme being explored is relevant to everyone. One specific strand of CA, known as institutional CA, focuses on communicative practices that are specific to par- ticular workplace settings. Drew and Heritage (1993) articu- lated three features of talk in workplace settings: it is goal orientated in relevant ways; it involves particular and special constraints on allowable contributions; and it is associated with particular frameworks and procedures. 3 Talk that Constructs Intersubjective Connection and Affiliation The findings demonstrate that MB inquiry has context-specific aspects within each of these features: inquiry has a particular direction and purpose which is aligned with the aims of the course and which are firmly maintained by the teacher; linked to this, the teacher shapes the norms of what content is included and excluded from the process; and there are particular interac- tional frameworks, methods, and structures which enable dialogic exchange to take place. Limitations and Future Directions Doing a conversation analysis reveals a lot of delicate subtle interactional work on the part of teachers and partic- ipants. The limitations of the scope of this study mean that only a small amount of this highly sophisticated and nuanced work could be selected for noticing and analysis here. In particular, the interactions of only three teachers were analyzed. Furthermore, we have only examined what is hearable within the teaching—there are clearly other (seeable and feelable) dimensions and processes that are taking place that are not included here. One dimension which could be included in a future study using CA methods is the visual aspects of communication. In the context of MB teaching, this could be a particularly useful way to examine how mindfulness is communicated through the teacher’s embodiment of process. The balance between creating an open welcoming atmo- sphere and steering the teaching process in a strongly direc- tional way is delicate. Participatory dialogic teaching requir- ing turn-taking needs to be carefully managed; learning tends to be more effective when it is co-constructed, and there is active involvement from participants to shape the direction of the discourse (Radford et al. 2011). The teacher is making continuous micro-judgements about how much floor space to give to a particular participant contribution. The overriding pedagogic goal is to lead an emergent process of dialogue that has enabled each contributor to deepen their understanding and that leads to a shared understanding that everyone has been party to (Skidmore and Murakami 2012). p y ( ) The pedagogical literature on MB approaches emphasizes the implicit qualities that MB teachers need to embody. The inquiry process rests not only on these deeply embodied understandings of mindfulness practice within the teacher, but it also relies on the teacher’s capacity to enact the multiple skills required to manage participatory classroom dialogue. No matter how profound any piece of MB teacher communi- cation is, it is only as effective as the response it generates and therefore needs to be understood in the conversational context it was made in. Through CA, these interactional skills can become tangible, visible, and explicit. Analysis of these ex- plicit competencies offers a view on how the implicit qualities of mindfulness emerge as learning themes within the teaching process. For example, mindfulness-based teachers seem to have a particular way of hearing experience. Discussion A key aim of this research was to explicate the context- specific structural organization of talk in MB inquiry and to pilot the potential of the CA method for this context. The analysis aimed to make visible some of the taken-for-granted, implicit, or unrecognized practices that take place during inquiry. It makes a unique contribution by using CA to reveal a particular perspective on the complex and multi-faceted moment-by-moment practice of inquiry in MBSR/MBCT Simultaneously, however, there is an ongoing process of improvisation taking place. In the process of repeated Mindfulness participation, the teacher continually responds to the process and modifies their methods to achieve the task in hand. In contrast with some other teacher-led whole class dialogue contexts, the MB teacher is not seeking a specific right re- sponse, is not the primary knower of information, genuinely does not know the participants’ experience, and cannot there- fore know in advance the exact learning themes that will emerge in the moment. The co-construction is therefore highly specific to the conditions of the moment. The teacher’s skill in being able to dance with the emergence of each moment while steering the learning process is of paramount importance. This underlines both the importance of planning and preparation before a class is taught and of letting go into the actuality of each moment (rather than being constrained by ideas of how it could or should be), so that it is possible to be responsive to the moment. The term “disciplined improvisation” coined by Sawyer (2004) aptly describes the creative tension inherent within the process. Psathas (1990) proposed “the interactional phenomena that are discovered…will enable us to state with greater certainty, what interactional competencies are requisite… And if members are lacking particular identifiable and describable interactional skills, we should be able to devel- op methods for teaching, demonstrating or training” (p. 21). Each moment of inquiry is unique; however, CA enables us to see that repeating patterns of interaction in inquiry can be made visible. Trainee MB teachers commonly share that leading the inquiry process in MB teaching is the most challenging and daunting aspect of the overall learning process. They could become more attuned to the practices at play within inquiry through the integration of perspec- tives from CA (using recorded examples of teaching) into training. Limitations and Future Directions That is to say that they are able to scan the detail of expressed experience and hone the focus of inquiry to a particular aspect that is relevant to the learning process. The particular skills and knowledge required to enact this competence are not well articulated in the literature, but in our view, much of it rests on a moment-by-moment connection to the teacher’s personal mindfulness practice, which they embody during teaching. In this study, we used CA in a relatively wide angle way to reveal some general characteristics of MB inquiry. This limit- ed the level of detailed analysis of each practice. We suggest that CA techniques offer the potential to move into narrow angle-detailed analysis of themes within each area. For exam- ple, the teacher has a range of options with the third-turn response. Detailed analysis of these and their consequences would be informative. For this study, we chose extracts which demonstrated typical features. It would be useful to investigate moments when the unexpected happens and when tensions arise in the learning process. The CA transcription system offers opportunity to study how the emotional climate is co- created in the classroom. We saw some shifts in the interac- tional practices employed by teachers and participants over the 8 weeks as the competencies of participants grew, but the scope of this study did not enable analysis of this. An explo- ration of how this happens would enable greater understand- ing of the tasks of the teacher at different stages in the program. Our study revealed that the MB teaching process is highly directional and teacher-led. There is an interesting Mindfulness tension between directional leadership and participatory co- construction that is at play in the teaching process. How this is navigated by the teacher could be a specific focus. A signif- icant portion of the learning process in MB courses takes place in dyads and small groups. The CA approach could be ex- tended to investigate discourse between participants without the direct intervention of the teacher. teacher training and development, which is much needed in the context of growing demand for training. It is a challenge that we hope the field will embrace so that there is a growing literature that represents the disciplined improvisation that is the work of the MB teacher. Acknowledgments RSC was funded by a Wellcome Trust Program Grant (067797/Z/02/A) during work on this research. Conclusions While it is essential to draw on theory and on the wisdom of expert teachers, it is also essential to build an empirically based body of knowledge about the MB teaching process and a collection of valid empirical methods for conducting research in this area. In this first study of MB teaching to examine naturally occurring material rather than retrospective accounts of participant experience or expert views of the teaching process, we have used CA to investigate what teachers and participants actually do with their talk in MBSR/MBCT. This revealed how turn-taking happens and how the teacher reformulates participant contributions; partic- ular participant competencies that are being trained through dialogue; and the atmosphere of affiliation that is created through the process of interaction in the group. Use of CA reveals the complexity of the interactional work that MB teachers are engaged in when leading participatory dialogue. Limitations and Future Directions The authors would like to thank Prof. Richard Hastings and Dr. Gemma Griffiths for helpful comments on this manuscript and for support in the conducting of this research. We are grateful to the MBSR/MBCT class participants for their participation in the research. Different research methods require the researcher to stand in different relationship to the object of study. The MB teach- ing process has been evaluated by third-person explorations, which aim to objectively describe and assess the pedagogical process (Crane et al. 2013). In this study, we have employed a second-person examination of intersubjective, interactional practices. There is further potential to use first-person research methods by examining teachers’ reflections on internal pro- cesses as they teach (e.g., via diaries/interviews). A research process which examined a piece of teaching by triangulating findings from these three perspectives has the potential to be particularly rich. Readers can refer to an online resource that accompanies this paper (http://www.bangor.ac.uk/mindfulness/documents/mindfulness- basedinquiryresource.pdf). This includes further details on the methodological approach to analysis and some additional transcripts with accompanying analytic commentary. The transcripts chosen include the full interaction sequence from session 2 from which extracts 1, 2, 3, and 5 were taken and a more extended version of extract 4 from session 4. Readers can thus choose to view context around the quoted extracts. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. References Antaki, C. (2011). Six kinds of applied conversational analysis. In C. Antaki (Ed.), Applied conversation analysis: intervention and change in institutional talk (pp. 1–15). Basingstoke: Palgrave Macmillan. Crane, R. S. (2009). Mindfulness-based cognitive therapy: distinctive features. London: Routledge. Crane, R.S., Soulsby, J.G., Kuyken, W., Williams, J.M. G. & Eames, C. (2012). The Bangor, Exeter & Oxford mindfulness-based interven- tions teaching assessment criteria (MBI-TAC) for assessing the competence and adherence of mindfulness-based class-based teach- ing. Retrieved 04/23, 2012, from http://www.bangor.ac.uk/ mindfulness/documents/MBI-TACJune2012.pdf. Crane, R. S., Eames, C., Kuyken, W., Hastings, R. P., Williams, J. M. G., Bartley, T., Evans, E., Sioverton, S., Soulsby, J. G., & Surawy, C. (2013). Development and validation of the mindfulness-based inter- ventions—teaching assessment criteria (MBI:TAC). Assessment. doi:10.1177/1073191113490790. 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N., Dimidjian, S., & Segal, Z. (2012). Collaboration in mindfulness-based cognitive therapy. Journal of Clinical Psychology: In Session, 68(2), 179–186. doi:10.1002/jclp.21832. Fjorback, L. O., Arendt, M., Ornbø, I. E., Fink, P., & Walach, H. (2011). Mindfulness-based stress reduction and mindfulness-based cogni- tive therapy: a systematic review of randomized controlled trials. Acta Psychiatrica Scandinavica, 124(2), 102–119. doi:10.1111/j. 1600-0447.2011.01704.x. Huijbers, M.J., Spijker, J., Rogier, A., Donders, T., van Schaik, D.J.F., van Oppen, P., Ruhe, H.G., Blom, M.B.J., Nolenm, W.A., Orme, J., vander Wilt, G.J., Kuyken, W., Spinhovenm, P., Specken, A.E.M., (2012). Preventing relapse in recurrent depression using mindfulness-based cognitive therapy, antidepressant medication or Mindfulness disorder: a systematic review and meta-analysis. 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Full catastrophe living, revised edition: how to cope with stress, pain and illness using mindfulness meditation. UK: Hachette. Santorelli, S. (2000). Healthy self: lessons on mindfulness in medicine. Random House. Kuyken, W., Watkins, E., Holden, E., White, K., Taylor, R. S., Byford, S., Evans, A., Radford, S., Teasdale, J., & Dalgleish, T. (2010a). How does mindfulness-based cognitive therapy work? Behaviour Research and Therapy, 48, 1105–1112. doi:10.1016/j.brat.2010.08.003. Sawyer, R. K. (2004). Creative teaching: collaborative discussion as disciplined improvisation. Educational Researcher, 33(2), 12–20. doi:10.3102/0013189X033002012. Segal, Z. V., Williams, J. M. G., & Teasdale, J. D. (2012). Mindfulness- based cognitive therapy for depression. New York: Guilford. Kuyken, W., Byford, S., Byng, R., Tim Dalgleish, T., Lewis, G., Taylor, R., Watkins, E.R., Hayes, R., Lanham, P., Kessler, D., Morant, N., Evans, A. (2010a). Study protocol for a randomized controlled trial comparing mindfulness-based cognitive therapy with maintenance anti-depressant treatment in the prevention of depressive relapse/ recurrence: the PREVENT trial. Trials, 11(99). Skidmore, D., & Murakami, K. (2012). Claiming our own space: polyph- ony in teacher–student dialogue. Linguistics and Education, 23, 200–210. doi:10.1016/j.linged.2012.02.003. van Aalderen, J. R., Breukers, W. J., Reuzel, R. P. B., & Speckens, A. E. M. (2012). The role of the teacher in mindfulness-based approaches: a qualitative study. Mindfulness. doi:10.1007/s12671-012-0162-x. Lee, Y. O. (2007). Third turn position in teacher talk: contingency and the work of teaching. Journal of Pragmatics, 39, 180–206. McCown, D., Reibel, D., & Micozzi, M. S. (2010). Teaching mindful- ness: a practical guide for clinicians and educators. New York: Springer. Williams, J. M. G., & Kabat-Zinn, J. (2011). Mindfulness: diverse per- spectives on its meaning, origins, and multiple applications at the intersection of science and dharma. Contemporary Buddhism, 12(01), 1–18. doi:10.1080/14639947.2011.564811. Peräkylä, A. (2004). Reliability and validity in research based on natu- rally occurring social interaction. In D. Silverman (Ed.), Qualitative research (pp. 283–304). London: Sage. Williams, J. References M. G., Russell, I. T., Crane, C., Russell, D., Whitaker, C., Duggan, D., Barnhofer, T., Fennell, M. J. V., Crane, R., & Silverton, S. (2010). The staying well after depression study: trial design and protocol. BMC Psychiatry, 10, 23. Piet, J., & Hougaard, E. (2011). The effect of mindfulness-based cognitive therapy for prevention of relapse in recurrent major depressive
https://openalex.org/W4386659369	https://zenodo.org/records/8340354/files/2..pdf	English	null	Prof. Dr. N. D. Patil Multidimentional Personality	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,898	Abstract The present paper shows the work of Prof Dr N.D. Patil in Maharashtra state. Dr .N.D.Patil a progressive and rational face of Maharashtra, who was a former co-operation minister, He was a leader of the peasants and workers party.Education is very important sector in rural Maharashtra. Rural education has provided opportunities for jobs in the field of Education sector, Co-operation sector, Private sector, Service sector etc. Development of Education is a key for economic growth and development. Development of Education has played very important role in achieving the goals of development in self-jobs in specific and rural development as a whole. Development of Education assists the people to improve their standard of living. The new economic policy that has been accepted by government of India in 24th July 1991. As a result education sector affected by this economic policy. Dr. N.D. Patil was devotee person he was devoted and contributed to the peasants and peasant worker. The result of Dr. N. D. Patil’s hireless contribution to the peasant and workers . He was honoured with various Awards and achievements. Key Words – Education, Development, Maharashtra, State, worker, political. peasant Introduction – Prof .Dr. N.D. Patil a progressive and rational face of Maharashtra. Narayan Dnyandeo Patil, popularly known as Dr. N. D. Patil , Was born on 15th July 1929 in Dhavali village in Sangli District (Maharashtra). Dr. N. D. Patil came from an impoverished background . His father was an illiterate farmer , as a result of his family background made him deficient and unstable . Dr. N. D. Patil was brought up very morally, and he grew up into a very respectable and well-behaved man. His childhood was no more but the ordinary life of a poor farmers son. He was well cultured and did well in his studies . his most outstanding values were kindness and love for everybody . Dr. N. D. Patil luckily managed to go to school and of course, with the help of his poor parents, he got his education successfully. Research Methodology – This Research Paper is based on Secondary data . Books, Newspaper, Magazine, Internet etc. Educational Work- Dr. N. D. Patil after completing his matriculation, he went for further education to Kolhapur at Rajaram college. Dr. N. D. Patil received an M.A. degree in Economics from Willingdon college Sangli. He also completed L.L.B. from Savitribai Phule Pune University. Prof. Dr. N. D. Patil Multidimentional Personality Dr. Sadashiv Rajaram Bankar Assistant Professor, Dept. Of. Economics, Shripatrao Kadam Mahavidyalaya Shirwal. Corresponding Author- Dr. Sadashiv Rajaram Bankar Email– sadashivbankar@gmail.com Dr. Sadashiv Rajaram Bankar Assistant Professor, Dept. Of. Economics, Shripatrao Kadam Mahavidyalaya Shirwal. Corresponding Author- Dr. Sadashiv Rajaram Bankar Email– sadashivbankar@gmail.com Abstract He has Worked as a professor at Chhatrapati Shivaji College Satara. He was also the chief and rector of the ‘Earn and Learn Scheme’. In 1960, Dr. N.D. Patil worked as a Principal at Karmaveer Bhaurao Patil college Uran- Islampur. While working at Shivaji University , he has served as member of various committies like the first advisory committee (1962). He was a member of the senate of Shivaji university Kolhapur in 1965. He was also former dean of humanism department of shivaji university Kolhapur in 1976 to 1978. he was a executive member of the shivaji university Kolhapur in 1962 to 1978. International Journal of Advance and Applied Research www.ijaar.co.in ISSN – 2347-7075 Impact Factor – 7.328 Peer Reviewed Bi-Monthly Vol.4 No.28 July-Aug 2023 International Journal of Advance and Applied Research www.ijaar.co.in ISSN – 2347-7075 Impact Factor – 7.328 Peer Reviewed Bi-Monthly Vol.4 No.28 July-Aug 2023 International Journal of Advance and Applied Research www.ijaar.co.in ISSN – 2347-7075 Patil’s hireless contribution to the peasant and workers, He was honoured with various Awards and Achievements like - in 1994 he was awarded with ‘Bhai Madhavrao Bagal Award. Swami Ramanand Teerth University Nanded honored him with D.Litt Degree in 1999. He has decorated the chairman position in National Seed Corporation Government of India in 1998 to 2000. Besides SRTMU Nanded , Dr. BAMU, Chhatrapati Sambhajinagar and Shivaji University Kolhapur also honored him with D. Litt Degree. When Dr. N.D. Patil was the co- operation minister (1978 to 1980) his son Suhas wanted to take admission in medical, but One or two marks were short. It was very easy task for him , but due to his moral upbringing he refused to take help from his maternal uncle Hon. Sharadchandraji Pawar ,then Chief Minister of Maharashtra and from his own father, Who was a Co-Operative Minister. But he stood in the queue and took admission in engineering, not in Medical stream. But the identity of uncle or father was not revealed by him. Dr. N. D. Patil’s wife Sarojmai share such a memory in her one of interview. Conclusion – Dr. N. D. Patil has played on important role in various sector like, education, politics, and in social stream also. At the same time he performed his duty to his family as the best husband and great father. Goddess Laxmi and Sarswati poured their blessings over him but his attachment with the soil root made him simple gentle and morally powerful person at the same time. He has devoted his entire life for the peasant, workers, Rayat Shikshan Sanstha and for the needy people of the society.Thus we come to know that he was a dynamic person and a multidimentional personality. Political Work – Dr. N. D. Patil was a devotee person, he has devoted and contributed to the peasants and peasant worker. At the early stage of his political career , in 1948 he joined the peasant workers party. Then he became the general secretary of Mumbai mill workers union in 1957. He was the only person in entire Political Work – Dr. N. D. Patil was a devotee person, he has devoted and contributed to the peasants and peasant worker. At the early stage of his political career , in 1948 he joined the peasant workers party. IJAAR IJAAR ISSN – 2347-7075 Then he became the general secretary of Mumbai mill workers union in 1957. He was the only person in entire 1. Principle Dr. Vijayrao Nalawde. Rayat Shiledar, ISSN: 2278-5914 first edition 22nd September 2014. 2. sarkarnama: a digital magizine 3. www. ndpatil.in 4. www.ndpatil education work.in. ISSN – 2347-7075 Karmaveer Bhaurao Patil founded Rayat Shikshan Sanstha in 1919 with a noble objective of bringing the socially and economically backward class in the mainstream by means of education. Dr. N. D. Patil proceeded to lead for over half- decade. He was attached with convening Democratic Alliance Government. He was the member of Maharashtra State Boundry Question Committee and prominent leader of Border Movement in 1999 to 2002. During his entire political career he extended his helping hand to the needy and poor workers, which shown his zest for the peasant and workers. He was a dynamic member of Rayat Shikshan Sanstha, He has been a piece of every formation held in Maharashtra right from the opportunity development. He was attached with convening Democratic Alliance Government. He was the member of Maharashtra State Boundry Question Committee and prominent leader of Border Movement in 1999 to 2002. During his entire political career he extended his helping hand to the needy and poor workers, which shown his zest for the peasant and workers. pp y p Marriage and Family – Dr . N. D. Patil got married to Sarojmai patil, she is a sister of NCP leader Hon. Sharadchandra Pawar . By Gods grace patil could see his grandchildren before he passed away. There is little that has been publicized about Dr N. D. Patil family. However ,he was a responsible father and husband. Dr. N. D. Patil was always working hard to ensure his family did not lack anything, he grew in poverty and thus did not wish his sons to grow under the same circumstances. Dr. N. D. Patil was a good family man and a strong pillar in society. He was constantly fighting and representing his people, especially those who were less privileged . That made him the favourite to everyone. Awards and Achievements – The result of Dr. N. D. Patil’s hireless contribution to the peasant and workers, He was honoured with various Awards and Achievements like - in 1994 he was awarded with ‘Bhai Madhavrao Bagal Award. Swami Ramanand Teerth University Nanded honored him with D.Litt Degree in 1999. He has decorated the chairman position in National Seed Corporation Government of India in 1998 to 2000. Besides SRTMU Nanded , Dr. BAMU, Chhatrapati Sambhajinagar and Shivaji University Kolhapur also honored him with D. Litt Degree. Awards and Achievements – The result of Dr. N. D. Vol.4 No.28 Vol.4 No.28 ISSN – 2347-7075 Objectives – 1. To Study the role of Dr. N. D. Patil in family. 2. To Study the role of Dr N.D.Patil in political sector in Maharashtra. 3. To Study the role of Dr. N.D. Patil in education development in Maharashtra. Rayat Shikshan Sanstha is one of the leading institutions in Asia. Dr . N .D. Patil 5 ISSN – 2347-7075 Indian politics who has donated his MLA remuneration to the poor and needy works during his 18 years of MLA term. He was became the MLA in 1960 to 1966, 1970 to 1976, and 1976 to 1982. Gradually he was honoured with the general secretary position in peasant workers party in 1969 to 1978 and 1985 to 2010. During the same year of span he became the minister of government, Maharashtra state in 1978 to 1980. In 1985 to 1990 he represented his region, i.e. Kolhapur constituency as a member of Maharashtra Legislative Assembly. He was attached with convening Democratic Alliance Government. He was the member of Maharashtra State Boundry Question Committee and prominent leader of Border Movement in 1999 to 2002. During his entire political career he extended his helping hand to the needy and poor workers, which shown his zest for the peasant and workers. Indian politics who has donated his MLA remuneration to the poor and needy works during his 18 years of MLA term. He was became the MLA in 1960 to 1966, 1970 to 1976, and 1976 to 1982. Gradually he was honoured with the general secretary position in peasant workers party in 1969 to 1978 and 1985 to 2010. During the same year of span he became the minister of government, Maharashtra state in 1978 to 1980. In 1985 to 1990 he represented his region, i.e. Kolhapur constituency as a member of Maharashtra Legislative Assembly. was the member of the managing council of Rayat Shikshan Sansthas Satara, from 1959 to till his death. Dr. N. D. Patil also worked as a Chairman of the Sanstha during the years from 1990 to 2008. Dr. N. D. Patil was a student of Rayat Shikshan Sanstha. Padamabhushan Dr. Karmaveer Bhaurao Patil founded Rayat Shikshan Sanstha in 1919 with a noble objective of bringing the socially and economically backward class in the mainstream by means of education. Dr. N. D. Patil proceeded to lead for over half- decade. was the member of the managing council of Rayat Shikshan Sansthas Satara, from 1959 to till his death. Dr. N. D. Patil also worked as a Chairman of the Sanstha during the years from 1990 to 2008. Dr. N. D. Patil was a student of Rayat Shikshan Sanstha. Padamabhushan Dr. Dr. Sadashiv Rajaram Bankar 6
https://openalex.org/W2605198980	https://tbiomed.biomedcentral.com/track/pdf/10.1186/s12976-017-0054-9	English	null	Establishment of a new initial dose plan for vancomycin using the generalized linear mixed model	Theoretical biology and medical modelling	2,017	cc-by	8,244	Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 DOI 10.1186/s12976-017-0054-9 Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 DOI 10.1186/s12976-017-0054-9 © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Establishment of a new initial dose plan for vancomycin using the generalized linear mixed model Yasuyuki Kourogi1,2, Kenji Ogata2, Norito Takamura2,3, Jin Tokunaga2, Nao Setoguchi2, Mitsuhiro Kai1, Emi Tanaka1 and Susumu Chiyotanda1 Correspondence: noritotaka@phoenix.ac.jp 2School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Nobeoka, Japan 3Second Department of Clinical Pharmacy, Graduate School of Clinical Pharmacy, Kyushu University of Health and Welfare, 1714-1 Yoshino, Nobeoka, Miyazaki 882-8508, Japan Abstract Background: When administering vancomycin hydrochloride (VCM), the initial dose is adjusted to ensure that the steady-state trough value (Css-trough) remains within the effective concentration range. However, the Css-trough (population mean method predicted value [PMMPV]) calculated using the population mean method (PMM) often deviate from the effective concentration range. In this study, we used the generalized linear mixed model (GLMM) for initial dose planning to create a model that accurately predicts Css-trough, and subsequently assessed its prediction accuracy. Full list of author information is available at the end of the article Methods: The study included 46 subjects whose trough values were measured after receiving VCM. We calculated the Css-trough (Bayesian estimate predicted value [BEPV]) from the Bayesian estimates of trough values. Using the patients’ medical data, we created models that predict the BEPV and selected the model with minimum information criterion (GLMM best model). We then calculated the Css-trough (GLMMPV) from the GLMM best model and compared the BEPV correlation with GLMMPV and with PMMPV. Results: The GLMM best model was {[0.977 + (males: 0.029 or females: -0.081)] × PMMPV + 0.101 × BUN/adjusted SCr – 12.899 × SCr adjusted amount}. The coefficients of determination for BEPV/GLMMPV and BEPV/PMMPV were 0.623 and 0.513, respectively. Conclusion: We demonstrated that the GLMM best model was more accurate in predicting the Css-trough than the PMM. Results: The GLMM best model was {[0.977 + (males: 0.029 or females: -0.081)] × PMMPV + 0.101 × BUN/adjusted SCr – 12.899 × SCr adjusted amount}. The coefficients of determination for BEPV/GLMMPV and BEPV/PMMPV were 0.623 and 0.513, respectively. Keywords: Vancomycin, Therapeutic drug monitoring, Initial dose planning, Generalized linear mixed model Background Vancomycin hydrochloride (VCM) is commonly used to treat methicillin-resistant Staphylococcus aureus (MRSA) infections but is known to have a narrow safe blood concentration range. To ensure safe and effective pharmacotherapy, the steady-state trough value (Css-trough) must be maintained at the effective blood concentration range of 10–20 μg/mL [1, 2]. The incidence of renal toxicity is known to increase when the Css-trough exceed 20 μg/mL [3, 4]. Therefore, the VCM dose must be adjusted using therapeutic drug monitoring (TDM) to keep the Css-trough within the effective blood concentration range (Fig. 1). This improves the cure rate of infections and the incidence of renal toxicity [5]. Because VCM has a high rate of renal excretion, the Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 2 of 16 Page 2 of 16 Several days later Several days later Immediately afterwards or several days later Yes No Initial dose planning using the PMM Taking a blood sample at trough Obtain result of measured trough value Bayesian estimation of the Css-trough Is the BEPV within the effective blood concentration range? Continue monitoring Revised dose planning Fig. 1 TDM protocol for VCM Is the BEPV within the effective blood concentration range? dose setting must be determined by the renal function of the patient. Therefore, the initial dose plan for VCM is set using the population mean method (PMM), which uses mean values for population pharmacokinetics parameters, creatinine clearance (CLcr) and weight to estimate the Css-trough (population mean method predicted value, PMMPV). Then, the estimated Css-trough is used to determine the VCM dose and infusion time and interval that would maintain an effective blood concentration range. VCM administration is commenced based on this initial dose plan and after several days the trough value is measured. We calculate the Css-trough (Bayesian estimate predicted value, BEPV) from the Bayesian estimate using the measured value and the population pharmacokinetics parameters. There is a large discrepancy between the PMMPV and BEPV in cases where the accuracy of the Css-trough determined from the initial dose plan as predicted by PMM is low. In that case, BEPV often deviates from the effective blood concentration range. Because the dose plan has to be changed in such cases, it requires sufficient time that Css-trough achieves the effective blood concentration range. As a result, the duration of infection is prolonged, the risk of adverse effects is higher. Subject extraction This study included 46 patients whose trough values were measured in 3–5 days from the start of drug administration and were selected from patients who received VCM (VANCOMYCIN HYDROCHLORIDE for I.V. Infusion “MEEK”, Meiji Seika Pharma, Tokyo, Japan) drip infusions between August 2008 and March 2015 at Chiyoda Hospital (Table 1). Exclusion criteria were receiving hemodialysis, outpatients, and under the age of 18 years. Background Therefore, in initial dose plan, it is necessary to devise to predict highly accurate Css-trough. However, because the predictive accuracy by the PMM is insufficient, a large discrepancy often exists between the PMMPV and BEPV [6]. PMM is a method of predicting the unknown Css-trough before commencing drug administration. Statistical modeling has attracted attention as a method of predicting unknown results using a formula (model) created by extracting only the necessary information from enormous amounts of data and is used in a variety of fields [7, 8]. One statistical modeling method is the generalized linear mixed model (GLMM), which is characterized by its ability to use multiple data (explanatory variables) to predict unknown outcomes (response variables). Medical facilities accumulate a variety of medical data, but when PMM is used to determine the initial VCM dose, only medical data such as the CLcr can be used. Therefore, we extracted that type of information Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 3 of 16 Page 3 of 16 that had a major impact on changes of Css-trough of VCM from patient’s medical data and created a model that predicts highly accurate Css-trough by applying that informa- tion to GLMM explanatory variable (Fig. 2). In this study, we created this model and assessed whether it could predict VCM Css-trough values that were closer to the BEPV than PMMPV, calculated using the model. Calculation of PMMPV and BEPV The PMMPV was calculated using CLcr at initial dose planning, weight, VCM dose conditions (dose, infusion time, and administration interval), and mean values for population pharmacokinetic parameters [11]. Furthermore, the calculations were based on the two-compartment model because the distribution of VCM is divided into the central compartment (blood and tissues which equilibrate rapidly with blood) and the peripheral compartment (tissues which equilibrate slowly with blood) [11]. The BEPV was calculated by CLcr at measuring trough value, weight, VCM adminis- tration conditions (dose, infusion time, and administration interval), and the estimating the patients’ pharmacokinetic parameters based on the two-compartment model using the Bayesian estimate. The PMMPV and BEPV were calculated using the TDM analytical software, Vancomycin MEEK Ver. 3.0 (Meiji Seika Pharma). CLcr mL= min ð Þ ¼ 140−Age years ð Þ ½ Weight kg ð Þ 72 SCr mg=dL ð Þ ð1BÞ CLcr mL= min ð Þ ¼ 140−Age years ð Þ ½ Weight kg ð Þ 72 SCr mg=dL ð Þ ð1BÞ ð1BÞ To calculate BEPV, the CLcr is calculated using Eq. 1 even when trough values are measured. If the SCr level has not reached 0.6 mg/dL then, it is adjusted accordingly. Calculation of CLcr The PMM requires the CLcr for calculating the VCM Css-trough values and, therefore, we first calculated the CLcr for each patient from their sex, age, weight, and serum creatinine (SCr) at initial dose planning using the Cockcroft-Gault formula (CG formula, Eq. 1) [9]. SCr was affected by the patient’s muscle mass. Therefore, because patients with low muscle mass have low SCr levels, we estimate that the CLcr calcu- lated using the CG formula would high, which overestimates the renal function. In Japan, to estimate CLcr calculated using the CG formula accurately, if the patient’s SCr is < 0.6 mg/dL, it is commonly adjusted to 0.6 mg/dL (adjusted SCr) [10]. Therefore, we used the same method here. Women CLcr mL= min ð Þ ¼ 140−Age years ð Þ ½ Weight kg ð Þ 72 SCr mg=dL ð Þ 0:85 ð1AÞ ð1AÞ Men A B Fig. 2 Changes in blood concentration after start of VCM administration. Changes in VCM blood concentration when initial dose planning is performed using (a) PMM and (b) GLMM. ●, Css-trough. The purpose of this study was to create the model that accurately predicts Css-trough at the initial VCM dose plan B A B Fig. 2 Changes in blood concentration after start of VCM administration. Changes in VCM blood concentration when initial dose planning is performed using (a) PMM and (b) GLMM. ●, Css-trough. The purpose of this study was to create the model that accurately predicts Css-trough at the initial VCM dose plan Page 4 of 16 Page 4 of 16 Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Table 1 Summary of patient characteristics Characteristic No. of patients (female/male) 46 (14/32) Age (years) 77.37 ± 8.79 Height (cm) 157.66 ± 8.59 Weight (kg) 46.66 ± 9.91 BMI (kg/m2) 18.70 ± 3.34 SCr (mg/dL) 0.82 ± 0.35 CLcr (mL/min) 45.37 ± 18.31 BUN (mg/dL) 19.15 ± 11.76 AST (IU/L) 34.70 ± 24.64 ALT (IU/L) 30.46 ± 36.94 CRP (mg/dL) 8.98 ± 7.32 The values are shown as the mean ± standard deviation The values are shown as the mean ± standard deviation CLcr mL= min ð Þ ¼ 140−Age years ð Þ ½ Weight kg ð Þ 72 SCr mg=dL ð Þ ð1BÞ Definition of difference (PMM prediction deviation quantity, PMMPDQ) between BEPV and PMMPV This study aimed to create a model that very accurately predicts the VCM Css-trough using patient medical data. We focused on the medical data having high correlation with the difference between BEPV and PMMPV. We could reduce the difference between BEPV and PMMPV by applying the medical data to the GLMM model as Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 5 of 16 Page 5 of 16 explanation variables. Thus, the difference between BEPV and PMMPV is defined as the PMM prediction deviation quantity (PMMPDQ, Eq. 2). ð2Þ PMMPDQ ¼ BEPV −PMMPV Establishing the basic model that the aimed model is based on Before creating the model, we first established the minimum configuration model (basic model) that formed its basis. Here, we attempted to use a model to very accurately predict the BEPV. Therefore, the response variable used in the model was the BEPV. Our investigation of the correlation between PMMPV and BEPV indicated that it was 0.702 (Spearman’s rank correlation coefficient). Guilford’s rule of thumb, which is commonly used as a standard for correlation coefficients, stipulates that correlation coefficients of 0–0.2, 0.2–0.4, 0.4–0.7, 0.7–0.9, and 0.9–1.0 are “almost none,” “weak,” “moderate,” “high,” and “extremely high” correlations, respectively [12]. Therefore, since PMMPV and BEPV are highly correlated, we believe that PMMPV is an appropri- ate explanatory variable for the model. Based on this, the basic model is expressed as Eq. 3 and is equivalent to the formula that predicts the Css-trough based on PMM. BEPV ¼ β1 PMMPV ð3Þ β1: PMMPV coefficient β1: PMMPV coefficient β1: PMMPV coefficient Creating the predictor model (GLMM best model) for VCM Css-trough based on GLMM Figure 3 shows the procedure we used to create the model (GLMM best model). To create a model with a high predictive accuracy, it is necessary to add effective explana- tory variables to the basic model. The use of medical data that is highly correlated to PMMPDQ in the model improves the predictive accuracy. Therefore, the explanatory Fig. 3 Procedure for creating GLMM best model for estimating Css-trough of VCM based on WAIC. Models with small WAIC have small predictive errors. We used WAIC to extract effective medical data (fixed and random effects) and determined the model that accurately predicts BEPV (GLMM best model) Fig. 3 Procedure for creating GLMM best model for estimating Css-trough of VCM based on WAIC. Models with small WAIC have small predictive errors. We used WAIC to extract effective medical data (fixed and random effects) and determined the model that accurately predicts BEPV (GLMM best model) Fig. 3 Procedure for creating GLMM best model for estimating Css-trough of VCM based on WAIC. Models with small WAIC have small predictive errors. We used WAIC to extract effective medical data (fixed and random effects) and determined the model that accurately predicts BEPV (GLMM best model) Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 6 of 16 variables added to the basic model must be medical data that is highly correlated to PMMPDQ. Extraction of medical data (fixed effect candidate 1) correlated to the difference between BEPV and PMMPV (PMMPDQ, Fig. 3, procedure 2) When using GLMM, multiple explanatory variables can be included in the model. However, the creation of a model including all the medical data we obtained would have produced an inordinate number of model types. Therefore, we first extracted the patient medical data (explanatory variables) that would be effective when added to the basic model. Thus, the two GLMM explanatory variables types were the fixed effect (equivalent to single and multiple regression analyses explanatory variables), which were elements that predict BEPV (response variable), and random effect, which were elements that chan- ged the fixed effect coefficient and the intercept values of the model. In accordance with the software specifications (Stan) used for the GLMM analysis, we used continuous variables (continuous, such as height and weight) for the fixed effect and discrete variables (qualitatively non-continuous, such as sex and all conditions) for random variables. Since the medical data that correlated highly with the PMMPDQ had an appropriate fixed effect for use in the model, we calculated the Spearman’s rank correlation coeffi- cient for all medical data and the PMMPDQ. Medical data (continuous variable) that correlated highly to the PMMPDQ (absolute value of the correlation coefficient of ≥0.2) were identified as fixed effect candidate 1. Collection of subject medical data (Fig. 3, procedure 1) To obtain medical data that can potentially be added to the basic model as explanatory variables, we collected the following subject data: Clinical findings (age, age range [10-year intervals], aged ≥75 or not, sex, height, weight, hospital days since drug administration commenced), blood test findings (total protein, serum albumin [Alb], aspartate transaminase [AST], alanine transaminase [ALT], lactate dehydrogenase [LDH], total bilirubin, blood urea nitrogen [BUN], SCr, adjusted SCr, BUN/SCr, BUN/ adjusted SCr, SCr adjustment amount, SCr adjusted or not, serum Na, serum K, serum Cl, blood glucose level, c-reactive protein [CRP], white blood cell [WBC], red blood cell [RBC], hemoglobin [Hb], hematocrit [Ht], platelet [PLT], mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concen- tration [MCHC]), and VCM administration schedule (initial dose, initial daily dose, single dose, daily dose, infusion time, and number of doses; whether doses were irregu- larly spaced; and number of days until blood concentration trough values were measured since drug administration commenced). Then, to extract effective explanatory variables from the medical data, we conducted the following investigation. Establishing the basic model that the aimed model is based on Thus, to identify medical data as potential explanatory variables that can be added to the basic model, we first obtained the subjects’ medical data. Determination of fixed effect model (Fig. 3, procedure 4) To determine the model (fixed effect model) composed of multiple fixed effects with the smallest predictive error, we created a model (Eq. 5) that included multiple fixed effect candidate 2 items in the basic model. Additionally, we calcu- lated the WAIC for all models. BEPV ¼ β1 PMMPV þ X n i¼1 βFE2i FE2i ð5Þ ð5Þ β1: PMMPV (fixed effect) coefficient, βFE2i: ith fixed effect candidate 2 coefficient, and FE2i: ith fixed effect candidate 2 (fixed effect). However, n is the upper limit of the number of medical data items corresponding to fixed effect candidate 2. Of all the models created, that with the smallest WAIC was selected as the fixed effect model. Extracting applicable medical data (random effect candidate) as random effect in the model (Fig. 3, procedure 5) Since medical data that is highly correlated to the PMMPDQ has a major effect on predictive accuracy, we calculated the intra-class correlation coefficient (ICC). Since medical data with a large ICC related to the PMMPDQ is a likely discrete variable that can be applied to the model [14], we identified the medical data (discrete variables) with the largest ICC as random effect candidates. BEPV ¼ β1 PMMPV þ βFE1 FE1 ð4Þ β1: PMMPV (fixed effect) coefficient, βFE1: each fixed effect candidate 1 (fixed effect) coefficients, and FE1: each fixed effect candidate 1 (fixed effect). WAIC is used to select the model with a high degree of predictive accuracy from multiple models and is an index for generalization errors (predictive error when making predictions using the model on unknown patients other than the subjects of this study). The smaller WAIC is, the higher predictive accuracy of a model is and it is determined to apply to unknown patients [13]. Therefore, fixed candidate 1 items used in a model made smaller WAIC than the basic model were considered an appropriate fixed effect in the GLMM best model and were designated as fixed effect candidate 2. Extraction of appropriate medical data (fixed effect candidate 2) for use as fixed effect in model (Fig. 3, procedure 3) We wanted to extract medical data from items identified as fixed effect candidate 1 that would be effective when added to the basic model (Eq. 3). Therefore, we created a model (Eq. 4) that included each fixed effect candidate 1 item as a fixed effect in the Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 7 of 16 Page 7 of 16 basic model and calculated the information criterion (Widely Applicable Information Criterion, WAIC) for each model. basic model and calculated the information criterion (Widely Applicable Information Criterion, WAIC) for each model. sessing predictive accuracy of GLMM best model Assessing predictive accuracy of GLMM best model First, we substituted the subjects’ medical data for all the explanatory variables (fixed and random effects) in the GLMM best model, which we used to calculate the Css-trough (GLMMPV). Next, to assess the predictive accuracy of the PMMPV and GLMMPV for BEPV, we set BEPV as the response variable and investigated the regression equation and coefficient of determination (R2) when the explanatory variable was either PMMPV or GLMMPV. The GLMM prediction deviation quantity (GLMMPDQ) was defined as the difference between BEPV and GLMMPV (Eq. 6). ð6Þ GLMMPDQ ¼ BEPV −GLMMPV GLMMPDQ ¼ BEPV −GLMMPV Data processing method We used the statistical analysis software R (ver. 3.2.3) and Microsoft Excel for Mac (ver. 15.22) to statistically analyze the data. We used functions included in R for our Spearman’s rank correlation coefficient calculations and Shapiro-Wilk test and the R package ICC (ver. 2.3.0) for ICC calculations. We used Excel for Mac for simple linear regression and R2 calculations. A P <0.05 was considered significant for all tests. We used R, Stan, the R packages rstan, and brms (ver. 2.9, 2.9.0-3, and 0.8.0, respect- ively) for GLMM analysis and WAIC calculations. We used the Bayesian estimation with Hamilton Monte Carlo to estimate the model coefficient. We used Rhat for the convergence test of the Bayesian estimation and determined that its convergence with Rhat was ≤1.1 [15]. The settings of brm function in brms package were as follows: Chains = 3, Iter = 30000 (100000 when random variables were included in the model), Warmup = 15000 (50000 when random variables were included in the model), Thin = 2, and Family = “normal.” When using the Shapiro-Wilk test on the BEPV, the null hypothesis that followed the normal distribution was not rejected (P = 0.19). Thus, the probability distribution for the response variable was a normal distribution. Determination of GLMM best model (Fig. 3, procedure 6) To determine the most appropriate predictive model (GLMM best model) with the smallest predictive error, we created multiple models including random effect candidate items in the fixed effect model (the model created using Procedure 4 in Fig. 3) and cal- culated WAIC for each model. Of the created models, that with the smallest WAIC was selected as the GLMM best model. Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 8 of 16 Page 8 of 16 GLMM best model construction Extraction of medical data (fixed effect candidate 1) that correlated with the difference (PMMPDQ) between BEPV and PMMPV (Fig. 3, procedure 2) Extraction of medical data (fixed effect candidate 1) that correlated with the difference (PMMPDQ) between BEPV and PMMPV (Fig. 3, procedure 2) Extraction of medical data (fixed effect candidate 1) that correlated with the differenc (PMMPDQ) between BEPV and PMMPV (Fig. 3, procedure 2) To increase the predictive accuracy of Css-trough when setting the VCM initial dose plan, the difference between BEPV and PMMPV, which is the absolute value of the PMMPDQ (Eq. 2), had to be reduced. Because the medical data items that correlated highly with the PMMPDQ had a large effect on predictive accuracy, their inclusion in the model would allow the predictive deviation to be reduced (that is, increase predict- ive accuracy). First, we investigated the correlation between the PMMPDQ and all the medical data items (continuous variables). The results indicated that 10 types of medical data with absolute correlation coefficient values with the PMMPDQ of ≥0.2 (BUN/adjusted SCr, BUN, BUN/SCr, AST, Age, SCr, CLcr, SCr amount adjusted, single dose, and daily dose, Table 2) were factors with a major effect on predictive accuracy. To determine whether they could be used as fixed effect items in the model we created, we conducted the following investigations on the 10 types of medical data as fixed effect candidate 1 items. Asterisks indicate p < 0.05 Results This study aimed to create a model (GLMM best model) that highly accurately predicts the Css-trough of the initial dose plan for VCM using patient medical data in the GLMM. Additionally, we assessed whether the VCM Css-trough values (GLMMPV) calculated using the GLMM best model were closer to the BEPV than the PMMPV. First, because we thought the medical data correlating to the differ- ence (PMMPDQ) between BEPV and PMMPV would decrease the predictive error, we extracted the medical data (fixed effect candidate 1) that was highly correlated with the PMMPDQ. Next, to extract the medical data that could be applied to the GLMM best model, we created a model (Eq. 4) including each the fixed effect can- didate 1 item in the basic model (Eq. 3). Then, we selected the medical data (fixed effect candidate 2) that made the WAIC of the model smaller (the smaller the WAIC, the higher the predictive accuracy of the model and the smaller the generalization error). Then, we created a model (Eq. 5) that included multiple fixed effect candidate 2 items in the basic model and selected the model with the smal- lest WAIC as the fixed effect model. We designated the medical data with the largest PMMPDQ-related ICC as the random effect candidate items. We created Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 9 of 16 Page 9 of 16 multiple models that included the random effect candidate items in the fixed effect model and selected the model with the smallest WAIC as the GLMM best model. Finally, in to assess the GLMMPV accuracy, we investigated the simple linear regression and R2 when the response variable was BEPV and the explanatory vari- able was either the PMMPV or the GLMMPV. Details of the results are below. Extracting medical data (fixed effect candidate 2) that was applicable as fixed effect (Fig. 3, procedure 3) To further extract the medical data that was applicable to the model from the fixed effect candidate 1 items, we created a model (Eq. 4) that included each of the fixed effect candidate 1 items in the basic model (Eq. 3) and calculated WAIC. Declines in WAIC indicate a reduced prediction error. Of the models with the fixed effect candidate 1, the one with a smaller WAIC than the basic model used the following medical data: BUN/adjusted SCr, BUN, BUN/SCr, age, and SCr ad- justed amount (Table 3). There is a high probability that these medical data items Table 2 Correlation coefficient for fixed effect candidate 1 and PMMPDQ Fixed effect candidate 1 (medical data) Correlation coefficient p-value BUN/adjusted SCr 0.398 0.006* BUN 0.372 0.011* BUN/SCr 0.332 0.024* AST 0.253 0.090 Age 0.248 0.096 SCr 0.215 0.152 CLcr -0.233 0.119 SCr adjusted amount -0.239 0.110 Single dose -0.263 0.078 Daily dose -0.279 0.060 Asterisks indicate p < 0 05 Page 10 of 16 Page 10 of 16 Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Table 3 WAIC and the Coefficients of the variables when all fixed effect candidate 1 items are included in basic model Fixed effect candidate 1 (medical data) Coefficient (l-95% CI, u-95% CI) WAIC None (Basic model) - 258.42 BUN/adjusted SCr 0.1 (0.02, 0.17) 254.52a BUN 0.09 (0.01, 0.17) 256.12a BUN/SCr 0.08 (0.00, 0.16) 256.15a AST 0.01 (-0.03, 0.05) 260.46 Age 0.04 (-0.01, 0.10) 257.51a SCr 1.13 (-1.48, 3.78) 259.73 CLcr -0.01 (-0.07, 0.04) 260.6 SCr adjusted amount -16.09 (-32.54, 0.26) 256.18a Single dose 0.00 (-0.01, 0.00) 260.6 Daily dose 0.00 (0.00, 0.00) 259.63 aWAIC of the model (Ep. 4) that included fixed effect candidate 1 item was smaller than the WAIC of the basic model (Ep. 3). Smaller WAIC indicates decreased predictive error in the model Table 3 WAIC and the Coefficients of the variables when all fixed effect candidate 1 items are aWAIC of the model (Ep. 4) that included fixed effect candidate 1 item was smaller than the WAIC of the basic model (Ep. 3). Smaller WAIC indicates decreased predictive error in the model can be used as an applicable fixed effect for the GLMM best model. Because the BUN/adjusted SCr and BUN/SCr are similar parameters, we used only the BUN/ adjusted SCr with a low WAIC value. Extracting medical data (fixed effect candidate 2) that was applicable as fixed effect (Fig. 3, procedure 3) Therefore, we used the BUN/adjusted SCr, BUN, age, and SCr adjusted amount as the fixed effect candidate 2 items in the following investigation. Fixed effect model determination (Fig. 3, procedure 4) The GLMM model can use multiple fixed effect items. Therefore, we created a model (Eq. 5) that included multiple fixed effect candidate 2 items in the basic model (Eq. 3) and calculated the WAIC. The results indicate that the WAIC of the model that simultaneously included BUN/adjusted SCr and SCr adjusted amount was the lowest (253.45, Table 4). Based on this, we designated this model as the fixed effect model. Table 4 WAIC when multiple fixed effect candidate 2 are included in the basic model Fixed effect candidate 2 (medical data) WAIC None (Basic model) 258.42 BUN/adjusted SCr and BUN 254.94 BUN/adjusted SCr and SCr adjusted amount 253.45a BUN/adjusted SCr and Age 256.26 BUN and SCr adjusted amount 254.33 BUN and Age 256.05 SCr adjusted amount and Age 256.03 BUN/adjusted SCr and BUN and SCr adjusted amount 254.58 BUN/adjusted SCr and BUN and Age 256.83 BUN/adjusted SCr and SCr adjusted amount and Age 255.25 BUN and SCr adjusted amount and Age 256.14 BUN/adjusted SCr and BUN and SCr adjusted amount and Age 256.56 aLowest WAIC above. Lower WAIC indicates decreased predictive error in the model Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 11 of 16 Page 11 of 16 Extracting medical data (random effect) that was applicable as random effect (Fig. 3, Extracting medical data (random effect) that was applicable as random effect (Fig. 3, Because medical data items (discrete variables) with a large PMMPDQ-related ICC affect the predictive accuracy considerably, it is highly likely that they can be used as applicable random effect items in the GLMM best model. Thus, we calculated the ICC for all PMMPDQ-related medical data items (Table 5). The item with the largest PMMPDQ-related ICC was sex (0.057) and, therefore, it was used as the random effect candidate item in the following investigation. GLMM best model determination (Fig. 3, procedure 6) To determine the optimum predictive model (GLMM best model) that reduces prediction error the most and includes fixed and random effects, we created mul- tiple models that included sex (random effect) in the fixed effect model (the model determined using Procedure 4 in Fig. 3) and calculated WAIC (Table 6). The results indicated that the model including sex (random effect) in the PMMPV (fixed effect) coefficient had the smallest WAIC (252.01). Therefore, we designated this model as the GLMM best model. Based on the above results, we determined the GLMM best model (Eq. 7) would predict the Css-trough with high accuracy when establishing the VCM initial dose plan. Women GLMMPV ¼ 0:977−0:081 ð Þ PMMPV þ 0:101 BUN=adjusted SCr −12:899 SCr adjusted amount ð7AÞ Men Men Assessing predictive accuracy of GLMM best model First, we investigated the simple linear regression and R2 when the response variable was BEPV, and the explanatory variable was either PMMPV or GLMMPV to assess the BEPV-related accuracy of PMMPV and GLMMPV. The single linear regression slope for PMMPV and GLMMPV was 0.902 and 1.060 respectively, and the intercept was 2.522 and -1.511 respectively. Furthermore, R2 was 0.513 and 0.623 respectively (Fig. 4). These results indicate that GLMMPV was closer to BEPV than PMMPV. Therefore, the GLMM best model may allow more accurate VCM Css-trough predictions. Men Men GLMMPV ¼ 0:977 þ 0:029 ð Þ PMMPV þ 0:101 BUN=adjusted SCr −12:899 ð7BÞ The coefficients and their credible intervals (CIs) for all explanatory variables (fixed and random effects) in the GLMM best model are shown in Table 7. Table 5 ICC for medical data (discrete variables) related to PMMPDQ Medical data (discrete variables) ICC l-95% CI u-95% CI Sex 0.057 -0.032 0.991 Adjusted SCr 0.036 -0.041 0.989 Aged 75 or above 0.023 -0.039 0.987 No. of days from start of administration to blood test for blood concentration trough -0.043 -0.065 0.484 Age group (10-year intervals) -0.044 -0.117 0.392 Irregular interval administration -0.047 -0.047 -0.037 No. of doses -0.071 -0.140 0.358 Medical data (discrete variables) with a large ICC in relation to PMMPDQ have a high likelihood of being random effect items suitable for use in the GLMM best model Page 12 of 16 Page 12 of 16 Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Table 6 WAIC when random effects are included in the fixed effect model Fixed effect including random effect (Sex) WAIC None (fixed effect model) 253.45 PMMPV 252.01a BUN/adjusted SCr 252.29 SCr adjusted amount 252.34 PMMPV and BUN/adjusted SCr 253.65 PMMPV and SCr adjusted amount 253.39 BUN/adjusted SCr and SCr adjusted amount 252.69 PMMPV and BUN/adjusted SCr and SCr adjusted amount 253.11 aLowest WAIC above. Lower WAIC indicates decreased predictive error in the model Discussion Figure 4b shows that the simple linear regression slope of BEPV and GLMMPV was closer to 1 than that of BEPV and PMMPV was (Fig. 4a, GLMMPV, 1.060 and PMMPV, 0.902). Additionally, the simple linear regression intercept of BEPV and GLMMPV was closer to 0 than that of the BEPV and PMMPV was (GLMMPV, -1.511 and PMMPV, 2.522). Additionally, because the R2 of BEPV and GLMMPV was higher than that of BEPV and PMMPV (GLMMPV, 0.623 and PMMPV, 0.513), we were able to determine that the GLMM best model created in this study predicted the VCM Css-trough with better accuracy than the PMM did for the study subjects. Table 6 shows that the WAIC of the GLMM best model (252.01) was smaller than that of the basic model (258.42, equivalent to the model that predicted Css-trough from the PMM). This indicates that generalization error is decreased in the GLMM best model. Therefore, we believe that the GLMM best model can predict the VCM Css-trough of unknown patients with greater accuracy than the PMM can. Figure 4 shows that 4.35% (2/46) of patients had PMMPDQ of ≥10 μg/mL, but none had GLMMPDQ of ≥10 μg/mL. Considering the effective blood concentration range of the VCM Css-trough, a difference of ≥10 μg/mL in Css-trough predictions would raise Table 7 All explanatory variables for the GLMM best model and their coefficient Explanatory variables Coefficient l-95% CI u-95% CI PMMPV (fixed effect) 0.977 0.314 1.960 BUN/adjusted SCr (fixed effect) 0.101 0.020 0.180 SCr adjusted amount (fixed effect) -12.899 -28.700 2.652 Sex: Female (random effect) -0.081 -1.201 0.592 Sex: Male (random effect) 0.029 -1.123 0.711 Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 13 of 16 A B Fig. 4 Comparison of correlation of PMMPV with BEPV and GLMMPV with BEPV. Fig. 4a indicates correlation of PMMPV and BEPV. Fig. 4b indicates correlation of GLMMPV and BEPV. Solid line is regression line with response variable as BEPV and explanatory variable as either PMMPV or GLMMPV. If Css-trough can be accurately predicted when establishing the initial dose plan, then all data plots will be located on the dotted line, and the solid and dotted lines will be identical. The letters a, b, and c indicate patients who showed improved accuracy in their VCM Css-trough predictions with the GLMM best model. Discussion The length of the dashed lines drawn vertically from a, b, and c indicates (a) PMMPDQ (the difference between PMMPV and BEPV) and (b) GLMMPDQ (the difference between GLMMPV and BEPV). The letters d, e, f, g and h indicate patients who showed the largest positive or negative deviation in their predictions A B B Fig. 4 Comparison of correlation of PMMPV with BEPV and GLMMPV with BEPV. Fig. 4a indicates correlation of PMMPV and BEPV. Fig. 4b indicates correlation of GLMMPV and BEPV. Solid line is regression line with response variable as BEPV and explanatory variable as either PMMPV or GLMMPV. If Css-trough can be accurately predicted when establishing the initial dose plan, then all data plots will be located on the dotted line, and the solid and dotted lines will be identical. The letters a, b, and c indicate patients who showed improved accuracy in their VCM Css-trough predictions with the GLMM best model. The length of the dashed lines drawn vertically from a, b, and c indicates (a) PMMPDQ (the difference between PMMPV and BEPV) and (b) GLMMPDQ (the difference between GLMMPV and BEPV). The letters d, e, f, g and h indicate patients who showed the largest positive or negative deviation in their predictions concerns that the drug may be less effective and cause adverse effects. However, we believe that the GLMM best model controls large prediction deviations like this. Next, we investigated patients with major improvements in predictive accuracy achieved by changing from the PMM to the GLMM best model in predicting VCM Css-trough. Patient a shown in Fig. 4 had a PMMPDQ and GLMMPDQ of 7.90 and 0.64 μg/mL (the length of the dashed lines in Fig. 4a and b, respectively). Based on this, the change from PMM to the GLMM best model allowed that predictive accuracy was improved 7.26 μg/mL (7.90–0.64 μg/mL). Similarly, patients b and c showed a 3.53 and 3.23 μg/mL (4.80–1.27 and 6.00–2.77 μg/mL) improvement, respectively. The graph of the changes in VCM blood concentration experienced by patient b (Fig. 5a) illustrates that the PMMPV deviated greatly from the BEPV (the absolute PMMPDQ value was large), which caused the blood concentration to fall outside the effective range. However, since the GLMM best model predicted the Css-trough with high accuracy, the GLMMPV was close to the BEPV (the absolute GLMMPDQ value was small) and achieved the effective blood concentration range (Fig. Discussion 5b). Similarly, if the Css-trough prediction accuracy can be increased and the achievement of an effective blood concen- tration range can be accurately predicted when establishing the initial dose plan, then a revised dose plan would be unnecessary. Furthermore, we found that the improvement in the Css-trough prediction accuracy for the patients achieved using the GLMM best model was related to high BUN/adjusted SCr values of these patients (patients a, b, and c: 71.35, 34.52, and 32.17, respectively). It has been reported that when the BUN/ SCr is > 20, the estimation of renal function (CLcr) using the CG formula results in overestimations [16]. Therefore, when establishing the initial dose plan using PMM for patients a, b, and c, we assessed the CLcr at a higher than actual level, which led to excessive VCM doses. We speculate that this further caused the deviation between the Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 14 of 16 A B Fig. 5 Blood concentration of VCM-time profiles in patient who was benefited more from GLMM best model than from PMM. Patient b in Fig. 4 showed BEPV of 13.20 μg/mL, PMMPV of 8.40 μg/mL, and GLMMPV of 11.93 μg/mL. a Patient’s PMMPV showed major differences with the BEPV, putting the level outside the effective blood concentration range. Therefore, PMM led to major disadvantages in prediction. However, the GLMMPV and BEPV in (b) were close and effective blood concentration range was reached, indicating the GLMM best model was appropriate for making these predictions A B B Fig. 5 Blood concentration of VCM-time profiles in patient who was benefited more from GLMM best model than from PMM. Patient b in Fig. 4 showed BEPV of 13.20 μg/mL, PMMPV of 8.40 μg/mL, and GLMMPV of 11.93 μg/mL. a Patient’s PMMPV showed major differences with the BEPV, putting the level outside the effective blood concentration range. Therefore, PMM led to major disadvantages in prediction. However, the GLMMPV and BEPV in (b) were close and effective blood concentration range was reached, indicating the GLMM best model was appropriate for making these predictions PMMPV and BEPV. However, when using the GLMM best model we included the BUN/adjusted SCr (fixed effect), which corrected the overestimated renal function in the CG formula and ultimately increased the VCM Css-trough prediction accuracy. Ethics approval and consent to participate pp p p This study was conducted after receiving approval from the Institutional Review Boards of Chiyoda Hospital and Kyushu University of Health and Welfare. Received: 8 September 2016 Accepted: 20 March 2017 Received: 8 September 2016 Accepted: 20 March 2017 Received: 8 September 2016 Accepted: 20 March 2017 Discussion Nevertheless, there were also cases where the GLMM best model created in this study did not improve the predictive accuracy of the Css-trough values. These patients had large deviations (PMMPDQ) between PMMPV and BEPV, and the GLMM best model did not improve the Css-trough prediction accuracy. For example, PMMPDQ and GLMMPDQ of patient d showed large positive deviations (Fig. 4a and b, 11.2 and 9.4 μg/mL, respectively), and those of patient e also showed positive deviations (Fig. 4a and b, 6.9 and 5.5 μg/mL, respectively). PMMPDQ and GLMMPDQ of patient f showed negative deviations (Fig. 4a and b, -7.0 and -8.8 μg/mL, respectively). We believe that these were likely attributable to the effect of changes in SCr after VCM administration commenced. Our results showed that SCr of patient d was 0.60 mg/dL before VCM administration, but rose to 0.85 mg/dL after VCM administration, and SCr of patient e was risen from 1.05 mg/dL to 1.52 mg/dL. We considered that whose renal functions were declined. Our results also showed that SCr of patient f was 1.20 mg/dL before VCM administration, but decreased to 0.82 mg/dL after VCM administration, which we considered that whose renal function was improved. There- fore, since the renal function of these patients changed after VCM administration started (change in CLcr), the Css-trough prediction accuracy worsened, and the abso- lute PMMPDQ and GLMMPDQ values increased. Furthermore, PMMPDQ and GLMMPDQ of patient g showed large positive deviations (Fig. 4a and b, 8.8 and 6.4 μg/mL, respectively), and those of patient h also showed large positive deviations (Fig. 4a and b, 10.3 and 8.5 μg/mL, respectively). We thought these were due mainly to involvement of hypoalbuminemia. It has been reported that kidney function is overesti- mated because of proximal tubule secretion of creatinine increases in patients with hypoalbuminemia [17]. The serum albumin levels of patients g and h were 2.4 and 2.0 g/dL, respectively. We considered that overestimation of kidney function in patients Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 Page 15 of 16 g and h led to excessive VCM doses, and rose Css-trough unexpectedly, resulting the absolute PMMPDQ and GLMMPDQ values increased. To solve these problems, new medical data must be extracted and included in the GLMM best model. Consent for publication Not applicable. Not applicable. Availability of data and materials Please contact author for data requests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Funding Funding Self-funded. Authors’ contributions l d d d YK conceptualized and developed the models for GLMM. YK, KO and NT wrote the manuscript. JT and NS contributed to the composition of the manuscript. MK and ET collected medical data and performed TDM. SC planed the clinical protocol. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Competing interests p g The authors declare that they have no competing interests. Conclusions This study demonstrated that the GLMM best model we created for use with the GLMM method in initial VCM dose planning allowed a more accurate Css-trough prediction than PMM did. The GLMM best model increased the rate of achieving the effective VCM blood concentration range. This may lead to reduce the revised dose planning requirement and increase the therapeutic effect of VCM safely. Author details 1 1Chiyoda Hospital, Social Medial Corporation Senwakai, Hyuga, Japan. 2School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Nobeoka, Japan. 3Second Department of Clinical Pharmacy, Graduate School of Clinical Pharmacy, Kyushu University of Health and Welfare, 1714-1 Yoshino, Nobeoka, Miyazaki 882-8508, Japan. Kourogi et al. Theoretical Biology and Medical Modelling (2017) 14:8 References 1. 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https://openalex.org/W4320508716	https://science.swsu.ru/jour/article/download/930/579	Russian	null	Two-step Approach to Corrosion Detection of Metal Structures Using Convolutional Neural Networks When Inspecting Industrial Facilities	Izvestiâ Ûgo-Zapadnogo gosudarstvennogo universiteta	2,022	cc-by	6,420	Оригинальная статья / Original article https://doi.org/10.21869/2223-1560-2021-25-3-152-166 https://doi.org/10.21869/2223-1560-2021-25-3-152-166 Информатика, вычислительная техника и управление / Computer science, computer engineering and control 152 Информатика, вычислительная техника и управление / Computer science, computer engineering and control 152 К. Д. Русаков 1 , А. В. Чехов 1 1 Институт проблем управления им. В.А. Трапезникова РАН, ул. Профсоюзная,д. 65, стр. 1, г. Москва 117342, Российская Феде 1 Институт проблем управления им. В.А. Трапезникова РАН, ул. Профсоюзная,д. 65, стр. 1, г. Москва 117342, Российская Федерация  e-mail: rusakov.msk@yandex.ru  e-mail: rusakov.msk@yandex.ru Двухэтапный подход к распознаванию коррозии металлических конструкций с использованием сверточных нейронных сетей в ходе проведения инспекций промышленных объектов К. Д. Русаков 1 , А. В. Чехов 1 Известия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021; 25(3): 152-166 Известия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2 Резюме Цель исследования: Распознавание коррозии на металлических конструкциях является серьезной проблемой в проведение инспекций промышленных объектов. Существующие подходы к анализу изображений имеют тенденцию использовать все изображения для распознавания участков, поврежденных коррозией, что не подходит как для структурного анализа, так как процент ошибок при таком подходе очень велик. В условиях прогнозирования коррозии по всему изображению возможны ошибки, связанные с прогнозируемой маской не на металлической конструкции. В связи с этим необходимо удалять результаты прогнозирования положитель- ного класса для участков, поврежденных коррозией, но не размещенных на металлической конструкции. Поэтому в данной работе авторы разработали двухэтапный подход к распознаванию коррозии металлических конструкций, тем самым достигая цель – повышение точности распознавания. Методы. В этой статье мы применяем две модели глубокого обучения, ориентированные на семанти- ческую сегментацию (DeepLabv3, BiSeNetV2) для обнаружения коррозии, которые работают лучше с точки зрения точности и времени и требуют меньшего количества аннотированных образцов по сравнению с другими глубокими моделями, например, Unet, FCN, Mask-RCNN. В работе предложен новый подход к распознаванию металлических участков, поврежденных коррозией, на основе совмещения двух сверточных нейронных сетей для более точного пиксельного предсказания глубинными моделями архитектуры DeepLabv3 и BiSeNetV2. Результаты. В ходе экспериментальных исследований проводился расчет точности и F1 меры с исполь- зованием моделей FCN, Unet, Mask-RCNN, а также предложенного подхода. На основании полученных результатов был сделан вывод о том, что предложенный подход состоящий в совмещении сетей DeepLabv3 и BiSeNetV2 на 3 % повышает точность и F1 меру для алгоритма Unet, на 10% точность и 2% F1 меру для Mask R-CNN и на 12 % точности и 4 % F1 меру для FCN сети. Экспериментальные резуль- таты и сравнения с реальными наборами данных подтверждают эффективность предложенной схемы даже для очень сложных изображений с множеством типов дефектов. Производительность оценивалась на базе данных, аннотированной экспертами. Заключение. В статье проведен анализ существующих решений в области распознавания металлических конструкций, поврежденных коррозией, и выявлены недостатки существующих решений, основанных либо на детекции очагов коррозии, либо на попиксельной сегментации полного изображения. В данной работе предложен новый подход к распознаванию металлических участков, поврежденных коррозией, на основе совмещения двух сверточных нейронных сетей для более точного пиксельного предсказания DeepLabv3 и BiSeNetV2. Производительность оценивается на базе данных, аннотированной экспертами по метрикам Precision и F1-score _______________________  Русаков К. Д., Чехов А. В., 2021 Двухэтапный подход к распознаванию коррозии металлических конструкций... 153 Русаков К. Д., Чехов А. В. Ключевые слова: вертикальные инспекции; семантическая сегментация; глубокое обучение; обнаруже- ние коррозии; сверточные нейронные сети. Резюме Конфликт интересов: Авторы декларируют отсутствие явных и потенциальных конфликтов инте- ресов, связанных с публикацией настоящей статьи. Для цитирования: Русаков К. Д., Чехов А. В. Двухэтапный подход к распознаванию коррозии металлических конструкций с использованием сверточных нейронных сетей в ходе проведения инспекций промышленных объектов // Известия Юго-Западного государственного университета. 2021; 25(3): 152-166. https://doi.org/ 10.21869/2223-1560-2021-25-3-152-166. Поступила в редакцию 24.08.2021 Подписана в печать 30.09.2021 Подписана в печать 30.09.2021 Опубликована 21.10.2021 Опубликована 21.10.2021 Опубликована 21.10.2021 Keywords: vertical inspections; semantic segmentation; deep learning; corrosion detection; convolutional neural networks. Abstract Purpose of research. Corrosion recognition on metal structures is a serious problem in conducting inspections of industrial facilities. Existing approaches to image analysis use all images to recognize areas damaged by corrosion, which is not suitable for structural analysis, since the percentage of errors in this approach is very large. Under condi- tions of corrosion prediction throughout the image, errors related to predictive mask not on metal structure are possi- ble. Therefore, it is necessary to delete the results of positive class prediction for areas damaged by corrosion but not placed on metal structure. Therefore, in this work, the authors have developed two-step approach for recognizing corrosion of metal structures, thereby achieving the goal of improving recognition accuracy. corrosion of metal structures, thereby achieving the goal of improving recognition accuracy. Methods. We implement two deep learning models focused on Semantic segmentation (DeepLabv3, BiSeNetV2) for corrosion detection that work better in terms of accuracy and time and require fewer annotated samples compared to other deep models, such as Unet, FCN, Mask-RCNN. A new detection approach to metal areas damaged by corro- sion, based on the combination of two convolutional neural networks for more accurate pixel prediction by depth ar- chitecture models: DeepLabv3 and BiSeNetV2. Results. Experimental studies have calculated the accuracy and F1 measures using FCN, Unet, Mask-RCNN mod- els as well as the proposed approach. Based on obtained results, it was concluded that proposed approach of com- bining DeepLabv3 and BiSeNetV2 networks increases accuracy and F1 measure for Unet algorithm by 3%, accuracy by 10% and 2% F1 measure for Mask R-CNN and by 12% accuracy and 4% F1 measure for FCN network. Experi- mental results and comparisons with real data sets confirm the effectiveness of proposed scheme even for very com- plex images with many different defects. Productivity was assessed based on data annotated by experts. Conclusion. Analyses of existing solutions in the field of recognition of metal structures damaged by corrosion is described. Shortcomings of existing solutions based either on detection of corrosion sites or on pixel segmentation of full image are identified. A new approach to the recognition of metal areas damaged by corrosion based on the com- bination of two convolutional neural networks for more accurate pixel prediction of DeepLabv3 and BiSeNetV2 is in- droduced. Production is evaluated based on data annotated by Precision and F1-score metrics experts. Konstantin D. Rusakov 1 , Anton V. Chekhov 1 1 V. A. Trapeznikov Institute of Control Sciences of Russian Academy of Sciences 65 Profsoyuznaya str., Moscow 117997, Russian Federation  e-mail: rusakov.msk@yandex.ru Keywords: vertical inspections; semantic segmentation; deep learning; corrosion detection; convolutional neural Abstract Известия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021; 25(3 Информатика, вычислительная техника и управление / Computer science, computer engineering and control 154 Conflict of interest. The authors declare the absence of obvious and potential conflicts of interest related to the publication of this article Информатика, вычислительная техника и управление / Computer science, computer engineering and control 154 Conflict of interest. The authors declare the absence of obvious and potential conflicts of interest related to the publication of this article. For citation: Rusakov K. D., Chekhov A. V. Two-step approach to corrosion detection of metal structures using convolutional neural networks when inspecting industrial facilities. Izvestiya Yugo-Zapadnogo gosudarstvennogo universiteta = Proceedings of the Southwest State University. 2021; 25(3): 152-166 (In Russ.). https://doi.org/ 10.21869/2223-1560-2021-25-3-152-166. For citation: Rusakov K. D., Chekhov A. V. Two-step approach to corrosion detection of metal structures using convolutional neural networks when inspecting industrial facilities. Izvestiya Yugo-Zapadnogo gosudarstvennogo universiteta = Proceedings of the Southwest State University. 2021; 25(3): 152-166 (In Russ.). https://doi.org/ 10.21869/2223-1560-2021-25-3-152-166. Published 21.10.2021 Received 24.08.2021 Accepted 30.09.2021 *** пример, площадь, соотношение сторон, максимальное расстояние). Таким обра- зом, нам нужна более точная классифи- кация на уровне пикселей. Распознава- ние коррозии в реальном времени необ- ходима для быстрого осмотра критиче- ски важной инфраструктуры, особенно в крупномасштабных структурах. Введение Металлические конструкции широ- ко используются в энергетике, напри- мер в линиях электропередач (ЛЭП), инфраструктуре связи (вышках сотовой связи), транспортных инфраструктурах (мосты). Ржавчина и коррозия могут привести к серьезным проблемам с без- опасностью. Следовательно, обнаруже- ние металлических дефектов является серьезной проблемой для обеспечения быстрого, эффективного, но также без- опасного осмотра, оценки и обслужива- ния инфраструктуры [1] и борьбы с яв- лениями разрушения материалов, кото- рые возникают из-за нескольких факто- ров, таких, как изменение климата и по- годные явления, а также проведения ин- спекций, в том числе с использованием БПЛА [2]. Современные подходы к ана- лизу изображений для обнаружения де- фектов основаны на ограничивающих рамках, размещаемых вокруг дефект- ных областей, чтобы помочь инженерам быстро сосредоточиться на поврежде- ниях [3]. Однако такие подходы не под- ходят для структурного анализа, по- скольку для оценки состояния дефекта требуются несколько показателей (на- В настоящее время наблюдается большой интерес к методам глубокого обучения [4, 5, 6, 7, 8], в том числе и в целях обнаружения коррозии. Авторы [9] применяли сверточные нейронные сети (CNN) для идентификации ржав- чины по полученным в качестве вход- ных данных 2D-изображениям. Другие подходы используют структуру свер- точных нейронных сетей для обнару- жения трещин в бетонной и стальной инфраструктуре [10], повреждений до- рог [11] и дефектов металлических по- верхностей в железных дорогах [12]. Авторы в работе [13] объединяют свер- точную нейронную сеть и схему объ- единения данных Байеса для обнаруже- ния трещин на атомных электростанци- ях. Основная проблема всех вышеупо- мянутых подходов состоит в том, что они используют традиционные глубин- ные модели, такие как сверточные ней- Известия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021; 25 .. 155 Двухэтапный подход к распознаванию коррозии металлических конструкций... 155 Русаков К. Д., Чехов А. В. тической сегментацией объектов, таких как люди, здания и автомобили в город- ской среде, где формы объектов пра- вильные и четко очерченные. Точно так же объекты на медицинских изображе- ниях хорошо изучены и обычно имеют четко определенные формы с некото- рыми вариациями. Дефекты, которые необходимо выявить, очень необычны, например, трещины, проколы и корро- зия [17]. Один из подходов к построе- нию архитектур сверточных нейронных сетей для семантической сегментации состоит в выборе структуры кодера и декодера. Предварительно обученная сверточная нейронная сеть, такая как ResNet, используется в качестве коди- ровщика и изучает отличительные осо- бенности данных. Затем полученный эм- беддинг (закодированный вектор) под- ключается к сети декодера, которая про- гнозирует фактические пиксели изобра- жения. Введение ронные сети, которые требуют большо- го количества аннотированных данных. В нашем случае такой сбор – сложная задача, так как аннотация должна вы- полняться экспертами на пиксельном уровне. По этой причине большинство существующих методов оценивают де- фектные области через граничные рам- ки. Кроме того, существующие подхо- ды попиксельного прогнозирования также не справляются с задачей, так как требуют большого количества изобра- жений для точной работы. Чтобы уст- ранить эти ограничения, мы используем двухэтапный подход к глубокому обу- чению, на основе нейронных сетей DeepLabv3 [14] и BiSeNetV2 [15] для семантической сегментации. Эффек- тивность конкретных методов уже под- тверждена медицинской визуализацией (например, обнаружение опухоли го- ловного мозга) [16]. Материалы и методы В условиях прогнозирования кор- розии по всему изображению, возмож- ны ошибки, связанные с прогнозируе- мой маской не на металлической кон- струкции. В связи с этим необходимо результаты прогнозирования положи- тельного класса для участков, повре- жденных коррозией, но не размещен- ных на металлической конструкции удалять. Для этих целей будем использо- вать дополнительную сверточную ней- ронную сеть для семантической сегмен- тации фона. С точки зрения визуального осмот- ра поверхность, поврежденная коррози- ей, более шероховатая, чем поверхность некоррозийного участка, и её цвет вы- глядит как оттенок между красным и коричневым. Поиск коррозийных участ- ков на металлической конструкции – это особый случай семантической сег- ментации. Во многих случаях обнару- жение коррозийных участков на метал- лических конструкциях гораздо более сложная задача по сравнению с семан- ия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021; 25(3): 152-16 Информатика, вычислительная техника и управление / Computer science, computer engineering and control 156 Рис. 1. Блок схема алгоритма детекции коррозии Fig. 1. Block diagram of corrosion detection algorithm Рис. 1. Блок схема алгоритма детекции коррозии Fig. 1. Block diagram of corrosion detection algorithm В задачах прогнозирования маски коррозии металлической конструкции очень важно находить границы повре- жденной области, а потому декодер должен работать максимально эффек- тивно. В качестве архитектуры прогно- зирования маски коррозии металличе- ской конструкции выбрана DeepLabv3+, содержащая эффективный модуль де- кодирования для уточнения результатов сегментации, в том числе по границам объектов [14] (рис. 2). сти и точности путем адаптации модели Xception [18] для задачи семантической сегментации и применения жесткой разделяемой свертки как к модулям пу- линга пространственных пирамид, так и к модулям декодера. Для создания входных изображе- ний были подготовлены 117 изображе- ний разрешением 4056х3040, на кото- рых была размечены все области, под- верженные коррозией. На первом шаге подготовки данных, в целях повышения качества все изображения были разбиты на множество мелких изображений размером 256х256. Семантическая информация зако- дирована в выходном эмбеддинге коде- ра DeepLabv3, а модуль декодера поз- воляет детально восстанавливать гра- ницы объекта. Авторы [14] демонстри- руют улучшение с точки зрения скоро- Данный шаг позволил извлечь мак- симальное количество информации из изображений высокой четкости для . 157 Русаков К. Д., Чехов А. В. Русаков К. Д., Чехов А. В. Двухэтапный подход к распознаванию коррозии металлических конструкций... 157 Двухэтапный подход к распознаванию коррозии металлических конструкций... 157 лучшего распознавания коррозии. На втором шаге была произведена следу- ющая аугментация данных: – Случайное изменение яркости и контраста на 10 %. – Оптическое искажение изображе- ния (кроволинейное). – Случайный поворот изображения на угол не более 30 градусов со сдвигом и масштабированием на 20 %. – Оптическое искажение сетки изображениях. – Случайное вертикальное и гори- зонтальное отображение изображений. – Случайные эластичные преобра- зования изображений. – Случайные эластичные преобра- зования изображений. Рис.2. Архитектура DeepLabv3+ Fig. 2. DeepLabv3+ architecture Рис.2. Архитектура DeepLabv3+ Сверточная нейронная сеть была настроена на обучение с размером паке- та 4 в течение 40 эпох. Механизм ранней остановки использовался для прекраще- ния обучения после того, как не было заметного улучшения в течение 5 после- довательных эпох. В результате общее время обучения составило 30 эпох. В конце обучения наша сеть дала среднее значение IoU 0.26 на валидации, а ко- эффициент Dice достиг 0.34 на валида- ции (рис. 4). Следующие обучающие изображе- ния были сгенерированы с использова- нием процесса аугментации и кропа (рис. 3): – 2700 изображений металлической конструкции с коррозийным поврежде- нием для обучения; – 500 изображений металлической конструкции с коррозийным поврежде- нием для валидации. Информатика, вычислительная техника и управление / Computer science, computer engineering and control 158 Рис. 3. Примеры аугментированных изображений Fig. 3. Examples of augmented images Рис. 3. Примеры аугментированных изображений Fig. 3. Examples of augmented images Рис. 3. Примеры аугментированных изображений Fig. 3. Examples of augmented images Рис. 3. Примеры аугментированных изображений Рис. 3. Примеры аугментированных изображений Fig. 3. Examples of augmented images Fig. 3. Examples of augmented images стия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021; 25(3): 1 Рис. 4. Графики качества обучения модели Fig. 4. Model Training Quality Graphs Рис. 4. Графики качества обучения модели Fig. 4. Model Training Quality Graphs Fig. 4. Model Training Quality Graphs Известия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021; 25(3): 152-166 .. 159 Русаков К. Д., Чехов А. В. Русаков К. Д., Чехов А. В. Двухэтапный подход к распознаванию коррозии металлических конструкций... 15 В задачах прогнозирования маски металлической конструкции и отделе- ния её от фона также важно находить границы самой металлической кон- струкции, при этом важна скорость прогнозирования маски, так как это влияет на общую скорость распознава- ния коррозии. представления объектов с высоким раз- решением; – семантическую ветвь с узкими каналами и глубокими слоями для по- лучения семантического контекста вы- сокого уровня. Двухэтапный подход к распознаванию коррозии металлических конструкций... 157 В основе структуры BiSeNetV2 три компонента: двухканальная магистраль в фиолетовой пунктирной рамке, слой агрегации в оранжевой пунктирной рамке и бустерная часть в желтой пунк- тирной рамке. Магистраль с двумя пу- тями имеет Детальную ветвь (синие ку- бики) и Семантическую ветвь (зеленые кубики). Между тем, числа в кубах – это отношение размера карты объектов к разрешению входных данных (рис. 5). С этой целью выбрана эффективная и действенная архитектура с хорошим компромиссом между скоростью и точ- ностью, называемая сетью двусторон- ней сегментации BiSeNetV2 [15]. Эта архитектура включает в себя: – детальную ветвь с широкими ка- налами и мелкими слоями для захвата низкоуровневых деталей и генерации Рис. 5. Архитектура BiSeNetV2 Fig. 5. BiSeNetV2 architecture В части уровня агрегации применя- ется двусторонний уровень агрегации. Кроме того, в бустерной части разрабо- таны несколько вспомогательных голов сегментации, чтобы улучшить произво- дительность сегментации без каких- либо дополнительных затрат на логиче- ский вывод. Рис. 5. Архитектура BiSeNetV2 Fig. 5. BiSeNetV2 architecture Рис. 5. Архитектура BiSeNetV2 Fig. 5. BiSeNetV2 architecture Fig. 5. BiSeNetV2 architecture сегментации, чтобы улучшить произво- дительность сегментации без каких- либо дополнительных затрат на логиче- ский вывод. В части уровня агрегации применя- ется двусторонний уровень агрегации. Кроме того, в бустерной части разрабо- таны несколько вспомогательных голов Известия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021; 25(3): 152-166 Информатика, вычислительная техника и управление / Computer science, computer engineering and control 160 Рис. 6. Аугментированное изображения для фильтрации фона Fig. 6. Augmented image for background filtering Рис. 6. Аугментированное изображения для фильтрации фона Fig. 6. Augmented image for background filtering Были подготовлены 280 изображе- ний разрешением 5472х3078, на кото- рых была размечена вся металлическая конструкция. Изображения для упро- щения разметки снимались только на фоне неба. На первом шаге подготовки данных, в целях повышения скорости все изображения были разбиты на 4 ровных части, при этом фон у каждой части менялся случайно, но, таким об- разом, чтобы внизу изображения всегда была земля, дома, лес, а вверху изобра- жения – небо. Экспериментально выяв- лено, что именно данная аугментация позволяет качественно решить задачу фильтрации фона. На втором шаге была произведена следующая аугментация данных: – случайное изменение яркости и контраста на 10%. Следующие обучающие изображе- ния были сгенерированы с использова- нием процесса аугментации и кропа: – 8462 изображения металлической конструкции для обучения; – 1256 изображений металлической конструкции для валидации. Сверточная нейронная сеть была настроена на обучение с размером па- кета 4 в течение 120 эпох. Известия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021 Двухэтапный подход к распознаванию коррозии металлических конструкций... 157 Механизм ранней остановки использовался для прекращения обучения после того, как не было заметного улучшения в течение 5 последовательных эпох. В результате общее время обучения составило 95 эпох. В конце обучения реализованная сеть дала среднее значение IoU 0.84 на валидации. Это значение указывает на то, что предсказанные маски покрыва- ют 84% истинной области коррозии в тестовом наборе. – случайный поворот изображения на угол не более 30 градусов со сдвигом и масштабированием на 20%; – случайный поворот изображения на угол не более 30 градусов со сдвигом и масштабированием на 20%; – случайное вертикальное и гори- зонтальное отображение изображений; Русаков К. Д., Чехов А. В. Русаков К. Д., Чехов А. В. . 161 Двухэтапный подход к распознаванию коррозии металлических конструкций... 161 Выводы В статье проведен анализ суще- ствующих решений в области распозна- вания металлических конструкций, по- врежденных коррозией, и выявлены не- достатки существующих решений, ос- нованных либо на детекции очагов кор- розии, либо на попиксельной сегмента- ции полного изображения. В данной работе предложен новый подход к распознаванию металлических участков, поврежденных коррозией, на основе совмещения двух сверточных нейронных сетей для более точного пиксельного предсказания. Глубинны- ми моделями были DeepLabv3 и BiSeNetV2. В данной работе предложен новый подход к распознаванию металлических участков, поврежденных коррозией, на основе совмещения двух сверточных нейронных сетей для более точного пиксельного предсказания. Глубинны- ми моделями были DeepLabv3 и BiSeNetV2. В дальнейшем исследовании пла- нируется оптимизировать [20] две от- дельные нейронные сети в одну, для ускорения вычислений. Результаты и их обсуждение На рис. 8 показана предсказанная маска из изображения в нашем тестовом наборе. На рис. 7 показана предсказанная маска из изображения в нашем тесто- вом наборе. Маска окрашена в красный цвет, чтобы выделяться на фоне метал- лической конструкции. Можно заме- тить, что прогноз учитывает сложную форму очага коррозии, выделяя только поврежденную коррозией область и иг- норируя «чистую» металлическую кон- струкцию. Вместе с тем, видно, что мо- дель ошибается на фоне изображения и прогнозирует на доме внизу коррозию. Сравнительные результаты точно- сти и F1 меры [19] показаны в табл. 1. Предлагаемый двухэтапный подход улучшает точность и показатель F1 по сравнению с существующими решени- ями попиксельной сегментации всего изображения. Метрики измерялись на валидационном наборе данных метал- лической конструкции – вышке сотовой связи. Рис. 7. Результат предсказания модели сегментации коррозии Fig. 7. Prediction result of corrosion segmentation model Рис. 7. Результат предсказания модели сегментации коррозии Fig. 7. Prediction result of corrosion segmentation model Рис. 8. Результат предсказания модели сегментации конструкции Fig. 8. Prediction result of design segmentation model Рис. 8. Результат предсказания модели сегментации конструкции Рис. 8. Результат предсказания модели сегментации конструкции Fig. 8. Prediction result of design segmentation model Fig. 8. Prediction result of design segmentation model ия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021; 25(3): 152-16 Информатика, вычислительная техника и управление / Computer science, computer engineering and control 162 Информатика, вычислительная техника и управление / Computer science, computer engineering and contr 162 Таблица 1 Table 1 Архитектура сети / Network architecture Precision F1-score FCN 0,7 0,71 Unet 0,79 0,72 Mask R-CNN 0,72 0,73 DeepLabv3 + BiSeNetV2 0,82 0,75 На первом этапе нейронная сеть DeepLabv3 прогнозирует наличие кор- розийных участков на металлической конструкции, а на втором этапе нейронная сеть BiSeNetV2 сегментиру- ет металлическую конструкцию. Экспе- риментальные результаты и сравнения с реальными наборами данных подтвер- ждают эффективность предложенной схемы даже для очень сложных изоб- ражений с множеством типов дефектов. Производительность оценивается на ба- зе данных, аннотированной экспертами. Известия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021; 25 Список литературы 1. Image processing algorithms for crack detection in welded structures via pulsed eddy current thermal imaging / Z. Liu, G. Lu, X. Liu, X. Jiang, and G. Lodewijks // IEEE Instru- mentation & Measurement Magazine. 2017. Vol. 20. N. 4. P. 34–44. 1. Image processing algorithms for crack detection in welded structures via pulsed eddy current thermal imaging / Z. Liu, G. Lu, X. Liu, X. Jiang, and G. Lodewijks // IEEE Instru- mentation & Measurement Magazine. 2017. Vol. 20. N. 4. P. 34–44. 2. An application of swarm of quadcopters for searching operations / Р.В. Мещеряков, П.М. Трефилов, А.В. Чехов, С.А. 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Fu-Chen Chen, Mohammad R Jahanshahi Nbcnn: Deep learning-based crack detec- tion using convolutional neural network and na¨ıve bayes data fusion. IEEE Transactions on Industrial Electronics, 2017, vol. 65, no. 5, pp. 4392–4400. 14. Chen LC., Zhu Y., Papandreou G., Schroff F., Adam H. Encoder-Decoder with Atrous Separable Convolution for Semantic Image Segmentation. In: Ferrari V., Hebert M., Sminchisescu C., Weiss Y. (eds) Computer Vision – ECCV 2018. ECCV 2018. Lecture Notes in Computer Science, 2018, vol 11211. Springer, Cham. 14. Chen LC., Zhu Y., Papandreou G., Schroff F., Adam H. Encoder-Decoder with Atrous Separable Convolution for Semantic Image Segmentation. In: Ferrari V., Hebert M., Sminchisescu C., Weiss Y. (eds) Computer Vision – ECCV 2018. ECCV 2018. Lecture Notes in Computer Science, 2018, vol 11211. Springer, Cham. ия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021; 25(3): 152-16 форматика, вычислительная техника и управление / Computer science, computer engineering and contr 15. Yu Changqian, Gao Changxin, Wang Jingbo, Yu Gang, Shen Chunhua, Sang Nong. BiSeNet V2: Bilateral Network with Guided Aggregation for Real-time Semantic Segmenta- tion. 2020. 15. Yu Changqian, Gao Changxin, Wang Jingbo, Yu Gang, Shen Chunhua, Sang Nong. BiSeNet V2: Bilateral Network with Guided Aggregation for Real-time Semantic Segmenta- tion. Информация об авторах / Information about the Authors Русаков Константин Дмитриевич, научный сотрудник, Институт проблем управления им. В.А. Трапезникова РАН, г. Москва, Российская Федерация, e-mail: rusakov.msk@yandex.ru, ORCID: https://orcid.org/ 0000-0002-1895-8001 Konstantin D. Rusakov, Research Associate, V. A. Trapeznikov Institute of Control Sciences of Russian Academy of Sciences, Moscow, Russian Federation, e-mail: rusakov.msk@yandex.ru, ORCID: https://orcid.org/ 0000-0002-1895-8001 Konstantin D. Rusakov, Research Associate, V. A. Trapeznikov Institute of Control Sciences of Russian Academy of Sciences, Moscow, Russian Federation, e-mail: rusakov.msk@yandex.ru, ORCID: https://orcid.org/ 0000-0002-1895-8001 Чехов Антон Валерьевич, ведущий эксперт, Институт проблем управления им. В.А. Трапезникова РАН, г. Москва, Российская Федерация, e-mail: achekhov@gmail.com Чехов Антон Валерьевич, ведущий эксперт, Институт проблем управления им. В.А. Трапезникова РАН, г. Москва, Российская Федерация, e-mail: achekhov@gmail.com Anton V. Chekhov, Leading Expert, V. A. Trapeznikov Institute of Control Sciences of Russian Academy of Sciences, Moscow, Russian Federation, e-mail: achekhov@gmail.com Известия Юго-Западного государственного университета / Proceedings of the Southwest State University. 2021
https://openalex.org/W4281384310	http://vestnik.vsmu.by/downloads/2022/vestnikVGMU-21-2-2022.pdf	Russian	null	null	Vestnik Vitebskogo gosudarstvennogo medicinskogo universiteta	2,022	cc-by	67,135	Главный редактор: Щастный Анатолий Тадеушевич – д.м.н., профессор. Редакционный совет: Адаскевич В.П. – д.м.н., профессор, г.Витебск, Беларусь; Алексеенко Ю.В. – к.м.н., доцент, г.Витебск, Беларусь; Бяловский Ю.Ю. – д.м.н., профессор, г. Рязань, Россия; Власов Т.Д. – д.м.н., профессор, г.С.-Петербург, Россия; Генералов И.И. – д.м.н., профессор, г.Витебск, Беларусь; Клочкова С.В. – д.м.н., профессор, г.Москва, Россия; Львов А.Н. – д.м.н., профессор, г.Москва, Россия; Мяделец О.Д. – д.м.н., профессор, г.Витебск, Беларусь; Никитюк Д.Б. – д.м.н., профессор, г.Москва, Россия; Осочук С.С. – д.м.н., профессор, г.Витебск, Беларусь; Пискун Д.В. – к.м.н., г.Херизау, Швейцария; Рубникович С.П. – д.м.н., профессор, г.Минск, Беларусь; Сиврев Д.П. – д.м.н., профессор, г.Стара Загора, Болгария; Таганович А.Д. – д.м.н., профессор, г.Минск, Беларусь; Юпатов Г.И. – д.м.н., профессор, г.Витебск, Беларусь. Том 21 №2 (март-апрель) ISSN 1607-9906 (print), ISSN 2312-4156 (online) Журнал является членом Cross Ref и Ассоциации научных редакторов и издателей (АНРИ) Адрес редакции: 210009, г. Витебск, пр-т Фрунзе, 27, тел. +375 (212) 33-90-95, http://vestnik.vsmu.by, e-mail: vestnik.vsmu@tut.by Журнал зарегистрирован в Министерстве информации Республики Беларусь, свидетельство № 108 от 22.04.2009 г. Рецензируемый научно-практический журнал Vestnik of Vitebsk State Medical University Peer-reviewed scientific-practical journal ISSN 1607-9906 (print) ISSN 2312-4156 (online) ISSN 1607-9906 (print) ISSN 2312-4156 (online) ISSN 1607-9906 (print) ISSN 2312-4156 (online) Рецензируемый научно-практический журнал Vestnik of Vitebsk State Medical University Peer-reviewed scientific-practical journal Редакционная коллегия: Редакционный совет: Адаскевич В.П. – д.м.н., профессор, г.Витебск, Беларусь; Алексеенко Ю.В. – к.м.н., доцент, г.Витебск, Беларусь; Бяловский Ю.Ю. – д.м.н., профессор, г. Рязань, Россия; Власов Т.Д. – д.м.н., профессор, г.С.-Петербург, Россия; Генералов И.И. – д.м.н., профессор, г.Витебск, Беларусь; Клочкова С.В. – д.м.н., профессор, г.Москва, Россия; Львов А.Н. – д.м.н., профессор, г.Москва, Россия; Мяделец О.Д. – д.м.н., профессор, г.Витебск, Беларусь; Никитюк Д.Б. – д.м.н., профессор, г.Москва, Россия; Осочук С.С. – д.м.н., профессор, г.Витебск, Беларусь; Пискун Д.В. – к.м.н., г.Херизау, Швейцария; Рубникович С.П. – д.м.н., профессор, г.Минск, Беларусь; Сиврев Д.П. – д.м.н., профессор, г.Стара Загора, Болгария; Таганович А.Д. – д.м.н., профессор, г.Минск, Беларусь; Юпатов Г.И. – д.м.н., профессор, г.Витебск, Беларусь. Алексанин С.С. – д.м.н., профессор, г.Санкт-Петербург, Россия; Бекиш В.Я. – д.м.н., профессор, г.Витебск, Беларусь; Глушанко В.С. – д.м.н., профессор, г.Витебск, Беларусь; Городецкая И.В. – д.м.н., профессор, г.Витебск, Беларусь; Власов Т.Д. – д.м.н., профессор, г.С.-Петербург, Россия; енералов И.И. – д.м.н., профессор, г.Витебск, Беларусь; Жданова О.Б. – д.б.н., профессор, г.Киров, Россия; Жебентяев А.И. – д.ф.н., профессор, г.Витебск, Беларусь; лочкова С.В. – д.м.н., профессор, г.Москва, Россия; Львов А.Н. – д.м.н., профессор, г.Москва, Россия; Карпук И.Ю. – д.м.н., профессор, г.Витебск, Беларусь; Мяделец О.Д. – д.м.н., профессор, г.Витебск, Беларусь; озловский В.И. – д.м.н., профессор, г.Витебск, Беларусь; Коневалова Н.Ю. – зам. главного редактора, д.б.н., профессор Никитюк Д.Б. – д.м.н., профессор, г.Москва, Россия; Осочук С.С. – д.м.н., профессор, г.Витебск, Беларусь г.Витебск, Беларусь; Конорев М.Р. – д.м.н., профессор, г.Витебск, Беларусь; Пискун Д.В. – к.м.н., г.Херизау, Швейцария; Луд Н.Г. – д.м.н., профессор, г.Витебск, Беларусь; Рубникович С.П. – д.м.н., профессор, г.Минск, Беларусь; Сиврев Д.П. – д.м.н., профессор, г.Стара Загора, Болгария; Лысенко О.В. – д.м.н., профессор, г.Витебск, Беларусь; Таганович А.Д. – д.м.н., профессор, г.Минск, Беларусь; Наркевич И.А. – д.ф.н., профессор, г.Санкт-Петербург, Россия; Юпатов Г.И. – д.м.н., профессор, г.Витебск, Беларусь. Пиманов С.И. – д.м.н., профессор, г.Витебск, Беларусь; Семенов В.М. – д.м.н., профессор, г.Витебск, Беларусь; Снежицкий В.А. – д.м.н., профессор, г.Гродно, Беларусь Сучков И.А. – д.м.н., доцент, г.Рязань, Россия; Усович А.К. – д.м.н., профессор, г.Витебск, Беларусь. Vol. 21 No. 2 (March-April) ISSN 1607-9906 (print), ISSN 2312-4156 (online) Peer-reviewed scientiﬁ c-practical journal. Founded in 2002. Frequency – 6 times per year. The founder and publisher – Educational Establishment «Vitebsk State Order of Peoples’ Friendship Medical Univers Editor-in-chief: Shchastniy Anatoliy Tadeushevich – PhD, MD (Medicine), professor. Editorial council: Adaskevich V.P. – PhD, MD (Medicine), professor, Belarus; Alekseyenko Yu.V. – PhD (Medicine), associate professor, Belaru Byalovsky Yu.Yu. – PhD, MD (Medicine), professor, Russia; Vlasov T.D. – PhD, MD (Medicine), professor, Russia; Generalov I.I. – PhD, MD (Medicine), professor, Belarus; Klochkova S.V. – PhD, MD (Medicine), professor, Russia; Lvov A.N. – PhD, MD (Medicine), professor, Russia; Myadelets O.D. – PhD, MD (Medicine), professor, Belarus; Nikityuk D.B. – PhD, MD (Medicine), professor, Russia; Osochuk S.S. – PhD, MD (Medicine), professor, Belarus; Piskun D.V. – PhD (Medicine), Switzerland; Rubnikovich S.P. – PhD, MD (Medicine), professor, Belarus; Sivrev D.P. – PhD, MD (Medicine), professor, Bulgaria; Tahanovich A.D. – PhD, MD (Medicine), professor, Belarus; Yupatov G.I. – PhD, MD (Medicine), professor, Belarus. Editorial oﬃ ce: 210009, Vitebsk, Frunze ave., 27, phone: (0212) 33-90-95, http://vestnik.vsmu.by, e-mail: vestnik.vsmu@tut.by The journal is registered in the Ministry of Information of the Republic of Belarus, certiﬁ cate of registration No 108, dated 22.04.2009. © Vitebsk State Order of Peoples’ Friendship Medical University, 2022 Editorial board: Editorial council: Adaskevich V.P. – PhD, MD (Medicine), professor, Belarus; Alekseyenko Yu.V. – PhD (Medicine), associate professor, Belarus Byalovsky Yu.Yu. – PhD, MD (Medicine), professor, Russia; Vlasov T.D. – PhD, MD (Medicine), professor, Russia; Generalov I.I. – PhD, MD (Medicine), professor, Belarus; Klochkova S.V. – PhD, MD (Medicine), professor, Russia; Lvov A.N. – PhD, MD (Medicine), professor, Russia; Myadelets O.D. – PhD, MD (Medicine), professor, Belarus; Nikityuk D.B. – PhD, MD (Medicine), professor, Russia; Osochuk S.S. – PhD, MD (Medicine), professor, Belarus; Piskun D.V. – PhD (Medicine), Switzerland; Rubnikovich S.P. – PhD, MD (Medicine), professor, Belarus; Sivrev D.P. – PhD, MD (Medicine), professor, Bulgaria; Tahanovich A.D. – PhD, MD (Medicine), professor, Belarus; Yupatov G.I. – PhD, MD (Medicine), professor, Belarus. Секретариат: Секретариат: Бебешко И.А.; Есипова Л.В.; Кадушко Р.В., к.филол.н., доцент; Ксениди И.Д., Лапусева И.Н.; Флоряну И.А., к.филол.н., доцент. : ; Есипова Л.В.; Кадушко Р.В., к.филол.н., доцент; Ксениди И.Д., Лапусева И.Н.; Флоряну И.А., к.филол.н., доцент. Адрес редакции: 210009, г. Витебск, пр-т Фрунзе, 27, тел. +375 (212) 33-90-95, http://vestnik.vsmu.by, e-mail: vestnik.vsmu@tut.by Журнал зарегистрирован в Министерстве информации Республики Беларусь, свидетельство № 108 от 22.04.2009 г. © Витебский государственный ордена Дружбы народов медицинский университет, 2022 Ministry of Public Health of the Republic of Belarus Vitebsk State Medical University Ministry of Public Health of the Republic of Belarus Vitebsk State Medical University Editorial council: Editorial board: Aleksanin S.S. – PhD, MD (Medicine), professor, Russia; Bekish V.Ya. – PhD, MD (Medicine), professor, Belarus; Glushanko V.S. – PhD, MD (Medicine), professor, Belarus; Gorodetskaya I.V. – PhD, MD (Medicine), professor, Belarus; Zhdanova O.B. – PhD, MD (Biology), professor, Russia; Zhebentyaev A.I. – PhD, MD (Pharmacy), professor, Belarus; Karpuk I.Y. – PhD, MD (Medicine), professor, Belarus; Kozlovskiy V.I. – PhD, MD (Medicine), professor, Belarus; Konevalova N.Yu. – PhD, MD (Biology), professor, deputy editor-in-chief, Belarus; Konorev M.R. – PhD, MD (Medicine), professor, Belarus; Lud N.G. – PhD, MD (Medicine), professor, Belarus; Lysenko O.V – PhD, MD (Medicine), professor, Belarus; Narkevich I.A. – PhD, MD (Pharmacy), professor, Russia; Pimanov S.I. – PhD, MD (Medicine), professor, Belarus; Semenov V.M. – PhD, MD (Medicine), professor, Belarus; Snezhitskiy V.A. – PhD, MD (Medicine), professor, Belarus Suchkov I.A. – PhD, MD (Medicine), associate professor, Russia; Usovich А.К. – PhD, MD (Medicine), professor, Belarus. Editorial board: Aleksanin S.S. – PhD, MD (Medicine), professor, Russia; Bekish V.Ya. – PhD, MD (Medicine), professor, Belarus; Glushanko V.S. – PhD, MD (Medicine), professor, Belarus; Gorodetskaya I.V. – PhD, MD (Medicine), professor, Belarus; Zhdanova O.B. – PhD, MD (Biology), professor, Russia; Zhebentyaev A.I. – PhD, MD (Pharmacy), professor, Belarus; Karpuk I.Y. – PhD, MD (Medicine), professor, Belarus; Kozlovskiy V.I. – PhD, MD (Medicine), professor, Belarus; Konevalova N.Yu. – PhD, MD (Biology), professor, deputy editor-in-chief, Belarus; Konorev M.R. – PhD, MD (Medicine), professor, Belarus; Lud N.G. – PhD, MD (Medicine), professor, Belarus; Lysenko O.V – PhD, MD (Medicine), professor, Belarus; Narkevich I.A. – PhD, MD (Pharmacy), professor, Russia; Pimanov S.I. – PhD, MD (Medicine), professor, Belarus; Semenov V.M. – PhD, MD (Medicine), professor, Belarus; Snezhitskiy V.A. – PhD, MD (Medicine), professor, Belarus Suchkov I.A. – PhD, MD (Medicine), associate professor, Russi Usovich А.К. – PhD, MD (Medicine), professor, Belarus. Nikityuk D.B. – PhD, MD (Medicine), professor, Russia; Osochuk S.S. – PhD, MD (Medicine), professor, Belarus; Piskun D.V. – PhD (Medicine), Switzerland; Secretariate: Bebeshko I.A.; Esipova L.V.; Kadushko R.V., PhD (Philology), associate professor; Ksenidi I.D.; Lapuseva I.N.; Floryanu I.А., PhD (Philology), associate professor. Editorial oﬃ ce: 210009, Vitebsk, Frunze ave., 27, phone: (0212) 33-90-95, http://vestnik.vsmu.by, e-mail: vestnik.vsmu@tut.by The journal is registered in the Ministry of Information of the Republic of Belarus, certiﬁ cate of registration No 108, dated 22.04.2009. Public health and health service 63 Editorial council: © Vitebsk State Order of Peoples’ Friendship Medical University, 2022 The journal is registered in the Ministry of Information of the Republic of Belarus, certiﬁ cate of registration No 108, dated 22.04.2009. © Vitebsk State Order of Peoples’ Friendship Medical University, 2022 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Review Fliuryk S.V., Dremza I.K. The mechanisms of mitochondrial neuron dysfunction under the inﬂ uence of arsenic and aluminium (review) Rzheussky S.E. Silver nanoparticles in medicine Dikareva E.A., Pimanov S.I., Makarenko E.V., Lahutchau V.V., Kukharau A.V. Adherence to therapy in rheumatology practice. Literature review Physiology Kuzhel O.P. Fish oil prevents the development of arterial hypotension in rats with post-traumatic stress disorder Radiodiagnosis and radiotherapy Zherko O.M. Diagnostic eﬀ ectiveness of a new method for determining kidney damage in chronic heart failure Public health and health service Tsygankov A.M., Lyatos I.A. The analysis of the disease incidence and labor losses in the Armed Forces of the Republic of Belarus from 2017 to 2020 Gavrilik A.A. The system of indicators and indices of foreign citizens’ conﬁ dence in the professional activity of Belarusian doctors (on the example of Grodno region) Dentistry Tserakhava T.N., Pohodenko-Chudakova I.O., Nijiati N., Yudina O.A. Comparison of the results of morphological examination at diﬀ erent periods of laser therapy integration into complex treatment for experimental periostitis Обзор Флюрик С.В., Дремза И.К. Механизмы митохондриальной дисфункции нейронов при воздействии мышьяка и алюминия (обзор) Ржеусский С.Э. Наночастицы серебра в медицине Дикарева Е.А., Пиманов С.И., Макаренко Е.В., Лагутчев В.В., Кухарев А.В. Приверженность терапии в ревматологической практике. Обзор литературы Физиология Кужель О.П. Рыбий жир предупреждает развитие артериальной гипотензии у крыс с посттравматическим стрессовым расстройством Лучевая диагностика, лучевая терапия Жерко О.М. Диагностическая эффективность нового метода определения повреждения почек при хронической сердечной недостаточности Общественное здоровье и здравоохранение Цыганков А.М., Лятос И.А. Анализ заболеваемости и трудопотерь в Вооруженных Силах Республики Беларусь за 2017-2020 годы Гаврилик А.А. Система показателей и индексов доверия иностранных граждан к профессиональной деятельности белорусских врачей (на примере Гродненской области) Стоматология Терехова Т. Н., Походенько-Чудакова И.О., Ницзяти Н., Юдина О.А. Сравнительное сопоставление результатов морфологического исследования при различных сроках интеграции лазеротерапии в комлексное лечение экспериментального периостита СОДЕРЖАНИЕ CONTENTS 7 55 70 45 15 25 35 63 Стоматология 5 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Педагогика и психология высшей школы Церковский А.Л., Гапова О.И., Скорикова Е.А., Петрович С.А. К вопросу о коммуникативных позициях в общении студентов лечебного факультета ВГМУ Голюченко О.А., Жильцов И.В., Скребло Е.И., Адаменко Г.П., Колядко Е.И. Разработка учебно-методического комплекса по учебной дисциплине «Доказательная и персонализированная медицина. Доказательная база диагностики и лечения Covid-19» (на английском языке) для студентов медицинского университета Василенко Н.И., Кадушко Р.В., Мясоедов А.М., Погоцкий А.К. Патриоты-медики Витебского государственного медицинского института в годы Великой Отечественной войны Юбилей К юбилею Олега Данииловича Мядельца. 70 лет со дня рождения Некролог Новости Правила для авторов Pedagogics and psychology of higher school Tserkovsky A.L., Gapova O.I., Skorikova E.A., Petrovich S.A. On the question of communicative positions in communication of VSMU medical students Haliuchenka V.A., Zhyltsou I.V., Skreblo Y.I., Adamenko G.P., Kaliadka Y.I. The development of educational and methodological complex in the discipline «Evidence-based and personalized medicine. Evidence base for diagnosing and treatment of Сovid-19» (in the English language) for the students of the medical university Vasilenko N.I., Kadushko R.V., Myasoedov A.M., Pogotsky A.K. Patriots-doctors of Vitebsk State Medical Institute during the years of the Great Patriotic War Jubilee To the 70th anniversary of O.D. Myadelets Obituary News Instructions for authors 85 94 102 79 100 103 108 Педагогика и психология высшей школы Церковский А.Л., Гапова О.И., Скорикова Е.А., Петрович С.А. К вопросу о коммуникативных позициях в общении студентов лечебного факультета ВГМУ Голюченко О.А., Жильцов И.В., Скребло Е.И., Адаменко Г.П., Колядко Е.И. Разработка учебно-методического комплекса по учебной дисциплине «Доказательная и персонализированная медицина. Доказательная база диагностики и лечения Covid-19» (на английском языке) для студентов медицинского университета Василенко Н.И., Кадушко Р.В., Мясоедов А.М., Погоцкий А.К. Патриоты-медики Витебского государственного медицинского института в годы Великой Отечественной войны Юбилей К юбилею Олега Данииловича Мядельца. 70 лет со дня рождения Некролог Новости Правила для авторов Pedagogics and psychology of higher school Tserkovsky A.L., Gapova O.I., Skorikova E.A., Petrovich S.A. On the question of communicative positions in communication of VSMU medical students Haliuchenka V.A., Zhyltsou I.V., Skreblo Y.I., Adamenko G.P., Kaliadka Y.I. The development of educational and methodological complex in the discipline «Evidence-based and personalized medicine. Evidence base for diagnosing and treatment of Сovid-19» (in the English language) for the students of the medical university Vasilenko N.I., Kadushko R.V., Myasoedov A.M., Pogotsky A.K. Стоматология Patriots-doctors of Vitebsk State Medical Institute during the years of the Great Patriotic War Jubilee To the 70th anniversary of O.D. Myadelets Obituary News Instructions for authors 85 94 102 79 100 103 108 85 94 THE MECHANISMS OF MITOCHONDRIAL NEURON DYSFUNCTION UNDER THE INFLUENCE OF ARSENIC AND ALUMINIUM (REVIEW) Grodno State Medical University, Grodno, Republic of Belarus Vestnik VGMU. 2022;21(2):7-14. Grodno State Medical University, Grodno, Republic of Belarus Vestnik VGMU. 2022;21(2):7-14. Резюме. Резюме. Воздействие нейротропных химических веществ (алюминий, мышьяк и др.) в результате загрязнения объектов окружающей среды может вызывать нарушение биоэнергетики нервных клеток. Цель исследования – анализ и обобщение данных литературы о механизмах воздействия мышьяка и алюминия на структуру и функции мито- хондрий нейронов. Источники данных: литературные источники, отражающие механизмы воздействия данных нейротоксикантов на митохондрии нейронов. р р р Методы. Основой данного исследования стал обзор литературы по данной теме. Методы. Основой данного исследования стал обзор литературы по данной теме. Результаты. Воздействие соединений мышьяка на нервные клетки вызывает митохондриальную дисфункцию за счет активации окислительного стресса, повышения внутриклеточного уровня Ca2+, снижения митохондриально- го мембранного потенциала и уровня кальпаина 1, а соединения алюминия увеличивают образование активных форм кислорода (АФК) и нарушают активность цитохром-с-оксидазы и энергообразующей функции митохон- дрий в различных типах нейронов. Дисфункция митохондрий, вызванная воздействием этих металлов, сопро- вождается снижением ресинтеза АТФ и активацией окислительного стресса, что в свою очередь еще больше снижает энергообразование в митохондриях по механизму порочного круга «CIRCULUS VITIOSUS» Заключение. Представленная информация углубляет знания о механизмах нарушений биоэнергетики нейронов при воздействии соединений мышьяка и алюминия, что является основой для дальнейших исследований с целью разработки эффективных методов профилактики и терапии при острых и хронических отравлениях соединения- ми мышьяка и алюминия и внедрения полученных результатов в практическое здравоохранение. Ключевые слова: митохондрии, биоэнергетика нейронов, нервная система, нейротоксичность мышьяка и алю- миния, электронтранспортная цепь, митохондрии. е од . Ос о о да о о сс едо а с а обзор ера ур о да о е е. Результаты. Воздействие соединений мышьяка на нервные клетки вызывает митохондриальную дисфункцию за счет активации окислительного стресса, повышения внутриклеточного уровня Ca2+, снижения митохондриально- го мембранного потенциала и уровня кальпаина 1, а соединения алюминия увеличивают образование активных форм кислорода (АФК) и нарушают активность цитохром-с-оксидазы и энергообразующей функции митохон- дрий в различных типах нейронов. Дисфункция митохондрий, вызванная воздействием этих металлов, сопро- вождается снижением ресинтеза АТФ и активацией окислительного стресса, что в свою очередь еще больше снижает энергообразование в митохондриях по механизму порочного круга «CIRCULUS VITIOSUS» З П ф б й б й МЕХАНИЗМЫ МИТОХОНДРИАЛЬНОЙ ДИСФУНКЦИИ НЕЙРОНОВ ПРИ ВОЗДЕЙСТВИИ МЫШЬЯКА И АЛЮМИНИЯ (ОБЗОР) Гродненский государственный медицинский университет, г. Гродно, Республика Беларусь Вестник ВГМУ. – 2022. – Том 21, №2. – С. 7-14. Некролог 6 ОБЗОР DOI: https://doi.org/10.22263/2312-4156.2022.2.7 Abstract. Exposure to neurotropic chemicals (aluminum, arsenic, etc.) as a result of pollution of environmental objects can cause disruption of the bioenergetics of nerve cells. Objectives. To analyze and summarize the literature data on the mechanisms of the effects of arsenic and aluminum on the structure and functions of neuronal mitochondria. Sources of data: literature sources reflecting the mechanisms of the influence of these neurotoxicants on neuronal mitochondria. Methods. The basis of this study was the review of literature on this topic. Results. The influence of arsenic compounds on nerve cells causes mitochondrial dysfunction due to the activation of oxidative stress, an increase in the intracellular level of Ca2+, a decrease in the mitochondrial membrane potential and the level of calpain 1, but aluminum compounds increase the formation of reactive oxygen species (ROS) and disrupt the activity of cytochrome c-oxidase and the energy-producing function of mitochondria in various types of neurons. Mitochondrial dysfunction, caused when exposed to these metals is accompanied by the decrease in ROS resynthesis and 7 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 the activation of oxidative stress, which in its turn decreases energy generation in mitochondria still to a greater extent according to the mechanism of a vicious circle («CIRCULUS VITIOSUS»). the activation of oxidative stress, which in its turn decreases energy generation in mitochondria still to a greater extent according to the mechanism of a vicious circle («CIRCULUS VITIOSUS»). Conclusions. The presented information deepens our knowledge about the mechanisms of neuronal bioenergetics disorders under the influence of arsenic and aluminum compounds, which is the basis for further research in order to develop effective methods of prevention, detoxification and antioxidant therapy for acute and chronic arsenic and aluminum poisoning and to implement the results obtained in practical healthcare. Key words: mitochondria, neuronal bioenergetics, nervous system, arsenic and aluminum neurotoxicity, el chain, mitochondria. Нарушение структурно-функциональных свойств митохондрий нейронов при токсическом воздействии химических веществ (солей тяже- лых металлов и др.) сопровождается развитием энергодефицита и гибелью нейронов по некроти- ческому, апоптотическому либо аутофагическому механизму и в дальнейшем приводит к нейроде- генерации, что требует разработки эффективных мер профилактики и лечения. Результаты и обсуждение Митохондрии играют ключевую роль в физиологических и патологических процессах в клетке, включая энергетический обмен, кальци- евый гомеостаз, биосинтез липидов и апоптоз [1]. Основной функцией митохондрий является синтез АТФ, что достигается путем сопряже- ния окисления и фосфорилирования. Окисление энергетических субстратов осуществляется в ма- триксе митохондрий и сопряжено с образованием НАДН+, который, в свою очередь, передает элек- троны и протоны в электрон-транспортную цепь (ЭТЦ), локализованную во внутренней мембране митохондрий. ЭТЦ состоит из четырех основных металл-содержащих белковых комплексов пере- носчиков электронов и протонов (I-IV). Электро- ны переносятся продольно мембране от I к IV комплексу и затем на молекулярный кислород, а протоны перемещаются в поперечном направ- лении в межмембранное пространство, что при- водит к формированию протонного градиента, энергия которого используется в АТФ-синтазном комплексе (комплекс V) для ресинтеза АТФ. Материал и методы р фу поскольку ядерная ДНК кодирует многочисленные митохондриальные белки. повреждение ядерной и незащищенной гистона- ми митохондриальной ДНК (рис. 1). Ранее было показано, что внутриклеточное накопление алю- миния быстро приводит к дозозависимому уве- личению разрывов двойных цепей ДНК, измене- нию числа хромосом (анеуплоидии) и остановке клеточного цикла в фазе G2/M [2], что, в ко- нечном итоге, и будет определять дальнейшую судьбу нейрона: его выживание либо гибель по некротическому, апоптотическому или аутофа- гическому механизму. Материал и методы Критерии приемлемости: исследование проводилось на основании сбора литературы. Рассматривались англо- и русскоязычные жур- нальные публикации, соответствующие заявлен- ной тематике. Источники информации. В качестве источ- ников информации использовались базы данных ресурсов PubMed, ЭБС «Лань» системы автома- тизации библиотек «Ирбис» с датами охвата с 01.01.2016 по 31.12.2021 гг. Поиск. Электронный поиск в указанных базах данных осуществлялся с использованием ключевых слов, представляющих из себя назва- ние того или иного процесса, происходящего в митохондриях. Задавался временной промежуток поиска (с 2016 по 2021 гг.), после чего давалась команда поиска. ( ) р В процессе функционирования ЭТЦ ми- тохондрий за счет так называемой «утечки» электронов на молекулярный кислород образу- ется побочный продукт – супероксид-анион ра- дикал (О2 ·-), который нестабилен и с участием митохондриальных супероксиддисмутаз (СОД) быстро превращается в пероксид водорода (H2O2), который, в свою очередь, в цитоплазме клетки трансформируется в другие активные формы кислорода (АФК). Чрезмерное образова- ние АФК в митохондриях может вызвать окис- лительный стресс, окислительные повреждения комплексов ЭТЦ, мембран митохондрий, а так- же клеточных белков, липидов и ДНК. Важным механизмом нейрональных нарушений при ин- токсикации мышьяком и алюминием является Отбор данных. Извлечение данных осу- ществлялось на основании соответствия описы- ваемых в статьях исследований с интересуемой авторов тематикой: особенностями действия алюминия и мышьяка на биоэнергетику нейро- цитов. Всего было извлечено 23 статьи. Иные пу- бликации, представленные системами в резуль- тате поиска, исключались ввиду несоответствия интересуемой тематики. Элементы данных: нервная система, мы- шьяк, алюминий, митохондрии, нейроны. 8 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Рисунок 1 – Общие патогенетические механизмы митохондрио-зависимой нейротоксичности. Рисунок 1 – Общие патогенетические механизмы митохондрио-зависимой нейротоксичности. Рисунок 1 – Общие патогенетические механизмы митохондрио-зависимой нейротоксичности. (A) Митохондриальная дисфункция – общий механизм, индуцированный многими токсикантами окружающей среды и нейродегенеративными болезнями, что приводит к каскаду взаимосвязанных клеточных дисфункций; (B) Воспалению, при котором микроглия и, в меньшей степени, астроциты высвобождают нейротоксические факторы, такие как цитокины, интерлейкины и АФК, что приводит к повреждению нейронов; (C) Ускорению деления и фрагментации митохондрий, что может инициировать высвобождение цитохрома С и апоптозную гибель клеток; (D) Активации аутофагии и убиквитин-протеасомной деградации поврежденных белков и клеточных органелл вследствие снижения уровня АТФ, поскольку эти механизмы являются АТФ-зависимыми и чувствительными к АФК; (E) Генерации АФК, что приводит к образованию токсичных олигомеров и белковых агрегатов (F), нарушает функцию убиквитин-протеасомной системы (G) и вызывает повреждение как ядерной, так и митохондриальной ДНК. Повреждение ДНК (H) приводит к изменению ядерной функции, нестабильности генома и митохондриальной дисфункции, поскольку ядерная ДНК кодирует многочисленные митохондриальные белки. некротическому, апоптотическому или аутофа- гическому механизму. Мозг человека в состоянии покоя использу- ет около 20% энергии АТФ, производимой мито- хондриями организма, в то время как на его долю приходится лишь около 2% массы тела. Основное количество производимой мозгом энергии тра- тится на поддержание мембранного потенциала нейронов. Важной функцией митохондрий яв- 9 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 ляется депонирование ионов кальция, которые в нейронах опосредуют динамику высвобождения нейромедиаторов. ные механизмы их повреждающего действия на митохондриальные процессы в нейронах окон- чательно не выяснены. В данном обзоре про- анализированы последние данные о механизмах митохондриальной дисфункции при нейроток- сичности, вызванной воздействием мышьяка и алюминия. Известно, что митохондриальная дисфунк- ция нейронов мозга является одной из причин ряда нейродегенеративных болезней: Альцгей- мера, Паркинсона, Гентингтона, аутизма, и боко- вого амиотрофического склероза и других форм нейродегенерации [3]. Мышьяк (As) – широко распространенный токсичный металлоид, который представляет опасность примерно для 200 миллионов человек в более чем 24 странах мира. Известными источни- ками загрязнения окружающей среды мышьяком являются электростанции, использующие бурый уголь, медеплавильные заводы. As также исполь- зуется при производстве полупроводников, стек- ла, красителей, инсектицидов, фунгицидов и др. Загрязнение окружающей среды достигло даже нетронутых мест. Ранее было обнаружено, что в образцах снега с Эвереста уровни As и Cd пре- вышали нормы для питьевой воды, а все образцы почвы были сильно загрязнены мышьяком. Мы- шьяк может поступать в кровь через кожу, желу- дочно-кишечный тракт и легкие при дыхании. В организме животных и человека As может нака- пливаться в различных органах, включая почки, легкие, печень и селезенку [5]. По данным ВОЗ, в организм человека с суточным рационом по- ступает в среднем 0,05-0,45 мг мышьяка. Допу- стимая суточная доза As 0,05 мг/кг массы тела. В зависимости от дозы мышьяк может вызывать острое или хроническое отравление; разовая доза As в количестве 30 мг смертельна для человека. Особенно опасно его накопление в различных об- ластях мозга [6]. Организм человека может периодически подвергаться токсическому воздействию различ- ных химических элементов, включая ионы тяже- лых металлов, таких как мышьяк (As), алюминий (Al), кадмий (Cd), свинец (Pb), медь (Cu), марга- нец (Mn) и др. Являясь естественными компонен- тами земной коры, они попадают в биосферу в результате разнообразной деятельности челове- ка. Основными путями их попадания в организм человека являются желудочно-кишечный тракт, легкие и кожа. Нейроны головного мозга способ- ны эффективно устранять негативные эффекты низких концентраций этих веществ путем акти- вации антиоксидантных ферментов, системы глу- татиона, механизмов репарации ДНК, биогенеза митохондрий и митофагии в случае необратимо- го повреждения отдельных митохондрий. некротическому, апоптотическому или аутофа- гическому механизму. головного мозга, что, в конечном итоге, приводи- ло к снижению продукции АТФ. Таким образом, проанализированные ис- точники литературы показали, что в индуциро- ванной As-нейродегенерации ключевую роль играют механизмы окислительного стресса, при- водящие к нарушению энергообразующей функ- ции митохондрий. Многочисленные исследова- ния показали, что наиболее важным механизмом As-нейротоксичности в ЦНС является митохон- дриальная дисфункция (рис. 1). Она включает на- рушение гомеостаза Ca2+, снижение мембранного потенциала, проницаемости митохондриальных мембран и митохондриального дыхания [12], что, в конечном итоге, приводит к повреждению и ги- бели нейронов по митохондриально-зависимым путям (рис. 1). у (р ) Алюминий (Al) является повсеместно рас- пространенным на Земле металлом. Он может легко всасываться при контакте с кожей, вдыха- нии и проглатывании. Значительная часть алю- миния поступает в организм человека с продук- тами питания (22 мг), из которых всасывается 1 мг. Сульфат алюминия широко используется для очистки воды, в пищевой и фармацевтической промышленности, в медицине и других отраслях производства, что создает условия для его по- падания в организм человека. Многочисленные исследования показывают, что Al может нака- пливаться в различных органах млекопитающих, включая кости, почки, легкие, печень, селезенку и головной мозг [13]. Растущее количество источ- ников литературы также свидетельствует о том, что накопление Al в различных областях мозга может вызывать симптомы нейротоксичности и ухудшение способности к обучению [13]. Иссле- дования на грызунах показали, что хроническое воздействие Al приводит к накоплению Al в гип- покампе и вызывает поведенческие нарушения [14]. В других исследованиях было показано, что Al вызывает дегенерацию нейрофибрилл. Эпи- демиологические исследования показали, что Al рассматривается как потенциальный фактор ри- ска развития нейродегенеративных заболеваний, таких как болезнь Альцгеймера, болезнь Паркин- сона и др. [15]. Хорошо известно, что митохондрия явля- ется основным источником и главной мишенью АФК [11]. Окислительный стресс, индуцирован- ный соединениями As, тесно связан с дисфунк- цией митохондрий. Так, увеличение уровней АФК и усиление перекисного окисления липи- дов после воздействия NaAsO2 в течение 28 дней сопровождалось снижением активности мито- хондриальных ферментов – марганец-зависи- мой супероксиддисмутазы (MnSOD) и каталазы в митохондриальной фракции разных областей мозга (включая стриатум, гиппокамп и лобную кору) крыс. Кроме того, в митохондриальной фракции головного мозга крыс при субтокси- ческом воздействии As снижались активности MnSOD, каталазы, глутатионпероксидазы, глу- татионредуктазы и глутатионтрансферазы. Более того, различные исследователи показали, что As напрямую нарушает тканевое дыхание посред- ством окислительного стресса. Так, индуциро- ванный соединениями As окислительный стресс ингибировал активность I, II и IV комплексов в митохондриях мозга крыс. некротическому, апоптотическому или аутофа- гическому механизму. Одним из наиболее важных механизмов нейротоксичности тяжелых металлов является их взаимодействие с сульфгидрильными группа- ми белков, что вызывает инактивацию жизненно важных для клетки макромолекул (структурных белков, ферментов), истощение запасов восста- новленного глутатиона, активацию окислитель- ного стресса, повреждение мембранных липидов и ДНК [2, 4]. С другой стороны, строго спец- ифичных механизмов защиты нейронов от по- вреждающего действия конкретных металлов не установлено. Тем не менее, их длительное посту- пление в организм в субтоксических дозах может привести к накоплению в ткани головного мозга до какого-то критического уровня, способного привести к окислительному стрессу и наруше- нию ресинтеза АТФ в митохондриях (рис. 1). При этом гибель нейронов может идти путем апопто- за и/или некроза, включая и механизм аутофагии, который еще окончательно не выяснен. Исследования in vivo показали, что чрез- мерное воздействие соединений мышьяка вы- зывает усиление апоптоза нейронов, приводя к нарушению развития нервной системы в онто- генезе и когнитивных функций у взрослых крыс [7]. Эпидемиологические исследования свиде- тельствуют, что у взрослых и пожилых людей, проживающих в сельской местности, при содер- жании в питьевой воде As в количестве 3-15 мкг/л нарушаются показатели когнитивных функций и памяти, что указывает на его нейротоксичность и является фактором риска болезни Альцгейме- ра [8]. Однако механизмы As-нейротоксичности до конца не выяснены. На сегодняшний день ее связывают с перепроизводством нейронами ами- лоида Aβ, воспалительными реакциями [9], де- фицитом тиамина, окислительным стрессом, на- рушением образования нейротрансмиттеров [10], Метаболизм нейротоксичных металлов в мозге и их роль в этиологии различных видов нейродегенерации в последнее время активно исследуются, о чем свидетельствует большое количество работ, посвященных этой проблеме. Однако эффекты различных металлов и конкрет- 10 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № нарушением экспрессии белков цитоскелета, митохондриальной дисфункцией и нарушением активности холинэстеразы. Митохондриальная дисфункция среди этих патогенетических факто- ров As-нейротоксичности играет ключевую роль (рис. 1). В экспериментах in vitro многочислен- ные исследования показали, что мышьяк может оказывать неблагоприятное воздействие на функ- ции митохондрий. Известно, что обработка куль- туры клеток A172 триоксидом мышьяка (As2O3, 50 мкМ в течение 8 часов) приводила к образо- ванию агрегатов белков и митохондрий (рис. 1). Впоследствии другие исследователи также пока- зали, что обработка арсенитом натрия (NaAsO2) или As2O3 вызывала митохондриальную дис- функцию вследствие повышения внутриклеточ- ного уровня Ca2+, снижения митохондриального мембранного потенциала и уровней кальпаина 1 в культуре клеток N2A, клетках SHSY-5Y, в пер- вичных астроцитах и нейроцитах крыс. Кроме того, исследования in vivo также подтвердили критическую роль окислительного стресса и ми- тохондриальной дисфункции при индуцирован- ной мышьяком нейротоксичности. некротическому, апоптотическому или аутофа- гическому механизму. Кроме того, хрони- ческое воздействие низких уровней As снижало экспрессию генов митохондриальных комплек- сов II, IV и V в мозге мышей. Таким образом, мы- шьяк уменьшал активность митохондриального дыхания и фосфорилирования в митохондриях Некоторые исследователи высказали пред- положение, что в Al-токсических эффектах, включая и нейротоксичность, митохондриальная дисфункция может играть решающую роль [16]. После добавления Al к глиальным клетках в тече- ние 24 часов возрастало образование АФК, снижа- 11 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Кроме того, было показано, что и другие природ- ные антиоксиданты, такие как кроцин, куркумин и полифенолы, обладают нейропротекторным действием при Al-нейротоксичности [23]. Эти исследования показывают, что ингибирование окислительного стресса и предотвращение мито- хондриальной дисфункции может быть главной терапевтической стратегией при вызванном Al повреждении нейронов. лись митохондриальная дыхательная активность и запасы восстановленного глутатиона. Также воздействие Al увеличивало образование АФК и нарушало активность цитохром-с-оксидазы и энергообразующей функции митохондрий в раз- личных типах нейронов, включая линию PC12 [17], клетки нейробластомы SH-SY5Y [18], а так- же мозжечковых гранулярных клеток крыс [19]. Митохондриальная дисфункция при воз- действии алюминия наблюдалась также и в ис- следованиях in vivo. Острое воздействие 50 мкМ мальтоната алюминия посредством интрацистер- нальной инъекции вызывало высвобождение из митохондрий цитохрома с (cyt-c), что сопро- вождалось снижением уровня антиапоптотиче- ских белков семейства Bcl-2 и активацией про- апоптотических белков: Bax, p53, эффекторной каспазы-3, а также фрагментацией ДНК в мито- хондриях мозга кролика. Воздействие Al в суб- токсических дозах в течение 12 недель приводило к повышенному образованию АФК и снижению синтеза АТФ и уровня цитохромов в мозге крыс, что предполагало нарушение функции митохон- дрий (рис. 1). Кроме того, воздействие Al снижа- ет активность MnSOD и аконитазы в различных областях мозга крыс. Результаты электронной микроскопии показали, что воздействие алюми- ния вызывает набухание митохондрий и их ваку- олизацию, что приводило к увеличению их диа- метра в нейронах гиппокампа мышей и крыс [16]. Наконец, воздействие Al повышало активность связанных с аутофагией белков LC3-II и Beclin-1 и, в то же время, подавляло экспрессию белка p62, что предполагало наличие связи между на- рушением обучения и памяти и митофагией [16]. Митохондриальная дисфункция сопрово- ждается повышением уровня внутриклеточного Ca2+ (I) за счет выхода из поврежденных митохон- дрий в цитозоль, тем самым увеличивая клеточ- ную эксайтотоксичность. Чрезмерная активация возбуждающих рецепторов приводит, в свою оче- редь, к поступлению в клетку экзогенного Са2+ и эксайтотоксичности, индуцируя митохондриаль- ную деполяризацию и выход Са2+ из митохон- дрий по механизму порочного круга. некротическому, апоптотическому или аутофа- гическому механизму. Как показывают литературные источники, все эти механизмы, потенцируя друг друга, до- стигают кульминации и приводят к нейродегене- рации. Заключение Анализ и обобщение современных литера- торных даннных о механизмах нейротоксично- сти соединений алюминия и мышьяка, которые могут накапливаться в организме вследствие за- грязнения окружающей среды, показывают, что ведущим механизмом их повреждающего дей- ствия является нарушение энергообразующей функции митохондрий нейронов, что, в свою оче- редь, предполагает разработку эффективных ми- тохондриотропных средств для профилактики и терапии этих нарушений. В частности, в качестве таких средств могут быть использованы антиок- сиданты растительного происхождения, доноры SH-групп, «хелаторы» (унитиол, липоевая кисло- та) и другие перспективные, в том числе адрес- ные митохондриопротекторные препараты или их комплексы. В последнее время окислительный стресс и митохондриальные нарушения рассматриваются в качестве основных мишеней нейротоксичности, вызванной алюминием. Так, использование анти- оксиданта кверцетина предотвращало вызванное Al набухание митохондрий и конденсацию хро- матина в гиппокампе крыс [20]. Нарингин также оказывал защитное действие на нарушение памя- ти у крыс при субтоксическом воздействии алю- миния, предотвращая активацию митохондриаль- ного окислительного повреждения в головном мозге [21]. 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Arsenic affects inflammatory cytokine expression in Gallus gallus brain tissues. BMC Vet Res. 2017 Jun;13(1):157. doi: 10.1186/s12917-017-1066-8 11. Wang Yi, Tang B, Long L, Luo P, Xiang W, Li X, et al. Improvement of obesity-associated disorders by a small- molecule drug targeting mitochondria of adipose tissue macrophages. Nat Commun. 2021;12:102. doi: 10.1038/ s41467-020-20315-9 18. Tsialtas I, Gorgogietas VA, Michalopoulou M, Komninou A, Liakou E, Georgantopoulos A, et al. Neurotoxic effects of aluminum are associated with its interference with estrogen receptors signaling. Neurotoxicology. 2020 Mar;77:114-126. doi: 10.1016/j.neuro.2020.01.004 12. References Liu J, Zhao H, Wang Y, Shao Y, Zong H, Zeng X, et al. Arsenic trioxide andor copper sulfate induced apoptosis and autophagy associated with oxidative stress and perturbation of mitochondrial dynamics in the thymus of Gallus gallus. Chemosphere. 2019 Mar;219:227-235. doi: 10.1016/j. chemosphere.2018.11.188 19. Wang P, Wu Q, Wu W, Li H, Guo Y, Yu P, et al. Mitochondrial Ferritin Deletion Exacerbates beta-Amyloid- Induced Neurotoxicity in Mice. Oxid Med Cell Longev. 2017;2017:1020357. doi: 10.1155/2017/1020357 20. Sharma DR, Wani WY, Sunkaria A, Kandimalla RJ, Sharma RK, Verma D, et al. Quercetin attenuates neuronal death against aluminum-induced neurodegeneration in the rat hippocampus. Neuroscience. 2016 Jun;324:163-76. doi: 10.1016/j.neuroscience.2016.02.055 13. Promyo K, Iqbal F, Chaidee N, Chetsawang B. Aluminum chloride-induced amyloid beta accumulation and endoplasmic reticulum stress in rat brain are averted by melatonin. Food Chem Toxicol. 2020 Dec;146:111829. doi: 10.1016/j.fct.2020.111829 21. Prakash A, Shur B, Kumar A. Naringin protects memory impairment and mitochondrial oxidative damage against aluminum- induced neurotoxicity in rats. Int J Neurosci. 2013 Sep;123(9):636-45. doi: 10.3109/00207454.2013.785542 14. Liu H, Zhang W, Fang Y, Yang H, Tian L, Li K, et al. Neurotoxicity of aluminum oxide nanoparticles and their mechanistic role in dopaminergic neuron injury involving p53-related pathways. J Hazard Mater. 2020 Jun;392:122312. doi: 10.1016/j.jhazmat.2020.122312 22. Prakash A, Kumar A. Еffect of Centellaasiatica against aluminum-induced neurotoxicity in rats: Possible relevance to its anti-oxidant and anti-apoptosis mechanism. Neurol Sci. 2013 Aug;34(8):1403-9. doi: 10.1007/s10072-012-1252-1 15. McLachlan DRC, Bergeron C, Alexandrov PN, Walsh WJ, Pogue AI, Percy ME, et al. Aluminum in Neurological and Neurodegenerative Disease. Mol Neurobiol. 2019 Feb;56(2):1531-1538. doi: 10.1007/s12035-018-1441-x 23. Wang C, Cai X, Hu W, Li Z, Kong F, Chen X, et al. Investigation of the neuroprotective effects of crocin via antioxidant activities in HT22 cells and in mice with Alzheimer’s disease. Int J Mol Med. 2019 Feb;43(2):956- 966. doi: 10.3892/ijmm.2018.4032 16. Huang T, Guo W, Wang Y, Chang L, Shang N, Chen J, et al. Involvement of Mitophagy in Aluminum Oxide Nanoparticle-Induced Impairment of Learning and Memory Submitted 31.01.2022 Accepted 21.04.2022 Сведения об авторах: д р Флюрик С.В. – преподаватель кафедры патологической физиологии им. Д.А. Маслакова, Гродненский государ- ственный медицинский университет; д ц у р ; Дремза И.К. – к.б.н., доцент кафедры патологической физиологии им. Д.А. Маслакова, Гродненский государ- ственный медицинский университет. Резюме. При написании обзора были обобщены и сопоставлены опубликованные данные по истории применения, ме- ханизму действия и эффективности применения в клинической практике наночастиц серебра и препаратов на их основе. Данный металл используют в медицинской практике с древних времен, но статистические данные о его эффективности были получены только в конце XIX века. С тех пор он получил широкое распространение в виде коллоидных растворов, солей, а в последние десятилетия – наночастиц. Особенно ярко проявляется инте- рес к препаратам серебра в связи с распространением устойчивых к антибиотикам микроорганизмов. Ионы и наночастицы прикрепляются к их клеточной стенке, нарушают ее функционирование, разрушают, проникают в клетку, где связываются с фосфор- и серосодержащими молекулами. Имея такой неспецифический механизм дей- ствия, наночастицы серебра обладают широким спектром противомикробной и противогрибковой активности. По данным материалов кохрейновской библиотеки можно сделать вывод о том, что наночастицы серебра имеют клинически доказанную эффективность при применении в хирургии, стоматологии, для изготовления изделий медицинского назначения, применяемых в хирургии и трансплантологии. Ключевые слова: наночастицы серебра, клиническая эффективность, токсичность. р рур р Ключевые слова: наночастицы серебра, клиническая эффективность, токсичность. RZHEUSSKY S.E. Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus НАНОЧАСТИЦЫ СЕРЕБРА В МЕДИЦИНЕ РЖЕУССКИЙ С.Э. Витебский государственный ордена Дружбы народов медицинский университет, г. Витебс Республика Беларусь Вестник ВГМУ. – 2022. – Том 21, №2. – С. 15-24. Information about authors: Information about authors: Fliuryk S.V. – lecturer of the Chair of Pathological Physiology named after D.A. Maslakov, Grodno State Medical University; Dremza I.K.– Candidate of Biological Sciences, associate professor of the Chair of Pathological Physiology named after D.A. Maslakov, Grodno State Medical University. Fliuryk S.V. – lecturer of the Chair of Pathological Physiology named after D.A. Maslakov, Grodno State Medical University; Dremza I.K.– Candidate of Biological Sciences, associate professor of the Chair of Pathological Physiology named after D.A. Maslakov, Grodno State Medical University. Fliuryk S.V. lecturer of the Chair of Pathological Physiology named after D.A. Maslakov, Grodno State Medical University; Dremza I.K.– Candidate of Biological Sciences, associate professor of the Chair of Pathological Physiology named after D.A. Maslakov, Grodno State Medical University. y; Dremza I.K.– Candidate of Biological Sciences, associate professor of the Chair of Pathological Physiology named after D.A. Maslakov, Grodno State Medical University. Адрес для корреспонденции: Республика Беларусь, 230009, г. Гродно, ул. Горького, 80, Гродненский государ- ственный медицинский университет, кафедра патологической физиологии им. Д.А. Маслакова. E-mail: sﬂ urik@ gmail.com – Флюрик Сергей Владимирович. Адрес для корреспонденции: Республика Беларусь, 230009, г. Гродно, ул. Горького, 80, Гродненский государ- ственный медицинский университет, кафедра патологической физиологии им. Д.А. Маслакова. E-mail: sﬂ urik@ gmail.com – Флюрик Сергей Владимирович. Correspondence address: Republic of Belarus, 230009, Grodno, 80 Gorky str., Grodno State Medical University, Chair of Pathological Physiology named after D.A. Maslakov. E-mail: sﬂ urik@gmail.com – Siarhei V. Fliuryk. Correspondence address: Republic of Belarus, 230009, Grodno, 80 Gorky str., Grodno State Medical University, Chair of Pathological Physiology named after D.A. Maslakov. E-mail: sﬂ urik@gmail.com – Siarhei V. Fliuryk. 14 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № DOI: https://doi.org/10.22263/2312-4156.2022.2.15 Abstract. Серебро в форме соли позволило со- кратить частоту данного заболевания с 10,8%, до 0,2-0,5% [3]. Их открытие и изучение стало возможным после того, как в середине ХХ века был изобре- тен электронный микроскоп и сами наночастицы удалось увидеть и измерить. Согласно современ- ному определению наночастица – это аморфная или полукристаллическая структура, имеющая хотя бы один характерный размер в диапазоне от 1 до 100 нм [8]. Однако, дело ограничивается не только размерами. При переходе на наноме- тровый уровень у материалов изменяются физи- ко-химические свойства и/или возникают новые явления, которые невозможно предсказать на ос- нове изучения вещества в виде более крупных ча- стиц [9]. Например, температура плавления нано- частиц золота с размером 5 нм составляет 800°С, а наночастиц с размером 2 нм – 300°С. И это при том, что обычно золото плавится при температу- ре 1065°С [10]. Другой пример – это появление у наночастиц некоторых благородных металлов спектрофотометрической активности, чего лише- ны как их соли, так и макрообразцы [11, 12]. Такие впечатляющие результаты послужи- ли поводом к дальнейшему изучению серебра и его соединений. В конце XIX – начале XX века был разработан целый ряд субстанций и лекар- ственных препаратов на их основе: колларгол, протаргол, альбаргил, эларгол, силаргель, арго- сульфан и др. Некоторые из них с успехом при- меняются до сих пор [4]. Некоторые специалисты считают, что до открытия антибиотиков именно соли серебра являлись одним из самых широко используемых средств с антимикробной активно- стью [5]. В 40-х годах XX века в арсенале врачей всего мира появляется пенициллин и интерес к серебру начинает ослабевать [6]. Действитель- но, кому нужны были токсичные соли серебра, если буквально нескольких уколов пеницилли- на было достаточно, чтобы справиться почти с любым бактериальным заболеванием. К сожале- нию, «всесилие» антибиотиков длилось недолго. Проблемы с этой группой лекарственных средств в своей нобелевской речи предсказал еще Алек- сандр Флеминг. Он говорил: «Настанут времена, когда любой сможет купить пенициллин в магази- не, поэтому есть опасность, что какой-нибудь не- сведущий человек может легко принять слишком малую дозу и вырастить в себе микроорганизмы под влиянием низких концентраций лекарства, которые будут устойчивы к пенициллину». Слова оказались пророческими, не прошло и полвека, как появилось понятие «антибиотикорезистент- ность бактерий». Интерес к медицинскому применению на- ночастиц серебра с каждым годом только усили- вается. Так, по данным сайта pubmed.gov, количе- ство публикуемых научных работ по данной теме к 2021 году выросло до 2807. Причем тенденция к росту сохраняется, несмотря на пандемию (рис. 1) [13]. Abstract. When writing this review, published data on the history of use, mechanism of action and the effectiveness of application of silver nanoparticles and preparations based on them in clinical practice were summarized and compared. This metal has been used in medical practice since ancient times, but statistical data on its effectiveness were obtained only at the end of the 19th century. Since then, it has become widely spread in the form of colloidal solutions, salts, and, in recent decades, nanoparticles. The interest to silver preparations is especially pronounced in connection with the spread of antibiotic- resistant microorganisms. Ions and nanoparticles attach to their cell wall, disrupt its functioning, destroy it, penetrate into the cell, where they bind to phosphorus and sulfur-containing molecules. Possessing such a non-specific mechanism of action, silver nanoparticles have a wide spectrum of antimicrobial and antifungal activity. According to the materials of the Cochrane Library, it can be concluded that silver nanoparticles possess clinically proven efficacy when used in surgery, dentistry, for manufacturing medical products that are used in surgery or transplantology. Key words: silver nanoparticles, clinical efficacy, toxicity. Древнекитайские источники говорят нам о том, что серебро в медицинских целях начало применяться еще за 2500 тысячи лет до нашей эры. Чего здесь было больше, первобытной ре- лигии или реальных наблюдений над его эффек- тивностью, сказать сложно. Однако Кир Великий и Александр Македонский во время своих заво- евательных походов хранили воду в серебряных кубках, древние индусы обеззараживали воду, помещая в нее раскаленное серебро, а Гиппократ 15 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 рингологии использовали раствор протаргола, для местного лечения ран применяли мягкие ле- карственные формы с сульфадиазином серебра. Однако сейчас особый интерес вызывают не сое- динения серебра, а нульвалентный металл в виде наночастиц. рекомендовал серебро для лечения трофических язв [1, 2]. Так или иначе, но первое статисти- ческое доказательство эффективности серебра было получено только в 1881 году, когда немец- кий акушер, доктор медицины Креде Карл Зиг- мунд Франц предложил использовать 1% раствор нитрата серебра для лечения бленнореи у ново- рожденных. Серебро в форме соли позволило со- кратить частоту данного заболевания с 10,8%, до 0,2-0,5% [3]. рекомендовал серебро для лечения трофических язв [1, 2]. Так или иначе, но первое статисти- ческое доказательство эффективности серебра было получено только в 1881 году, когда немец- кий акушер, доктор медицины Креде Карл Зиг- мунд Франц предложил использовать 1% раствор нитрата серебра для лечения бленнореи у ново- рожденных. Abstract. Стоит отметить, что в указанной междуна- родной базе данных первые публикации, посвя- щенные изучению и медицинскому применению наночастиц серебра, появляются в 1995 году. К этому же году относятся первые публикации в русскоязычных изданиях и даты подачи первых патентов на способы лечения гнойных пораже- ний с помощью первой российской фармацевти- ческой субстанции на основе наночастиц серебра – повиаргола [14, 15]. К концу XX века эта проблема приобрела значительный размах, в результате чего активи- зировались работы по поиску новых или совер- шенствованию старых антимикробных препара- тов. Свое внимание исследователи обратили и на серебро [6, 7]. Существует много способов получения наночастиц. Самым распространенным являет- ся химическое восстановление ионов серебра до нульвалентных наночастиц. Этот метод довольно прост, но зачастую при его применении исполь- зуются токсичные растворители, а для получения частиц с заданными характеристиками требуется очень жесткое соблюдение параметров реакции Нельзя сказать, что с 40-х годов оно было забыто. В медицине все эти годы в виде глазных капель применяли раствор нитрата серебра, до- вольно широко в офтальмологии и оторинола- 16 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № 0 500 1000 1500 2000 2500 3000 1995 2000 2005 2010 2015 2020 Количество публикаций Годы Рисунок 1 – Количество статей, посвященных изучению наночастиц серебра, по данным сайта pubmed.gov. Рисунок 1 – Количество статей, посвященных изучению наночастиц серебра, по данным сайта pubmed.gov. [16]. Физические методы, такие как механиче- ское измельчение [17], лазерная абляция [18] или конденсация пара [19], являются высокопроизво- дительными и позволяют получить наночастицы с узким распределением по размерам, однако тре- буют специфического сложного оборудования и являются энергоемкими [16]. Биологический син- тез лишен многих вышеописанных недостатков. С использованием бактерий, грибов, актиномице- тов, водорослей или растений получают хорошо растворимые, стабильные наночастицы [20-22]. Однако, как и любой биотехнологический метод, данный способ синтеза довольно дорог. заряд E. coli – с -28,5 до -3,5 mV [31]. Чем силь- нее положительный заряд наночастиц, тем силь- нее они оказывают свой антимикробный эффект. Изменение заряда мембраны и связывание сере- бра с транспортными белками или белками дыха- тельной цепи влияет на ее проницаемость, работу дыхательной цепи, деление клеток, транспорт ио- нов, выработку энергии [32]. Зачастую при воз- действии наночастиц на бактериальную клетку наблюдается утечка клеточного содержимого и неконтролируемый транспорт через цитоплазмо- тическую мембрану [33]. Часть наночастиц и ионов проникает внутрь клетки, связываясь с биомолекулами и клеточными структурами (липиды, белки, ДНК) [24]. Это приводит к остановке или замедлению процессов транскрипции, трансляции, синтеза белка, денатурации рибосом, что ведет к наруше- нию жизнедеятельности клетки [34, 35]. Abstract. Тапальского по разработке антибак- териальных покрытий для титановых имплантов было показано, что эффективно подавляют рост микроорганизмов и образование биопленок толь- ко биодеградируемые покрытия [40, 41]. Такое по- крытие постепенно разрушается, высвобождая на- ночастицы. Схожие выводы в своей работе сделал Yun'an Qing с коллективом [42]. Другой вариант – это нанесение раствора наночастиц на поверх- ность материала, которому хотят придать анти- микробные свойства [43]. торое никак отрицательно не сказывается на здо- ровье человека. В случае, если же он подверга- ется воздействию большого количества серебра, у него может развиться редкое заболевание ар- гирия, характеризующееся изменением окраски кожи в серый или синий цвет [47]. Данное забо- левание наблюдается у ювелиров с многолетним стажем, или работников химических предпри- ятий, или у пациентов, бесконтрольно употре- бляющих соединения серебра [47, 48]. И здесь ключевым словом является «бесконтрольно», по- скольку многочисленные исследования показа- ли, что при использовании серебросодержащих препаратов с соблюдением рекомендаций врача никаких побочных реакций не наблюдается. Так, Smock, K.J. с коллегами в результате плацебо- контролируемого, простого слепого, перекрест- ного исследования с контролируемой дозой на 18 добровольцах выяснил, что после перорального приема в течение двух недель не наблюдается усиления активации тромбоцитов [49]. Munger M.A. с коллегами в результате слепого, контро- лируемого, перекрестного исследования на 60 здоровых добровольцах выяснил, что после при- ема коммерческих растворов серебра не происхо- дит клинически значимых изменений в метабо- лических или гематологических показателях, не обнаруживаются морфологические изменения в легких, сердце, органах брюшной полости [50]. В составе другого научного коллектива этот же автор установил, что после 14 дней приема пре- парата, содержащего наночастицы серебра, этот металл обнаруживается в крови, однако это не вызывает клинически значимых изменений ме- таболических, гематологических, физических показателей. Также это не сказывается на актив- ности цитохрома Р450 [51]. Во-вторых, действие наночастиц серебра строго дозозависимо. Их минимальная подавля- ющая концентрация полностью останавливает рост микроорганизмов. Однако серебро в более низких концентрациях, возможно, слегка замед- ляет, но не может остановить размножение [38, 39]. В связи с этим странно смотрятся зубные пасты, косметические кремы и другие средства с «коллоидным серебром» или «наночастицами серебра» с неизвестными концентрациями. Осо- бенно это важно в связи с тем, что, по некоторым данным, к действию наночастиц серебра бакте- рии Escherichia coli и Pseudomonas aeruginosa могут вырабатывать устойчивость путем выра- ботки адгезивного белка флагеллина, который за- пускает агрегацию наночастиц [44]. В-третьих, активность наночастиц серебра значительно снижается в присутствии гноя или биологических жидкостей. Также снижает их ак- тивность биопленка. По информации коллектива под руководством М. Abstract. Серебро снижает синтез и нарушает работу антиоксидант- ных ферментов, что приводит к накоплению в клетке активных форм кислорода [36]. Изначально считалось, что наночастицы серебра проявляют свою активность только за счет высвобождения ионов, выступая в качестве депо. Современная точка зрения говорит о том, что активность проявляют как ионы, так и сами наночастицы [23-25]. В основе их фармакологи- ческой активности лежит то, что серебро являет- ся кислотой Льюиса, то есть является акцептором электронной пары [26, 27]. А значит, имеет хи- мическое сродство с фосфором и серосодержа- щими биомолекулами, которые, в свою очередь, являются основными компонентами клеточной мембраны, белков, ДНК. За счет химических и электростатических сил ионы и наночастицы се- ребра прикрепляются к клеточной стенке [28]. Это приводит к сжатию цитоплазмы, отслоению мембраны, изменению ее формы [29, 30]. Кроме того, изменяется поверхностный заряд бакте- рий. Так, например, установлено, что наночасти- цы способны изменять поверхностный заряд P. aeruginosa c -29,6 до -5,4 mV, а поверхностный Дальше наночастицы, ионы и свободные радикалы связываются с ДНК, препятствуя ее репликации и размножению клеток, изнутри раз- рушают цитоплазматическую мембрану, вызывая в конечном итоге гибель [37]. Механизм действия наночастиц серебра не специфический, поэтому они практически одина- ково действуют на грамположительную и грамо- трицательную микрофлору. С меньшей активно- стью, но оказывают подавляющее и фунгицидное действие на микроскопические грибы [38,39]. С этим связана большая популярность наночастиц 17 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 серебра. Их используют при производстве кос- метики, биологически активных добавок к пище, изделий медицинского назначения и так далее. Однако существует ряд сложностей, которые огра- ничивают применение серебра. Во-первых, для того, чтобы наночастицы смогли выделить ионы или прикрепиться к клеточной стенке микроор- ганизма, они должны находиться во взвешенном состоянии, т.е. находиться в растворе. Например, в работах Д.В. Тапальского по разработке антибак- териальных покрытий для титановых имплантов было показано, что эффективно подавляют рост микроорганизмов и образование биопленок толь- ко биодеградируемые покрытия [40, 41]. Такое по- крытие постепенно разрушается, высвобождая на- ночастицы. Схожие выводы в своей работе сделал Yun'an Qing с коллективом [42]. Другой вариант – это нанесение раствора наночастиц на поверх- ность материала, которому хотят придать анти- микробные свойства [43]. серебра. Их используют при производстве кос- метики, биологически активных добавок к пище, изделий медицинского назначения и так далее. Однако существует ряд сложностей, которые огра- ничивают применение серебра. Во-первых, для того, чтобы наночастицы смогли выделить ионы или прикрепиться к клеточной стенке микроор- ганизма, они должны находиться во взвешенном состоянии, т.е. находиться в растворе. Например, в работах Д.В. Abstract. Saravanan, концентрация наночастиц серебра, которая убивает планктон- ную культуру, не вызывает 100% потери жизне- способности бактерий в биопленке [45]. Основное количество опубликованных от- четов о клинических испытаниях средств на ос- нове наночастиц серебра касаются их эффектив- ности. Чаще всего подобные средства применяют с целью лечения ран и ожогов. В результате ис- следования, в котором принял участие 281 паци- ент, установлено, что при лечении трофических язв нижних конечностей препарат нанокристал- лического серебра оказал более быстрое и пол- ное действие, чем препарат с кадексомером йода [52]. Эффективность препаратов или повязок на основе наночастиц серебра доказана при лече- нии пролежней, язв, травматических и хирурги- ческих ран у пациентов со средним возрастом 80 лет [53], при лечении остаточных ожоговых ран В-четвертых, серебро может быть токсич- ным. Точнее, серебро в больших количествах ока- зывает токсический эффект. Это микроэлемент. Ежедневно с пищей и водой человек потребляет 0,0014-0,08 мг серебра, которое частично выво- дится из организма, а частично откладывается в железах внутренней секреции, печени, почках, костях [46]. Это очень маленькое количество, ко- 18 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № [54, 55], при заживлении ран после обрезаний [56] и т.д. В исследовании с участием 92 женщин с кесаревым сечением [57] и в исследовании с 34 пациентами с апикальным перидонтитом до- казано наличие обезболивающего эффекта [58]. В исследовании 2017 года гель с наночастицами серебра показал такую же эффективность при ле- чении вульгарных угрей, как и гель с клиндами- цином [59]. антимикробная активность серебросодержащих лекар- ственных средств / С. Э. Ржеусский, В. В. Кугач, М. А. Ва- луева // Вестн. фармации. – 2013. – № 2. – С. 25–30. 5. Antimicrobial silver: uses, toxicity and potential for resistance / K. Mijnendonckx [et al.] // Biometals. – 2013 Aug. – Vol. 26, N 4. – Р. 609–621. 6. Антибактериальная активность некоторых коллоидных форм наносеребра в отношении неферментирующих гра- мотрицательных бактерий / О. А. Качанова [и др.] // Со- времен. проблемы науки и образования. – 2014. – № 2. – С. 215–222. В стоматологии нанофторид серебра пока- зал свою эффективность для предотвращения об- разования зубной биопленки [60], а зубные щет- ки, содержащие серебро, – «многообещающие результаты в качестве альтернативы стандартным стратегиям гигиены полости рта» [61]. Серебря- ное покрытие доказало свою актуальность при изготовлении наружных дренажных катетеров желудочков [62], эндотрахеальных трубок [63], стоматологических ретейнеров [64]. 7. Благитко, Е. М. О целесообразности введения нанопрепа- ратов серебра как антибактериальных противовирусных средств в медицинскую практику в Российской Федера- ции / Е. М. Abstract. Благитко // Нанотехнологии и наноматериалы для биологии и медицины : науч.-практ. конф. с междунар. участием, 11–12 окт. 2007 г. : в 2 ч. / СибУПК [и др.]. – Но- восибирск, 2007. – Ч. 2. – С. 36–39. 8. Гусев, А. И. Наноматериалы, наноструктуры, нанотехно- логии / А. И. Гусев. – Москва : Физматлит, 2005. – 416 с. 9. К вопросу о токсичности наночастиц серебра при перо- ральном введении коллоидного раствора / Е. Н. Петрицкая [и др.] // Альм. клин. медицины. – 2011, № 25. – С. 9–12. Однако есть и исследования с отрицатель- ным результатом. Так, Fries C.A. с коллегами пока- зал, что повязка с наночастицами серебра не имеет преимуществ перед простой марлей при лечении ран после хирургических манипуляций [65], а Vermeulen H. с коллегами не нашел достаточных доказательств преимущества подобных средств для лечения инфицированных хронических ран [66]. Неудачными были исследования венозных катетеров, пропитанных наночастицами серебра. Их использование не оказало значительного влия- ния на частоту колонизации катетера, возникнове- ние инфекций, связанных с ним, или смерти паци- ентов в критическом состоянии [67]. 10. Не так страшен черт, как его малютка // Нанотехнологии. – 2008. – № 3. – С. 9–12. 11. Карпов, С. Оптические эффекты в металлических на- ноколлоидах / С. Карпов // Фотоника. – 2012. – № 2. – С. 40–51. 12. Ржеусский, С. Э. Валидация спектрофотометрической ме- тодики количественного определения наночастиц серебра в водных растворах / С. Э. Ржеусский // Вестн. фармации. – 2019. – № 1. – С. 21–25. 13. PubMed : National Library of Medicine [Electronic resource]. – Mode of access: https://pubmed.ncbi.nlm.nih. gov/?term=Silver+nanoparticles. – Date of access: 31.03.2022. 14. Водорастворимая серебросодержащая бактерицидная композиция и способ ее получения : пат. 2128047 RU : МПК A61K31/79, A61K33/38 / Г. Е. Афиногенов, В. В. Ко- пейкин, Е. Ф. Панарин ; заявитель и патентообладатель Г. Е. Афиногенов. – № 95119636/14 ; заявл. 21.06.95 ; опубл. 27.03.99. Как видно из представленных данных, се- ребро имеет хорошие перспективы в качестве противомикробного, ранозаживляющего и про- тивовоспалительного агента. Однако это далеко не панацея и подход к его использованию в меди- цине должен быть взвешенным и продуманным. 15. Способ лечения гнойных ран : пат. RU 2142279 C1 : МПК A61K 33/38, A61N 7/00 / А. М. Гнетнев, Б. Я. Поздняко- ва, Р. Д. Либерзон ; заявитель и патентообладатель Са- рат. науч.-исслед. ин-т травматологии и ортопедии. – № 95109614/14 ; заявл. 07.06.95 ; опубл. 10.12.99. 16. 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Selective cytotoxicity of green synthesized silver nanoparticles against the MCF-7 tumor cell line and their enhanced antioxidant and antimicrobial properties. Int J Nanomedicine. 2018 Nov;13:8013-8024. doi: 10.2147/IJN.S189295 42. Qing Y, Cheng L, Li R, Liu G, Zhang Y, Tang X, et al. Potential antibacterial mechanism of silver nanoparticles and the optimization of orthopedic implants by advanced modification technologies. Int J Nanomedicine. 2018 Jun;13:3311-3327. doi: 10.2147/IJN.S165125 29. Ponarulselvam S, Panneerselvam C, Murugan K, Aarthi N, Kalimuthu K, Thangamani S. Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn. G. Don and their 43. Frolova IuV, Kirsh IA, Beznaeva OV, Pomogova DA, 22 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 Tikhomirov AA. Creation of packaging polymeric materials with antimicrobial properties. Izv Vuzov Priklad Khimiia Biotekhnologiia. 2017;7(3):145-52. (In Russ.) 57. Boroumand Z, Golmakani N, Mazloum SR, Dadgar S, Golmohamadzadeh S. The effect of spray silver nanoparticles (Nivasha) on intensity of cesarean wound pain; A randomized clinical trial. Iran J Obstet Gynecol Infertil. 2018;21(9):83-92. doi: 10.22038/ijogi.2018.12138 44. Panáček A, Kvítek L, Smékalová M, Večeřová R, Kolář M, Röderová M, et al. Bacterial resistance to silver nanoparticles and how to overcome it. Nat Nanotechnol. 2018;13(1):65-71. 58. Abbasy FZ, Salsabyl I, Olfat S, Geraldine A. Intra-canal medication containing silver nanoparticle versus calcium hydroxide in reducing postoperative pain: A randomized clinical trial. Avaiable from: https://f1000research.com/ articles/7-1949/v1/. [Accessed 01th Apr 2022]. 45. Saravanan M, Arokiyaraj S, Lakshmi T, Pugazhendhi A. Synthesis of silver nanoparticles from Phenerochaete chrysosporium (MTCC-787) and their antibacterial activity against human pathogenic bacteria. 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Сведения об авторах: Ржеусский С.Э. – к.ф.н., доцент кафедры менеджмента и маркетинга фармации, Витебский государственный ор- дена Дружбы народов медицинский университет. References Vermeulen H, van Hattem JM, Storm-Versloot MN, Ubbink DT, Westerbos SJ. Topical silver for treating infected wounds. In: Cochrane. Trusted evidence. Informed decisions. Better health. Avaiable from: https://www.cochrane.org/CD005486/ WOUNDS_topical-silver-for-treating-infected-wounds. [Accessed 01th Apr 2022]. 55. Erring M, Gaba S, Mohsina S, Tripathy S, Sharma RK. Comparison of efficacy of silver-nanoparticle gel, nano-silver- foam and collagen dressings in treatment of partial thickness burn wounds. Burns. 2019 Dec;45(8):1888-1894. doi: 10.1016/j.burns.2019.07.019 67. Antonelli M, De Pascale G, Ranieri VM, Pelaia P, Tufano R, Piazza O, et al. Comparison of triple-lumen central venous catheters impregnated with silver nanoparticles (AgTive®) vs conventional catheters in intensive care unit patients. J Hosp Infect. 2012 Oct;82(2):101-7. doi: 10.1016/j.jhin.2012.07.010 56. Balzarro M, Rubilotta E, Mancini V, Pastore A, Soldano A, Trabacchin N, et al. Early and late efficacy on wound healing of silver nanoparticles gel (PeonilÂ®) in males underwent circumcision. J Urol. 2019 Oct;18(9 suppl):e3320. Submitted 22.02.2022 Accepted 21.04.2022 Submitted 22.02.2022 Accepted 21.04.2022 23 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Резюме. Проблема приверженности терапии продолжает оставаться одной из наиболее существенных для современной медицины. Цель работы состояла в анализе литературных данных о приверженности терапии в ревматологиче- ской практике. В настоящее время применяются различные определения приверженности терапии: «комплайнс», «приверженность», «согласование». Несмотря на значительный прогресс в создании новых высокоэффективных патогенетических лекарственных средств в ревматологии, эффективность назначенной терапии остается недо- статочно удовлетворительной. Нередко такая ситуация связана с низкой приверженностью лечению. При лечении ревматоидного артрита болезнь модифицирующими лекарственными средствами приверженность составляла около 65%. При остеоартрите отмечено снижение приверженности не только к лекарственной терапии, но и к другим методам консервативного лечения. Увеличение риска переломов на 16% было выявлено у тех пациентов с остеопорозом, у которых имела место плохая приверженность режиму лечения. При лечении нестероидными противовоспалительными лекарственными средствами риск неблагоприятных событий со стороны верхних от- делов желудочно-кишечного тракта у пациентов с низкой приверженностью терапии ингибиторами протонной помпы возрастал в 4,0 раза. От уровня приверженности лечению зависит скорость прогрессирования заболева- ния, степень функциональных нарушений и уровень качества жизни пациентов. Ключевые слова: приверженность терапии, ревматоидный артрит, остеоартрит, остеопороз, комплайнс. фу ру ур ые слова: приверженность терапии, ревматоидный артрит, остеоартрит, остеопороз, комплайн ПРИВЕРЖЕННОСТЬ ТЕРАПИИ В РЕВМАТОЛОГИЧЕСКОЙ ПРАКТИКЕ. ОБЗОР ЛИТЕРАТУРЫ 1Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus 2Vitebsk Regional Clinical Hospital, Vitebsk, Republic of Belarus Vestnik VGMU. 2022;21(2):25-34. DOI: https://doi.org/10.22263/2312-4156.2022.2.25 ПРИВЕРЖЕННОСТЬ ТЕРАПИИ В РЕВМАТОЛОГИЧЕСКОЙ ПРАКТИКЕ. ОБЗОР ЛИТЕРАТУРЫ ДИКАРЕВА Е.А.1, ПИМАНОВ С.И.1, МАКАРЕНКО Е.В.1, ЛАГУТЧЕВ В.В.1, КУХАРЕВ А.В.2 1Витебский государственный ордена Дружбы народов медицинский университет, г. Витебск, Республика Беларусь 2Витебская областная клиническая больница, г. Витебск, Республика Беларусь Вестник ВГМУ. – 2022. – Том 21, №2. – С. 25-34. ADHERENCE TO THERAPY IN RHEUMATOLOGY PRACTICE. LITERATURE REVIEW DIKAREVA E.A.1, PIMANOV S.I.1, MAKARENKO E.V.1, LAHUTCHAU V.V.1, KUKHARAU A.V.2 1Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus 2Vitebsk Regional Clinical Hospital, Vitebsk, Republic of Belarus Vestnik VGMU 2022;21(2):25-34 DOI: https://doi.org/10.22263/2312-4156.2022.2.25 Information about authors: f Rzheussky S.E. – Candidate of Pharmaceutical Sciences, associate professor of the Chair of Management & Marketing of Pharmacy, Vitebsk State Order of Peoples’ Friendship Medical University. Адрес для корреспонденции: Республика Беларусь, 210009, г. Витебск, пр. Фрунзе, 27, Витебский государствен- ный ордена Дружбы народов медицинский университет, кафедра менеджмента и маркетинга фармации. E-mail: sirrr@inbox.ru – Ржеусский Сергей Эдуардович. Correspondence address: Republic of Belarus, 210009, Vitebsk, 27 Frunze ave., Vitebsk State Order of Peoples’ Friendship Medical University, Chair of Management & Marketing of Pharmacy. E-mail: sirrr@inbox.ru – Sergey E. Rzheussky. 24 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Abstract. The problem of adherence to therapy continues to be one of the most significant for modern medicine. The aim of the work was to analyze the literature data on adherence to therapy in rheumatological practice. Currently, various definitions of adherence to therapy are used: «compliance», «adherence», «concordance». Despite significant progress in the creation of new highly effective pathogenetic drugs in rheumatology, the effectiveness of the prescribed therapy remains insufficiently satisfactory. Often this situation is associated with low adherence to treatment. In the treatment of rheumatoid arthritis with disease modifying drugs, adherence made up about 65%. In osteoarthritis, there was a decrease in adherence not only to drug therapy, but also to other methods of conservative treatment. An increase in fracture risk by 16% was found in those osteoporotic patients who had poor adherence to treatment. When treated with non-steroidal anti-inflammatory drugs, the risk of adverse events from the upper gastrointestinal tract in patients with low adherence to therapy with proton pump inhibitors increased 4.0 times. The rate of progression of the disease, the degree of functional disorders and the level of patients’ life quality depend on the level of adherence to treatment. d dh h h d h h h b l 25 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 За последние 20 лет улучшилась доступ- ность и эффективность лекарственных средств (ЛС), применяемых при ревматологических забо- леваниях, таких как ревматоидный артрит (РА), остеоартрит (ОА) и остеопороз. Это привело к уменьшению количества обострений и замедле- нию прогрессирования заболеваний. Для получе- ния максимальной пользы от назначенного лече- ния пациенты должны соблюдать предписанные им рекомендации. Однако в связи с тем, что схе- мы назначенной терапии могут быть сложными, а лечение необходимо продолжать в течение дли- тельного времени, возможны различные нару- шения режима. При терапии ревматологических заболеваний выявляется низкая приверженность лечению, которая приводит к ухудшению резуль- татов терапии. Причины потери контроля над за- болеванием многоплановые и сложные. Одним из факторов, который может повлиять на конеч- ный фармакологический результат, является при- верженность лечению. Приверженность лечению определяется в настоящее время как степень со- ответствия приема ЛС рекомендациям врача [1]. Была изучена база данных PubMed с ключе- выми словами «комплайнс», «приверженность» и «согласование» с 2000 по 2020 гг. Было найдено 219937 опубликованных работ, в которых употре- блялось слово «приверженность», 151463 статей с термином «комплайнс» и 55755 работ с терми- ном «согласование». Abstract. Такое большое внимание к этому вопросу говорит о значимости привержен- ности терапии в современной медицине и обще- стве. Цель работы состояла в анализе литератур- ных данных о приверженности терапии в ревма- тологической практике. Различия в терминологии В настоящее время применяются разноо- бразные определения приверженности терапии, что может приводить к возникновению трудно- стей при проведении сравнений исследований. В литературе встречаются различные определе- ния: комплайнс (compliance), приверженность (adherence), согласование (concordance). Комплайнс (compliance) – действия паци- ента, основанные на рекомендациях врача. Ис- пользование термина «комплайнс» предполагает отсутствие обсуждения назначенного лечения между пациентом и врачом. Назначенная терапия устанавливается врачом без согласования с паци- ентом. Несоблюдение режима лечения можно раз- делить на два подтипа: непреднамеренное (из-за забывчивости, сложности режима или физиче- ских проблем) и преднамеренное (основанное на решении пациента не принимать или принимать меньше лекарств). В случае преднамеренного несоблюдения режима пациенты, вероятно, про- водят анализ пользы и риска, взвешивая предпо- лагаемые риски лечения и возможные преимуще- ства. На это могут влиять убеждения пациента о ЛС, предыдущая эффективность лечения паци- ента и его осведомленность о заболевании. Это означает, что, помимо устранения практических препятствий, врачи должны быть внимательны к личным убеждениям пациента, которые могут повлиять на приверженность назначенному лече- нию [2]. Приверженность (adherence) – использова- ние ЛС, выполнение диетических рекомендаций, а также модификация образа жизни согласно ме- дицинским назначениям. Применение термина «приверженность» означает, что врач проинфор- мировал о лечении пациента, а он дал согласие на выполнение предписанных рекомендаций. Данный термин используют для того, чтобы определить, как пациенты следуют медицинским рекомендациям. Выполнение назначенной тера- пии имеет большое значение в контроле над за- болеваниями [4]. Когда пациенты не соблюдают назначен- ную лекарственную терапию, то может создаться впечатление, что лечение не помогает. Если при- чиной плохого ответа на проводимую терапию является низкая приверженность лечению, иг- норирование приема ЛС и несоблюдение пред- писанного режима лечения, то это приведет к увеличению дозы лекарства или к добавлению других ЛС. Все это в дальнейшем сопровожда- ется увеличением риска развития нежелательных побочных эффектов и возрастанием стоимости лечения [3]. Согласование (concordance) – это достиже- ние после совместного обсуждения соглашения между медицинским работником и пациентом в отношении назначенной терапии. В данном слу- чае врачом принимаются во внимание пожелания пациента, касающиеся дальнейшего лечения, а также учитываются возможности и убеждения пациента. Стоит отметить, что в такой ситуации пациент намерен выполнять предписанные на- значения [5]. Термин «согласование» использу- 26 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № возрастанию частоты модификаций лечения [10, 11]. ется в основном в Великобритании. В литерату- ре последних лет часто применяется в качестве синонима для терминов «приверженность» или «комплайнс». При РА приверженность к лечению сильно варьирует и, как правило, является неоптималь- ной. Различия в терминологии При этом данные о приверженности к БМЛС варьируют от 30% (недостаточное использова- ние) до 107% (чрезмерное использование) [12]. Несоблюдение режима лечения БМЛС связано с ухудшением клинической картины и увеличени- ем инвалидности [13], а высокая приверженность сопровождается снижением активности заболе- вания [14]. Особую роль приверженность терапии играет среди пожилых пациентов, которые часто имеют сопутствующие заболевания и принимают сразу несколько ЛС. Отсутствие приверженно- сти назначенной терапии может сопровождаться прогрессированием заболеваний, учащением обострений и приводить к инвалидизации паци- ентов. Все это в дальнейшем сопровождается на- значением дополнительных ЛС или увеличением количества госпитализаций [6]. Метотрексат рекомендуется в качестве БМЛС как терапия первой линии у пациентов с РА, а также он может использоваться в комбина- ции с другими БМЛС или в сочетании с биологи- ческими генно-инженерными ЛС [3, 15]. Несоблюдение режима лечения в первые 6 месяцев терапии является важным предиктором более высокой активности ревматологического заболевания [7]. В исследовании C.A. Waimann и соавт. проводилась количественная оценка привержен- ности пероральной терапии у пациентов с РА из различных этнических групп и у малоимущих пациентов с помощью электронного мониторин- га приема ЛС (данная система предназначена для отслеживания приверженности лечению и представляет собой крышку, которая устанав- ливается на стандартные флаконы с лекарства- ми и записывает время и дату каждый раз, ког- да флакон открывается и закрывается), а также осуществлялось изучение клинических послед- ствий низкой приверженности терапии. В двух- летнее проспективное когортное исследование вошло 107 пациентов с РА из трех государствен- ных амбулаторных ревматологических клиник в Хьюстоне штата Техас. Все участники исследо- вания согласились на электронный мониторинг приема пероральной лекарственной терапии РА с использованием системы мониторинга меди- каментозных событий. Приверженность БМЛС и преднизолону определялась как процент дней (или недель для метотрексата), в течение кото- рых пациент принимал правильную дозу, пред- писанную врачом. В работе проводилась оценка здоровья, активности заболевания по системе DAS28 (Disease Activity Score), качества жизни и рентгенологические изменения. Соблюдение схемы лечения определялось процентным соот- ношением правильных доз. В ходе выполненного исследования было обнаружено, что соблюдение схемы лечения составило 64% для БМЛС и 70% для преднизолона. Было показано, что пациенты с лучшим психическим здоровьем статистиче- ски более склонны к приверженности. Правиль- В ревматологической практике необходимо как можно скорее достичь ремиссии для того, что- бы избежать необратимого повреждения тканей и попасть в так называемое «окно возможностей». Несоблюдение режима лечения требует особого внимания особенно в первый год лечения. Различия в терминологии Рев- матологи прежде всего должны знать, что низкая приверженность терапии является важным фак- тором, который следует учитывать при лечении пациентов и оценке эффективности болезнь мо- дифицирующими лекарственными средствами (БМЛС). Совместное принятие решений (согласова- ние) рассматривается как важный всеобъемлю- щий принцип лечения, который был добавлен к рекомендациям Европейской лиги против ревма- тизма (EULAR) по ведению РА в 2010 году [8]. Это действенный способ, при помощи которого можно улучшить приверженность лечению. В по- вседневной практике ревматолог должен строить открытые и доверительные отношения с пациен- том, в которых можно открыто обсуждать режим лечения [9]. Приверженность лечению пациен- тов с ревматоидным артритом Для лечения пациентов с РА доступны вы- сокоэффективные фармакотерапевтические вари- анты лечения. Тем не менее, многим пациентам не удается достичь ремиссии заболевания, что увеличивает вероятность функционального ухуд- шения, частоту повторных лабораторных иссле- дований и госпитализаций, а также приводит к 27 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 но было принято менее двух третей предписан- ных доз БМЛС. Только 23 пациента (21%) имели среднюю приверженность к БМЛС ≥ 80%. Через 2 года наблюдения у этих пациентов были значи- тельно лучшие средние значения DAS28, чем у тех, кто был менее привержен лечению (3,28 про- тив 4,09; р=0,02). Рентгенологические показатели также были хуже у пациентов с отсутствием при- верженности. При помощи пошагового регресси- онного анализа было выявлено, что привержен- ность была связана с такими показателями как нахождение в браке (p<0,01), низкая активность заболевания (p<0,05) и хорошее психическое здо- ровье (p<0,05) [16]. Имеются данные о том, что наличие ОА в 1,6 раза увеличивает риск общей смертности в сравнении с общей популяцией. В исследовании R.J. Cleveland и соавт. указывалось, что устране- ние функциональных ограничений и боли, на- блюдаемых при ОА, может потенциально сни- зить повышенную смертность, наблюдаемую у этих людей [20]. но было принято менее двух третей предписан- ных доз БМЛС. Только 23 пациента (21%) имели среднюю приверженность к БМЛС ≥ 80%. Через 2 года наблюдения у этих пациентов были значи- тельно лучшие средние значения DAS28, чем у тех, кто был менее привержен лечению (3,28 про- тив 4,09; р=0,02). Рентгенологические показатели также были хуже у пациентов с отсутствием при- верженности. При помощи пошагового регресси- онного анализа было выявлено, что привержен- ность была связана с такими показателями как нахождение в браке (p<0,01), низкая активность заболевания (p<0,05) и хорошее психическое здо- ровье (p<0,05) [16]. Результат лечения ОА зависит от многих факторов (медикаментозное и немедикаментоз- ное лечение, уровень образования, масса тела). Особое значение проблема низкой приверженно- сти приобретает у пациентов с ОА при назначе- нии ЛС с отсроченным эффектом. В ретроспек- тивном исследовании Ю.В. Поляковой и соавт. проводилась оценка состояния костно-мышечной системы и потребности в нестероидных противо- воспалительных средствах (НПВС) у пациентов с ОА коленных суставов в зависимости от дли- тельности периода терапии ЛС на основе неомы- ляемых соединений авокадо и сои. Приверженность лечению пациен- тов с ревматоидным артритом Было показа- но, что у пациентов, завершивших прием только одного курса терапии, выявлялось выраженное ухудшение функционального состояния суставов и увеличение потребности в НПВС в сравнении с теми пациентами, которые использовали данные ЛС в различных режимах (регулярный и курсо- вой режим) в течение пяти лет [21]. В исследовании A. Pasma и соавт. было показано снижение приверженности лечению БМЛС с увеличением продолжительности лече- ния. Только при приеме преднизолона не наблю- далось снижение приверженности. Показатели несоблюдения режима лечения были самыми высокими для сульфасалазина. Приверженность терапии сульфасалазином снизилась с 80% через 3 месяца до 53,8% через 12 месяцев. Привержен- ность метотрексату уменьшилась с 91,2% через 3 месяца до 69,3% через 12 месяцев [7]. В исследовании I. Contreras-Yáñez и соавт. было показано, что плохая приверженность тера- пии у пациентов с РА ассоциирована с приемом более 3 БМЛС (отношение шансов (ОШ) 31,5; 95% доверительный интервал (ДИ): 2,3–433,3; p=0,009)) [13]. При ОА отмечается снижение привержен- ности не только к лекарственной терапии, но и к другим методам консервативного лечения. По данным Максимова Д.М., приверженность к ле- чебной гимнастике составила только 37% [22]. Плохое долгосрочное соблюдение режима при- верженности к лечебной физкультуре продемон- стрировало негативное влияние на терапевтиче- ский эффект этого метода лечения [18]. Исследование, проведенное Ахунова Р.Р. и соав., показало, что только 65,6% пациентов с РА были привержены терапии БМЛС. При этом при- верженными лечению считались те пациенты, которые принимали БМЛС более 80% времени с момента их назначения. Снижение приверженно- сти наблюдалось при увеличении длительности суставного синдрома (р<0,05) [17]. Приверженность приему ингибито- ров протонной помпы НПВС обладают анальгетическими и про- тивовоспалительными свойствами, благодаря которым приобрели широкое распространение среди пациентов с ревматологическими заболева- ниями [28, 29]. Применение НПВС может приве- сти к появлению симптомов со стороны верхних отделов желудочно-кишечного тракта (ЖКТ), та- ких как диспепсия, гастродуоденальные эрозии и язвы, а также кровотечение [30, 31]. Для профи- лактики гастропатии, индуцированной приемом НПВС (НПВС-гастропатия), необходимо со- вместно с данными ЛС использовать ингибиторы протонной помпы (ИПП) [32, 33]. Исследование IMPACT, в котором участво- вало 2302 женщины с постменопаузальным осте- опорозом, показало, что клинически значимое снижение (>50%) уровней биохимических мар- керов резорбции костной ткани можно ожидать только у пациентов с приверженностью приему бисфосфонатов более 60% [25]. Фактическое одновременное использова- ние ИПП с НПВС может быть проблематичным по двум причинам: неоптимальное назначение врачом и неправильное использование пациен- том. Хотя ситуация в последнее время улучшает- ся, в исследованиях указывается, что пациентам с факторами риска НПВС-гастропатии не всегда назначаются ИПП, или же пациенты их не еже- дневно принимают [34, 35]. Связь между неправильным использовани- ем бисфосфонатов и неблагоприятным воздей- ствием на минеральную плотность кости была продемонстрирована в исследовании R.A. Yood и соавт. В исследование было включено 176 жен- щин с остеопорозом, которые ранее не получали лечения. Было выявлено, что пациенты, которые принимали не менее двух третьих доз бисфосфо- натов, показали увеличение минеральной плот- ности кости в поясничном отделе позвоночника и бедре на 3,8% и 2,6% соответственно. Это увели- чение было значительно выше, чем у пациентов, которые были менее привержены назначенной терапии [26]. В работе K. Henriksson и соавт. изучал- ся прием ИПП при одновременном назначении НПВС. В ходе этого ретроспективного исследо- вания историй болезней были выявлены паци- енты с диагнозом «ОА», «РА» или «анкилози- рующий спондилоартрит», которым назначены ИПП вместе с НПВС. Все пациенты нуждались в лечении гастропротективными средствами на основании клинической оценки врачей и были проинструктированы о необходимости всегда совместно с НПВС использовать ИПП. Фактиче- ское потребление НПВП и ИПП было ретроспек- тивно зарегистрировано с использованием анке- ты самооценки. Приверженность к лечению ИПП оценивалась с использованием описательной статистики. Всего было включено 96 пациентов (72% с ОА, 16% с РА, 12% с анкилозирующим спондилоартритом). Среднее значение привер- женности пациентов назначенным ИПП состави- ло 73-81%. Доля пациентов с самооценкой при- верженности лечению ≤80% была 26%. Никаких прогностических факторов низкой приверженно- сти выявить в данной работе не удалось [36]. В исследовании J.J. Caro и соавт. Приверженность лечению пациен- тов с остеоартритом Остеопороз – это заболевание, которое свя- зано с нарушением архитектуры кости, уменьше- нием минеральной плотности кости и увеличени- ем риска переломов [23]. ОА является одним из наиболее распро- страненных заболеваний суставов, приводящих к разрушению хряща и значительному ухудшению качества жизни пациентов [18]. ОА является важ- ной медико-социальной проблемой в связи с тем, что имеет высокую распространенность, приво- дит к временной и стойкой утрате трудоспособ- ности и занимает лидирующие позиции в инва- лидизации пациентов [19]. Низкая приверженность приему бисфосфо- натов является огромной проблемой при лечении постменопаузальнго остеопороза, приводит к увеличению риска переломов и негативно влия- ет на качество жизни. J.A. Cramer и соавт. оце- нивали прием бисфосфонатов с 1997 по 2002 гг. у женщин с остеопорозом в постменопаузе (>45 28 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 ВЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, лет). В данной работе изучалось несколько ва- риантов назначения бисфосфонатов: один раз в неделю (алендронат в дозе 35 мг или 70 мг) или ежедневное использование (алендронат 5 мг или 10 мг или ризедронат 5 мг). Пациенты с разными вариантами приема ЛС наблюдались в течение 12 месяцев. Было выявлено, что у участников иссле- дования, принимавших бисфосфонаты один раз в неделю, была значительно более высокая привер- женность к лечению, чем у пациентов, осущест- влявших ежедневный прием данных ЛС (69,2% против 57,6%, p≤0,0001). Следовательно, умень- шение частоты перорального приема бисфосфо- натов является одной из доступных мер для по- вышения приверженности терапии [24]. лет). В данной работе изучалось несколько ва- риантов назначения бисфосфонатов: один раз в неделю (алендронат в дозе 35 мг или 70 мг) или ежедневное использование (алендронат 5 мг или 10 мг или ризедронат 5 мг). Пациенты с разными вариантами приема ЛС наблюдались в течение 12 месяцев. Было выявлено, что у участников иссле- дования, принимавших бисфосфонаты один раз в неделю, была значительно более высокая привер- женность к лечению, чем у пациентов, осущест- влявших ежедневный прием данных ЛС (69,2% против 57,6%, p≤0,0001). Следовательно, умень- шение частоты перорального приема бисфосфо- натов является одной из доступных мер для по- вышения приверженности терапии [24]. Приверженность приему ингибито- ров протонной помпы с участи- ем 11249 женщин с остеопорозом значительно больший риск переломов был сопряжен с плохой приверженностью режиму лечения по сравнению с пациентами, у которых определялась хорошая приверженность (на 16% более высокий риск; 95% ДИ: 5–25%). В этом исследовании также со- общалось о значительной связи между плохой приверженностью режиму и увеличением часто- ты госпитализаций, которые приводили к возрас- танию затрат на лечение на 14% [27]. Таким образом, многочисленные исследо- вания показали, что пациенты, которые не при- нимают адекватное количество бисфосфонатов, подвергаются большему риску переломов [24, 25, 27]. В другом исследовании оценивалось вли- яние приверженности приема ИПП на частоту развития повреждений ЖКТ среди пациентов с РА при длительном приеме НПВС. В соответ- 29 ESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 ствии с приемом ИПП все участники исследо- вания были разделены на три группы: высокой, средней и низкой приверженностью к гастропро- тективной терапией. Было показано, что только 36,7% обследованных, которым для профилак- тики возникновения НПВС-гастропатии были назначены ИПП, строго следовали назначениям врача. НПВС-гастропатия была обнаружена у 50% пациентов с низкой приверженностью лече- нию ИПП, у 12,5% участников исследования со средней приверженностью и у 4,5% пациентов с высокой приверженностью гастропротектив- ной терапии. При низкой приверженности при- ему ИПП эрозивно-язвенные повреждения ЖКТ встречались в 11 раз чаще, чем при высокой при- верженности (χ2=7,77; p=0,005) [34]. ствии с приемом ИПП все участники исследо- вания были разделены на три группы: высокой, средней и низкой приверженностью к гастропро- тективной терапией. Было показано, что только 36,7% обследованных, которым для профилак- тики возникновения НПВС-гастропатии были назначены ИПП, строго следовали назначениям врача. НПВС-гастропатия была обнаружена у 50% пациентов с низкой приверженностью лече- нию ИПП, у 12,5% участников исследования со средней приверженностью и у 4,5% пациентов с высокой приверженностью гастропротектив- ной терапии. При низкой приверженности при- ему ИПП эрозивно-язвенные повреждения ЖКТ встречались в 11 раз чаще, чем при высокой при- верженности (χ2=7,77; p=0,005) [34]. В другой работе указывалось, что при уменьшении приверженности гастропротектив- ной терапии на 10% отмечали увеличение часто- ты гастродуоденальных осложнений на 16%. По сравнению с пациентами с приверженностью те- рапии ИПП > 80%, пациенты с приверженностью 20–80% и <20% имели увеличение риска в 2,5 (95% ДИ: 1,0-6,7) и 4,0 (95% ДИ: 1,2-13,0) раза соответственно [38]. Подобные результаты были получены в других исследованиях [40, 41]. Немногие паци- енты получают сопутствующую гастропротек- тивную терапию при назначении НПВС, хотя ее применение увеличивается при наличии гастро- интестинальных факторов риска. Заключение Приверженность пациентов лечению оста- ется одной из важных проблем в повседневной клинической практике. Несоблюдение режима приема ЛС может сильно повлиять как на кра- ткосрочные, так и на долгосрочные результаты, привести к увеличению частоты обострений и го- спитализаций пациентов с ревматологическими заболеваниями. Особое значение низкий уровень приверженности терапии имеет при хронических заболеваниях. От уровня приверженности лече- нию зависит скорость прогрессирования забо- левания, степень функциональных нарушений и уровень качества жизни пациентов. Приверженность приему ингибито- ров протонной помпы Соблюдение режима приема ИПП при использовании НПВС имеет первостепенное значение для уменьшения числа гастродуоденальных повреждений [42]. J.L. Goldstein и соавт. оценивали прием гастропротективных средств (мизопростол или ИПП) среди пациентов, которым впервые были назначены НПВС. Анализ проводился среди 144 203 пациентов. Было выявлено, что только 1,8% получали лечение гастропротекторами. Частота использования мизопростола или ИПП повыша- лась при наличии следующих факторов риска: возраст старше 65 лет (ОШ 1,40; 95% ДИ: 1,3- 1,5), наличие в анамнезе язвенной болезни (ОШ 2,5; 95% ДИ: 1,8-3,3), эзофагит/гастроэзофаге- альный рефлюкс (ОШ 3,8; 95% ДИ: 3,5-4,1), язва/ кровотечение из верхних отделов ЖКТ (ОШ 1,4; 95% ДИ: 1,2-1,5) или гастрит (ОШ 2,5; 95% ДИ: 2,2-2,8). Среди участников исследования, полу- чавших сопутствующую гастропротективную терапию, 68% имели показатели приверженности 80% и более. Значительно более высокий риск развития язв/осложнений со стороны верхних от- делов ЖКТ наблюдался у пациентов, принимав- ших НПВС и имевших приверженность к гастро- протективной терапии менее 80% по сравнению с участниками исследования с приверженностью 80% или более (ОШ 2,4; 95% ДИ: 1,0-5,6) [35]. Литература Compliance and persistence with bisphosphonate dosing regimens among women with postmenopausal osteoporosis / J. A. Cramer [et al.] // Curr. Med. Res. 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Effect of monitoring bone turnover markers on persistence with risedronate treatment of postmenopausal osteoporosis. J Clin Endocrinol Metab. 2007 Apr;92(4):1296-304. doi: 10.1210/jc.2006-1526 14. Cannon GW, Mikuls TR, Hayden CL, Ying J, Curtis JR, Reimold AM, et al. Merging Veterans Affairs rheumatoid arthritis registry and pharmacy data to assess methotrexate adherence and disease activity in clinical practice. Arthritis Care Res (Hoboken). 2011 Dec;63(12):1680-90. doi: 10.1002/acr.20629 26. Yood RA, Emani S, Reed JI, Lewis BE, Charpentier M, Lydick E. Compliance with pharmacologic therapy for osteoporosis. Osteoporos Int. 2003 Dec;14(12):965-8. doi: 10.1007/s00198-003-1502-4 15. Singh JA, Furst DE, Bharat A, Curtis JR, Kavanaugh AF, Kremer JM, et al. 2012 update of the 2008 American College of Rheumatology recommendations for the use of disease-modifying antirheumatic drugs and biologic agents in the treatment of rheumatoid arthritis. 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Сведения об авторах: Дикарева Е.А. – к.м.н., доцент кафедры внутренних болезней и ультразвуковой диагностики ФПК и ПК, Витеб- ский государственный ордена Дружбы народов медицинский университет; Дикарева Е.А. – к.м.н., доцент кафедры внутренних болезней и ультразвуковой диагностики ФПК и ПК, Витеб- ский государственный ордена Дружбы народов медицинский университет; Пиманов С.И. – д.м.н., профессор, заведующий кафедрой внутренних болезней и ультразвуковой диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет; Макаренко Е.В. – д.м.н., профессор кафедры внутренних болезней и ультразвуковой диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет; Лагутчев В.В. – к.м.н., доцент кафедры внутренних болезней и ультразвуковой диагностики ФПК и ПК, Витеб- ский государственный ордена Дружбы народов медицинский университет; Кухарев А.В. – врач-эндоскопист, Витебская областная клиническая больница. Пиманов С.И. – д.м.н., профессор, заведующий кафедрой внутренних болезней и ультразвуковой диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет; Макаренко Е.В. – д.м.н., профессор кафедры внутренних болезней и ультразвуковой диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет; Лагутчев В.В. – к.м.н., доцент кафедры внутренних болезней и ультразвуковой диагностики ФПК и ПК, Витеб- ский государственный ордена Дружбы народов медицинский университет; Кухарев А.В. – врач-эндоскопист, Витебская областная клиническая больница. References Prevalence of osteoporosis and osteopenia among women over fifty years of age, from the city of Durango, Mexico, diagnosed by forearm-DEXA. Gac Med Mex. 2007 Sep- Oct;143(5):365-9. 37. van Soest EM, Valkhoff VE, Mazzaglia G, Schade R, Molokhia M, Goldstein JL, et al. Suboptimal gastroprotective coverage of NSAID use and the risk of upper gastrointestinal bleeding and ulcers: an observational study using three European databases. Gut. 2011 Dec;60(12):1650-9. doi: 10.1136/gut.2011.239848 24. Cramer JA, Amonkar MM, Hebborn A, Altman R. Compliance and persistence with bisphosphonate dosing regimens among women with postmenopausal osteoporosis. Curr Med Res Opin. 2005 Sep;21(9):1453- 60. doi: 10.1185/030079905X61875 38. van Soest EM, Sturkenboom MCJM, Dieleman JP, 33 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Verhamme KMC, Siersema PD, Kuipers EJ. Adherence to gastroprotection and the risk of NSAID-related upper gastrointestinal ulcers and haemorrhage. Aliment Pharmacol Ther. 2007 Jul;26(2):265-75. doi: 10.1111/j.1365- 2036.2007.03358.x Verhamme KMC, Siersema PD, Kuipers EJ. Adherence to gastroprotection and the risk of NSAID-related upper gastrointestinal ulcers and haemorrhage. Aliment Pharmacol Ther. 2007 Jul;26(2):265-75. doi: 10.1111/j.1365- 2036.2007.03358.x during cyclooxygenase 2 inhibitor treatment and the risk of upper gastrointestinal tract events: a population-based study. Arthritis Rheum. 2012 Aug;64(8):2792-802. doi: 10.1002/art.34433 during cyclooxygenase 2 inhibitor treatment and the risk of upper gastrointestinal tract events: a population-based study. Arthritis Rheum. 2012 Aug;64(8):2792-802. doi: 10.1002/art.34433 during cyclooxygenase 2 inhibitor treatment and the risk of upper gastrointestinal tract events: a population-based study. Arthritis Rheum. 2012 Aug;64(8):2792-802. doi: 10.1002/art.34433 41. Jonasson C, Hatlebakk JG, Lundell L, Kouri JP, Andersen M, Granath F. Association between adherence to concomitant proton pump inhibitor therapy in current NSAID users and upper gastrointestinal complications. Eur J Gastroenterol Hepatol. 2013 May;25(5):531-8. doi: 10.1097/MEG.0b013e32835d5acd 39. Sturkenboom MCJM, Burke TA, Tangelder MJD, Dieleman JP, Walton S, Goldstein JL. Adherence to proton pump inhibitors or H2-receptor antagonists during the use of non-steroidal anti-inflammatory drugs. Aliment Pharmacol Ther. 2003 Dec;18(11-12):1137-47. doi: 10.1046/j.1365- 2036.2003.01795.x 42. Pimanov SI, Dikareva EA, Makarenko EV. Commitment to pharmacotherapy is a prerequisite for effective treatment. Lecheb Delo. 2014;(5):47-52. (In Russ.) 40. Valkhoff VE, van Soest EM, Mazzaglia G, Molokhia M, Schade R, Trifiro G, et al. Adherence to gastroprotection Submitted 23.11.2021 Accepted 21.04.2022 Submitted 23.11.2021 Accepted 21.04.2022 Резюме. Ключевые слова: посттравматическое стрессовое расстройство, рыбий жир, артериальное давление, оксид азота, прооксидантно-антиоксидантный статус. р р , у р р ру Заключение. Введение рыбьего жира крысам предупреждает развитие артериальной гипотензии, предотвращает изменение концентрации еNOS и iNOS, препятствует нарушению прооксидантно-антиоксидантного статуса сы- воротки крови и уменьшает выраженность системного воспаления у животных с ПТСР. р р у р у Ключевые слова: посттравматическое стрессовое расстройство, рыбий жир, артериальное давление, оксид азота, прооксидантно-антиоксидантный статус. FISH OIL PREVENTS THE DEVELOPMENT OF ARTERIAL HYPOTENSION IN RATS WITH POST-TRAUMATIC STRESS DISORDER Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus Information about authors: Dikareva E.A. – Candidate of Medical Sciences, associate professor of the Chair of Internal Diseases & Ultrasound Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Pimanov S.I. – Doctor of Medical Sciences, professor, head of the Chair of Internal Diseases & Ultrasound Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Makarenko E.V. – Doctor of Medical Sciences, professor of the Chair of Internal Diseases & Ultrasound Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Lahutchau V.V. – Candidate of Medical Sciences, associate professor of the Chair of Internal Diseases & Ultrasound Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Kukharau A.V. – endoscopist, Vitebsk Regional Clinical Hospital. Kukharau A.V. – endoscopist, Vitebsk Regional Clinical Hospital. Адрес для корреспонденции: Республика Беларусь, 210009, г. Витебск, пр. Фрунзе, 27, Витебский государствен- ный ордена Дружбы народов медицинский университет, кафедра внутренних болезней и ультразвуковой диагно- стики ФПК и ПК. E-mail: ruselikelena@mail.ru – Дикарева Елена Александровна. Correspondence address: Republic of Belarus, 210009, Vitebsk, 27 Frunze ave., Vitebsk State Order of Peoples’ Friendship Medical University, Chair of Internal Diseases & Ultrasound Diagnostics of the Faculty for Advanced Training & Retraining. E-mail: ruselikelena@mail.ru – Elena A. Dikareva. 34 ВЕСТНИ ФИЗИОЛОГИЯ DOI: https://doi.org/10.22263/2312-4156.2022.2.35 КУЖЕЛЬ О.П. Витебский государственный ордена Дружбы народов медицинский университет, г. Витебск, Республика Беларусь Витебский государственный ордена Дружбы народов медицинский университет, г. Витебск, Республика Беларусь Вестник ВГМУ. – 2022. – Том 21, №2. – С. 35-44. Резюме. Резюме. Цель исследования – оценить характер и механизмы протекторного действия рыбьего жира (РЖ) на системную гемодинамику у крыс с экспериментальным аналогом посттравматического стрессового расстройства (ПТСР). Материал и методы. Исследования проведены на 50 беспородных белых крысах- самцах массой 210-240 г, рас- пределенных по группам: «Контроль», «ПТСР», «РЖ», «РЖ+ПТСР». Аналог ПТСР воспроизводили посредством контакта крыс с кошачьими экскрементами в течение 10 дней по 15 мин ежедневно. Рыбий жир вводили внутри- желудочно по 0,2 мл (100 мг/кг массы тела эйкозапентаеновой (ЭПК) и докозагексаеновой (ДГК) кислот, ЗАО «Биосола», Литва) за 3 дня до моделирования ПТСР и в течение 10 дней действия стрессора. Спустя 2 недели измеряли систолическое, диастолическое, среднее артериальное давление (САД, ДАД, СрАД) и частоту сердеч- ных сокращений (ЧСС) неинвазивным методом. В сыворотке крови методом иммуноферментного анализа (ИФА) определяли концентрацию эндотелиальной NO-синтазы (eNOS), индуцибельной NO-синтазы (iNOS), кортикосте- рона, С-реактивного белка (С-РБ), интерлейкина-1β (ИЛ-1β); спектрофотометрическим методом – концентрацию малонового диальдегида (МДА), диеновых конъюгатов (ДК), а также активность каталазы (КАТ) и супероксид- дисмутазы (СОД). Результаты. У животных с ПТСР наблюдалось снижение САД, ДАД и СрАД на 18-23% и увеличение ЧСС на 20%. Концентрация eNOS в крови крыс с ПТСР снижалась на 28%, а iNOS увеличилась в 2,28 раза, наряду с увеличением содержания ДК и МДА в 3,3 и 3,6 раза на фоне снижения активности СОД на 27%, КАТ на 59% и увеличения концентрации С-РБ и ИЛ-1β в 1,6 и 3 раза соответственно, по сравнению с контролем. Введение РЖ либо предотвращало, либо существенно ограничивало выраженность обнаруженных изменений. Результаты. У животных с ПТСР наблюдалось снижение САД, ДАД и СрАД на 18-23% и увеличение ЧСС на 20%. Концентрация eNOS в крови крыс с ПТСР снижалась на 28%, а iNOS увеличилась в 2,28 раза, наряду с увеличением содержания ДК и МДА в 3,3 и 3,6 раза на фоне снижения активности СОД на 27%, КАТ на 59% и увеличения концентрации С-РБ и ИЛ-1β в 1,6 и 3 раза соответственно, по сравнению с контролем. Введение РЖ либо предотвращало, либо существенно ограничивало выраженность обнаруженных изменений. Заключение. Введение рыбьего жира крысам предупреждает развитие артериальной гипотензии, предотвращает изменение концентрации еNOS и iNOS, препятствует нарушению прооксидантно-антиоксидантного статуса сы- воротки крови и уменьшает выраженность системного воспаления у животных с ПТСР. р д р щ , ущ р р ру Заключение. Введение рыбьего жира крысам предупреждает развитие артериальной гипотензии, предотвращает изменение концентрации еNOS и iNOS, препятствует нарушению прооксидантно-антиоксидантного статуса сы- воротки крови и уменьшает выраженность системного воспаления у животных с ПТСР. Abstract. The concentration of eNOS in the blood of rats with PTSD decreased by 28%, and iNOS increased 2.28 times, along with 3.3 and 3.6 times increase in the content of DC and MDA against the background of the decreased SOD activity by 27%, CAT by 59% and 1.6 and 3 times increase in the concentration of C-RP and IL-1β, respectively, compared with the control. The introduction of FO either prevented or significantly limited the severity of the detected changes. p g y y g Conclusions. The introduction of fish oil to rats prevents the development of arterial hypotension, as well as the changes in the concentration of eNOS and iNOS, it also prevents the disruption of the prooxidant-antioxidant status of blood serum, and reduces the severity of systemic inflammation in animals with PTSD. y y Key words: post-traumatic stress disorder, fish oil, arterial blood pressure, nitric oxide, prooxidant-antioxidant status. Посттравматическое стрессовое расстрой- ство (ПТСР) возникает как отсроченная реакция организма на стрессовое событие исключительно угрожающего или катастрофического характера (боевые действия, несчастные случаи, авто- и авиакатастрофы, стихийные бедствия, присут- ствие при насильственной смерти других, раз- бойное нападение, пытки, изнасилование, смерть близкого человека), которое способно вызвать нарушение психики практически у любого че- ловека. Частыми проявлениями ПТСР являют- ся случаи навязчивых воспоминаний о психо- травмирующем событии, из-за чего появляются ощущения эмоционального притупления, без- различие к окружающим, отчуждение от других людей, равнодушное отношение к житейским ситуациям, которые ранее приносили радость, а также избегание ситуаций, которые могут напом- нить о травмирующем событии [1]. Первые сим- птомы изменений высшей нервной деятельности и соматические расстройства при ПТСР были описаны во время гражданской войны в США (Da-Costa, 1871). Тогда это состояние обознача- лось как «солдатское сердце», так как основное внимание военных врачей привлекали кардиоло- гические симптомы. Во время первой мировой войны ПТСР называли «снарядным шоком», но особенно большой интерес к этому синдрому по- явился у врачей после войны во Вьетнаме, а поз- же в Афганистане. По современным данным, на протяжении жизни от данного расстройства стра- дают около 13% всего населения земного шара. Отдаленные последствия ПТСР проявляются в различных вариантах психосоматической пато- логии, включая заболевания сердечно-сосуди- стой системы (ССС) [2]. Если физиологическим ответом организ- ма на острый стресс является выброс гормонов стресса (адреналина и норадреналина), повыше- ние продукции кортизола, адренокортикотропно- го гормона с активацией гипофизарно-тиреоид- но-надпочечниковой оси (стадия тревоги общего адаптационного синдрома, согласно модели Г. Abstract. Objectives. To evaluate the nature and mechanisms of the protective effect of fish oil (FO) on systemic hemodynamics in rats with an experimental analogue of post-traumatic stress disorder (PTSD). Material and methods. The studies were carried out on 50 outbred white male rats weighing 210-240 g, divided into groups: «Control», «PTSD», «FO», «FO+PTSD». An analogue of PTSD was reproduced by rats contacting with cat feces for 10 days for 15 minutes daily. Fish oil was administered intragastrically in doses of 0.2 ml (100 mg/kg body weight of 35 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, CJSC «Biosola», Lithuania) 3 days before modelling PTSD and during 10 days of stressor action. After 2 weeks, systolic, diastolic, mean arterial blood pressure (SBP, DBP, MAP) and heart rate (HR) were measured by a non-invasive method. The concentration of endothelial NO-synthase (eNOS), inducible NO-synthase (iNOS), corticosterone, C-reactive protein (C-RP), interleukin-1β (IL-1β) was determined in blood serum by enzyme-linked immunosorbent assay (ELISA); spectrophotometric method was used to determine the concentration of malondialdehyde (MDA), diene conjugates (DC), as well as the activity of catalase (CAT) and superoxide dismutase (SOD). ( ) Results. In animals with PTSD, there was a decrease in SBP, DBP and MAP by 18-23% and an increase in heart rate by 20%. The concentration of eNOS in the blood of rats with PTSD decreased by 28%, and iNOS increased 2.28 times, along with 3.3 and 3.6 times increase in the content of DC and MDA against the background of the decreased SOD activity by 27%, CAT by 59% and 1.6 and 3 times increase in the concentration of C-RP and IL-1β, respectively, compared with the control. The introduction of FO either prevented or significantly limited the severity of the detected changes. Conclusions. The introduction of fish oil to rats prevents the development of arterial hypotension, as well as the changes in the concentration of eNOS and iNOS, it also prevents the disruption of the prooxidant-antioxidant status of blood serum, and reduces the severity of systemic inflammation in animals with PTSD. Key words: post-traumatic stress disorder, fish oil, arterial blood pressure, nitric oxide, prooxidant-antioxidant status. Results. In animals with PTSD, there was a decrease in SBP, DBP and MAP by 18-23% and an increase in heart rate by 20%. Материал и методы Исследования проводились на базе НИЛ УО «Витебский государственный ордена Друж- бы народов медицинский университет» (ВГМУ) в соответствии с Хельсинской Декларацией о гу- манном обращении с животными (1986) и Женев- ской конвенцией «Internetional Guiding Principals for Biomedical Involving Animals (Geneva, 1990), а также в соответствии с рекомендациями Кон- венции Совета Европы по охране позвоночных животных, которые используются в эксперимен- тальных и других научных целях, Директивой Европейского парламента и Совета Европейского Союза 2010/63/ЕС от 22.09.2010 о защите живот- ных, использующихся для научных целей, ТКП 125-2008. Протокол проведения экспериментов был утвержден Комиссией по биоэтике и гуман- ному обращению с лабораторными животными ВГМУ. Артериальное давление (АД) измеряли не- инвазивным способом с использованием системы NIBP (non-invasive blood pressure) фирмы Panlab (Harvard Bioscience Group). Животных предвари- тельно адаптировали к этой процедуре в течение 2-х недель. Крыс помещали в прозрачные пеналы и закрепляли в автоматическом нагревателе при температуре 29°С, затем подсоединяли датчик- манжету к хвосту таким образом, чтобы он рас- полагался точно в проекции хвостовой артерии. Спустя 20 мин проводили измерения систоличе- ского, диастолического, среднего артериально- го давления и частоты сердечных сокращений (САД, ДАД, СрАД, ЧСС соответственно). В сыворотке крови крыс спектрофотоме- трически определяли содержание малонового диальдегида (МДА) [8], диеновых конъюгатов (ДК) [9], а также активность антиоксидантных ферментов каталазы (КАТ) и супероксиддисму- тазы (СОД) [10]. Из 50 беспородных самцов крыс (массой 210-240 г) были сформированы 4 группы: «Кон- троль» (n=10), «ПТСР» (n=20), «Рыбий жир» (n=10), «Рыбий жир+ПТСР» (n=10). Крысам групп «Рыбий жир», «Рыбий жир+ПТСР» вводи- ли внутрижелудочно с помощью металлического зонда с круглым наконечником по 0,2 мл рыбьего жира (100 мг/кг массы тела эйкозапентаеновой (ЭПК) и докозагексаеновой (ДГК) кислот, ЗАО «Биосола», Литва) в течение 13 дней (за 3 дня до и в течение 10 дней действия стрессового фак- тора). Крыс содержали в стандартных условиях вивария при контролируемой температуре (18- 22°C) и влажности (65%), на стандартном пище- вом рационе. Концентрацию эндотелиальной (eNOS) и индуцибельной (iNOS) NO-синтаз, кор- тикостерона, С-реактивного белка (С-РБ), интерлейкина-1β (ИЛ-1β) в сыворотке кро- ви определяли методом иммуноферментного анализа (ИФА), используя наборы реактивов Elabscience Biotechnology Inc. (Китай). Характе- ристики наборов представлены в таблице 1. Оп- тическую плотность проб измеряли фотометром универсальным Ф300ТП (ОАО «Витязь», РБ) при рабочей длине волны 450±10. Статистическую обработку результатов проводили с помощью программы «Statistica 10.0» (StatSoftinc. STA999K347156-W), исполь- зуя непараметрический метод – U-критерий Ман- на-Уитни для независимых групп. Характер ча- стотных распределений представляли в виде Me (25%; 75%). Статистически достоверными счита- ли различия при р<0,05. Abstract. Селье), то при длительном воздействии стрес- соров, как при ПТСР, организм начинает терять способность бороться со стрессом, что приводит в итоге к истощению [3]. На экспериментальной модели ПТСР нами ранее было показано сниже- ние тонуса коронарных сосудов, а также нару- шение функционирования потенциал-зависимых калиевых каналов (Кv-каналов), расположенных в гладких миоцитах сосудов. В основе таких на- рушений лежит гиперпродукция оксида азота, ко- торую катализирует индуцибельная NO-синтаза. Однако литературные данные о характере изме- нения артериального давления (АД) при ПТСР весьма противоречивы и требуют дальнейшего изучения, что и сформировало интерес к этой проблеме [4]. Поскольку современному человеку избе- жать воздействий стресса не представляется воз- можным, особенно остро ставится вопрос о по- иске способов ограничения или предотвращения негативных последствий ПТСР. Имеющиеся дан- ные указывают на то, что некоторые микронутри- енты, такие как рыбий жир, содержащий омега-3 36 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 полиненасыщенные жирные кислоты и витамин D, оказывают благотворное влияние на сердеч- но-сосудистую систему (ССС) [5], но возможные механизмы его действия раскрыты не полностью. Цель исследования – оценить характер и механизмы протекторного действия рыбьего жира (РЖ) на системную гемодинамику у крыс с экспериментальным аналогом посттравматиче- ского стрессового расстройства (ПТСР). полиненасыщенные жирные кислоты и витамин D, оказывают благотворное влияние на сердеч- но-сосудистую систему (ССС) [5], но возможные механизмы его действия раскрыты не полностью. в клетки животных на 15 мин в течение 10 дней, в разное время суток. Следующие 14 дней крысы не подвергались действию стрессора. На 24-ый день у животных определяли поведение в тесте «Открытое поле». О наличии ПТСР у крыс су- дили по снижению их исследовательской актив- ности и повышению тревожности (уменьшение количества вертикальных стоек на 50%, увели- чение времени замирания на 60% и увеличение времени пребывания их в периферической зоне на 19%). Признаки ПТСР наблюдались у 55% животных, у которых имитировали присутствие хищника [7]. Цель исследования – оценить характер и механизмы протекторного действия рыбьего жира (РЖ) на системную гемодинамику у крыс с экспериментальным аналогом посттравматиче- ского стрессового расстройства (ПТСР). Материал и методы У этой же группы экспери- ментальных животных определялось увеличение концентрации продуктов перекисного окисления липидов: так, содержание ДК и МДА увеличива- лось в 3,3 и 3,6 раза соответственно, в сравнении с аналогичными показателями контрольной груп- пы животных (рис. 3, 4). Увеличение содержания продуктов ПОЛ происходило на фоне уменьше- ния активности СОД и КАТ на 27 и 59% соответ- ственно (рис. 5, 6), и увеличения концентрации С-реактивного белка в 1,6 раза, ИЛ-1β в 3 раза (табл. 3), по сравнению с группой «Контроль». В сыворотке крови животных группы «ПТСР» на- блюдалась тенденция к снижению уровня корти- костерона (p=0,72) (табл. 3). Материал и методы Для воспроизведения экспериментального аналога посттравматического стрессового рас- стройства у крыс использовали модифицирован- ную модель «имитации присутствия хищника», которая в настоящее время считается адекватной моделью ПТСР [6]. Присутствие хищника ими- тировали путем размещения экскрементов кошек 37 ESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Таблица 1 – Описание используемых наборов для иммуноферментного анализа Определяемое вещество Производитель, наименование набора Минимальная определяемая концентрация вещества eNOS Elabscience Rat NOS3/eNOS (Nitric Oxide Synthase 3, Endothelial) ELISA Kit Catalog No: E-EL- R0367 9,38 пг/мл iNOS Elabscience Rat NOS2/iNOS (Nitric Oxide Synthase 2, Inducible) ELISA Kit Catalog No: E-EL- R0520 0,19 нг/мл С-РБ Elabscience Rat hs-CRP (high-sensitivity C-Reactive Protein) ELISA Kit Catalog No: E-EL- R3002 4,69 пг/мл ИЛ-1β Elabscience Rat IL-1β (Interleukin 1 Beta) ELISA Kit Catalog No: E-EL- R0012 18,75 пг/мл Кортикостерон Elabscience Rat CORT (Corticosterone)ELISA Kit Catalog No: E-EL-R0269 46,88 пг/мл ица 2 – Изменение артериального давления и частоты сердечных сокращений у крыс с пос еским стрессовым расстройством на фоне использования рыбьего жира Таблица 2 – Изменение артериального давления и частоты сердечных сокращений у крыс с пост- травматическим стрессовым расстройством на фоне использования рыбьего жира Группы животных Показатели САД, мм рт. ст ДАД, мм рт. ст СрАД, мм рт. ст ЧСС, уд / мин Контроль (n=10) 120,0 (110,0;124,0) 86,0 (75,0;99,0) 102,0 (95,0;108,0) 296,0 (289,0;312,0) ПТСР (n=11) 99,0 (90,0;104,0)* 66,0 (60,0;75,0)* 84,0 (75,0;87,0)* 356,0 (343,0;369,0)* Рыбий жир (n=10) 116,0 (110,0;126,0) 80,0 (73,0;90,0) 97,0 (94,0;10,4,0) 323,0 (318,0;336,0) Рыбий жир + ПТСР (n=10) 114,0 (108,0;119,0) 82,0 (74,0;103,0) 95,0 (92,0;110,0) 316,0 (303,0;323,0) Примечание: * р<0,05 – различия по сравнению с контрольной группой животных. Примечание: * р<0,05 – различия по сравнению с контрольной группой животных. концентрации индуцибельной NO-синтазы в 2,28 раза (р=0,03), по сравнению с контрольными зна- чениями (рис. 1, 2). У этой же группы экспери- ментальных животных определялось увеличение концентрации продуктов перекисного окисления липидов: так, содержание ДК и МДА увеличива- лось в 3,3 и 3,6 раза соответственно, в сравнении с аналогичными показателями контрольной груп- пы животных (рис. 3, 4). Увеличение содержания продуктов ПОЛ происходило на фоне уменьше- ния активности СОД и КАТ на 27 и 59% соответ- ственно (рис. 5, 6), и увеличения концентрации С-реактивного белка в 1,6 раза, ИЛ-1β в 3 раза (табл. 3), по сравнению с группой «Контроль». В сыворотке крови животных группы «ПТСР» на- блюдалась тенденция к снижению уровня корти- костерона (p=0,72) (табл. 3). концентрации индуцибельной NO-синтазы в 2,28 раза (р=0,03), по сравнению с контрольными зна- чениями (рис. 1, 2). Результаты Median 25%-75% ДИ минимум; максимум 1 2 3 4 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 Рисунок 2 – Содержание индуцибельной NO-синтазы (iNOS) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация iNOS: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,03, по сравнению с группой «Контроль»; # – р=0,012, по сравнению с группой «ПТСР». р ру р ; р , р Median 25%-75% ДИ минимум; максимум 1 2 3 4 A 200 300 400 500 600 700 800 900 1000 Рисунок 1 – Содержание эндотелиальной NO-синтазы (eNOS) в сыворотке крови животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация eNOS: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,018, по сравнению с группой «Контроль». Median 25%-75% ДИ минимум; максимум 1 2 3 4 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 Median 25%-75% ДИ минимум; максимум 1 2 3 4 A 200 300 400 500 600 700 800 900 1000 Рисунок 1 – Содержание эндотелиальной NO-синтазы (eNOS) в сыворотке крови животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация eNOS: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,018, по сравнению с группой «Контроль». Рисунок 1 – Содержание эндотелиальной NO-синтазы (eNOS) в сыворотке крови животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация eNOS: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,018, по сравнению с группой «Контроль». Рисунок 2 – Содержание индуцибельной NO-синтазы (iNOS) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация iNOS: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,03, по сравнению с группой «Контроль»; # – р=0,012, по сравнению с группой «ПТСР». пы «ПТСР» (рис. 3, 4). При введении рыбьего жира у животных с ПТСР активность СОД и КАТ стати- стически значимо не отличалась от таковых у кон- трольных животных (рис. 5, 6). Результаты У крыс с ПТСР наблюдалось развитие ар- териальной гипотензии, что характеризовалось снижением САД на 18%, ДАД на 23%, СрАД на 18% и тахикардией (повышением ЧСС на 20%), по сравнению с группой «Контроль» (табл. 2). Все эти системные гемодинамические изменения были статистически значимыми (p<0,05). Показа- тели САД, ДАД, СрАД и ЧСС у животных груп- пы «Рыбий жир» не отличались от контрольных значений. У крыс группы «Рыбий жир+ПТСР» АД и ЧСС не отличались от таковых в группе «Контроль». Следовательно, использование ры- бьего жира предупреждало изменения системной гемодинамики (снижение САД, ДАД, СрАД и увеличение ЧСС у крыс с ПТСР. В сыворотке крови крыс группы «Рыбий жир» концентрация NO-синтаз (eNOS и iNOS, рис. 1, 2); продуктов ПОЛ (ДК и МДА, рис. 3, 4); В сыворотке крови животных с ПТСР на- блюдалось снижение концентрации эндотелиаль- ной NO-синтазы на 28% (р=0,018) и увеличение 38 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Таблица 3 – Концентрации интерлейкина-1β (ИЛ-1β), С-реактивного белка (С-РБ) и кортикосте- рона в сыворотке крови экспериментальных животных Таблица 3 – Концентрации интерлейкина-1β (ИЛ-1β), С-реактивного белка (С-РБ) и кортикосте- рона в сыворотке крови экспериментальных животных Концентрации Группы животных Контроль ПТСР Рыбий жир Рыбий жир +ПТСР С-РБ, пг/мл 2,55 (2,54;3,57) 4,25* (3,57;4,92) 2,38 (1,53;2,55) 1,87# (1,53;3,23) ИЛ-1β, пг/мл 1,71 (1,43;2,38) 5,34* (2,29;6,76) 1,60 (1,43;3,72) 3,05# (2,38;3,62) Кортикостерон, пг/мл 187,19 (138,75; 264,54) 151,13 (134,91; 202,66) 173,27 (142,59; 318,54) 253,71 (159,69;317,58) Примечание: цифровые показатели представлены в виде медианы, 25-го и 75-го процентилей; р<0,05 – по сравнению с группой «Контроль»; # р<0,05 – по сравнению с группой «ПТСР». Примечание: цифровые показатели представлены в виде медианы, 25-го и 75-го процентилей; * р<0,05 – по сравнению с группой «Контроль»; # р<0,05 – по сравнению с группой «ПТСР». Примечание: цифровые показатели представлены в виде медианы, 25-го и 75-го процентилей; * р<0,05 – по сравнению с группой «Контроль»; # р<0,05 – по сравнению с группой «ПТСР». Примечание: цифровые показатели представлены в виде медианы, 25-го и 75-го процентилей; * р<0,05 – по сравнению с группой «Контроль»; # р<0,05 – по сравнению с группой «ПТСР». ю с ру о С . Результаты Рисунок 6 – Концентрация каталазы (КАТ) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация КАТ: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,007, по сравнению с группой «Контроль». Рисунок 6 – Концентрация каталазы (КАТ) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация КАТ: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,007, по сравнению с группой «Контроль». Рисунок 5 – Концентрация супероксиддисмутазы (СОД) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир + ПТСР» по оси ординат – концентрация СОД: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,017, по сравнению с группой «Контроль». давления у крыс. На первый взгляд, может пока- заться, что такие результаты несколько противо- речат результатам исследований, показывающих, что хронический стресс вызывает артериаль- Результаты В сыворотке крови животных группы «Рыбий жир+ПТСР» изменений концентрации С-реактивного белка не было выяв- лено, по сравнению с группой «Контроль»; содер- жание ИЛ-1β в сыворотке крови животных группы «Рыбий жир+ПТСР» снижалось на 42%, по срав- нению с таковым у крыс с ПТСР, не получавших рыбий жир. Уровень кортикостерона в сыворотке крови животных с ПТСР, получавших РЖ, не от- личался от содержания этого гормона в сыворотке крови контрольных животных (табл. 3). пы «ПТСР» (рис. 3, 4). При введении рыбьего жира у животных с ПТСР активность СОД и КАТ стати- стически значимо не отличалась от таковых у кон- трольных животных (рис. 5, 6). В сыворотке крови животных группы «Рыбий жир+ПТСР» изменений концентрации С-реактивного белка не было выяв- лено, по сравнению с группой «Контроль»; содер- жание ИЛ-1β в сыворотке крови животных группы «Рыбий жир+ПТСР» снижалось на 42%, по срав- нению с таковым у крыс с ПТСР, не получавших рыбий жир. Уровень кортикостерона в сыворотке крови животных с ПТСР, получавших РЖ, не от- личался от содержания этого гормона в сыворотке крови контрольных животных (табл. 3). антиоксидантная активность (СОД и КАТ, рис. 5, 6); уровень кортикостерона и маркеров воспале- ния (С-реактивного белка и ИЛ-1β) не отличались от контрольных значений (табл. 3). У крыс группы «Рыбий жир+ПТСР» концентрация эндотелиаль- ной NO-синтазы не отличалась от контрольного по- казателя; содержание индуцибельной NO-синтазы снижалось на 35%, по сравнению с содержанием данного фермента в сыворотке крови животных группы «ПТСР» (рис. 1, 2). В группе животных с ПТСР, получавших рыбий жир, статистически зна- чимых изменений в концентрации ДК не наблюда- лось, а концентрация МДА снижалась на 39%, по сравнению с соответствующим показателем груп- антиоксидантная активность (СОД и КАТ, рис. 5, 6); уровень кортикостерона и маркеров воспале- ния (С-реактивного белка и ИЛ-1β) не отличались от контрольных значений (табл. 3). У крыс группы «Рыбий жир+ПТСР» концентрация эндотелиаль- ной NO-синтазы не отличалась от контрольного по- казателя; содержание индуцибельной NO-синтазы снижалось на 35%, по сравнению с содержанием данного фермента в сыворотке крови животных группы «ПТСР» (рис. 1, 2). В группе животных с ПТСР, получавших рыбий жир, статистически зна- чимых изменений в концентрации ДК не наблюда- лось, а концентрация МДА снижалась на 39%, по сравнению с соответствующим показателем груп- 39 ESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. Результаты Рисунок 3 – Концентрация диеновых коньюгатов (ДК) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация ДК: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,03, по сравнению с группой «Контроль». Рисунок 3 – Концентрация диеновых коньюгатов (ДК) Рисунок 4 – Концентрация малонового диальдегида (МДА) в сыворотке крови экспериментальных Рисунок 3 – Концентрация диеновых коньюгатов (ДК) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация ДК: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,03, по сравнению с группой «Контроль». по сравнению с группой «Контроль»; # – р=0,002, по сравнению с группой «ПТСР». по сравнению с группой «Контроль»; # – р=0,002, по сравнению с группой «ПТСР». Median 25%-75% ДИ минимум; максимум 1 2 3 4 -100 0 100 200 300 400 500 600 700 800 Рисунок 5 – Концентрация супероксиддисмутазы (СОД) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир + ПТСР» по оси ординат – концентрация СОД: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,017, по сравнению с группой «Контроль». Median 25%-75% ДИ минимум; максимум 1 2 3 4 -100 0 100 200 300 400 500 600 700 800 Median 25%-75% ДИ минимум; максимум 1 2 3 4 -100 0 100 200 300 400 500 600 700 800 Median 25%-75% ДИ минимум; максимум 1 2 3 4 -1 0 1 2 3 4 5 6 Median 25%-75% ДИ минимум; максимум 1 2 3 4 -1 0 1 2 3 4 5 6 Рисунок 5 – Концентрация супероксиддисмутазы (СОД) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; Рисунок 6 – Концентрация каталазы (КАТ) Рисунок 6 – Концентрация каталазы (КАТ) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация КАТ: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,007, по сравнению с группой «Контроль». Результаты 21, N2 Median 25%-75% ДИ минимум; максимум 1 2 3 4 A 0 2 4 6 8 10 12 Median 25%-75% ДИ минимум; максимум 1 2 3 4 A 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 Рисунок 3 – Концентрация диеновых коньюгатов (ДК) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация ДК: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,03, по сравнению с группой «Контроль». Рисунок 4 – Концентрация малонового диальдегида (МДА) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация МДА: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,03, по сравнению с группой «Контроль»; # – р=0,002, по сравнению с группой «ПТСР». Median 25%-75% ДИ минимум; максимум 1 2 3 4 -100 0 100 200 300 400 500 600 700 800 Median 25%-75% ДИ минимум; максимум 1 2 3 4 -1 0 1 2 3 4 5 6 Рисунок 5 – Концентрация супероксиддисмутазы (СОД) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир + ПТСР» по оси ординат – концентрация СОД: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,017, по сравнению с группой «Контроль». Рисунок 6 – Концентрация каталазы (КАТ) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация КАТ: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,007, по сравнению с группой «Контроль». Median 25%-75% ДИ минимум; максимум 1 2 3 4 A 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 Median 25%-75% ДИ минимум; максимум 1 2 3 4 A 0 2 4 6 8 10 12 Рисунок 3 – Концентрация диеновых коньюгатов (ДК) в сыворотке крови экспериментальных животных: по оси абсцисс – группы экспериментальных животных: 1 – «Контроль»; 2 – «ПТСР»; 3 – «Рыбий жир»; 4 – «Рыбий жир+ПТСР» по оси ординат – концентрация ДК: [25%-75%] – интерквартильный интервал, ДИ – доверительный интервал; * – р=0,03, по сравнению с группой «Контроль». Обсуждение В нашем исследовании аналог ПТСР при- водил к снижению системного артериального 40 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № ную гипертензию (АГ) [11, 12]. Анализ моделей ПТСР различных экспериментаторов показал, что в таких исследованиях использовали либо однократное воздействие стрессора – хищника [11] или хроническое воздействие относитель- но «мягких» стрессоров, таких как ограничение двигательной активности – гипокинезию или со- циальную изоляцию [12]. Также во многих экспе- риментах, в которых при ПТСР наблюдалась АГ, использовались особые породы крыс, склонные к повышению АД [13]. Модель ПТСР, которую использовали мы в своих исследованиях, была основана на многократных воздействиях стрес- сора, и, согласно концепции Ганса Селье, можно полагать, что у крыс имелась тенденция к пере- ходу общего адаптационного синдрома в стадию истощения, что подтверждается как снижением АД, так и тенденцией к снижению концентрации кортикостерона в сыворотке крови [3]. ную гипертензию (АГ) [11, 12]. Анализ моделей ПТСР различных экспериментаторов показал, что в таких исследованиях использовали либо однократное воздействие стрессора – хищника [11] или хроническое воздействие относитель- но «мягких» стрессоров, таких как ограничение двигательной активности – гипокинезию или со- циальную изоляцию [12]. Также во многих экспе- риментах, в которых при ПТСР наблюдалась АГ, использовались особые породы крыс, склонные к повышению АД [13]. Модель ПТСР, которую использовали мы в своих исследованиях, была основана на многократных воздействиях стрес- сора, и, согласно концепции Ганса Селье, можно полагать, что у крыс имелась тенденция к пере- ходу общего адаптационного синдрома в стадию истощения, что подтверждается как снижением АД, так и тенденцией к снижению концентрации кортикостерона в сыворотке крови [3]. уровня глюкокортикоидов возможно усиление эффектов провоспалительных цитокинов, кото- рые способны индуцировать экспрессию iNOS [14]. Как правило, глюкокортикоиды подавляют секрецию цитокинов [15], а при ПТСР сниже- ние уровня глюкокортикоидов и окислительный стресс могут способствовать развитию систем- ного воспаления, что и подтверждается повыше- нием концентрации в сыворотке крови ИЛ-1β и С-реактивного белка – маркеров воспаления. Эти результаты согласуются с клиническими данны- ми. Было продемонстрировано, что содержание C-реактивного белка имеет положительную кор- реляцию с тяжестью посттравматического стрес- сового расстройства у людей, выживших после террористических актов 11 сентября 2011 года [16]. Кроме того, недавние экспериментальные данные показали, что при ПТСР снижается уро- вень витамина D [17], что может повлечь за со- бой развитие воспаления низкой интенсивности в сосудистой стенке и, как следствие, усиленную продукцию NO, продуцируемого iNOS. Более ранние исследования показали, что гиперпро- дукция монооксида азота, катализируемого ин- дуцибельной NO-синтазой, являлась причиной постстрессорного ослабления базального тонуса коронарных сосудов [18]. Литература 1. Хоженко, Е. В. Нейрональные механизмы формирования основных клинических синдромов при посттравматиче- ском стрессовом расстройстве / Е. В. Хоженко // Клин. медицина. – 2009. – Т. 87, № 4. – С. 4–9. Использование рыбьего жира у животных группы «Рыбий жир+ПТСР» ограничивало: про- грессирование дисфункции эндотелия (снижение концентрации еNOS и частичное ограничение прироста содержания iNOS на 35% в сравнении с группой «ПТСР»); развитие окислительного стресса (снижение содержания МДА на 39%); системное воспаление низкой интенсивности (уменьшение концентрации ИЛ-1β на 42%). Вве- дение рыбьего жира крысам с ПТСР полностью предупредило снижение активности антиокси- дантной системы (восстановление активности СОД и КАТ до контрольных значений), развитие артериальной гипотензии и снижение концентра- ции кортикострерона в сыворотке крови крыс с посттравматическим стрессовым расстройством. 2. Близнюк, А. И. Посттравматическое стрессовое рас- стройство у комбатантов: клиника, диагностика, коррек- ция / А. И. Близнюк // Воен. медицина. – 2006. – № 1. – С. 31–40. 2. Близнюк, А. И. Посттравматическое стрессовое рас- стройство у комбатантов: клиника, диагностика, коррек- ция / А. И. Близнюк // Воен. медицина. – 2006. – № 1. – С. 31–40. 3. Selye, H. The Physiology and Pathology of Exposure to Stress. A Treatise Based on the Concept of the General Adaptation Syndrome and the Diseases of Adaptation / H. Selye // Inc. Med. Gaz. – 1952 Sep. – Vol. 87, N 9. – Р. 431. 4. Richter-Levin, G. Animal models of PTSD: a challenge to be met / G. Richter-Levin, O. Stork, M. V. Schmidt // Mol. Psychiatry. – 2019. – Vol. 24. – Р. 1135–1156. 5. Коркушко, О. В. Применение омега-3 полиненасыщен- ных жирных кислот для нормализации эндотелиальной функции и реологических показателей крови при пато- логии сердечно-сосудистой системы / О. В. Коркушко, В. Б. Шатило, В. А. Ищук // Укр. мед. часоп. – 2010. – № 2. – С. 46–49. Протекторные эффекты рыбьего жира, ве- роятно, обусловлены входящими в его состав ЭПК, ДГК и витамином D. Известно, что ЭПК и ДГК способны активировать экспрессию гена eNOS, с последующим улучшением биодоступ- ности NO, а также снижать экспрессию гена iNOS [20, 21], ограничивать активацию свобод- норадикального окисления и увеличивать общую антиоксидантную активность крови [22]. Кроме того, показано, что рыбий жир подавляет образо- вание провоспалительных цитокинов и обладает 6. Adrenal insufficiency in rats after prolonged exposure to the predator cue: A new animal model of post-traumatic stress disorder / O. B. Tseilikman [et al.] // Psychoneuroendocrinol. – 2017. – Vol. 83. – Р. 1–83. . 7. Обсуждение ство сопровождается увеличением содержа- ния продуктов перекисного окисления липидов (ПОЛ) на фоне снижения активности антиокси- дантной системы (АОА) [19]. При уменьшении уровня кортикостерона у крыс с ПТСР наблюда- ется повышение концентрации маркеров воспа- ления (С-реактивного белка и ИЛ-1β). Развивше- еся воспаление низкой интенсивности при ПТСР способно увеличить образование индуцибельной NO-синтазы, а также привести к так называемо- му «разобщению» эндотелиальной NO-синтазы (eNOS). В таких условиях eNOS, наряду с моно- оксидом азота, способна продуцировать и супе- роксидный анион. Более того, активные формы кислорода способствуют уменьшению биодо- ступности NO. Образующийся пероксинитрит способен повышать концентрацию индуцибель- ной NO-синтазы. Увеличенное образование NO, продуцируемого iNOS, может стать причиной снижения системного артериального давления у крыс с ПТСР. Предполагаемые точки приложе- ния РЖ: повышение экспрессии eNOS, ингиби- рование экспрессии iNOS, подавление развития окислительного стресса и системной воспали- тельной реакции. Обсуждение В нашем исследовании снижение концен- трации эндотелиальной NO-синтазы и увеличе- ние содержания индуцибельной NO-синтазы в сыворотке крови могут косвенно свидетельство- вать о развитии дисфункции эндотелия кровенос- ных сосудов крыс при ПТСР. Кроме того, у крыс с посттравматическим стрессовым расстрой- ством наблюдалось снижение концентрации ан- тиоксидантных ферментов (СОД и КАТ) на фоне увеличения содержания продуктов перекисного окисления липидов (ДК и МДА), что может опос- редованно указывать на развитие в этих условиях окислительного стресса. В условиях сниженного Предположительный механизм снижения артериального давления при экспериментальном посттравматическом стрессовом расстройстве представлен на рисунке 7. Посттравматическое стрессовое расстрой- Рисунок 7 – Предположительный механизм развития артериальной гипотензии при посттравматическом стрессовом расстройстве и возможные механизмы протекторного действия рыбьего жира в этих условиях. Воспаление Окислительное повреждение РЖ ПТСР РЖ ↑ ПОЛ (ДК; МДА) ↓ АОА (СОД; КАТ) ↓ eNOS РЖ ↓ Кортикостерон ↑ С-RB ИЛ-1β Разобщение ↑ О2 - ↓NO ONOO- ↑ iNOS РЖ ↑NO ↓ Артериальное давление активация Окислительное повреждение ПТСР ↓ АОА (СОД; КАТ) ↑ ПОЛ (ДК; МДА) ↓ Кортикостерон ↓NO Разобщение ONOO- ↑ iNOS активация ↑NO ↓ Артериальное давление Рисунок 7 – Предположительный механизм развития артериальной гипотензии при посттравматическом стрессовом расстройстве и возможные механизмы протекторного действия рыбьего жира в этих условиях. 41 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 противовоспалительным действием [23]. Не ис- ключаем и роли витамина D, входящего, хоть и в небольших концентрациях (на 100 г рыбьего жира – 0,00025 г витамина D), в состав рыбьего жира. Доказано, что витамин D способен пода- влять воспаление, тормозить избыточное обра- зование активных форм кислорода и активацию индуцибельной NO-синтазы, тем самым улучшая функцию эндотелия и биодоступность NO, об- разуемого конституциональной изоформой NO- синтазы [24]. ство сопровождается увеличением содержа- ния продуктов перекисного окисления липидов (ПОЛ) на фоне снижения активности антиокси- дантной системы (АОА) [19]. При уменьшении уровня кортикостерона у крыс с ПТСР наблюда- ется повышение концентрации маркеров воспа- ления (С-реактивного белка и ИЛ-1β). Развивше- еся воспаление низкой интенсивности при ПТСР способно увеличить образование индуцибельной NO-синтазы, а также привести к так называемо- му «разобщению» эндотелиальной NO-синтазы (eNOS). В таких условиях eNOS, наряду с моно- оксидом азота, способна продуцировать и супе- роксидный анион. Более того, активные формы кислорода способствуют уменьшению биодо- ступности NO. Образующийся пероксинитрит способен повышать концентрацию индуцибель- ной NO-синтазы. Увеличенное образование NO, продуцируемого iNOS, может стать причиной снижения системного артериального давления у крыс с ПТСР. Предполагаемые точки приложе- ния РЖ: повышение экспрессии eNOS, ингиби- рование экспрессии iNOS, подавление развития окислительного стресса и системной воспали- тельной реакции. Заключение Введение рыбьего жира крысам до и во время моделирования ПТСР предупреждает раз- витие артериальной гипотензии, системного вос- паления; предотвращает снижение концентрации еNOS и частично ограничивает прирост содержа- ния iNOS, а также препятствует нарушению про- оксидантно-антиоксидантного баланса сыворот- ки крови этих животных. 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Beliaeva LE, Pavliukevich AN. Early human disease programming and the use of nutraceuticals for preventive purposes: a focus on fish oil. Literature review. Vestn VGMU. 2019;18(5):12-25. (In Russ.) Submitted 23.12.2021 Accepted 21.04.2022 Резюме. Резюме. Цель исследования ‒ оценить диагностическую эффективность комплексной ультразвуковой методики определе- ния повреждения почек у пациентов с хронической сердечной недостаточностью (ХСН). Цель исследования ‒ оценить диагностическую эффективность комплексной ультразвуковой методики определе- ния повреждения почек у пациентов с хронической сердечной недостаточностью (ХСН). Материал и методы. В 2017–2021 гг. на базе УЗ «1 городская клиническая больница» г. Минска выполнено кли- нико-инструментальное исследование 203 пациентов в возрасте 66 (59; 75) лет, из них 111 (54,7%) женщин и 92 (45,3%) мужчины. Динамическое ультразвуковое исследование почек проведено у 80 пациентов с ХСН спустя ≥3 месяца. Критерии включения: синусовый ритм у пациентов; эссенциальная (первичная) артериальная гипертен- зия; хроническая ишемическая болезнь сердца; застойная сердечная недостаточность; информированное добро- вольное согласие пациента на участие в исследовании. Критерии исключения: гломерулярные, тубулоинтерсти- циальные заболевания почек, обструктивные уропатии, стенозы почечных артерий, врожденные болезни почек, злокачественные новообразования почек в анамнезе. Результаты. Метод определения повреждения почек при ХСН строится на концепции установления комплекса ультразвуковых признаков: включает оценку индекса суммарного объема почек, толщины паренхимы, конечной диастолической скорости кровотока, пульсационного индекса, индекса резистентности в сегментарных почечных артериях и характеризует повреждение почек у пациентов с нормальной или незначительно сниженной скоро- стью клубочковой фильтрации (СКФ) (категории С1‒С2), хронической болезнью почек (ХБП) с категориями СКФ С3А‒С4 и ХСН. Заключение. Метод имеет высокую точность ‒ 98,3%, что позволяет рекомендовать его применение при установ- лении ХБП у пациентов с ХСН. Ключевые слова: хроническая сердечная недостаточность, повреждение почек, хроническая болезнь почек, уль- тразвуковая диагностика. DIAGNOSTIC EFFECTIVENESS OF A NEW METHOD FOR DETERMINING KIDNEY DAMAGE IN CHRONIC HEART FAILURE Belarusian Medical Academy of Post-Graduate Education, Minsk, Republic of Belarus Information about authors: Kuzhel O.P. – senior lecturer of the Chair of Normal Physiology, Vitebsk State Order of Peoples’ Friendship Medical University. Адрес для корреспонденции: Республика Беларусь, 210009, г. Витебск, пр. Фрунзе, 27, Витебский государствен- ный ордена Дружбы народов медицинский университет, кафедра нормальной физиологии. E-mail: kdworks@mail. ru – Кужель Ольга Петровна. Correspondence address: Republic of Belarus, 210009, Vitebsk, 27 Frunze ave., Vitebsk State Order of Peoples’ Friendship Medical University, Chair of Normal Physiology. E-mail: kdworks@mail.ru – Olga P. Kuzhel. 44 ВЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО ЛУЧЕВАЯ ДИАГНОСТИКА, ЛУЧЕВАЯ ТЕРАПИЯ DOI: https://doi.org/10.22263/2312-4156.2022.2.45 ДИАГНОСТИЧЕСКАЯ ЭФФЕКТИВНОСТЬ НОВОГО МЕТОДА ОПРЕДЕЛЕНИЯ ПОВРЕЖДЕНИЯ ПОЧЕК ПРИ ХРОНИЧЕСКОЙ СЕРДЕЧНОЙ НЕДОСТАТОЧНОСТИ Белорусская медицинская академия последипломного образования, г. Минск, Республика Беларусь Вестник ВГМУ. – 2022. – Том 21, №2. – С. 45-54. VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Results. The method for determining kidney damage in CHF is based on the concept of establishing a complex of ultrasound signs. It includes an assessment of the total volume of the kidneys, parenchymal thickness, end diastolic blood flow velocity, pulsation index, resistance index in segmental renal arteries, and characterizes kidney damage in patients with normal or slightly reduced glomerular filtration rate (GFR) (categories C1 ‒ C2), chronic kidney disease (CKD) with categories of GFR C3A ‒ C4 and CHF. g Conclusions. The method has a high accuracy – 98.3%, which makes it possible to recommend its use when establishing CKD in patients with CHF. p Key words: chronic heart failure, kidney damage, chronic kidney disease, ultrasound diagnostics. Частота развития хронической болезни по- чек (ХБП) у пациентов с болезнями сердца значи- тельно выше, чем в общей популяции: сочетание факторов кардиоваскулярного риска – сахарного диабета (СД) 2-го типа, эссенциальной артери- альной гипертензии (АГ), атеросклеротической болезни и других – повышает вероятность по- вреждения почек и развития ХБП в 4 раза. Нару- шение функции почек при хронической сердеч- ной недостаточности (ХСН), формирующейся в исходе большинства сердечно-сосудистых забо- леваний, является неблагоприятным прогности- ческим маркером [1-3]. гемодинамики у пациентов с ХСН позволит при- менить соответствующие доступные вмешатель- ства на ранней стадии ХБП. Целью исследования явилась оценка диа- гностической эффективности комплексной уль- тразвуковой методики определения повреждения почек у пациентов с хронической сердечной не- достаточностью. Abstract. Objectives. To evaluate diagnostic effectiveness of a complex ultrasound method for determining kidney damage in patients with chronic heart failure (CHF). Material and methods. In 2017-2021 on the basis of the Healthcare Institution «1st City Clinical Hospital» in Minsk, a clinical and instrumental study was performed on 203 patients aged 66 (59; 75) years, of which 111 (54.7%) were women and 92 (45.3%) men. Dynamic ultrasound investigation of the kidneys was carried out in 80 patients with CHF after ≥3 months. Inclusion criteria: sinus rhythm in patients; essential (primary) arterial hypertension; chronic ischemic heart disease; congestive heart failure; informed voluntary consent of the patient to participate in the study. Exclusion criteria: glomerular, tubulointerstitial kidney disease, obstructive uropathy, renal artery stenosis, congenital kidney diseases, history of renal malignancy. 45 Результаты Для разработки комплексного метода опре- деления повреждения почек при ХСН выборка, состоящая из 203 пациентов, разделена на кон- трольную группу (n=32), куда отнесены данные пациентов без ХСН и ХБП, основную группу – с ХСН (n=171). Контрольная и основная группы не имели статистически значимых отличий по возрасту, полу, индексу массы тела (ИМТ), рас- пространенности СД 2-го типа, эссенциальной АГ и ее степени, статистически значимо отли- чались по уровню заболеваемости хронической ИБС (табл. 1). Далее основная группа была раз- делена на группы сравнения: в 1-ю группу (n=93) включены пациенты, имевшие ХСН и нормаль- ную или незначительно сниженную СКФ, ко 2-й группе (n=78) отнесены пациенты с ХСН и ХБП с категориями СКФ С3А‒С4. У пациентов с ХСН определены статистически значимые отли- чия значений креатинина, мочевины в сыворотке крови, показателей СКФ, МАУ в суточной моче, относительной частоты распространения высо- кой и очень высокой альбуминурии, суммарного объема почек, индекса суммарного объема почек, толщины паренхимы, индексов, характеризую- щих периферическое сосудистое сопротивление в сегментарных артериях, от контрольной груп- пой (табл. 2). Частота распространения ХБП с СКФ <60 мл/мин/1,73 м2 у обследованных паци- ентов с ХСН составила 45,6%. Расчет скорости клубочковой фильтрации у обследованных пациентов в мл/мин/1,73 м2 вы- полняли по формуле CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration Formula). ХБП со сниженной СКФ (категории СКФ ≥С3а) диагностировали на основании ретроспектив- ного анализа медицинской документации, при наличии СКФ <60 мл/мин/1,73 м2, маркеров по- вреждения почек, сохраняющихся 3 месяца и бо- лее [7]. Для статистического анализа создана в сре- де Excel-2013 и зарегистрирована база данных обследованных пациентов (регистрационное сви- детельство Государственного регистра информа- ционных ресурсов от 26.02.2019 г. № 1761917812), ее обработку осуществляли с помощью пакета прикладных программ STATISTICA (v8.0). Для большинства количественных переменных рас- пределение отличалось от нормального, в связи с чем результаты оценивали непараметрически- ми методами. Количественные значения изучае- мых показателей представляли в виде медианы и интерквартильного размаха: Ме (LQ; UQ). Для сравнения по количественным признакам двух независимых выборок использовали U-критерий Манна-Уитни, для сравнения зависимых групп – Т-критерий Вилкоксона, для сравнения по но- минальным признакам выполняли построение таблиц сопряженности по методу максимально- го правдоподобия χ2. Для оценки взаимосвязей между рассматриваемыми признаками рассчи- тывали коэффициент корреляции Спирмена (R). Для прогнозирования развития повреждения по- чек и отбора наиболее сильных связей использо- вали метод бинарного логистического регресси- онного анализа с обратным пошаговым отбором показателей, имевших статистически значимые регрессионные коэффициенты, и построени- ем логит-уравнения. ROC-анализ использовали для оценки чувствительности, специфичности и точности нового метода определения поврежде- ния почек при ХСН. Материал и методы В 2017-2021 гг. на базе УЗ «1 городская клиническая больница» г. Минска выполнено клинико-инструментальное исследование 203 пациентов в возрасте 40-86 (66 (59; 75)) лет, из них 111 (54,7%) женщин и 92 (45,3%) мужчины, в том числе динамическое ультразвуковое иссле- дование почек у 80 пациентов с ХСН спустя ≥3 месяца. При первичном, хроническом или остром нарушении функции сердца страдает функция почек. Одновременное формирование у пациента дисфункции или недостаточности сердца и почек определено как кардиоренальный синдром [3-6]. Развитию кардиоренального синдрома 2-го типа при ХСН способствуют гемодинамические рас- стройства, активация нейрогуморальных регу- ляторных систем, эндотелиальная дисфункция, атеросклеротическая ангиопатия, воспаление, оксидативный стресс [1-6]. Критерии включения в исследование: си- нусовый ритм у пациентов; эссенциальная (пер- вичная) АГ; хроническая ишемическая болезнь сердца; застойная сердечная недостаточность; информированное добровольное согласие паци- ента на участие в исследовании. Критерии исклю- чения: гломерулярные, тубулоинтерстициальные заболевания почек, обструктивные уропатии, стенозы почечных артерий, врожденные болезни почек, злокачественные новообразования почек в анамнезе. Хроническая болезнь почек (ХБП) со ско- ростью клубочковой фильтрации (СКФ) <60 мл/ мин/1,73м2 формируется у 32-77% пациентов с ХСН, у 90,3% больных с ХСН с низкой фрак- цией выброса левого желудочка [3]. К маркерам повреждения почек относят альбуминурию ≥30 мг/сут, отношение альбумин/креатинин мочи ≥30 мг/г (≥3 мг/ммоль), изменения осадка мочи, электролитные и другие нарушения вследствие канальцевой дисфункции, гистологические из- менения, структурные нарушения по данным визуализирующих методов исследования, транс- плантацию почки в анамнезе. Нарушения струк- туры почек, как маркеры повреждения, обычно предшествуют снижению почечной функции [7], поэтому своевременное установление структур- ных повреждений почек и аномалий почечной У пациентов определяли уровень N-концевого предшественника мозгового на- трийуретического пептида (NT-proBNP). Уровень микроальбуминурии устанавливали в суточной порции мочи, отношение альбумин/креатинин ‒ в утренней порции мочи. Ультразвуковое исследо- вание почек, дуплексное сканирование почечных артерий на экстра- и интраренальном уровнях выполняли на ультразвуковых аппаратах Siemens Acuson S1000 (Германия), Siemens Acuson Х300 (Германия). Ультразвуковую визуализацию почек 46 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № и дуплексное сканирование почечных артерий при первичном и динамическом исследовани- ях выполняли согласно методике исследования, описанной в инструкции по применению № 025- 0421 «Метод определения повреждения почек при сердечной недостаточности», утвержденной Министерством здравоохранения Республики Беларусь 21.05.2021 [8]. Динамическую соногра- фию почек выполняли спустя не менее 3 месяцев после первичного ультразвукового исследования. (ОШ), относительного риска (ОР) и их 95% до- верительных интервалов (ДИ) разработанных ультразвуковых признаков повреждения почек, ROC-анализ выполняли программой MedCalc@ Version14.8.1. Статистически значимыми считали различия при р<0,05. Результаты Расчет отношения шансов На основании данных корреляционного анализа Спирмена выделены ультразвуковые по- казатели, характеризующие повреждение почек, у пациентов с ХСН: индекс суммарного объема почек (R=0,35, p<0,001), толщина паренхимы (R=0,36, p<0,001), конечная диастолическая ско- рость кровотока (R=0,43, p<0,001), индекс ре- зистентности (RI) (R=-0,52, p<0,001) и пульса- ционный индекс (PI) в сегментарных артериях (R=-0,51, p<0,001) [9, 10]. Динамическое ультразвуковое исследова- ние почек было выполнено 40 пациентам с ХСН, нормальной и незначительно сниженной СКФ (категории С1‒С2) спустя 12 (9; 14) месяцев; 47 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. Y= χ2=43,17, р<0,001, где: 40 пациентам с ХСН и ХБП со сниженной СКФ (категории С3А‒С4) спустя 9 (5; 12) месяцев. На основании Т-критерия Вилкоксона определены статически значимые отличия СКФ, МАУ в су- точной моче, ультразвуковых показателей, харак- теризующих индексы суммарного объема почек и внутрипочечную гемодинамику, полученных при первичном и повторном ультразвуковом исследо- вании, характеризующих увеличение выражен- ности повреждения почек при нормальной и не- значительно сниженной СКФ и ХБП у пациентов с ХСН в динамике (табл. 3, 4), что согласуется с понятием «хронизации» процесса. За три и более месяца острая дисфункция почек, вызванная по- вреждающими влияниями, завершается выздо- ровлением или переходит в фазу хронического процесса со стойкими лабораторными, структур- ными и другими признаками [2-4, 6, 7]. Y= χ2=43,17, р<0,001, где: Y – повреждение почек с нормальной и не- значительно сниженной СКФ при ХСН. Чувствительность разработанной модели составила 72,7%, специфичность – 92,7%, число верно классифицированных случаев поврежде- ния почек с нормальной и незначительно сни- женной СКФ при ХСН согласно разработанной модели прогнозирования – 87,8% при пороге от- сечения р=0,5, площадь поля под кривой (AUC) 0,92 (95% ДИ 0,84-0,96). Для разработки многофакторной модели прогнозирования повреждения почек и развития ХБП со сниженной СКФ при ХСН, определения наиболее сильных взаимосвязей с диагностиче- скими ультразвуковыми показателями выполнен логистический регрессионный анализ с построе- нием логит-уравнения: В целях разработки многофакторной мо- дели прогнозирования повреждения почек с нормальной и незначительно сниженной СКФ (категории С1‒С2) при ХСН, определения наибо- лее сильных взаимосвязей с диагностическими ультразвуковыми показателями выполнен логи- стический регрессионный анализ с построением логит-уравнения (табл. 5): Y= Y= χ2=43,23, р<0,001, где: χ2=43,23, р<0,001, где: Y – повреждение почек с развитием ХБП при ХСН. Результаты 21, N2 Таблица 1 – Клиническая характеристика пациентов с хронической сердечной недостаточностью Показатель Группа сравнения р контрольная основная (пациенты с ХСН) Возраст, лет 67,0 (64,0; 75,0) 70,0 (64,0; 78,0) U=5854,0 p=0,25 Пол женский, % (n) 50,0 (16) 55,6 (95) χ2=0,61 р=0,43 ИМТ, кг/м2 30,84 (26,93; 34,06) 30,27 (27,05; 34,31) U=6048,5 p=0,64 Эссенциальная АГ: % (n) 100,0 (32) 100,0 (171) - степень 2 (2; 3) 2 (2; 3) χ2=4,18 p=0,38 Хроническая ИБС, % (n) 46,9 (15) 95,9 (164) χ2=111,79 p<0,001 СД 2-го типа, % (n) 31,3 (10) 29,8 (51) χ2=3,53 p=0,06 NT-proBNP, пг/мл 51,8 (31,1; 75,1) 454,0 (270,0; 1306,0) U=0,00 р<0,001 Таблица 2 – Клинико-лабораторные, инструментальные характеристики контрольной группы и групп сравнения а 2 – Клинико-лабораторные, инструментальные характеристики контрольной группы и ния Таблица 2 – Клинико-лабораторные, инструментальные характеристики контрольной группы и групп сравнения Показатель Группа сравнения р контрольная 1-я группа 2-я группа Креатинин, мкмоль/л 74,0 (68,0; 86,0) 93,0 (83,0; 103,5) 129,0 (112,0; 152,0) Н=120,89 р<0,001 Мочевина, ммоль/л 5,50 (4,60; 6,50) 5,90 (5,10; 7,00) 8,00 (6,00; 10,60) Н=37,09 р=0,00001 СКФ, мл/мин/1,73 м2 96,0 (93,0; 99,0) 74,0 (69,0; 80,5) 45,0 (40,0; 54,0) Н=165,57 р<0,001 МАУ в суточной моче, мг/ сутки 13,2 (7,0; 29,5) 19,6 (11,3; 59,1) 23,6 (13,0; 57,5) Н=11,27 р=0,004 Альбуминурия высокая и очень высокая, % (n) 0 (0) 41,9 (39) 41,0 (32) χ2=20,45 р<0,001 Альбумин/креатинин в моче, мг/ммоль 1,62 (0,87; 2,73) 1,72 (1,13; 3,59) 1,82 (1,24; 3,82) Н=1,26 р=0,53 Суммарный объем почек, см3 274,35 (233,85; 332,91) 272,32 (231,15; 320,94) 221,03 (194,60; 272,24) Н=20,09 р<0,001 Индекс суммарного объема почек, см3/м2 143,2 (127,6; 168,4) 139,2 (124,8; 154,0) 115,0 (104,2; 133,5) Н=27,80 р<0,001 Толщина паренхимы, см 1,76 (1,49; 1,91) 1,66 (1,45; 1,84) 1,41 (1,28; 1,59) Н=29,46 р<0,001 Vps в сегментарных артериях, см/сек 42,6 (38,1; 47,8) 43,3 (37,4; 50,2) 46,2 (36,4; 48,6) Н=0,38 р=0,83 Ved в сегментарных артериях, см/сек 12,7 (10,1; 16,6) 12,0 (10,3; 14,2) 9,4 (8,1; 10,3) Н=16,19 р=0,0003 ТАМХ в сегментарных артериях, см/сек 22,50 (20,90; 25,80) 23,70 (20,20; 27,70) 18,60 (17,60; 21,30) Н=22,53 р<0,001 PI в сегментарных артериях 1,17 (1,02; 1,44) 1,20 (1,05; 1,34) 1,75 (1,44; 2,02) Н=31,89 р<0,001 RI в сегментарных артериях 0,65 (0,61; 0,70) 0,66 (0,61; 0,71) 0,78 (0,72; 0,81) Н=35,49 р<0,001 48 ВЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 40 пациентам с ХСН и ХБП со сниженной СКФ (категории С3А‒С4) спустя 9 (5; 12) месяцев. Результаты На основании Т-критерия Вилкоксона определены статически значимые отличия СКФ, МАУ в су- точной моче, ультразвуковых показателей, харак- теризующих индексы суммарного объема почек и внутрипочечную гемодинамику, полученных при первичном и повторном ультразвуковом исследо- вании, характеризующих увеличение выражен- ности повреждения почек при нормальной и не- значительно сниженной СКФ и ХБП у пациентов с ХСН в динамике (табл. 3, 4), что согласуется с понятием «хронизации» процесса. За три и более месяца острая дисфункция почек, вызванная по- вреждающими влияниями, завершается выздо- ровлением или переходит в фазу хронического процесса со стойкими лабораторными, структур- ными и другими признаками [2-4, 6, 7]. Y= χ2=43,17, р<0,001, где: 21, N2 Таблица 4 – Ультразвуковые показатели, характеризующие повреждение почек хронической сер- дечной недостаточностью и хронической болезнью почек со сниженной скоростью клубочковой филь- трации (категории С3‒С4) Таблица 4 – Ультразвуковые показатели, характеризующие повреждение почек хронической сер- дечной недостаточностью и хронической болезнью почек со сниженной скоростью клубочковой филь- трации (категории С3‒С4) трации (категории С3‒С4) Ультразвуковой показатель Исследование р первичное повторное СКФ, мл/мин/1,73 м2 41,0 (40,0; 43,0) 41,0 (39,0; 43,0) Т=0,00 р<0,001 МАУ в суточной моче, мг/сутки 21,4 (13,0; 397,1) 21,4 (15,0; 401,5) Т=0,00 р<0,001 Индекс суммарного объема почек, см3/м2 111,8 (96,8; 126,2) 111,8 (96,8; 124,6) Т=0,00 р<0,001 Толщина паренхимы, см 1,45 (1,31; 1,49) 1,45 (1,31; 1,53) Т=1334,5 р=0,17 Vps в сегментарных артериях, см/сек 47,3 (37,4; 48,9) 47,3 (37,4; 48,9) Т=0,00 р<0,001 Ved в сегментарных артериях, см/сек 9,7 (8,1; 10,3) 9,4 (8,1; 10,3) Т=0,00 р<0,001 ТАМХ в сегментарных артериях, см/сек 19,2 (18,3; 20,9) 19,2 (18,3; 20,4) Т=0,00 р<0,001 RI в сегментарных артериях 0,79 (0,77; 0,82) 0,80 (0,77; 0,82) Т=0,00 р<0,001 PI в сегментарных артериях 1,94 (1,60; 2,10) 2,00 (1,60; 2,08) Т=624,0 р<0,001 Таблица 5 – Итоговая таблица логистической регрессии прогнозирования повреждения почек с нормальной и незначительно сниженной коростью клубочковой фильтрации при хронической сердеч- ной недостаточности Переменная Коэффициент Стандартная ошибка р Индекс суммарного объема почек -0,03 0,01 0,04 RI 28,46 6,19 <0,001 Постоянная -14,78 а 5 – Итоговая таблица логистической регрессии прогнозирования повреждения почек с незначительно сниженной коростью клубочковой фильтрации при хронической сердеч- очности р<0,001), ОР ‒ 5,58 (95% ДИ 2,20-14,13, р<0,001); при величине конечной диастолической скоро- сти кровотока в сегментарных артериях почек ≤14,3 см/сек ОШ равно 9,71 (95% ДИ 2,08-45,37, р=0,004), ОР – 2,74 (95% ДИ 1,40-5,38, р=0,003), при PI >1,26 ОШ определено 6,00 (95% ДИ 2,08- 45,37, р=0,01), ОР – 3,00 (95% ДИ 1,16-7,73, р=0,02), при RI >0,68 ОШ установлено 9,71 (95% ДИ 2,08-45,37, р=0,004), ОР – 2,74 (95% ДИ 1,40- 5,38, р=0,003); для толщины паренхимы ≤1,68 см ОШ ‒ 3,58 ( 95% ДИ 1,34-9,52, р=0,01), ОР – 3,42 (95% ДИ 1,68-6,93, р=0,0007). Чувствительность разработанной моде- ли составила 100,0%, специфичность – 100,0%, число верно классифицированных случаев по- вреждения почек с развитием ХБП со сниженной СКФ при ХСН согласно разработанной модели прогнозирования равно 100,0% при пороге отсе- чения р=0,5, AUC 1,00 (95% ДИ 0,89-1,00). Полу- ченные данные положены в основу дальнейшей разработки алгоритма определения повреждения почек при ХСН. Y= χ2=43,17, р<0,001, где: Таблица 3 – Диагностические показатели, характеризующие повреждение почек с нормальной и незначительно сниженной скорости клубочковой фильтрации (категории С1‒С2) при хронической сердечной недостаточности, при первичном и повторном исследовании Ультразвуковой показатель Исследование р первичное повторное СКФ, мл/мин/1,73 м2 64,5 (54,0; 80,5) 64,5 (54,0; 80,0) Т=0,00 р<0,001 МАУ в суточной моче, мг/сутки 28,9 (15,5; 48,6) 29,2 (15,1; 52,6) Т=0,00 р<0,001 Индекс суммарного объема почек, см3/м2 124,0 (107,4;136,1) 130,0 (114,1; 139,2) Т=0,00 р<0,001 Толщина паренхимы, см 1,49 (1,37; 1,57) 1,50 (1,37; 1,61) Т=1503,5 р=0,58 Vps в сегментарных артериях, см/сек 41,9 (38,1; 51,0) 41,9 (38,1; 51,0) Т=0,00 р<0,001 Ved в сегментарных артериях, см/сек 11,4 (10,1; 13,7) 11,4 (9,8; 13,7) Т=0,00 р<0,001 ТАМХ в сегментарных артериях, см/сек 21,3 (17,9; 24,0) 21,3 (17,9; 24,0) Т=0,00 р<0,001 RI в сегментарных артериях 0,71 (0,66; 0,76) 0,71 (0,68; 0,77) Т=0,00 р<0,001 PI в сегментарных артериях 1,34 (1,15; 1,72) 1,38 (1,23; 1,72) Т=1420,5 р=0,34 Таблица 3 – Диагностические показатели, характеризующие повреждение почек с нормальной и незначительно сниженной скорости клубочковой фильтрации (категории С1‒С2) при хронической сердечной недостаточности, при первичном и повторном исследовании 49 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. ливающее нарушение функции [3, 4, 11]. (95% ДИ 4,51-49,36, р<0,001), ОР – 5,22 (95% ДИ 2,06-13,22, р<0,001); для толщины паренхи- мы ≤1,68 см ОШ – 14,39 (95% ДИ 4,67-44,39, р<0,001), ОР – 2,44 (95% ДИ 1,51-3,93, р=0,0003); при значении конечной диастолической скорости кровотока в сегментарных артериях ≤12,0 см/сек ОШ установлено 79,17 (95% ДИ 12,01-521,97, р<0,001), ОР – 9,38 (95% ДИ 2,49-35,25, р<0,001); для PI >1,55 ОШ определено 92,37 (95% ДИ 5,04-1691,87, р=0,002), ОР – 30,93 (95% ДИ 1,97- 485,45, р=0,015); при RI>0,75 ОШ равно 92,37 (95% ДИ 5,04-1691,87, р=0,002), ОР – 30,93 (95% ДИ 1,97-485,45, р=0,015). Ультразвуковые крите- рии повреждения почек должны быть получены первично и при повторном исследовании по ис- течении 3 месяцев и более. ливающее нарушение функции [3, 4, 11]. ру фу Уменьшение размеров, объема и индексов объема почек является признаками необратимого структурного повреждения почек. Симметрич- ное уменьшение объема почек неспецифично и может быть обусловлено пожилым возрастом пациента, гипертензивной нефропатией, хрони- ческой ишемией, хроническим гломерулонеф- ритом, диабетическим гломерулосклерозом на поздних стадиях и другими заболеваниями [12]. Нами установлены граничные значения индексов суммарного объема, толщины паренхимы почек при повреждении почек с различными категория- ми СКФ у пациентов с ХСН, без известных ранее болезней почек. В норме почечные артерии на экстра- и ин- траренальном уровнях характеризуются низким периферическим сосудистым сопротивлением. Почечный кровоток может динамически изме- няться при повышении давления в правых каме- рах сердца, центрального венозного давления, ве- нозном застое в большом круге кровообращения, диастолическом обкрадывании и ишемии почки [12-15]. Изменения индексов периферического сопротивления в сегментарных артериях почек у пациентов с ХСН являются неспецифическими. Различная почечная патология может приводить к их повышению: отечный синдром в острой ста- дии пиелонефрита, острая обструкция мочевыво- дящих путей, тромбоз почечной вены, острое или хроническое отторжение трансплантата почки, хронические диффузные заболевания почек, диа- бетическая микроангиопатия, ангиопатии иного генеза [12, 15, 16]. ROC-анализ показал высокую диагности- ческую эффективность нового метода определе- ния повреждения почек при ХСН с отличным ка- чеством модели: площадь поля под кривой (AUC) 0,946, чувствительность метода составила 98,3%, специфичность 90,9%, р<0,001, точность ‒ 98,3% (рис. 1). Y= χ2=43,17, р<0,001, где: Разработанные граничные значения уль- тразвуковых диагностических показателей [8-10] имеют высокие показатели ОШ и ОР развития повреждения почек при нормальной и незна- чительно сниженной СКФ (категории С1‒С2) у пациентов с ХСН: при индексе суммарного объ- ема почек ≤126,38 см3/м2 ОШ развития повреж- дения почек составило 18,91 (95% ДИ 5,39-66,39, Определенные в более ранних исследова- ниях [8-10] пороговые значения ультразвуковых диагностических показателей имеют высокие по- казатели ОШ и ОР развития ХБП с категориями СКФ С3А‒С4 при ХСН: для индекса суммарного объема почек ≤126,38 см3/м2 ОШ составляет 14,91 50 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 Литература 1. Congestion in chronic systolic heart failure is related to renal dysfunction and increased mortality / K. Damman [et al.] // Eur. J. of Heart Fail. – 2010 Sep. – Vol. 12, N 9. – P. 974–982. 2. Evaluation of kidney function throughout the heart failure trajectory – a position statement from the Heart Failure Association of the European Society of Cardiology / W. Mullens [et al.] // Eur. J. of Heart Fail. – 2020 Apr. – Vol. 22, N 4. – P. 584–603. Таким образом, разработанный метод со- держит ультразвуковые критерии повреждения почек, обладающие высокой прогностической способностью, как показано на основании анали- за ОШ и ОР развития повреждения почек, позво- ляет определить повреждение почек у пациентов с различными категориями СКФ, формирование ХБП при ХСН с высокой точностью. 3. Резник, Е. В. Кардиоренальный синдром у больных с сердечной недостаточностью как этап кардиоренально- го континуума (часть I): определение, классификация, патогенез, диагностика, эпидемиология (обзор литера- туры) / Е. В. Резник, И. Г. Никитин // Арх. внутр. меди- цины. – 2019. ‒ Т. 9, № 1. – С. 5–22. 4. Cardiorenal syndrome: pathophysiology and potential targets for clinical management / P. Hatamizadeh [et al.] // Nat. Rev. Nephrol. – 2013 Feb. – Vol. 9, N 2. – Р. 99–111. 4. Cardiorenal syndrome: pathophysiology and potential targets for clinical management / P. Hatamizadeh [et al.] // Nat. Rev. Nephrol. – 2013 Feb. – Vol. 9, N 2. – Р. 99–111. 5. Target organ cross talk in cardiorenal syndrome: animal models / L. G. Bongartz [et al.] // Am. J. Physiol. Renal Physiol. – 2012 Nov. – Vol. 303, N 9. – P. F1253–F1263. 5. Target organ cross talk in cardiorenal syndrome: animal models / L. G. Bongartz [et al.] // Am. J. Physiol. Renal Physiol. – 2012 Nov. – Vol. 303, N 9. – P. F1253–F1263. Обсуждение Хроническая болезнь почек формируется вследствие персистирующего в течение не менее 3 месяцев повреждения почек. Морфологической основой формирования ХБП является замещение нормальных структур почек фиброзом, обуслав- Рисунок 1 – Результаты проверки операционных характеристик диагностической эффективности метода определения повреждения почек при ХСН по данным ROC-анализа. Снижение диастолической фазы кровото- ка, повышение периферического сопротивления в почечном артериальном сосудистом бассейне являются отражением гемодинамических влия- ний у пациентов с ХСН, повреждающих почки, вызывающих снижение давления перфузии, ише- мию почек и обуславливающих падение СКФ. Давление перфузии почек определяется как раз- ница между средним артериальным давлением и центральным венозным давлением. У пациентов с ХСН и низким системным давлением, перегруз- кой объемом, повышенным легочным артери- альным давлением или центральным венозным давлением может нарушаться давление перфу- зии почек, обуславливающее снижение СКФ. Общее снижение давления наполнения артерий вызывает выброс нейротрансмиттеров, запуска- ет производство вазоконстрикторов – адренали- Рисунок 1 – Результаты проверки операционных характеристик диагностической эффективности метода определения повреждения почек при ХСН по данным ROC-анализа. 51 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 на, эндотелина и ренин-ангиотензин-альдосте- роновый каскад [17, 18]. Вазоактивные агенты увеличивают почечную и периферическую ва- зоконстрикцию, снижают почечный кровоток, СКФ. Результатами эндогенного нейротрансмит- тер-опосредованного сужения сосудов являются высвобождение цитокинов, воспаление, окис- лительный стресс, почечная гипоксия, апоптоз, которые вследствие долгосрочного влияния вы- зывают потерю структурной и функциональной целостности почек [1-5, 17, 18]. Задержка на- трия и воды почками, повышенная артериальная сосудистая жесткость, системное воспаление, нарушение фосфорно-кальциевого обмена, ги- перкоагуляция в свою очередь вызывают про- грессирование ХСН, формируется порочный круг [18]. Установленные нами пороговые диагности- ческие значения скоростных характеристик диа- столической фазы кровотока, индексов RI и PI в сегментарных почечных артериях характеризуют гемодинамические механизмы повреждения по- чек при различных категориях СКФ у пациентов с ХСН. Благодарности. Благодарности выража- ются Олиферко Н. П., заведующему кардиологи- ческим отделением № 3 УЗ «1-я городская кли- ническая больница» г. Минска, за предоставление пациентов для участия в исследовании и их лече- ние, анонимным рецензентам статьи. Acknowledgements. The author expresses her gratitude to the head of the cardiology department No. 3 of the Healthcare Institution «1st City Clinical Hospital» in Minsk Oliferko N.P. for enabling the participation of patients in the research and their treatment, as well as to anonymous reviewers of this article. References from: https://kdigo.org/wp-content/uploads/2017/02/ KDIGO_2012_CKD_GL.pdf. [Accessed 15th Apr 2022]. from: https://kdigo.org/wp-content/uploads/2017/02/ KDIGO_2012_CKD_GL.pdf. [Accessed 15th Apr 2022]. 1. Damman K, Voors AA, Hillege HL, Navis G, Lechat P, van Veldhuisen DJ, et al. Congestion in chronic systolic heart failure is related to renal dysfunction and increased mortality. Eur J Heart Fail. 2010 Sep;12(9):974-82. doi: 10.1093/eurjhf/hfq118 8. 8. Zherko OM, Chukanov AN, Oliferko NP, Gankova IV, Ivanovskaia MI. Method for Determining Renal Damage in Heart Failure: instrukcija po primeneniju № 025-0421: utv M-vom zdravoohranenija Resp Belarus' 21.05.2021 g. Minsk, RB; 2021. 8 р. (In Russ.) 2. Mullens W, Damman K, Testani JM, Martens P, Mueller C, Lassus J, et al. Evaluation of kidney function throughout the heart failure trajectory – a position statement from the Heart Failure Association of the European Society of Cardiology. Eur J Heart Fail. 2020 Apr;22(4):584-603. doi: 10.1002/ejhf.1697 9. Zherko OM. Renal damage in chronic heart failure. Kardiologija Belarusi. 2019;11(5):765-74. (In Russ.) 10. Zherko OM. Ultrasound diagnosis of kidney damage in patients with chronic heart failure. Zdravoohranenie. 2020;(4):73-8. (In Russ.) 11. Ronco C, Haapio M, House AA, Anavekar N, Bellomo R. Cardiorenal syndrome. J Am Coll Cardiol. 2008 Nov;52(19):1527-39. doi: 10.1016/j.jacc.2008.07.051 3. Reznik EV, Nikitin IG. Cardiorenal syndrome in patients with heart failure as a stage of cardiorenal continuum (part I): definition, classification, pathogenesis, diagnosis, epidemiology (literature review). Arh Vnutr Mediciny. 2019;9(1):5-22. (In Russ.) 12. Kapustin SV, Ouen R, Pimanov SI. Ultrasound in urology and nephrology. 2-e izd, ispr i dop. Moscow, RF: Umnyj doktor; 2017. 176 р. (In Russ.) 4. Hatamizadeh P, Fonarow GC, Budoff MJ, Darabian S, Kovesdy CP, Kalantar-Zadeh K. Cardiorenal syndrome: pathophysiology and potential targets for clinical management. Nat Rev Nephrol. 2013 Feb;9(2):99-111. doi: 10.1038/nrneph.2012.279 13. Kuntcevich GI, Belolopatko EA, Gavrilin AV, Zhestovskaia SI, Zhurenkova TV, Kusova FU, i dr. Ultrasound diagnostics in abdominal and vascular surgery. Minsk, RB: Kavaler Pablishers; 1999. 256 р. (In Russ.) 14. Leliuk VG, Leliuk SE. Ultrasound angiology: prakt posobie. 2-e izd, ispr i dop. Moscow, RF: Real'noe vremja; 2003. 322 р. (In Russ.) 5. Bongartz LG, Braam B, Gaillard CA, Cramer MJ, Goldschmeding R, Verhaar MC, et al. Target organ cross talk in cardiorenal syndrome: animal models. Am J Physiol Renal Physiol. 2012 Nov;303(9):F1253-63. doi: 10.1152/ ajprenal.00392.2012 15. Kulikov VP. Basics of vascular ultrasound. Moscow, RF: Vidar-M; 2015. 387 р. (In Russ.) 16. Kalachik OV, Fedoruk AM. Заключение Разработанный метод определения по- вреждения почек при ХСН строится на концеп- ции установления комплекса ультразвуковых признаков, характеризующих структурные и ге- модинамические аномалии почек, персистирую- щих 3 месяца и более: включает оценку индекса суммарного объема почек, толщины паренхимы, конечной диастолической скорости кровотока, пульсационного индекса и индекса резистентно- сти в сегментарных почечных артериях. Точность разработанного метода определения поврежде- ния почек составляет 98,3% (AUC 0,946, чув- ствительность метода – 98,3%, специфичность ‒ 90,9%, р<0,001), что позволяет рекомендовать его применение в паттерне диагностики повреж- дения и хронической болезни почек с различны- ми категориями СКФ у пациентов с хронической сердечной недостаточностью. 6. Pathophysiology of cardiorenal syndrome type 2 in stable chronic heart failure: workgroup statements from the eleventh consensus conference of the Acute Dialysis Quality Initiative (ADQI) / D. N. Cruz [et al.] // Contrib. Nephrol. – 2013. – Vol. 182. – P. 117–136. 7. KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease [Electronic resource] / G. Eknoyan [et al.] // Kidney Int. Suppl. – 2013. – Vol. 3, № 1. – Mode of access: https://kdigo.org/wp- content/uploads/2017/02/KDIGO_2012_CKD_GL.pdf. – Date of access: 15.04.2022. 8. Метод определения повреждения почек при сердеч- ной недостаточности : инструкция по применению № 025-0421 : утв. М-вом здравоохранения Респ. Беларусь 21.05.2021 г. / О. М. Жерко [др.]. – Минск, 2021. – 8 с. 9. Жерко, О. М. Повреждение почек при хронической сер- дечной недостаточности / О. М. Жерко // Кардиология в Беларуси. – 2019. – Т. 11, № 5. – С. 765–774. 10. Жерко, О. М. Ультразвуковая диагностика повреждения почек у пациентов с хронической сердечной недоста- точностью / О. М. Жерко // Здравоохранение. – 2020. 52 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 – № 4. – С. 73–78. – № 4. – С. 73–78. сосудов / В. П. Куликов. – Москва : Видар-М, 2015. – 387 с. 11. Cardiorenal syndrome / C. Ronco [et al.] // J. Am. Coll. Cardiol. – 2008 Nov. – Vol. 52, N 19. – P. 1527–1539. 16. Калачик, О. В. Трансплантация почки: основные хирур- гические методы, ультразвуковая визуализация и мини- мально инвазивная коррекция патологии аллографта почки / О. В. Калачик, А. М. Федорук. – Минск : Пара- докс, 2016. – 160 с. 12. Капустин, С. В. Ультразвуковое исследование в уроло- гии и нефрологии / С. В. Капустин, Р. Оуен, С. И. Пима- нов. – 2-е изд., испр. и доп. ‒ Москва : Умный доктор, 2017. ‒ 176 с. Заключение 17. Jois, P. Cardio-Renal Syndrome Type 2: Epidemiology, Pathophysiology, and Treatment / P. Jois, A. Mebazaa // Semin. Nephrol. – 2012 Jan. – Vol. 32, N 1. – P. 26–30. 13. Ультразвуковая диагностика в абдоминальной и сосуди- стой хирургии / Г. И. Кунцевич [и др.]. – Минск : Кава- лер Паблишерс, 1999. – 256 с. 18. Мацкевич, С. А. Хроническая болезнь почек: кардио- ренальные взаимоотношения / С. А. Мацкевич // Лечеб. дело. – 2017. – № 1. – С. 4–10. 14. Лелюк, В. Г. Ультразвуковая ангиология : практ. посо- бие / В. Г. Лелюк, С. Э. Лелюк. – 2-е изд., испр. и доп. – Mосква : Реальное время, 2003. – 322 с. 15. Куликов, В. П. Основы ультразвукового исследования Поступила 29.10.2021 г. Принята в печать 21.04.2022 г. Поступила 29.10.2021 г. Принята в печать 21.04.2022 г. Поступила 29.10.2021 г. Принята в печать 21.04.2022 г. Поступила 29.10.2021 г. Принята в печать 21.04.2022 г. References Kidney transplantation: basic surgical techniques, ultrasound imaging, and minimally invasive correction of renal allograft pathology. Minsk, RB: Paradoks; 2016. 160 р. (In Russ.) 6. 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Submitted 29.10.2021 Accepted 21.04.2022 Submitted 29.10.2021 Accepted 21.04.2022 53 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Сведения об авторах: Жерко О.М. – к.м.н., доцент, заведующая кафедрой ультразвуковой диагностики, Белорусская медицинская ака- демия последипломного образования, ORCID: https://orcid.org/0000-0001-5752-0988. ORCID: https://orcid.org/0000-0001-5752-0988. Резюме. Резюме. Вопрос изучения уровня заболеваемости и трудопотерь в Вооруженных Силах Республики Беларусь всегда стоял остро. Это связано с реализацией мероприятий по ранней и точной диагностике, ограниченным выбором эффек- тивных средств специфической профилактики и лечения. Особенно актуальным этот вопрос возник сейчас, в период пандемии COVID-19. Наслоение COVID-19 на сезонный подъём заболеваемости органов дыхания при- водит к увеличению числа госпитализаций (военнослужащие, проходящие службу по призыву, подлежат стацио- нарному лечению по эпидемическим показаниям при любой степени тяжести заболевания). Для госпитализации приходится использовать возможности государственных учреждений здравоохранения, которые сталкиваются с той же проблемой. Учитывая заинтересованность в изучении данной проблемы Министерства обороны и Мини- стерства здравоохранения Республики Беларусь, нами был проведен анализ медицинских отчетов Министерства обороны Республики Беларусь по форме 3/МЕД за 2017–2020 годы статистическими методами пакета статистики SSPP 22. Был сделан акцент на I, X, XXI классах по Международной классификации болезней, травм и причин смерти 10-го пересмотра (МКБ-10). В результате анализа статистических данных был наглядно продемонстри- рован рост заболеваемости и трудопотерь военнослужащих Вооруженных Сил Республики Беларусь в 2020 году, что объясняется влиянием COVID-19. В структуре заболеваемости военнослужащих за 2020 год COVID-19 мог быть установлен в 10292 (15,41%) случаях и 76502 дней (22,60%) в структуре трудопотерь. Это диктует необходи- мость в переработке документов, касающихся деятельности военно-медицинских учреждений и подразделений в условиях пандемий. у Ключевые слова: COVID-19, заболеваемость, трудопотери, военнослужащие. THE ANALYSIS OF THE DISEASE INCIDENCE AND LABOR LOSSES IN THE ARMED FORCES OF THE REPUBLIC OF BELARUS FROM 2017 TO 2020 Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus Vestnik VGMU. 2022;21(2):55-62. Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus АНАЛИЗ ЗАБОЛЕВАЕМОСТИ И ТРУДОПОТЕРЬ В ВООРУЖЕННЫХ СИЛАХ РЕСПУБЛИКИ БЕЛАРУСЬ ЗА 2017-2020 ГОДЫ ЦЫГАНКОВ А.М., ЛЯТОС И.А. Витебский государственный ордена Дружбы народов медицинский университет, г. Витебск, Республика Беларусь Вестник ВГМУ. – 2022. – Том 21, №2. – С. 55-62. Information about authors: Information about authors: Zherko O.M. – Candidate of Medical Sciences, associate professor, head of the Chair of Ultrasound Diagnosis, Belarusian Medical Academy of Post-Graduate Education, ORCID: https://orcid.org/0000-0001-5752-0988. Адрес для корреспонденции: Республика Беларусь, 220013, г. Минск, ул. П. Бровки, д.3, корп. 3, Белорусская ме- дицинская академия последипломного образования, кафедра ультразвуковой диагностики. E-mail: zherco@mail. ru – Жерко Ольга Михайловна. Correspondence address: Republic of Belarus, 220013, Minsk, 3-3 P. Brovki str., Belarusian Medical Academy of Post- Graduate Education, Chair of Ultrasound Diagnosis. E-mail: zherco@mail.ru – Olga М. Zherko. 54 ВЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕД ОБЩЕСТВЕННОЕ ЗДОРОВЬЕ И ЗДРАВООХРАНЕНИЕ DOI: https://doi.org/10.22263/2312-4156.2022.2.55 DOI: https://doi.org/10.22263/2312-4156.2022.2.55 DOI: https://doi.org/10.22263/2312-4156.2022.2.55 ESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Belarusian Armed Forces in 2020 was clearly demonstrated, which explained by the impact of COVID-19. COVID-19 could be established in the structure of morbidity of servicemen for 2020 in 10292 (15.41%) cases and 76502 days (22.60%) in the structure of labor losses. This calls for revising documents concerning the activities of military medical institutions and units under the conditions of pandemics. Key words: COVID-19, morbidity, labor losses, servicemen. В 2020 году медицинская служба Воору- женных Сил Республики Беларусь, как и весь мир, начала борьбу с пандемией COVID-19. Новая бо- лезнь COVID-19 потребовала внести коррективы по кодированию в рамках МКБ-10. Учитывая тот факт, что подтверждение COVID-19 проводится, как правило, с помощью ПЦР (реже ИФА), не- обходимо говорить о высокой достоверности ла- бораторной диагностики. Согласно документам Министерства здравоохранения COVID-19 мо- жет кодироваться как B 34.2 (коронавирусная ин- фекция неуточненная); B 97.2 (коронавирусы как причина болезней других рубрик), Z20 (контакт с больным и возможность заражения инфекцион- ными болезнями); Z20.8 (контакт с больным и воз- можность заражения другими инфекционными болезнями) и J12.8 (другая вирусная пневмония) [1]. Это послужило целью проверить гипотезу о том, что окончательный диагноз COVID-19 был зашифрован в рамках I и ХХI классов отчетов 3/ МЕД. Подтверждение тому – результаты анализа заболеваемости и трудопотерь военнослужащих Вооруженных Сил Республики Беларусь по I, X, XXI классам МКБ-10 за 2017-2020 годы. Несмотря на усилия в Вооруженных Си- лах Республики Беларусь по раннему выявлению больных и контактов 1 уровня, по недопущению заноса инфекции в воинские коллективы, по раз- рыву передачи возбудителя, был зафиксирован рост заболеваемости и, следственно, госпитали- заций военнослужащих всех категорий. Причем перепрофилирование коечного фонда не всегда позволяет справиться с возросшим количеством больных силами военно-медицинских учреж- дений и подразделений. Рост заболеваемости COVID-19 можно объяснить влиянием трудно корригируемых факторов SARS-CoV-2: пато- генность, вирулентность, контагиозность и им- муногенность со способностью уклоняться от иммунного надзора [3, 4]. Также трудно влиять на факторы окружающей среды (температура, влажность, скорость ветра, снижение инсоляции в осенне-весенний период), индивидуальные и популяционные особенности (HLA репертуар, иммунный статус, коллективный иммунинитет), социально-экономический уровень государства (уровень оказания медицинской помощи, уровень доходов граждан), медицинской грамотности на- селения [5, 6]. А также влияет стратегия органов власти в пандемию (карантинные мероприятия различной степени жесткости): ограничение пе- редвижений, проведения массовых мероприятий и т.д. Благодаря проведенной работе в Вооружен- ных Силах Республики Беларусь эпидемиологи- ческая ситуация сейчас находится под контролем (за счет двукратной вакцинации военнослужа- щих воинских частей и подразделений). Abstract. The issue of studying the level of morbidity and labor losses in the Armed Forces of the Republic of Belarus has always been thorny. It is connected with the implementation of measures aimed at early and proper diagnosis, limited choice of effective means of specific prophylaxis and treatment. This issue is particularly pressing now, during the COVID-19 pandemic. The layering of COVID-19 on the seasonal rise of respiratory diseases leads to an increase in the number of hospitalizations (conscripts are subject to inpatient treatment according to epidemic indications for any degree of the disease severity). For hospitalization, they have to use the capacities of state healthcare institutions, which face the same problem. Considering the interest of the Ministry of Defense and the Ministry of Health of the Republic of Belarus in studying this problem, we have analyzed the medical reports of the Ministry of Defense of the Republic of Belarus on Form 3/MED during 2017-2020 using statistical methods of SSPP 22 statistics package. The emphasis was placed on I, X, XXI classes according to the International Classification of Diseases, Injuries and Causes of Death of the 10th revision (ICD-10). As a result of the statistical data analysis, the growth of morbidity and labor losses of the servicemen in the 55 ESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Характерным проявлением роста заболева- емости органов дыхания в воинских коллективах являются массовые вспышки острых респира- торных инфекций, причина которых объясняет- ся особенностью проживания военнослужащих (скученность) и питания (столовая как вероят- ное место передачи), интенсивной боевой под- готовкой (при любых погодных условиях занятия на полигонах, совершение маршей, проведение учений), обновлением воинских коллективов (поступление в воинскую часть молодого по- полнения) и его «перемешиванием» (перемеще- ние военнослужащих из одного подразделения в другое). Отдельно стоит отметить, что в периоды интенсивной подготовки военнослужащих может возникнуть дисбаланс между трудозатратами и энергетической ценностью суточного рациона питания, между отдыхом и физическими и пси- хоэмоциональными нагрузками [2]. Успешность мероприятий по снижению заболеваемости в воинских частях зависит от грамотных управленческих решений в условиях ограниченности ресурсов, что возможно только при выполнении комплекса мер, направленных на выявление источника заболевания, разрыв ме- ханизма передачи возбудителя и снижения вос- приимчивости организма. Четкое выполнение комплекса противоэпидемических мероприятий позволит контролировать эпидемиологическую 56 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № возрасте от 18 до 27 лет (средний возраст около 20 лет), которые выполняют физические нагруз- ки, редко покидают коллектив на протяжении службы, признаны военно-врачебной комиссией годными к военной службе и редко имеют хрони- ческие заболевания. Военнослужащие, проходя- щие службу по контракту, и офицеры – это лица обоих полов (преобладают мужчины) в возрасте от 20 до 55 лет (средний возраст около 30 лет), которые чаще подвержены психоэмоциональ- ным нагрузкам, могут свободно перемещаться в пределах страны (и при необходимости за ее пре- делы), признаны военно-врачебной комиссией годными к военной службе и чаще имеют хрони- ческие заболевания. Поэтому военнослужащие были разделены следующим образом: военнос- лужащие, проходящие военную службу по при- зыву, – первая группа (далее 1 группа); офицеры и военнослужащие, проходящие военную службу по контракту, – вторая группа (далее 2 группа). Общее количество дней трудопотерь в 2017 г. со- ставило 280 850, в 2018 г. – 298 365, в 2019 г. – 225 128, в 2020 г. – 338 526. ситуацию и в дальнейшем снизить заболевае- мость. Одним из главных направлений деятель- ности по снижению заболеваемости является вакцинация с учетом охвата не менее 70% насе- ления в 2022 году (ВОЗ, 2021). В Вооруженных Силах Республики Беларусь охват должен стре- миться к 95%. Исключение составляют лица с медицинскими противопоказаниями. Вышеизло- женное обуславливает военно-эпидемиологиче- скую значимость COVID-19 для Вооруженных Сил Республики Беларусь. Материал и методы Исходными данными служила отчетная документация Министерства обороны Республи- ки Беларусь (форма 3/МЕД за 2017-2020 годы), материалы публикаций по изучаемой пробле- матике. Для обработки полученных результатов использовался пакет статистики SSPP 22. Ста- тистическую обработку полученных результатов исследования проводили в соответствии с тре- бованиями, предъявляемыми к проведению ис- следований, при этом качественные показатели представлены абсолютными и относительными величинами. Для 1 и 2 группы дни трудопотерь состави- ли соответственно: в 2017 г. – 223 947 или 79,74% (95% ДИ: 79,59-79,89) и 56903 или 20,26% (95% ДИ: 20,11-20,41); в 2018 г. – 223 606 или 74,94% (95% ДИ: 74,79-75,10) и 74759 или 25,06 % (95% ДИ: 24,90-25,21); в 2019 г. – 169944 или 75,49% (95% ДИ: 75,31-75,67) и 55184 или 24,51% (95% ДИ: 24,33-24,69); в 2020 г. – 217 165 или 64,15% (95% ДИ: 63,99-64,31) и 121361 или 35,85% (95% ДИ: 35,69-36,01). Результаты и обсуждение Военнослужащие при прохождении во- енной службы неоднородны по возрасту, полу, выполняемым нагрузкам, по возможности пере- мещений, по состоянию здоровья. Так, военнос- лужащие по призыву – это молодые мужчины в ) На рисунке 1 виден рост трудопотерь в Рисунок 1 – Трудопотери в днях за 2017-2020 годы. 0 50000 100000 150000 200000 250000 300000 350000 400000 2017 год 2018 год 2019 год 2020 год 1 группа 2 группа Рисунок 1 – Трудопотери в днях за 2017-2020 годы. 57 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Таблица 1 – Трудопотери среди военнослужащих за 2017-2020 гг. Группа / класс по 1МКБ-10 2017 год 2018 год 2019 год 2020 год 1 гр. Х кл. 116594 (52,06%; 95% ДИ: 51,86-52,27) 121556 (54,36%; 95% ДИ: 54,16-54,57) 84885 (49,95%; 95% ДИ: 49,71-50,19) 108452 (49,94%; 95% ДИ: 49,73-50,15) I кл. 5922 (2,64%; 95% ДИ: 2,58-2,71) 6459 (2,89%; 95% ДИ: 2,82-2,96) 8244 (4,85%; 95% ДИ: 4,75-4,95) 13327 (6,14%; 95% ДИ: 6,04-6,24) XXI кл. 6733 (3,01%; 95% ДИ: 2,94-3,08) 6090 (2,72%; 95% ДИ: 2,66-2,79) 3948 (2,32%; 95% ДИ: 2,25-2,39) 19319 (8,9%; 95% ДИ: 8,78-9,02) 2 гр. Х кл. 24281 (42,67%; 95% ДИ: 42,26-43,08) 33642 (45,0%; 95% ДИ: 44,64-45,36 22208 (40,24%; 95% ДИ: 39,83-40,65 55276 (45,55%; 95% ДИ: 45,27-45,83) I кл. 244 (0,11%; 95% ДИ: 0,10,-0,12) 1355 (0,61%; 95% ДИ: 0,57-0,64) 253 (0,15%; 95% ДИ: 0,13-0,17) 23432 (10,79%; 95% ДИ: 10,66-10,92) XXI кл. 2823 (1,66%; 95% ДИ: 1,60-1,72) 3355 (1,5%; 95% ДИ: 1,45-1,55) 4385 или 2,58% (95% ДИ: 2,50-2,66) 7688 (3,54%; 95% ДИ: 3,46-3,62) Рисунок 2 – Трудопотери для 1 группы по I, XXI классам. 0 5000 10000 15000 20000 25000 2017 год 2018 год 2019 год 2020 год Класс I Класс XXI Рисунок 2 – Трудопотери для 1 группы по I, XXI классам. 0 5000 10000 15000 20000 25000 2017 год 2018 год 2019 год 2020 год Класс I Класс XXI Класс I Класс XXI 2020 году за счет 2 группы. 2020 году за счет 2 группы. Для 1 группы трудопотери по Х классу не превышали средние значения за исследуемый пе- риод (табл. 1). Основным показателем уровня заболева- емости в Вооруженных Силах Республики Бе- ларусь является количество обращений. Общее количество первичных обращений (заболева- емость) военнослужащих составило в 2017 г.- 50332, в 2018 г. – 60776, в 2019 г. – 44872, в 2020 г. – 66780. Можно заключить, что рост заболева- емости в 2020 году произошел за счет двух групп по сравнению с предыдущими годами (рис. 4). Результаты и обсуждение При детальном анализе трудопотерь для 1 группы было показано, что наблюдался рост в 2020 году по I, XXI классам в сравнении с преды- дущими годами, что видно на рисунке 2. При ана- лизе трудопотерь для 2 группы было показано, что наблюдался рост по всем изучаемым классам в 2020 году (рис. 3). Для 1 и 2 группы заболеваемость составила в 2017 г. 41348 или 82,15% (95% ДИ: 81,82-82,49) и 8984 или 17,85 (95% ДИ: 17,51-18,18); в 2018 г. – 46527 или 76,55% (95% ДИ: 76,22-76,89) и 14249 или 23,45% (95% ДИ: 23,11-23,78); в 2019 г. – 36047 или 80,33% (95% ДИ: 79,97-80,7) и Количество дней трудопотерь для 1 и 2 группы в 2020 г. составило 3459,6‰ и 2314,85‰ соответственно. Это значит, что условно каждый военнослужащий 1 группы в течение года имел до 4 дней трудопотерь, а военнослужащий 2 58 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Рисунок 3 – Трудопотери за 2017-2020 годы для 2 группы. Рисунок 4 – Первичная заболеваемость за 2017-2020 годы. 0 10000 20000 30000 40000 50000 60000 2017 год 2018 год 2019 год 2020 год Класс X Класс I Класс XXI 0 10000 20000 30000 40000 50000 60000 70000 2017 год 2018 год 2019 год 2020 год 1 группа 2 группа 0 10000 20000 30000 40000 50000 60000 2017 год 2018 год 2019 год 2020 год Класс X Класс I Класс XXI Рисунок 3 – Трудопотери за 2017-2020 годы для 2 группы. Рисунок 3 – Трудопотери за 2017-2020 годы для 2 группы. Рисунок 3 – Трудопотери за 2017-2020 годы для 2 группы. Рисунок 3 – Трудопотери за 2017-2020 годы для 2 группы. Рисунок 4 – Первичная заболеваемость за 2017-2020 годы. 0 10000 20000 30000 40000 50000 60000 70000 2017 год 2018 год 2019 год 2020 год 1 группа 2 группа Рисунок 4 – Первичная заболеваемость за 2017-2020 годы. Таблица 2 – Заболеваемость среди военнослужащих за 2017-2020 гг. Группа и класс МКБ-10 2017 год 2018 год 2019 год 2020 год 1 гр. Х класс 16772 18741 13719 17722 I класс 805 644 510 1333 XXI класс 155 424 919 2630 2 гр. Х класс 4626 6231 3632 8709 I класс 40 92 34 1437 XXI класс 185 284 745 1297 Таблица 2 – Заболеваемость среди военнослужащих за 2017-2020 гг. возросла за исследуемый период (табл. 2). 8825 или 19,67% (95% ДИ: 19,3-20,03); в 2020 г. Результаты и обсуждение – 50407 или 75,48% (95% ДИ: 75,16-75,81) и 16373 или 24,52% (95% ДИ: 24,19-24,84). возросла за исследуемый период (табл. 2). Виден рост заболеваемости в 2020 году по I, XXI классам для военнослужащих 1 группы по сравнению с предыдущими годами на рисунке 5. Виден рост заболеваемости в 2020 году по сравнению с предыдущими годами по I, XXI клас- 8825 или 19,67% (95% ДИ: 19,3-20,03); в 2020 г. – 50407 или 75,48% (95% ДИ: 75,16-75,81) и 16373 или 24,52% (95% ДИ: 24,19-24,84). Для 1 группы заболеваемость по Х классу не превышала средние значения за исследуемый период, для 2 группы заболеваемость по Х классу Для 1 группы заболеваемость по Х классу не превышала средние значения за исследуемый период, для 2 группы заболеваемость по Х классу 59 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Рисунок 5 – Первичная заболеваемость по I, XXI классам за 2017-2020 годы для 1 группы. Рисунок 6 – Первичная заболеваемость по I, XXI классам за 2017-2020 годы для 2 группы. 0 500 1000 1500 2000 2500 3000 2017 год 2018 год 2019 год 2020 год Класс I Класс XXI 155 424 919 2630 0 500 1000 1500 2000 2500 3000 2017 год 2018 год 2019 год 2020 год Класс XXI Класс I Рисунок 5 – Первичная заболеваемость по I, XXI классам за 2017-2020 годы для 1 группы. 155 424 919 2630 0 500 1000 1500 2000 2500 3000 2017 год 2018 год 2019 год 2020 год Класс XXI Класс I Рисунок 5 – Первичная заболеваемость по I, XXI классам за 2017-2020 годы для 1 группы. Рисунок 6 – Первичная заболеваемость по I, XXI классам за 2017-2020 годы для 2 группы. 0 500 1000 1500 2000 2500 3000 2017 год 2018 год 2019 год 2020 год Класс I Класс XXI Рисунок 6 – Первичная заболеваемость по I, XXI классам за 2017-2020 годы для 2 группы. 0 500 1000 1500 2000 2500 3000 2017 год 2018 год 2019 год 2020 год Класс I Класс XXI Рисунок 6 – Первичная заболеваемость по I, XXI классам за 2017-2020 годы для 2 группы. Таблица 3 – Расчет трудопотерь (в днях) и заболеваемости (случаев) Группа / класс МКБ-10 Трудопотери / заболеваемость за 2017-2019 годы Среднее значение трудопотерь / заболеваемости Итоговая разница трудопотерь / заболеваемости (доля в 2020 году) 1 гр. Х класс 323035/49182 107678/16394 774/1328 (0,36%/2,63%) I класс 16771/1959 5590/653 13729/680 (6,32%/1,34%) XXI класс 20625/1498 6875/499 62454/13548 (28,76%/26,87%) 2 гр. Заключение 3. Gustine, J. N. Immunopathology of Hyperinflammation in COVID-19 / J. N. Gustine, D. Jones // Am. J. Pathol. – 2021 Jan. – Vol. 191, N 1. – P. 4–17. 3. Gustine, J. N. Immunopathology of Hyperinflammation in COVID-19 / J. N. Gustine, D. Jones // Am. J. Pathol. – 2021 Jan. – Vol. 191, N 1. – P. 4–17. В результате анализа отчетных медицин- ских документов в отчетном периоде показан рост заболеваемости и трудопотерь военнослу- жащих в обеих изучаемых группах, обусловлен- ный COVID-19. Особенностью заболеваемости в 2020 году среди военнослужащих, проходя- щих службу по контракту, стал значительный ее подъем по I, XXI классу в сравнении с военнос- лужащими, проходящими службу по призыву. В структуре заболеваемости военнослужащих Во- оруженных Сил Республики Беларусь за 2020 год COVID-19 мог быть установлен в 10292 (15,41%) случая; в структуре трудопотерь 76502 дней (22,60%). Снижение заболеваемости и трудопо- терь можно достичь в первую очередь за счет планируемой вакцинации против вирусов SARS 4. Breadth of concomitant immune responses prior to patient recovery: a case report of non-severe COVID-19 / I. Thevarajan [et al.] // Nat. Med. – 2020 Apr. – Vol. 26, N 4. – P. 453–455. 4. Breadth of concomitant immune responses prior to patient recovery: a case report of non-severe COVID-19 / I. Thevarajan [et al.] // Nat. Med. – 2020 Apr. – Vol. 26, N 4. – P. 453–455. 5. Brodin, P. Immune determinants of COVID-19 disease presentation and severity / P. Brodin // Nat. Med. – 2021 Jan. – Vol. 27, N 1. – P. 28–33. 5. Brodin, P. Immune determinants of COVID-19 disease presentation and severity / P. Brodin // Nat. Med. – 2021 Jan. – Vol. 27, N 1. – P. 28–33. 6. Цыганков, А. М. Защитные механизмы человека про- тив вирусов, вызывающих острые респираторные ин- фекции / А. М. Цыганков, В. В. Янченко, И. А. Лятос // Клин. инфектология и паразитология. – 2021. – Т. 10, № 1. – С. 88–103. 7. Медико-статистические показатели заболеваемости военнослужащих по призыву Вооруженных сил Респу- блики Беларусь и Российской Федерации (2003–2016 гг.) / В. И. Евдокимов [и др.] // Мед.-биол. и соц.-психол. проблемы безопасности в чрезвычайн. ситуациях. – 2018. – № 2. – С. 26–50. 7. Медико-статистические показатели заболеваемости военнослужащих по призыву Вооруженных сил Респу- блики Беларусь и Российской Федерации (2003–2016 гг.) / В. И. Евдокимов [и др.] // Мед.-биол. и соц.-психол. проблемы безопасности в чрезвычайн. ситуациях. – 2018. – № 2. Литература 1. О некоторых вопросах порядка проведения экспертизы временной нетрудоспособности, оформления листков нетрудоспособности лицам с инфекцией COVID-I9 и лицам, относящимся к контактам 1 и 2 уровня по ин- фекции COVID-19 : письмо М-ва здравоохранения Респ. Беларусь, 6 апр. 2020 г., № 3-2-8/6133 // Консуль- тант Плюс : Беларусь [Электронный ресурс] / ООО «ЮрСпектр», Нац. центр правовой информ. Респ. Бела- русь. – Минск, 2022. 1. О некоторых вопросах порядка проведения экспертизы временной нетрудоспособности, оформления листков нетрудоспособности лицам с инфекцией COVID-I9 и лицам, относящимся к контактам 1 и 2 уровня по ин- фекции COVID-19 : письмо М-ва здравоохранения Респ. Беларусь, 6 апр. 2020 г., № 3-2-8/6133 // Консуль- тант Плюс : Беларусь [Электронный ресурс] / ООО «ЮрСпектр», Нац. центр правовой информ. Респ. Бела- русь. – Минск, 2022. 2. Особенности формирования заболеваемости военнос- лужащих острыми респираторными инфекциями верх- них дыхательных путей / Р. М. Аминев [и др.] // Изв. Рос. Воен.-мед. акад. – 2021. – Т. 40, № S2. – С. 9–17. Заключение – С. 26–50. Поступила 12.01.2022 г. Принята в печать 21.04.2022 г. Поступила 12.01.2022 г. Принята в печать 21.04.2022 г. Результаты и обсуждение Х класс 80131/14489 26710/4830 28566/3879 (23,53%/23,69%) I класс 1852/166 617/55 22815/1382 (18,79%/8,44%) XXI класс 10563/1214 3521/405 4167/892 (3,43%/5,45%) 1 и 2 гр. I, Х, XXI классы 452967/68508 150993/22836 76517/10294 (22,60%/15,41%) Таблица 3 – Расчет трудопотерь (в днях) и заболеваемости (случаев) сам для военнослужащих 2 группы на рисунке 6. Таким образом, наблюдался рост трудопо- терь и первичной обращаемости военнослужа- щих по I, X, XXI классам МКБ-10 в 2020 году. Это можно объяснить вкладом заболевания COVID-19 как инфекционного заболевания, ко- дируемого в I классе, и кодируемым в ХХI клас- се контактом с больным. И, предположительно, 60 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 COVID-19 как болезнь органов дыхания, кодиру- емая в Х классе. COV-2 (гриппа) при охвате до 95% военнослу- жащих с учетом выполнения комплекса противо- эпидемических мероприятий. В соответствии с вышеизложенным возникла необходимость в пересмотре и корректировке руководящих доку- ментов, регламентирующих вопросы снабжения, штатной структуры, непосредственной работы военно-медицинских учреждений и подразделе- ний в условиях пандемии. Количество обращений и трудопотерь во- еннослужащих по поводу COVID-19 невозможно точно оценить в отчетах 3/МЕД. Тем не менее, можно оценить долю следующим образом: вы- честь разницу между первичными обращениями и трудопотерями в 2020 году и среднегодовыми за 2017-2019 года, что нашло отражение в таблице 3. В результате получили для 1 группы диапазон возможных случаев заболеваемости COVID-19: от 680 (0,36%) в I классе до 13548 (28,76%) в ХХI классе; для 2 группы: от 892 (5,45%) в XXI классе до 3879 (23,69%) в X классе. Число случаев COVID-19 в Вооруженных Силах Республики Беларусь могло составить 10294 (15,41%; 95 ДИ: 15,14-15,69) и 76502 дней трудопотерь (22,60%; 95 ДИ: 22,46-22,74). Таким образом, COVID-19 у военнослужащих всех ка- тегорий мог составить 10294 (15,41%) случая и 76502 дня трудопотерь (22,60%). Количество обращений и трудопотерь во- еннослужащих по поводу COVID-19 невозможно точно оценить в отчетах 3/МЕД. Тем не менее, можно оценить долю следующим образом: вы- честь разницу между первичными обращениями и трудопотерями в 2020 году и среднегодовыми за 2017-2019 года, что нашло отражение в таблице 3. Поступила 12.01.2022 г. Принята в печать 21.04.2022 г. ( ) j jp 4. Thevarajan I, Nguyen THO, Koutsakos M, Druce J, Caly 2. Aminev RM, Smirnov AV, Kuzin AA, Zobov AE, Nikishov ON. Peculiarities of formation of morbidity of servicemen with acute respiratory infections of the upper respiratory tract. Izv Ros Voen-med Akad. 2021;40(S2):9-17. (In Russ.) Сведения об авторах: Цыганков А.М. – старший преподаватель кафедры военной подготовки и медицины экстремальных ситуаций, Витебский государственный ордена Дружбы народов медицинский университет; Цыганков А.М. – старший преподаватель кафедры военной подготовки и медицины экстремальных ситуаций, Витебский государственный ордена Дружбы народов медицинский университет; Лятос И.А. – к.м.н., заместитель начальника кафедры – начальник учебной части кафедры военной подготовки и медицины экстремальных ситуаций, Витебский государственный ордена Дружбы народов медицинский универ- ситет. Лятос И.А. – к.м.н., заместитель начальника кафедры – начальник учебной части кафедры военной подготовки и медицины экстремальных ситуаций, Витебский государственный ордена Дружбы народов медицинский универ- ситет. References resurs]. Minsk, RB: 2022. (In Russ.) 2. Aminev RM, Smirnov AV, Kuzin AA, Zobov AE, Nikishov ON. Peculiarities of formation of morbidity of servicemen with acute respiratory infections of the upper respiratory tract. Izv Ros Voen-med Akad. 2021;40(S2):9-17. (In Russ.) 3. Gustine JN, Jones D. Immunopathology of Hyperinflammation in COVID-19. Am J Pathol. 2021 Jan;191(1):4-17. doi: 10.1016/j.ajpath.2020.08.009 4 Th j I N THO K t k M D J C l 2. Aminev RM, Smirnov AV, Kuzin AA, Zobov AE, Nikishov ON. Peculiarities of formation of morbidity of servicemen with acute respiratory infections of the upper respiratory tract. Izv Ros Voen-med Akad. 2021;40(S2):9-17. (In Russ.) 1. On some issues of the procedure of examination of temporary disability, registration of disability certificates for persons with COVID-I9 infection and persons related to level 1 and 2 contacts of COVID-19 infection: pis'mo M-va zdravookhraneniia Resp Belarus', 6 apr 2020 g, № 3-2- 8/6133. V: OOO «IurSpektr», Nats tsentr pravovoi inform Resp Belarus'. Konsul'tant Plius: Belarus' [Elektronnyi 3. Gustine JN, Jones D. Immunopathology of Hyperinflammation in COVID-19. Am J Pathol. 2021 Jan;191(1):4-17. doi: 10.1016/j.ajpath.2020.08.009 61 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 L, van de Sandt CE, et al. Breadth of concomitant immune responses prior to patient recovery: a case report of non- severe COVID-19. Nat Med. 2020 Apr;26(4):453-455. doi: 10.1038/s41591-020-0819-2 L, van de Sandt CE, et al. Breadth of concomitant immune responses prior to patient recovery: a case report of non- severe COVID-19. Nat Med. 2020 Apr;26(4):453-455. doi: 10.1038/s41591-020-0819-2 infections. Klin Infektologiia Parazitologiia. 2021;10(1):88- 103. infections. Klin Infektologiia Parazitologiia. 2021;10(1):88- 103. 7. Evdokimov VI, Chernov DA, Sivashchenko PP, Eskov AS. Medico-statistical indicators of morbidity among conscripts of the Armed Forces of the Republic of Belarus and the Russian Federation (2003-2016). Med-biol Sots- psikhol Problemy Bezopasnosti Chrezvychain Situatsiiakh. 2018;(2):26-50. (In Russ.) 5. Brodin P. Immune determinants of COVID-19 disease presentation and severity. Nat Med. 2021 Jan;27(1):28-33. 6. Tcygankov AM, Ianchenko VV, Liatos IA. Human defense mechanisms against viruses causing acute respiratory Submitted 12.01.2022 Accepted 21.04.2022 Submitted 12.01.2022 Accepted 21.04.2022 Резюме. Общий индекс доверия иностранных граждан к профессиональной деятельности белорусских врачей составляет 40,4%, что соответствует среднему уровню доверия. Заключение. Результаты говорят о достаточном уровне доверия белорусским врачам со стороны иностранных пациентов. Векторы для повышения доверия представлены аспектами расширения опыта врачей по работе с ан- глоязычными пациентами и вопросами этики и коммуникации в здравоохранении. Заключение. Результаты говорят о достаточном уровне доверия белорусским врачам со стороны иностранных пациентов. Векторы для повышения доверия представлены аспектами расширения опыта врачей по работе с ан- глоязычными пациентами и вопросами этики и коммуникации в здравоохранении. Ключевые слова: доверие, система здравоохранения, социология медицины, экспорт медицинских GAVRILIK A.A. Multidisciplinary medical organization «LODE», Grodno, Republic of Belarus Vestnik VGMU. 2022;21(2):63-69. Information about authors: Tsygankov A.M. – senior lecturer of the Chair of Military Training & Emergency Medicine, Vitebsk State Order of Peoples’ Friendship Medical University; Lyatos I.A. – Candidate of Medical Sciences, deputy head of the Chair – head of the educational department of the Chair of Military Training & Emergency Medicine, Vitebsk State Order of Peoples’ Friendship Medical University. Адрес для корреспонденции: Республика Беларусь, 210009, г. Витебск, пр. Фрунзе, 27, Витебский государствен- ный ордена Дружбы народов медицинский университет, кафедра военной подготовки и медицины экстремальных ситуаций. E-mail: 87senka@gmail.com – Арсений Михайлович Цыганков. Correspondence address: Republic of Belarus, 210009, Vitebsk, 27 Frunze ave., Vitebsk State Order of Peoples’ Friendship Medical University, Chair of Military Training & Emergency Medicine. E-mail: 87senka@gmail.com – Arseniy M. Tsygankov. 62 62 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № DOI: https://doi.org/10.22263/2312-4156.2022.2.63 Резюме. Резюме. Для решения задачи по оценке текущего состояния доверия иностранных граждан к профессиональной деятель- ности белорусских врачей предлагается использовать индекс доверия к белорусским врачам, определяемый на основе индексов обобщенного доверия к белорусским врачам (трех видов), значения которых рассчитываются, исходя из значений двух показателей: показателя обобщенного доверия и показателя меры личного доверия. Цель – внедрение нового метода оценки доверия к белорусским врачам у иностранных пациентов. внедрение нового метода оценки доверия к белорусским врачам у иностранных пациентов. Материал и методы. С использованием принципов социологии медицины разработан подход к математической оценке уровня доверия иностранных потребителей медицинских услуг к белорусским врачам, система внедре- на в 2021 году в работу ряда организаций здравоохранения Гродненской области (учреждение здравоохранения «Гродненская университетская клиника», учреждение здравоохранения «Городская клиническая больница ско- рой медицинской помощи г.Гродно, учреждение здравоохранения «Островецкая центральная районная клиниче- ская больница»). Выборка составила 297 англоговорящих иностранцев: 184 (61,95%) женщины и 113 (38,05%) мужчин. Каждый третий иностранец – 34,24% женщин {27,77;41,35}% и 30,97% мужчин {23,19;40,01}% – имели опыт получения медицинской помощи в других странах, кроме Беларуси и страны проживания. Для обработки и представления результатов применены программа Microsoft Excel 2010, пакет программ «Statistica 10», серийный номер AXAR207F394425FA-Q. р р ру р у р Материал и методы. С использованием принципов социологии медицины разработан подход к математической оценке уровня доверия иностранных потребителей медицинских услуг к белорусским врачам, система внедре- на в 2021 году в работу ряда организаций здравоохранения Гродненской области (учреждение здравоохранения «Гродненская университетская клиника», учреждение здравоохранения «Городская клиническая больница ско- рой медицинской помощи г.Гродно, учреждение здравоохранения «Островецкая центральная районная клиниче- ская больница»). Выборка составила 297 англоговорящих иностранцев: 184 (61,95%) женщины и 113 (38,05%) мужчин. Каждый третий иностранец – 34,24% женщин {27,77;41,35}% и 30,97% мужчин {23,19;40,01}% – имели опыт получения медицинской помощи в других странах, кроме Беларуси и страны проживания. Для обработки и представления результатов применены программа Microsoft Excel 2010, пакет программ «Statistica 10», серийный номер AXAR207F394425FA-Q. Результаты. Метод включает социологическое анкетирование с последующим расчётом индекса доверия к бело- русским врачам, определяемого на основе трех индексов обобщенного доверия, отражающих три характеристики – опыт, профессионализм и этика – отличаются, превалирует доверие профессионализму белорусских врачей (p<0,01). Общий индекс доверия иностранных граждан к профессиональной деятельности белорусских врачей составляет 40,4%, что соответствует среднему уровню доверия. русским врачам, определяемого на основе трех индексов обобщенного доверия, отражающих три характеристики – опыт, профессионализм и этика – отличаются, превалирует доверие профессионализму белорусских врачей (p<0,01). Abstract. The method includes a sociological survey followed by the calculation of the index of trust in Belarusian doctors, determined on the basis of generalized trust indices (three types). The indices of generalized trust in three characteristics – experience, professionalism, ethics – differ, trust in the professionalism of Belarusian doctors prevails (p<0.01), the general index of trust of foreign citizens in the professional activities of Belarusian doctors makes up 40.4%, which corresponds to the average level of trust. – experience, professionalism, ethics – differ, trust in the professionalism of Belarusian doctors prevails (p<0.01), the general index of trust of foreign citizens in the professional activities of Belarusian doctors makes up 40.4%, which corresponds to the average level of trust. Conclusions. The results indicate a sufficient level of trust in Belarusian doctors on the part of foreign patients. Vectors for increasing trust are represented by the aspects of expanding the experience of doctors working with English-speaking patients and issues of ethics and communication in healthcare. Conclusions. The results indicate a sufficient level of trust in Belarusian doctors on the part of foreign patients. Vectors for increasing trust are represented by the aspects of expanding the experience of doctors working with English-speaking patients and issues of ethics and communication in healthcare. p Key words: trust, healthcare system, sociology of medicine, export of medical services. показатели доверия [2-4]. Однако не существует универсального метода измерения доверия, одно- мерные шкалы чаще направлены на изучение до- верия к конкретной организации или группе това- ров и не могут быть использованы для измерения доверия в социальной сфере. Представляется актуальным создание и внедрение инструмента, с помощью которого можно было бы наладить конструктивную систему оценки и мониторинга доверия иностранных пациентов белорусским врачам. Безусловно, первым и необходимым ша- гом является разработка показателей, характери- зующих уровень доверия к профессиональной деятельности врачей. Принципиальным момен- том в социологической интерпретации индексов доверия является представление о нем в инте- гральном и аналитическом аспектах. Так, показа- тели доверия призваны стать объективной мерой оценки доверия с возможностью мониторинго- вый исследований для различных групп [3, 5]. Попыток оценить доверие иностранных потреби- телей медицинских услуг в Беларуси не проводи- лось. Недостаточность научных исследований в области оценки доверия иностранных пациентов для повышения экспортного потенциала бело- русских медицинских услуг стала определяющей в выборе темы исследования. Государственной программой «Здоровье народа и демографическая безопасность» (2021- 2025 гг.) запланировано увеличение экспорта ме- дицинских услуг за пять лет более чем на 40%. Abstract. В соответствии с Планом совместных действий Министерства здравоохранения Республики Бе- ларусь с Министерством иностранных дел Ре- спублики Беларусь по развитию торгово-эконо- мического и инвестиционного сотрудничества сохранение экспортного потенциала белорусско- го здравоохранения, доверия иностранных паци- ентов, оценка их удовлетворенности остаются приоритетами даже в условиях борьбы с рас- пространением COVID-19 [1]. Проблема доверия пациентов давно находится в центре внимания организаторов здравоохранения. Так, взаимо- отношения врача и пациента формируются как вариант межличностных отношений (социаль- но-психологический аспект), при этом зависят от ожиданий пациента в отношении уровня про- фессионализма врача и качества медицинской по- мощи (индивидуально-психологический аспект). Экономистами установлено, что самым надеж- ным активом при продаже услуг является именно доверие, т.е. высокий уровень доверия иностран- ных пациентов будет способствовать высоко- му уровню лояльности, повышению спроса на белорусские медицинские услуги. Для оценки уровня доверия сегодня как зарубежными, так и российскими исследователями разработаны и достаточно широко применяются «одномерные» Abstract. Introduction. To solve the problem of assessing the current state of foreign citizens’ trust in the professional activities of Belarusian doctors, it is proposed to use the index of confidence in Belarusian doctors, determined on the basis of the indices of generalized trust in Belarusian doctors (three types), the values of which are calculated based on the values of two indicators: the indicator of generalized trust and the indicator of the measure of personal trust. 63 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Objectives.To introduce a new method of assessing the trust in Belarusian doctors among foreign patients. Material and methods. Using the principles of sociology of medicine, an approach has been developed to mathematically assess the level of trust of foreign consumers of medical services in Belarusian doctors, the system was introduced in 2021 into the work of a number of healthcare organizations in Grodno region (healthcare institution «Grodno University Clinic», healthcare institution «Grodno City Clinical Hospital of Emergency Medical Care», healthcare institution «Ostrovets Central District Clinical Hospital»). The sample consisted of 297 English-speaking foreigners: 184 (61.95%) women and 113 (38.05%) men. Every third foreigner – 34.24% of women {27.77; 41.35}% and 30.97% of men {23.19;40.01}% – had already an experience of receiving medical care in countries other than Belarus and the country of their residence. The Microsoft Excel 2010 program, the Statistica 10 software package, serial number AXAR207F394425FA-Q were used to process and present the results. p p Results. The method includes a sociological survey followed by the calculation of the index of trust in Belarusian doctors, determined on the basis of generalized trust indices (three types). The indices of generalized trust in three characteristics – experience, professionalism, ethics – differ, trust in the professionalism of Belarusian doctors prevails (p<0.01), the Results. The method includes a sociological survey followed by the calculation of the index of trust in Belarusian doctors, determined on the basis of generalized trust indices (three types). The indices of generalized trust in three characteristics – experience, professionalism, ethics – differ, trust in the professionalism of Belarusian doctors prevails (p<0.01), the general index of trust of foreign citizens in the professional activities of Belarusian doctors makes up 40.4%, which corresponds to the average level of trust. Results. Материал и методы С использованием принципов социологии С использованием принципов социологии 64 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № медицины разработан подход к математической оценке уровня доверия иностранных потребите- лей медицинских услуг к белорусским врачам, система внедрена в 2021 году в работу ряда ор- ганизаций здравоохранения Гродненской обла- сти (учреждение здравоохранения «Гродненская университетская клиника», учреждение здра- воохранения «Городская клиническая больница скорой медицинской помощи г. Гродно, учреж- дение здравоохранения «Островецкая централь- ная районная клиническая больница»). Выборка настоящего социологического опроса составила 297 англоговорящих иностранцев: 184 (61,95%) женщины и 113 (38,05%) мужчин. Каждый третий иностранец – 34,24% женщин {27,77;41,35}% и 30,97% мужчин {23,19;40,01}% – имели опыт получения медицинской помощи в других стра- нах, кроме Беларуси и страны проживания. Ав- торская социологическая анкета была создана на основании результатов исследований ряда из- вестных социологов (Э. Гидденса, Ф. Фукуямы, П. Штомпки, М. Флуджельманаи др.) [6, 7-9], текст (инструкция, вопросы, варианты ответов) подвергался экспертной оценке, анкета была переведена на английский язык, на русскоязыч- ный и англоязычный варианты получены поло- жительные рецензии кандидатов социологиче- ских наук, сотрудников кафедры социологии и специальных социологических дисциплин ГрГУ им. Янки Купалы. Объем выборки составил 297 англоговорящих иностранцев: 184 (61,95%) женщины и 113 (38,05%) мужчин, которые об- ращались за медицинской помощью в организа- ции здравоохранения г. Гродно в течение 2020 года. С использованием принципов эконометри- ческого анализа автором был создан многопара- метрический подход к оценке доверия, система предложенных индексов доверия внедрена в 2021 году в работу ряда организаций здраво- охранения Гродненской области: учреждение здравоохранения «Гродненская университетская клиника», учреждение здравоохранения «Город- ская клиническая больница скорой медицинской помощи г.Гродно, учреждение здравоохранения «Островецкая центральная районная клиниче- ская больница». Цель исследования – внедрить многопа- раметрический подход и оценить доверие ино- Результаты и обсуждение Для оценки доверия иностранных пациен- тов белорусским врачам предложено использо- вать ряд индексов, отражающих уровень дове- рия по трем характеристикам: профессионализм врача, опыт и этические аспекты коммуникации врач-пациент. Система показателей и индексов доверия иностранных граждан к профессиональ- ной деятельности белорусских врачей приведена на рисунке 1. Методологическими предпосылками соз- дания индекса является постулат, что доверие граждан формируется под влиянием личного опыта иностранных потребителей медицинских услуг при взаимодействии с белорусскими вра- чами (личное доверие) и оценки ими обществен- ного доверия (социальное или общественное до- верие) [5]. р ) Показатель обобщенного доверия к бело- русским врачам определялся по ответам респон- дентов на вопрос: «Оцените, в какой степени Ваши знакомые доверяют белорусским врачам по следующим характеристикам». В формули- ровке вопроса были уточнены три изучаемые характеристики: профессионализм, этика, опыт. Ответ испытуемых мог принимать пять значе- ний: 5 – полностью доверяют, а 1 – совершенно не доверяют. Для расчета значений показателя обобщенного доверия иностранных потребите- лей медицинских услуг к белорусским врачам использована следующая формула: где: i – номер характеристики врача, i = 1, 2, 3; i – номер характеристики врача, i = 1, 2, 3; – оценка доверия отдельной характери- стики врача, данная j-тым иностранным гражда- нином, потребителем медицинской помощи; – объем выборки (только респон- денты, ответившие на вопрос анкеты); – показатель обобщенного доверия. Зна- чения данного показателя могут быть в интерва- ле от 0 до 1 [1]. Показатель личного доверия к белорусским врачам. Цель исследования – внедрить многопа- раметрический подход и оценить доверие ино- странных потребителей медицинских услуг к бе- лорусским врачам. Показатель меры личного доверия ино- странных потребителей медицинских услуг к белорусским врачам определялся на основе от- ветов на вопрос: «Оцените, в какой степени Вы 65 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Рисунок 1 – Система показателей и индексов доверия иностранных граждан к профессиональной деятельности белорусских врачей [1]. Рисунок 1 – Система показателей и индексов доверия иностранных граждан к профессионально деятельности белорусских врачей [1]. доверяете белорусским врачам по аналогичным показателю обобщенного доверия характеристи- кам (5 – полностью доверяю, 1 – совершенно не доверяю)». Для расчета значений использована идентичная по методологии формула: На основании представленных двух пока- зателей с учетом трех изучаемых характеристик были рассчитаны: индекс обобщенного доверия, так же по характеристикам, как среднее ариф- метическое из двух показателей (обобщенного доверия и меры личного доверия), выраженный в процентах и индекс доверия иностранных по- требителей медицинских услуг к белорусским врачам. Примечание: для построения индекса обобщенного доверия значение показателя обобщенного доверия переведено в процентный формат: 0–20% – низкое значение показателя; 21–40% – ниже среднего; 41–60% – среднее; 61–80% – выше среднего; выше 81% – высокое; – p<0,01. Результаты и обсуждение где: где: Для обработки и представления результа- тов применены программа Microsoft Excel 2010, пакет программ «Statistica 10», серийный номер AXAR207F394425FA-Q. i – номер характеристики врача, i = 1, 2, 3; – оценка доверия отдельной характери- стики врача, данная j-тым иностранным гражда- нином, потребителем медицинской помощи; Результаты расчётов приведены в таблице 1. – показатель меры личного доверия; Все характеристики меры обобщенного доверия у иностранных пациентов находятся в диапазоне ниже среднего – от 21 до 40%, что свидетельствует о недостаточно эффективной – объем выборки (только респон- денты, ответившие на вопрос анкеты). Значения данного показателя могут быть в интервале от 0 до 1 [1]. Таблица 1 – Характеристики обобщенного доверия иностранных пациентов белорусским врачам Характеристика Показатель обобщенного доверия, , % Показатель личного доверия, % Индекс обобщенного доверия, % Профессионализм 40,7 46,8 43,8* Этика 39,4 38,7 39,1 Опыт 39,2* 37,4 38,3 Среднее значение 39,8 41,0 40,4 – Характеристики обобщенного доверия иностранных пациентов белорусским врачам Таблица 1 – Характеристики обобщенного доверия иностранных пациентов белору Примечание: для построения индекса обобщенного доверия значение показателя обобщенного доверия переведено в процентный формат: 0–20% – низкое значение показателя; 21–40% – ниже среднего; 41–60% – среднее; 61–80% – выше среднего; выше 81% – высокое; – p<0,01. 66 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № рекламной деятельности по продвижению меди- цинских услуг и необходимости поиска имидж- формирующих технологий для нужд экспорта. Так, очевидно, что необходимо повышать обще- ственное доверие иностранных пациентов за счет формирования позитивного общественного мнения о белорусских врачах за рубежом – сре- ди потенциальных англоязычных потребителей медицинских услуг, для Гродненского региона в этом ключе особый интерес представляла воз- можность сотрудничества с туристическими агентствами в связи с возможностью безвизового порядка въезда и выезда иностранных граждан на территорию Гродно (Указ Президента Белару- си № 300 от 7 августа 2019 г. «Об установлении безвизового порядка въезда и выезда иностран- ных граждан»). Анализ таблицы 2 показывает очевидные векторы для повышения информированности иностранных пациентов о белорусских медицин- ских услугах, о рекламе для иностранных паци- ентов в зависимости от пола: пока и для мужчин, и для женщин основным таким источником ин- формации являются друзья и знакомые, одна- ко статистически установлено, что иностранцы мужчины чаще, чем женщины, обращались за медицинской помощью в нашей стране по реко- мендации другого врача и посредством поиска информации через интернет. В этом ключе по- мимо коммуникационных барьеров между по- тенциальными иностранными пациентами и вра- чами отсутствуют штатные переводчики, не во всех организациях здравоохранения переведены информационные страницы веб-сайтов. Литература 1. Гаврилик, А. А. Клиент-ориентированные векторы для продвижения экспорта медицинских услуг в Грод- ненской области / А. А. Гаврилик // Здравоохранение (Минск). – 2021. – № 12. – С. 13–20. 2. Гаврилик, А. А. О новых методах оценки доверия ино- странных потребителей медицинских услуг к белорус- ским врачам / А. А. Гаврилик // Журн. Гродн. гос. мед. ун-та. – 2020. – № 6. – С. 727–731. 3. Купрейченко, А. Б. Психология доверия и недоверия / А. Б. Купрейченко. – Москва : Ин-т психологии РАН, 2008. – 571 с. 4. Мерсиянова, И. В. Доверие граждан к деятельности го- сударственных служащих / И. В. Мерсиянова, В. Н. Яки- мец, Е. И. Пахомова // Вопр. гос. и муницип. упр. – 2012. – № 4. – С. 98–118. Заключение 5. Селигмен, А. Проблема доверия / А. Селигмен. – Москва : Идея-Пресс, 2002. – 256 с. Оценка обратной связи от иностранных англоязычных пациентов по полученным в Ре- спублике Беларусь медицинским услугам явля- ется стратегическим вектором по продвижению бренда белорусского здравоохранения на между- народном рынке. Методика расчета индексов доверия апробирована в организациях здраво- охранения Гродненской области Республики Беларусь с целью оценки доверия иностранных потребителей медицинских услуг белорусским врачам. Уровень доверия иностранных пациен- тов – 40,4%, что соответствует нижней границе среднего уровня обобщенного доверия. Струк- турный анализ полученных результатов показал, что больше всего иностранные пациенты доверя- 6. Сурмач, М. Ю. Социология медицины: предмет, методо- логия и сферы применения в Республике Беларусь / М. Ю. Сурмач. – Гродно : ГрГМУ, 2016. – 316 с. 7. Tang, L. The influences of patient’s trust in medical service and attitude towards health policy on patient’s overall satisfaction with medical service and sub satisfaction in China / L. Tang // BMC Public Health. – 2011 Jun. – Vol. 11. – Art. 472. 8. Trust in the referring physician reduces anxiety in an integrated community-to-hospital care system / M. Y. Flugelman [et al.] // Isr. J. Health Policy Res. – 2020 May. – Vol. 9, N 1. – Art. 7. 9. Niv-Yagoda, A. Association between trust in the public healthcare system and selecting a surgeon in public hospitals in Israel: a cross-sectional population study / A. Niv-Yagoda // Isr. J. Health Policy Res. – 2020 Jul. – Vol. 9, N 1. – Art. 38. 9. Niv-Yagoda, A. Association between trust in the public healthcare system and selecting a surgeon in public hospitals in Israel: a cross-sectional population study / A. Niv-Yagoda // Isr. J. Health Policy Res. – 2020 Jul. – Vol. 9, N 1. – Art. 38. Поступила 01.02.2022 г. Принята в печать 21.04.2022 г. Результаты и обсуждение Видимо, это связано с определенными трудностями в коммуникациях врач-пациент, в том числе из-за языкового барьера. ют профессионализму белорусских врачей, а ре- зервы для повышения доверия – это медицинская коммуникация, вопросы языка, этики и экспорт- ориентированная пропаганда позитивного опыта белорусских специалистов в лечении иностран- ных пациентов. Таким образом, предложенные характери- стики доверия врачу – профессионализм, этика, опыт – и распределение доверия на личное и общественное были апробированы в ряде орга- низаций здравоохранения Гродненской области и используются для цифровой оценки доверия иностранных пациентов с целью повышения экс- портного потенциала белорусских медицинских услуг, очевидный вектор развития – формиро- вание позитивного внешнего имиджа системы здравоохранения и белорусских врачей через ин- тернет-ресурсы. 1. Gavrilik AA. Client-oriented vectors to promote exports of medical services in Grodno region. Zdravookhranenie (Minsk). 2021;(12):13-20. (In Russ.) Результаты и обсуждение Считаем присутствие белорусских организаций здравоох- ранения с описанием экспортных позиций в сети интернет и в социальных сетях перспективным направлением продвижения белорусских меди- цинских услуг. Также в результате опроса установлено, что в абсолютном большинстве – в 69,02% слу- чаев обращения за медицинской помощью в Ре- спублике Беларусь информацию о той или иной организации здравоохранения иностранные па- циенты получили от друзей и знакомых, в каж- дом десятом случае – 12,12% – по рекомендации другого врача и только в 8,08% источником стал интернет, а в 3,03% – социальные сети органи- зации здравоохранения. Мы наблюдаем низкую информированность иностранных граждан о воз- можностях получения медицинских услуг в Бе- ларуси, однако закономерен вопрос: кто должен выступить субъектом такого информирования? Основные источники информации о возможно- сти получения медицинской помощи в Беларуси, по мнению иностранцев мужчин и женщин, при- ведены в таблице 2. Обращает на себя внимание, что среди компонентов показателя личного доверия после контакта с белорусскими врачами у иностранных пациентов статистически достоверно выше дове- рие профессионализму врачей, – p<0,01. Уста- новлено, что именно личное доверие является образующим для индекса обобщенного доверия, который составляет для англоговорящих паци- ентов 40,4%, что соответствует нижней границе среднего уровня доверия. Белорусские врачи добросовестно выпол- няют свои обязанности по отношению к ино- странным гражданам, что указали при опросе Таблица 2 – Источники информации для иностранцев о медицинских услугах организаций здра- воохранения г. Гродно Источники информации % мужчин Доверительный интервал, % % женщин Доверительный интервал, % Друзья, знакомые 62,83 53,64;71,18 72,83* 65,98;78,74 Родственники 6,19 3,03;12,24 1,09 0,3;3,88% Рекомендация другого врача 15,93* 10,32;23,78 9,78 6,28;14,93 Интернет 10,62* 6,18;17,65 6,52 3,77;11,05 Буклеты, бланковая реклама 8,85 4,88;15,53 5,98 3,37;10,39 Давно является клиентом этой клиники 0,88 0,16;4,84 1,09 0,3;3,88 Социальные сети 3,54 1,39;8,75 2,72 1,17;6,2 Другое 32,74 24,78;41,84 32,07 25,75;39,12 Примечание: * – p<0,05. 2 – Источники информации для иностранцев о медицинских услугах организаций здра- Гродно Таблица 2 – Источники информации для иностранцев о медицинских услугах орга воохранения г. Гродно 67 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 82,84% пациентов. Однако перед проведением различных процедур в 85,19% {80,7;88,78}% слу- чаев ни врач, ни медсестра не объясняли, с какой целью, каким образом будет проводиться про- цедура. Видимо, это связано с определенными трудностями в коммуникациях врач-пациент, в том числе из-за языкового барьера. 82,84% пациентов. Однако перед проведением различных процедур в 85,19% {80,7;88,78}% слу- чаев ни врач, ни медсестра не объясняли, с какой целью, каким образом будет проводиться про- цедура. ВЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТО 2020 May;9(1):7. doi: 10.1186/s13584-020-00365-6 9. Niv-Yagoda A. Association between trust in the public healthcare system and selecting a surgeon in public hospitals in Israel: a cross-sectional population Policy Res. 2020 Jul;9(1):38. doi: 1 00396-z 2020 May;9(1):7. doi: 10.1186/s13584-020-00365-6 in Israel: a cross-sectional population study. Isr J Health Policy Res. 2020 Jul;9(1):38. doi: 10.1186/s13584-020- 00396-z in Israel: a cross-sectional population study. Isr J Health Policy Res. 2020 Jul;9(1):38. doi: 10.1186/s13584-020- 00396-z 2020 May;9(1):7. doi: 10.1186/s13584-020-00365-6 9. Niv-Yagoda A. Association between trust in the public healthcare system and selecting a surgeon in public hospitals Submitted 01.02.2022 Accepted 21.04.2022 References 5. Seligmen A. The Problem of Trust. Moscow, RF: Ideia-Press; 2002. 256 р. (In Russ.) 1. Gavrilik AA. Client-oriented vectors to promote exports of medical services in Grodno region. Zdravookhranenie (Minsk). 2021;(12):13-20. (In Russ.) 2. Gavrilik AA. On new methods of assessing the confidence of foreign consumers of medical services in Belarusian doctors. Zhurn Grodn Gos Med Un-ta. 2020;(6):727-31. (In Russ.) 3. Kupreichenko AB. The Psychology of Trust and Mistrust. Moscow, RF: In-t psikhologii RAN; 2008. 571 р. (In Russ.) 4. Mersiianova IV, Iakimetc VN, Pakhomova EI. The confidence of citizens in the activities of civil servants. Vopr Gos Munitsip Upr. 2012;(4):98-118. (In Russ.) 1. Gavrilik AA. Client-oriented vectors to promote exports of medical services in Grodno region. Zdravookhranenie (Minsk). 2021;(12):13-20. (In Russ.) 6. Surmach MIu. Sociology of Medicine: Subject Matter, Methodology, and Spheres of Application in the Republic of Belarus. Grodno, RB: GrGMU; 2016. 316 р. (In Russ.) 2. Gavrilik AA. On new methods of assessing the confidence of foreign consumers of medical services in Belarusian doctors. Zhurn Grodn Gos Med Un-ta. 2020;(6):727-31. (In Russ.) 7. Tang L. The influences of patient's trust in medical service and attitude towards health policy on patient's overall satisfaction with medical service and sub satisfaction in China. BMC Public Health. 2011 Jun;11:472. doi: 10.1186/1471-2458-11- 472 3. Kupreichenko AB. The Psychology of Trust and Mistrust. Moscow, RF: In-t psikhologii RAN; 2008. 571 р. (In Russ.) 3. Kupreichenko AB. The Psychology of Trust and Mistrust. Moscow, RF: In-t psikhologii RAN; 2008. 571 р. (In Russ.) p g р ( ) 4. Mersiianova IV, Iakimetc VN, Pakhomova EI. The confidence of citizens in the activities of civil servants. Vopr Gos Munitsip Upr. 2012;(4):98-118. (In Russ.) 8. Flugelman MY, Jaffe R, Luria G, Yagil D. Trust in the referring physician reduces anxiety in an integrated community-to-hospital care system. Isr J Health Policy Res. 68 Резюме. Резюме. Цель исследования – осуществить сравнительную оценку результатов морфометрического исследования инфек- ционно-воспалительного очага и плотности сосудов микроциркуляторного русла при различных сроках примене- ния лазерного излучения в послеоперационном лечении экспериментального периостита челюстей. Цель исследования – осуществить сравнительную оценку результатов морфометрического исследования инфек- ционно-воспалительного очага и плотности сосудов микроциркуляторного русла при различных сроках примене- ния лазерного излучения в послеоперационном лечении экспериментального периостита челюстей. Материал и методы. Эксперимент выполнен на 56 кроликах. Из них у 54 был смоделирован острый гнойный периостит (ОГП) нижней челюсти. Все животные были разделены на серии следующим образом. Серия 1 – 12 кроликов, которым после создания модели ОГП применяли лазеротерапию на 1, 3 и 5 сутки после хирургической обработки воспалительного очага. Серия 2 – 16 объектов, которым лазеротерапию применяли на 3, 5 и 7 сутки после операции. Серия 3 (контрольная) – 26 кроликов, которым после создания модели ОГП никакого лечения не проводили. Серия 4 – 2 животных, фрагменты челюсти которых забирали как эталон. Материал и методы. Эксперимент выполнен на 56 кроликах. Из них у 54 был смоделирован острый гнойный периостит (ОГП) нижней челюсти. Все животные были разделены на серии следующим образом. Серия 1 – 12 кроликов, которым после создания модели ОГП применяли лазеротерапию на 1, 3 и 5 сутки после хирургической обработки воспалительного очага. Серия 2 – 16 объектов, которым лазеротерапию применяли на 3, 5 и 7 сутки после операции. Серия 3 (контрольная) – 26 кроликов, которым после создания модели ОГП никакого лечения не проводили. Серия 4 – 2 животных, фрагменты челюсти которых забирали как эталон. Результаты. Во всех микропрепаратах серии 1 при завершении эксперимента изменения можно охарактеризовать как минимально выраженное остаточное воспаление. Комплексное лечение периостита, включающее лазерное воздействие, примененное с первых суток послеоперационного периода, является эффективным для купирования инфекционного воспаления и создания оптимальных условий для регенерации тканей. Результаты указывают, что при более позднем включении в состав лечения лазеротерапии получение позитивного эффекта возможно, одна- ко оно запаздывает, что создает условия для более длительного периода интоксикации, а, следовательно, может явиться причиной генерализации инфекционного процесса. Заключение. Несмотря на сходство схем комплексного послеоперационного лечения экспериментального пери- остита с использованием лазерного воздействия на очаг воспаления в сериях 1 и 2, установлено, что еe поло- жительный эффект обусловлен большей степенью комплементарности при применении в максимально ранние сроки после операции. Ключевые слова: одонтогенная инфекция, острый гнойный периостит, эксперимент, комплексно ротерапия, морфологическое исследование. COMPARISON OF THE RESULTS OF MORPHOLOGICAL EXAMINATION AT DIFFERENT PERIODS OF LASER THERAPY INTEGRATION INTO COMPLEX TREATMENT FOR EXPERIMENTAL PERIOSTITIS TSERAKHAVA T.N., POHODENKO-CHUDAKOVA I.O., NIJIATI N., YUDINA O.A. Belarusian State Medical University, Minsk, Republic of Belarus Vestnik VGMU. 2022;21(2):70-78. Сведения об авторах: Сведения об авторах: р Гаврилик А.А. – директор филиала, Многопрофильная медицинская организация «ЛОДЭ». р Гаврилик А.А. – директор филиала, Многопрофильная медицинская организация «ЛОДЭ». Information about authors: Information about authors: Gavrilik A.A. – director of the branch, Multidisciplinary medical organization «LODE». Адрес для корреспонденции: Республика Беларусь, 230023, г. Гродно, ул. Большая Троицкая, 51, филиал ООО «ЛОДЭ». E-mail: alexandergavrilik@yandex.ru – Гаврилик Александр Анатольевич. Correspondence address: Republic of Belarus, 230023, Grodno, 51 Bolshaya Troitskaya str., branch of Multidisciplinary medical organization «LODE». E-mail: alexandergavrilik@yandex.ru – Alexander A. Gavrilik. 69 VESTNIK VITEBSKOGO СТОМАТОЛОГИЯ DOI: https://doi.org/10.22263/2312-4156.2022.2.70 Abstract. Одним из таких мето- дов является лазерное излучение, имеющее ряд преимуществ перед стандартными методами ле- чения ИВП: мощный бактерицидный эффект; минимальное число осложнений; неспособность микроорганизмов вырабатывать устойчивость к данному воздействию [8]. значительно уменьшает поступление продуктов аутолиза в кровь и снижает уровень интоксика- ции. В то же время в послеоперационной ране и воспалительном инфильтрате остается некоторое количество микроорганизмов, способных под- держивать дальнейшее течение патологического процесса. В специальной литературе имеются сведения, что более чем в 50,0% наблюдений после проведения первичной хирургической об- работки инфекеционно-воспалительного очага и его дренирования может развиться рецидив. В связи с указанным большее число известных и вновь разрабатываемых методов лечения при ИВП направлены на ликвидацию обусловившей его микрофлоры [7]. При этом следует подчер- кнуть, что на сегодня даже самая современная противомикробная терапия не способна обеспе- чить абсолютный результат и достичь желаемо- го эффекта. В связи с чем остается актуальным поиск новых путей эффективного воздействия на патогенную микрофлору. Одним из таких мето- дов является лазерное излучение, имеющее ряд преимуществ перед стандартными методами ле- чения ИВП: мощный бактерицидный эффект; минимальное число осложнений; неспособность микроорганизмов вырабатывать устойчивость к данному воздействию [8]. Инфекционно-воспалительные процессы (ИВП) челюстно-лицевой области и шеи у детей на современном этапе представляют наиболее распространенные заболевания в профильных отделениях детских клинических больниц, доля которых составляет 40-55% от общего числа го- спитализированных лиц с патологией головы и шеи [1]. Последние годы ознаменованы тем, что число детей с острыми одонтогенными ИВП че- люстно-лицевой области и шеи, которым потре- бовалась госпитализация, значительно возросло [2], что, с одной стороны, обусловлено высокой распространенностью и интенсивностью кари- озного поражения зубов, неизменно ведущих к формированию очагов хронической одонтоген- ной инфекции [3], а, с другой стороны, это обу- словлено анатомо-физиологическими особенно- стями тканей лица и шеи у детей, возрастными особенностями детского организма (незрелостью нервной, иммунной, гипоталамо-гипофизарно- надпочечниковой систем) [4]. Указанные пато- логические процессы часто имеют негативные последствия: нарушение роста и развития челю- стей, невриты, деформации альвеолярного от- ростка на верхней челюсти и альвеолярной части – на нижней челюсти [5], поражение отдаленно расположенных органов и их систем, что указы- вает на генерализацию ИВП [6]. В то же время известно, что разработка и научное обоснование тех или иных методов ле- чения и их комплексов невозможны без экспе- риментальных исследований, имеющих в своем составе морфологическую часть. Abstract. Objectives. To carry out a comparative assessment of the results of morphometric study of infectious and inflammatory focus and the density of vessels of the microcirculatory bed at different periods of laser radiation application in the postoperative treatment for experimental periostitis of the jaws. Material and methods. The experiment was performed on 56 rabbits. 54 of them had simulated acute pu 70 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № (APP) of the lower jaw. All the animals were divided into the following series. Series 1 – 12 rabbits which, after the creation of the APP model, were treated with laser therapy on the 1st, the 3rd and the 5th days after surgery. Series 2 – 16 rabbits which underwent laser therapy treatment on the 3rd, the 5th and the 7th days after surgery. Series 3 (control) – 26 rabbits which were not treated after the creation of the APP model. Series 4 – 2 animals whose jaw fragments were taken as a standard. Results. In all series 1 micropreparations, after completing the experiment the changes can be characterized as minimally residual inflammatory ones. Complex treatment of periostitis, including laser treatment, applied from the first day of the postoperative period, is effective for relieving infectious inflammation and creating optimal conditions for tissue regeneration. g Conclusions. Despite the similarity of the schemes of complex postoperative treatment for experimental periostitis using the effect of laser on the focus of inflammation in series 1 and 2, it has been found that its positive influence is due to a greater degree of complementarity when used as early as possible after surgery. Key words: odontogenic infection, acute purulent periostitis, experiment, complex treatment, laser therapy, morphological study. значительно уменьшает поступление продуктов аутолиза в кровь и снижает уровень интоксика- ции. В то же время в послеоперационной ране и воспалительном инфильтрате остается некоторое количество микроорганизмов, способных под- держивать дальнейшее течение патологического процесса. В специальной литературе имеются сведения, что более чем в 50,0% наблюдений после проведения первичной хирургической об- работки инфекеционно-воспалительного очага и его дренирования может развиться рецидив. В связи с указанным большее число известных и вновь разрабатываемых методов лечения при ИВП направлены на ликвидацию обусловившей его микрофлоры [7]. При этом следует подчер- кнуть, что на сегодня даже самая современная противомикробная терапия не способна обеспе- чить абсолютный результат и достичь желаемо- го эффекта. В связи с чем остается актуальным поиск новых путей эффективного воздействия на патогенную микрофлору. Abstract. Данные иссле- дования важны не только в связи с тем, что имеют направленный практический выход – разработан- ные с их помощью методы лечения, лекарствен- ные средства, изделия медицинского назначения, Известным является факт, что успех ле- чения ИВП различной локализации, в том чис- ле и челюстно-лицевой области одонтогенного генеза, определяет адекватная хирургическая тактика с обеспечением оптимальных условий для оттока гнойного отделяемого, эффективная антибактериальная терапия (АБТ). Интраопера- ционная эвакуация экссудата из очага воспаления 71 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 риостит (ОГП) нижней челюсти во фронтальном ее отделе. Все экспериментальные объекты были разделены на серии следующим образом. хирургическая тактика, а также возможность оценить их эффективность, но и не менее важной фундаментальной составляющей, позволяющей глубже раскрыть патогенез заболевания [9, 10]. Серия 1 включала 12 (21,4%) эксперимен- тальных животных, которым после создания модели ОГП нижней челюсти применяли лазе- ротерапию на 1, 3 и 5 сутки после ПХО инфекци- онно-воспалительного очага. Все перечисленные факты в совокупно- сти свидетельствуют об актуальности избранной темы и убеждают в необходимости проведения данного исследования. Серия 2 включала 16 (28,6%) эксперимен- тальных объектов, которым лазеротерапию при- меняли на 3, 5 и 7 сутки после ПХО инфекцион- но-воспалительного очага. Цель исследования – осуществить сравни- тельную оценку результатов морфометрическо- го исследования инфекционно-воспалительного очага и плотности сосудов микроциркуляторного русла при различных сроках применения лазер- ного излучения в послеоперационном лечении экспериментального периостита челюстей. После ПХО инфекционно-воспалительно- го очага в соответствии со сроками, указанными для каждой из серий животных, рану инстилли- ровали раствором фурацилина (1:5000). Затем на рану накладывали влажную повязку, пропитан- ную указанным антисептиком, после чего в тече- ние 1 минуты воздействовали лазерным излуче- нием при помощи аппарата АЛТ Жэнь-Шень М, спектральный диапазон которого соответствовал спектру электронного поглощения фурацилина в течение 3 минут. Далее рану дренировали по- лоской из перчаточной резины. Замену дренажей осуществляли ежедневно при перевязках до пол- ного прекращения отделения гнойного экссудата. Курс лазеротерапии включал три процедуры. Материал и методы Гистологическую проводку материала осуществляли в автоматическом режи- ме с использованием гистопроцессора карусель- ного типа LeicaTP 1020 по стандартной (спирты – ксилол – парафиновая среда) методике [12]. Обезвоженный материал заливали в парафино- вую среду для изготовления серийных срезов тол- щиной 3 мкм при помощи ротационного электро- механического микротома «Microm HM340E». Срезы монтировали на предметные стекла, за- тем депарафинировали в трех сменах ксилола, пяти сменах спиртов нисходящей концентрации и окрашивали гематоксилином и эозином. На по- следнем этапе срезы заключали в монтирующую среду на основе полистирола. процесса декальцинации варьировала от 30 до 45 суток. Декальцинированные объекты промывали в течение нескольких дней в часто сменяемом 70% спирте во избежание набухания волокон со- единительной ткани. Гистологическую проводку материала осуществляли в автоматическом режи- ме с использованием гистопроцессора карусель- ного типа LeicaTP 1020 по стандартной (спирты – ксилол – парафиновая среда) методике [12]. Обезвоженный материал заливали в парафино- вую среду для изготовления серийных срезов тол- щиной 3 мкм при помощи ротационного электро- механического микротома «Microm HM340E». Срезы монтировали на предметные стекла, за- тем депарафинировали в трех сменах ксилола, пяти сменах спиртов нисходящей концентрации и окрашивали гематоксилином и эозином. На по- следнем этапе срезы заключали в монтирующую среду на основе полистирола. альвеолярные части, мягкие ткани и слизистую оболочку. Было установлено, что после приме- нения модифицированной методики кислотной декальцинации и зубы, и связанная с ними аль- веолярная кость, не имевшая признаков патоло- гических изменений, равномерно, в соответствии с вариантными структурными особенностями, демонстрировали четкий паттерн окрашивания. Оксифильный линейный паттерн окрашивания преобладал для тела челюстной кости и альве- олярной части. Для цемента был характерным гомогенный оксифильный паттерн, для крове- носных сосудов – оранжевая окраска внутри- просветных эритроцитов. Базофильный паттерн окрашивания, вариабельный по интенсивности и плотности клеточных элементов, наблюдали для связок, нервных стволиков, дентина и пульпы (рис. 1 (А, Б)). При анализе микропрепаратов серии кон- троля было определено следующее. Острый гнойный экспериментальный периостит ниж- ней челюсти в 46,4% (26) наблюдений протекал в виде ограниченного воспаления надкостницы альвеолярной части с вовлечением нескольких зубов (рис. 1 (В-Е)). В 3,6% (2) наблюдений вос- палительный процесс распространялся и на над- костницу тела челюсти. Окрашенные микропрепараты исследовали в проходящем свете с помощью светового микро- скопа Leica DM 2500. Микрофотосъемку про- водили c увеличением х12,5-400 с разрешением 1920х1080 пикселей микрофотокамерой Leica DFC425. Материал и методы В рамках патогистологического иссле- дования микропрепаратов, полученных от живот- ных с экспериментальной моделью ОГП с приме- нением различных методов лечения, оценивали такие показатели, как локализация остаточного воспалительного инфильтрата (наружный слой надкостницы, слизистая оболочка, околочелюст- ные мягкие ткани, костная ткань, периодонт ря- дом стоящих зубов), морфологические изменения в надкостнице (некроз внутреннего слоя надкост- ницы, инфильтрация лейкоцитами, полнокровие в сосудистом русле, стаз в сосудах внутреннего слоя надкостницы), перифокальные изменения в прилежащей кости (отек, гиперемия костного мозга, расширение костномозговых пространств, замещение костного мозга грубоволокнистой тканью, резорбция кортикального слоя челюсти, тромбоз кровеносных сосудов кости) с ранговой оценкой признаков. В начальном периоде заболевания во всех наблюдениях надкостница была утолщена, от- ечна, инфильтрирована лейкоцитами. В сосудах отмечали полнокровие и стаз, также были выяв- лены кровоизлияния в мягкие ткани. Скопление гнойного экссудата в 3,6% (2) наблюдений вызва- ло отслойку надкостницы. Постепенно в экссу- дате нарастало число клеточных элементов, и он приобрел гнойный характер. В 5,4% (3) наблюде- ний в центре очага воспаления возникал некроз с гнойным расплавлением. Абсцедирование сопро- вождалось нарушением целостности надкостни- цы и распространением воспалительного процес- са в окружающие ткани. Во всех микропрепаратах серии 1 на мо- мент завершения эксперимента изменения мож- но охарактеризовать как минимально остаточные воспалительные или поствоспалительные. Оча- говое воспаление было полностью локализовано (рис. 2 (А, Б)). Слабая лимфоплазмоцитарная ин- фильтрация определялась в надкостнице в 21,4% (12) наблюдений и костной ткани в 16,1% (9). В костной ткани соседних костных структур выя- вить воспалительную инфильтрацию не предста- вилось возможным, однако их питающие каналы Материал и методы Экспериментальные исследования выпол- нены в соответствии с принципами биоэтики (GLP – надлежащая лабораторная практика), в том числе «Европейской конвенцией по защи- те прав позвоночных животных», принятой в г. Страсбурге (Франция) 18.03.1986 и «Всемир- ной декларацией прав животных» (Universal Declaration of Animal Rights», принятой Между- народной лигой прав животных в г. Лондоне (Ве- ликобритания) 23.09.1977) [11]. Животных указанных серий наблюдения выводили из эксперимента после достижения клинического выздоровления (полного купирова- ния ИВП и заживления послеоперационной раны в полости рта). В серии 1 этот срок равнялся 10 суткам после проведения ПХО, а в серии 2 – 13 суткам наблюдения. Экспериментальным исследованиям пред- шествовало положительное заключение био- этической комиссии учреждения образования «Белорусский государственный медицинский университет». Животные, выделенные для экс- периментальных исследований, находились на стандартном рационе питания в виварии науч- но-исследовательской лаборатории учреждения образования «Белорусский государственный ме- дицинский университет» со свободным доступом к пище и воде. Перед началом проведения экспе- римента животных выдерживали в выделенном боксе в течение одной недели для адаптации к но- вым условиям и с целью прохождения карантина. Всех животных взвешивали, тщательно осматри- вали на наличие признаков заболевания. Особей с выявленной патологией выбраковывали. Они в исследование не включались. Серия 3 включала 26 (46,4%) кроликов, которым после создания экспериментальной мо- дели ОГП нижней челюсти, никаких лечебных манипуляций не проводили. Забор материала для патогистологического исследования осуществля- ли через 3 суток после начала моделирования па- тологического процесса. Данная серия являлась контрольной. Серия 4 состояла из 2 (3,6%) эксперимен- тальных животных без какой-либо патологии, фрагменты нижней челюсти которых забирали как эталон. В эксперименте были использованы 56 самцов кроликов породы Шиншилла. Из указан- ного числа животных у 54 (96,4%) в соответствии со способом, предложенным И.О. Походенько- Чудаковой, Т.Н. Тереховой, Н. Ницзяти и соавт. (2020), был смоделирован острый гнойный пе- В эксперименте были использованы 56 самцов кроликов породы Шиншилла. Из указан- ного числа животных у 54 (96,4%) в соответствии со способом, предложенным И.О. Походенько- Чудаковой, Т.Н. Тереховой, Н. Ницзяти и соавт. (2020), был смоделирован острый гнойный пе- Забранные макропрепараты в течение 72 часов фиксировали в 10% нейтральном формали- не. На этапе для удаления солей кальция из кост- ной ткани и зубов использовали концентрирован- ную муравьиную кислоту, разбавленную равным количеством 70% спирта. Продолжительность 72 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 процесса декальцинации варьировала от 30 до 45 суток. Декальцинированные объекты промывали в течение нескольких дней в часто сменяемом 70% спирте во избежание набухания волокон со- единительной ткани. Результаты и обсуждение При исследовании образцов тканей челю- сти кроликов серии эталона оценивали пульпо- периапикальный комплекс, включающий пульпу, апикальный периодонт с цементом, кортикаль- ную пластинку и губчатое вещество, окружаю- щее верхушку корня зуба, а также соседние зубы, 73 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Рисунок 1 – Микрофото: патогистологические изменения в челюстной кости при моделировании острого гной- ного периостита: А – декальцинированный зуб и альвеолярная кость без патологических изменений. Ув. х12,5; Б – периодонтальные связки без патологии. Ув. х100; В – периостит (фрагментация края альвеолярной части). Ув. х200; Г – периостит (деструкция коллагеновых волокон периодонтальной связки, выраженная ангиоэктазия и парез сосудов микроциркуляторного русла). Ув. х50; Д – периостит (нарушение ориентации коллагеновых волокон периодонтальной связки). Ув. х50; Е – периостит (воспалительная инфильтрация в периваскулярных пространствах с инвагинацией в альвеолярную кость). Ув. х400. Окраска гематоксилин и эозин. Очевидно, что комплексное лечение экс- периментального периостита, включающее ла- зерное воздействие, примененное с первых суток послеоперационного периода, является эффек- тивным для купирования инфекционно-воспали- тельного процесса, а также создает оптимальные условия для регенерации поврежденных им тка- ней, что согласуется со сведениями E. Larionova et al. (2019) [8]. были умеренно сдавлены за счет активных репа- ративных процессов (рис. 2 (Д)). Регенерация была констатирована во всех микропрепаратах данной серии (рис. 2 (Е)). Мак- симальная выраженность ее наблюдалась в над- костнице и костномозговых пространствах. Вы- явлено запустевание новообразованных сосудов, уменьшение их диаметра, пролиферация фибро- бластов, появление тонких коротких волоконец (рис. 2 (Г)). Патогистологическое исследование микро- 74 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Рисунок 2 – Микрофото: изменения в челюсти при применении в послеоперационном периоде лазеротерапии на 1, 3 и 5 сутки: А – фокус остаточного воспаления. Ув. х12,5; Б – минимальная воспалительная инфильтрация периоста. Ув. х50; В – начальный склероз костномозговых пространств. Ув. х100; Г – фибробласты и волоконца в костномозговых пространствах. Ув. х100; Д – сдавление питающих каналов. Ув. х50; Е – регенераторные изменения. Ув. х50. Окраска гематоксилин и эозин. Рисунок 2 – Микрофото: изменения в челюсти при применении в послеоперационном периоде лазеротерапии на 1, 3 и 5 сутки: А – фокус остаточного воспаления. Ув. х12,5; Б – минимальная воспалительная инфильтрация периоста. Ув. х50; В – начальный склероз костномозговых пространств. Ув. х100; Г – фибробласты и волоконца в костномозговых пространствах. Ув. х100; Д – сдавление питающих каналов. Ув. х50; Е – регенераторные изменения. Ув. х50. Окраска гематоксилин и эозин. тальных животных констатировали деформацию костных структур за счет гипертрофических про- цессов (рис. 3 (А)). Результаты и обсуждение Разрастание соедини- 76 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 Литература 1. Воспалительные заболевания челюстно-лицевой обла- сти у детей / А. О. Изюмов [и др.] // J. Siberian Med. Sci. – 2015. – № 5. – С. 43–49. 2. Характеристика одонтогенных и неодонтогенных вос- палительных процессов челюстно-лицевой области у детей, проходивших лечение в челюстно-лицевом ста- ционаре / П. А. Железный [и др.] // J. Siberian Med. Sci. – 2018. – № 3. – С. 31–39. Полученные результаты указывают, что при более позднем включении в состав ком- плексного послеоперационного лечения ИВП в области фронтального отдела нижней челюсти лазеротерапии получение позитивного эффекта возможно, однако оно запаздывает, что создает условия для более длительного периода интокси- кации, а, следовательно, может явиться причиной генерализации инфекционного процесса, что со- гласуется с данными И.О. Походенько-Чудаковой и соавт. (2018) [9]. 3. Clinical consequences of untreated dental caries in German 5- and 8-year-olds / K. Grund [et al.] // BMC Oral Health. – 2015 Nov. – Vol. 15, N 1. – P. 140. 4. Железный, П. А. Эффективность препаратов, обладаю- щих антисептическими и остеостимулирующими свой- ствами при лечении осложненного кариеса / П. А. Же- лезный, А. П. Железная, К. О. Самойлов // Рос. стоматол. журн. – 2015. – Т. 19, № 2. – С. 9–12. 5. Surgical management of garre’s osteomyelitis in an 8-year- old child / L. M. N. Philip [et al.] // Afr. J. Paediatr. Surg. – 2021 Apr-Jun. – Vol. 18, N 2. – P. 111–113. 6. Васильев, А. В. Хроническая одонтогенная инфекция и септический эндокардит / А. В. Васильев, К. П. Пименов, А. Ф. Керзиков // Пародонтология. – 2019. – Т. 24, № 1. – С. 11–14. Заключение 7. Результаты лечения инфекции в области хирургического вмешательства методом фотодинамической терапии / Г. М. Исмаилов [и др.] // Эндоскоп. хирургия. – 2016. – Т. 22, № 3. – С. 28–36. Несмотря на сходство схем комплексного послеоперационного лечения экспериментально- го периостита с использованием лазерного воз- действия на инфекционно-воспалительную рану и очаг воспаления в сериях 1 и 2, установлено, что еe положительный эффект обусловлен боль- шей степенью комплементарности при начале применения в максимально ранние сроки после оперативного вмешательства. Это позволяет до- стичь положительного эффекта – купирования инфекционно-воспалительного процесса на 3 суток раньше по отношению к серии сравнения. Полученные результаты являются обоснованием для применения данного лечебного воздействия в условиях клиники с целью оптимизации ком- плексного послеоперационного лечения у паци- ентов-детей с острыми одонтогенными инфекци- 8. Use of erbium laser in the treatment of a patient with acute purulent periostitis and a resistant form of primary immune thrombocytopenia / E. Larionova [et al.] // Case Rep. Dent. – 2019 Aug. – Vol. 2019. – 8260605. 9. Морфологические изменения при применении рефлек- сотерапии в лечении хронического синусита верхнече- люстной пазухи в эксперименте / И. О. Походенько-Чу- дакова [и др.] // Стоматолог. – 2018. – № 1. – С. 64–68. 10. Evaluation of a collagen matrix in a mandible defect in rats submitted to the use of bisphosphonates / V. V. Cunha [et al.] // Acta Cir. Bras. – 2020 Nov. – Vol. 35, N 10. – e202001005. 11. Чадаев, В. Е. Этические принципы при работе с лабо- раторными животными / В. Е. Чадаев // Вiсн. проблем бiологii і медицини. – 2012. – Т. 1, вип. 2. – С. 113‒115. 12. Колтовой, Н. А. Методы контрастирования и микроско- пии : монография / Н. А. Колтовой, С. А. Краевой. – Мо- сква : Boolvika.ru, 2014. – 112 с. Поступила 02.02.2022 г. Принята в печать 21.04.2022 г. Поступила 02.02.2022 г. Принята в печать 21.04.2022 г. онно-воспалительными процессами челюстей. тельной ткани было значительным по площади и характеризовалось плотным расположением веретенообразных и полигональных фибробла- стов с единичными гигантскими многоядерными остеокластами с неровным цитоплазматическим контуром. Костное вещество имело глубокие за- падения, а отходящие от него сохранившиеся коллагеновые волокна связок имели разную тол- щину, в части полей зрения – в виде «отрубков» и сегментов. тельной ткани было значительным по площади и характеризовалось плотным расположением веретенообразных и полигональных фибробла- стов с единичными гигантскими многоядерными остеокластами с неровным цитоплазматическим контуром. Костное вещество имело глубокие за- падения, а отходящие от него сохранившиеся коллагеновые волокна связок имели разную тол- щину, в части полей зрения – в виде «отрубков» и сегментов. Результаты и обсуждение В других 8,9% (5) наблюде- ний микропрепаратов были выявлены тонкие, хаотично ориентированные костные балочки, прилежащие к «старой» костной ткани, в которой визуализировались ленты базофильного костно- го вещества с четкой границей с более светлым, гомогенным оксифильным межуточным веще- ством, содержащим узкие, с пикнотичными ядра- ми остеоциты. препаратов серии 2 позволило выявить измене- ния, которые не были выявлены в препаратах серии 1. Воспалительный очаг был локализован во всех наблюдениях данной серии, что состави- ло 28,6% (16) от общего числа анализируемых фактов. Однако плотность воспалительного ин- фильтрата в образцах варьировала как по степени выраженности (от слабой до умеренной), так и по распространенности. Так, в 10,7% (6) наблю- дений экспериментальных животных указанные изменения имели место и в соседних к фокусу структурах. В 8,9% (5) наблюдений эксперимен- В 16,1% (9) наблюдений эксперименталь- 75 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Рисунок 3 – Микрофото: изменения в челюсти при применении в послеоперационном периоде лазеротерапии на 3, 5 и 7 сутки: А – очаг склероза периоста. Ув. х12,5; Б – воспалительная инфильтрация в очаге склероза. Ув. х200; В – зрелая грануляционная ткань (отрезок между стрелками). Ув. х50; Г – заместительный склероз межбалочных пространств. Ув. х50; Д – воспалительный инфильтрат межбалочных пространств. Ув. х100; Е – пролиферация клеток адвентиции питательных кровеносных сосудов. Ув. х100. Окраска гематоксилин и эозин. Рисунок 3 – Микрофото: изменения в челюсти при применении в послеоперационном периоде лазеротерапии на 3, 5 и 7 сутки: А – очаг склероза периоста. Ув. х12,5; Б – воспалительная инфильтрация в очаге склероза. Ув. х200; В – зрелая грануляционная ткань (отрезок между стрелками). Ув. х50; Г – заместительный склероз межбалочных пространств. Ув. х50; Д – воспалительный инфильтрат межбалочных пространств. Ув. х100; Е – пролиферация клеток адвентиции питательных кровеносных сосудов. Ув. х100. Окраска гематоксилин и эозин. ных, неправильной формы полостей (рис. 3 (Г)). В расширенных межбалочных пространствах вы- явили гиперемированный жировой костный мозг, который в 17,9% (10) наблюдений был замещен грубой соединительной тканью с лимфоплазмо- цитарной инфильтрацией (рис. 3 (Д)). ных животных была отмечена выраженная про- лиферация клеток адвентиции питательных кровеносных сосудов, резорбирующих костное вещество (рис. 3 (Е)). В 5,4% (3) микропрепаратов животных диаметр отдельных питательных кана- лов приближался к межбалочным пространствам губчатого вещества (рис. 3 (Д)). Межбалочные пространства в 7,1% (4) микропрепаратов были с резко утолщенным эндостом. Отмечали разру- шение костного вещества с образованием круп- Параллельно резорбции костной ткани были определены признаки репаративных про- цессов, которые по своей интенсивности отли- чались вариабельностью. ( ) ( ) 5. Philip LMN, Akkara F, Khwaja T, Narayan T, Kamath AG, Jose References Siberian Med Sci. 2018;(3):31-9. (In Russ.) 3. Grund Katrin, Goddon Inka, Schüler IM, Lehmann T, Heinrich-Weltzien R. Clinical consequences of untreated dental caries in German 5- and 8-year-olds. BMC Oral Health. 2015 Nov 4;15(1):140. doi: 10.1186/s12903-015-0121-8 1. Iziumov AO, Noskova EV, Kolybelkin MV, Apraksina EIu, Borodina TV, Klimova IV, i dr. Inflammatory diseases of the maxillofacial region in children. J Siberian Med Sci. 2015;(5):43-9. (In Russ.) 4. Zheleznyi PA, Zheleznaia AP, Samoilov KO. Effectiveness of drugs with antiseptic and osteostimulating properties in the treatment of complicated caries. Ros Stomatol Zhurn. 2015;19(2):9-12. (In Russ.) 4. Zheleznyi PA, Zheleznaia AP, Samoilov KO. Effectiveness of drugs with antiseptic and osteostimulating properties in the treatment of complicated caries. Ros Stomatol Zhurn. 2015;19(2):9-12. (In Russ.) 2. Zheleznyi PA, Kolybelkin MV, Iziumov AO, Apraksina EIu, Zheleznaia AP. Characteristics of odontogenic and neodontogenic inflammatory processes of the maxillofacial region in children treated in the maxillofacial hospital. J ( ) ( ) 5. Philip LMN, Akkara F, Khwaja T, Narayan T, Kamath AG, Jose 77 ESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 NP. Surgical management of garre's osteomyelitis in an 8-year- old child. Afr J Paediatr Surg. 2021 Apr-Jun;18(2):111-113. doi: 10.4103/ajps.AJPS_66_20 Adolf EV. Morphological changes in the application of reflexotherapy in the treatment of chronic sinusitis of the maxillary sinus in experiment. Stomatolog. 2018;(1):64-8. (In Russ.) Adolf EV. Morphological changes in the application of reflexotherapy in the treatment of chronic sinusitis of the maxillary sinus in experiment. Stomatolog. 2018;(1):64-8. (In Russ.) Adolf EV. Morphological changes in the application of reflexotherapy in the treatment of chronic sinusitis of the maxillary sinus in experiment. Stomatolog. 2018;(1):64-8. (In Russ.) 6. Vasilev AV, Pimenov KP, Kerzikov AF. Chronic odontogenic infection and septic endocarditis. Parodontologiia. 2019;24(1):11-4. (In Russ.) 10. Cunha VV, de Barros Silva PG, Lemos JVM, Martins JOL, Freitas MO, Avelar RL. Evaluation of a collagen matrix in a mandible defect in rats submitted to the use of bisphosphonates. Acta Cir Bras. 2020 Nov;35(10):e202001005. doi: 10.1590/ s0102-865020200100000005 7. Ismailov GM, Slovokhodov EK, Iarema VI, Polsachev VI, Nikolaev NM, Chekanov VN. Results of photodynamic therapy treatment of surgical infection. Endoskop Khirurgiia. 2016;22(3):28-36. (In Russ.) 11. Chadaev VE. Ethical principles in working with laboratory animals. Visn Problem Biologii Meditsini. 2012;1(вип 2):113- 5. (In Russ.) 8. Larionova E, Tarasenko S, Morozova E, Diachkova E. Сведения об авторах: р Терехова Т.Н. – д.м.н., профессор кафедры стоматологии детского возраста, Белорусский государственный меди- цинский университет, Терехова Т.Н. – д.м.н., профессор кафедры стоматологии детского возраста, Белорусский государственный меди- цинский университет, у р ORCID: http://orcid.org/0000-0002-2647-5082; ORCID: http://orcid.org/0000-0002-2647-5082; p g ; Походенько-Чудакова И.О. – д.м.н., профессор, заведующая кафедрой хирургической стоматологии, Белорусский государственный медицинский университет, ORCID: http://orcid.org/0000-0002-0353-0125; p g ; Ницзяти Н. – аспирант кафедры стоматологии детского возраста, Белорусский государственный медицинский университет, ORCID: http://orcid.org/0000-0003-4895-2281; ORCID: http://orcid.org/0000-0003-4895-2281; Юдина О.А. – к.м.н., доцент кафедры патологической анатомии, Белорусский государственный медицинский ORCID: http://orcid.org/0000-0001-7623-0601. ORCID: http://orcid.org/0000-0001-7623-0601. References Use of erbium laser in the treatment of a patient with acute purulent periostitis and a resistant form of primary immune thrombocytopenia. Case Rep Dent. 2019 Aug;2019:8260605. doi: 10.1155/2019/8260605 12. Koltovoi NA, Kraevoi SA. Contrast and microscopy techniques: monografiia. Moscow, RF: Boolvika.ru; 2014. 112 р. (In Russ.) 9. Pokhodenko-Chudakova IO, Surin AV, Gerasimovich AI, 9. Pokhodenko-Chudakova IO, Surin AV, Gerasimovich AI, Submitted 02.02.2022 Accepted 21.04.2022 Submitted 02.02.2022 Accepted 21.04.2022 Резюме. Цель исследования – изучить коммуникативные позиции (КП) в общении студентов лечебного факультета ВГМУ и проанализировать их динамические особенности. р методы. В исследовании приняли участие 532 студента лечебного факультета. Из них: студентов человек (117 юношей и 269 девушек); 6 курса – 146 человек (33 юноши и 113 девушек). р р Материал и методы. В исследовании приняли участие 532 студента лечебного факультета. Из них: студентов 2 курса – 386 человек (117 юношей и 269 девушек); 6 курса – 146 человек (33 юноши и 113 девушек). Изучение коммуникативных позиций в общении проводилось с помощью методики Е.И. Рогова «Трансактный анализ общения». Результаты. У студентов выявлены:более высокая представленность детской («зависимой») позиции у студентов 2ЛФ, доминирование взрослой позиции у студентов 6ЛФ, наименьшая представленность родительской позиции в общей выборке,более выраженный динамический рост формулы «взрослый – дитя – родитель» (ВДР) и снижение формулы «взрослый – родитель – дитя» (ВРД) у девушек в сравнении с юношами. Заключение. Выявленные общие и гендерные особенности КП в общении свидетельствуют о том, что созданная в ВГМУ информационная и образовательная среда в целом оказывает благоприятное влияние на формирование коммуникативных навыков будущих врачей, что проявляется в гибком выборе студентами лечебного факультета КП в общении, адекватных требованиям образовательного процесса. Результаты исследования могут быть использованы в образовательном процессе ВГМУ, деятельности социально- педагогической и психологической службы, работе кураторов академических групп и тьюторов. Результаты исследования могут быть использованы в образовательном процессе ВГМУ, деятельности социально- педагогической и психологической службы, работе кураторов академических групп и тьюторов. Ключевые слова: трансактный анализ, коммуникативные позиции, общение студентов, лечебный факультет. Результаты исследования могут быть использованы в образовательном процессе ВГМУ, деятельности социально- педагогической и психологической службы, работе кураторов академических групп и тьюторов. Ключевые слова: трансактный анализ, коммуникативные позиции, общение студентов, лечебный факультет. педагогической и психологической службы, работе кураторов академических групп и тьюторов. Ключевые слова: трансактный анализ, коммуникативные позиции, общение студентов, лечебный факультет. лова: трансактный анализ, коммуникативные позиции, общение студентов, лечебный факультет. TSERKOVSKY A.L., GAPOVA O.I., SKORIKOVA E.A., PETROVICH S.A. SERKOVSKY A.L., GAPOVA O.I., SKORIKOVA E.A., PETROVICH S.A. Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus Vestnik VGMU. 2022;21(2):79-84. Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus Information about authors: Tserakhava T.N. – Doctor of Medical Sciences, professor of the Chair of Pediatric Dentistry, Belarusian State Medical University, ORCID: http://orcid.org/0000-0002-2647-5082; Pohodenko-Chudakova I.O. – Doctor of Medical Sciences, professor, head of the Chair of Oral Surgery, Belarusian State Medical University, ORCID: http://orcid.org/0000-0002-0353-0125; p g Nijiati N. – postgraduate of the Chair of Pediatric Dentistry, Belarusian State Medical University, ORCID: http://orcid.org/0000-0003-4895-2281; Nijiati N. – postgraduate of the Chair of Pediatric Dentistry, Belarusian State Medical University, ORCID h // id /0000 0003 4895 2281 p g Yudina O.A. – Candidate of Medical Sciences, associate professor of the Chair of Pathological Anatomy, Belarusian State Medical University, y ORCID: http://orcid.org/0000-0001-7623-0601. y ORCID: http://orcid.org/0000-0001-7623-0601. Адрес для корреспонденции: Республика Беларусь, 220116, г. Минск, пр. Дзержинского, 83, Белорусский го- сударственный медицинский университет, кафедра хирургической стоматологии. E-mail: ip-c@yandex.ru – По- ходенько-Чудакова Ирина Олеговна. Correspondence address: Republic of Belarus, 220116, Minsk, 83 Dzerzhinskogo ave., Belarusian State Medical University, Chair of Oral Surgery. E-mail: ip-c@yandex.ru – Irina O. Pohodenko-Chudakova. 78 ВЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕ ПЕДАГОГИКА И ПСИХОЛОГИЯ ВЫСШЕЙ ШКОЛЫ DOI: https://doi.org/10.22263/2312-4156.2022.2.79 Abstract. Objectives.To study the communicative positions (CP) in the communication of medical faculty students of VSMU and to analyze their dynamic features. Material and methods.The study involved 532 students of the medical faculty. The 2nd-year students numbered 386 people (117 boys and 269 girls); the 6th-year students were represented by 146 persons (33 boys and 113 girls).The study of communicative positions in communication was carried out using the methodology of E.I. Rogov «Transactional analysis of communication». Results.The students showed a higher representation of a child’s («dependent») position among the 2nd-year medical students, the dominance of an adult’s position among the 6th-year medical students, the lowest representation of the parental position in the total sample, a more pronounced dynamic increase in the «adult – child – parent» (ACP) formula and a decrease in the «adult – parent – child » (APC) formula in girls compared to young men. Conclusions.The identified general and gender features of CP in communication indicate that the information and educational environment created at VSMU on the whole has a beneficial effect on the formation of communication skills of future doctors, which is manifested in the flexible choice of CP in communication, adequate to the requirements of 79 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 the educational process by the medical faculty students.The results of the study can be used in the educational process of VSMU, the activities of the socio-pedagogical and psychological services, the work of academic group tutors. Key words: transactional analysis, communicative positions, communication of students, medical faculty. уважения, признания равенства со своим собе- седником. Она характеризуется четким разграни- чением ответственности, признанием независи- мости другого человека. уважения, признания равенства со своим собе- седником. Она характеризуется четким разграни- чением ответственности, признанием независи- мости другого человека. В настоящее время кафедра психологии и педагогики с курсом ФПК и ПК проводит ис- следование коммуникативной деятельности (КД) студентов ВГМУ. Особое внимание мы уделяем личностному компоненту КД. С нашей точки зре- ния, он является системообразующим компонен- том КД [1]. Цель исследования – изучить коммуника- тивные позиции в общении студентов лечебного факультета ВГМУ и проанализировать особенно- сти их динамики. Кроме этого, его необходимо рассматри- вать как важный фактор формирования комму- никативных навыков, обеспечивающих более эффективную регуляцию профессиональной де- ятельности будущих врачей. Материал и методы В исследовании приняли участие 532 сту- дента лечебного факультета. Из них: студентов 2 курса – 386 человек (117 юношей и 269 девушек); 6 курса – 146 человек (33 юноши и 113 девушек). Одним из этапов изучения личностного компонента КД является исследование коммуни- кативных позиций в общении [2]. Изучение коммуникативных позиций в общении проводилось с помощью методики Е.И. Рогова «Трансактный анализ общения» [4]. Важность коммуникативной позиции (КП) заключается в том, что она существенным образом влияет на характер коммуникативного взаимодей- ствия между субъектами общения. При этом КП во многом определяет его исход. С нашей точки зрения, выбор определенной позиции необходимо рассматривать как важный компонент коммуника- тивной компетентности личности. Методика включает в себя 21 высказыва- ние. Каждое высказывание оценивается в баллах от 0 до 10. Подсчитывается отдельно сумма бал- лов по строкам: 1, 4, 7, 10, 13, 16, 19 (Д – «Дитя»); 2, 5, 8, 11, 14, 17, 20 (В – «Взрослый»); 3, 6, 9, 12, 15, 18, 21 (Р – «Родитель»). При анализе КП в общении используется модель трансактного анализа Эрика Берна [3]. Согласно этой модели субъекты общения осоз- нанно или неосознанно обмениваются действия- ми, направленными на изменение и регулирова- ние их КП. Анализ результатов производится в за- висимости от полученных формул (ДВР, ДРВ, ВДР, ВРД, РВД, РДВ) и возможного количествен- ного равенства нескольких формул (например, ДВР=ВДР). Э. Берн выделил три основные КП в об- щении: родительскую («Родитель»), взрослую («Взрослый») и детскую («Ребенок»). Для изучения динамических особенностей КП нами были выбраны, наряду со студентами выпускного курса, студенты-второкурсники. Этот выбор обусловлен тремя обстоятель- ствами: во-первых, студенты 2 курса прошли пе- риод социально-психологической адаптации к но- вым условиям жизнедеятельности; во-вторых, они усвоили специфику информационной и образова- тельной среды медицинского университета и сво- ей новой социальной роли «студент»; в-третьих, эти изменения могут повлиять на выбор КП при общении с преподавателями и студентами. Родительская КП – это позиция «превос- ходства». Она отражает такое состояние наше- го «Я», такие чувства, установки (внутреннюю готовность к принятию решения) и привычное поведение, которые относятся к роли родителя: ощущение власти, морализаторство, требова- тельность, поучение. Детская КП проявляется в «зависимости» от субъекта в общении. Для нее характерны: склонность к переживанию и эгоцентризму, по- вышенная эмоциональность, большая импуль- сивность и малая осознанность поведения. Результаты и обсуждение Результаты исследования КП студентов 2 Взрослая КП – это позиция равноправия, 80 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 он преобладает у девушек 2ЛФ и юнощей 6ЛФ. (2ЛФ) и 6 (6ЛФ) курсов лечебного факультета от- ражены в таблице. Наименее представленным является вари- ант ДВР=ДРВ. Он полностью отсутствует у сту- дентов 6ЛФ и юношей 2ЛФ. При анализе КП студентов, с нашей точки зрения, особое внимание необходимо обращать на первый символ в формуле, так как именно он в наибольшей степени отражает влияние и выбор КП в общении. С учетом сложности анализа полученных результатов, по нашему мнению, наиболее целе- сообразно использовать суммарный вес каждой из коммуникативных позиций с учетом формулы (например, детская позиция = ДВР+ДРВ), а так- же курса и пола студентов. Так, в формулах ДВР и ДРВ доминирует детская позиция в общении. Эта позиция наибо- лее характерна для студентов 2ЛФ. Кроме этого, у студентов 6 курса отсутствует формула ДРВ. Сравнительный анализ гендерных особенностей студентов 2ЛФ и 6ЛФ указывает на преобладание детской позиции у девушек обоих курсов. Более высокая представленность детской («зависимой») позиции у студентов 2ЛФ в срав- нении со студентами 6ЛФ может быть объяснена теорией поколений Уильяма Штрауса и Нейла Хауа (1991). Студенты исследуемой выборки от- носятся к поколению Z. Преобладание взрослой позиции в обще- нии характерно для формул ВДР и ВРД. Формула ВДР в наибольшей степени представлена у сту- дентов 6ЛФ, а формула ВРД – у студентов 2ЛФ. При анализе гендерных особенностей выявлены следующие особенности: 1) формула ВДР доми- нирует у юношей 2ЛФ и девушек 6ЛФ; 2) форму- ла ВРД преобладает у юношей обоих курсов. На младших курсах существенное влияние на поведение студентов оказывают внутренние личностные факторы, обусловленные особенно- стями личности молодых людей, относящихся к поколению Z. Важнейшей особенностью пред- ставителя этого поколения является отсутствие «авторитетов и каких-то четких ориентиров... Из-за этой неспособности чувствовать авторитет других людей представители этого поколения ча- сто ощущают себя потерянными и дезориенти- рованными. У представителей этого поколения востребованы своего рода «кураторы» – люди, на которых они возлагают ответственность за ре- зультат и которые используются как средство до- стижения целей» [5]. Отличительной особенностью формул РВД и РДВ является их отсутствие у студентов 6ЛФ. Что касается студентов 2ЛФ, то здесь отмечается преобладание РВД у юношей и РДВ – у девушек. При анализе представленности формул КП при общении студентов нами получены также три варианта количественного равенства двух формул. Так, вариант ДВР=ВДР чаще встречается у студентов 6ЛФ. При этом он преобладает у юно- шей 2ЛФ и девушек 6ЛФ. Примечание: 1) О – общее количество студентов; Ю – юноши; Д – девушки; 2) Д – детская позиция; В – взрослая позиция; Р – родительская позиция; 3) «=» – количественное равенство двух формул. Результаты и обсуждение Кроме этого, преобладанию детской по- зиции у студентов 2ЛФ способствует сложность образовательного процесса в медицинском уни- верситете. При этом высокая социальная значи- Вариант ВДР=ВРД также наиболее часто обнаруживается у студентов 6ЛФ. Кроме этого, Таблица – Коммуникативные позиции студентов 2 и 6 курсов лечебного факультета (%) Формула коммуникативной позиции Курсы 2ЛФ 6ЛФ О Ю Д О Ю Д ДВР 24,4 15,4 28,3 16,4 6,0 19,5 ДРВ 1,0 0,9 1,1 – – – ВДР 48,4 51,7 46,9 67,1 63,8 68,1 ВРД 20,5 24,8 18,6 10,3 24,2 6,2 РВД 1,3 1,8 1,1 – – – РДВ 1,0 0,9 1,1 – – – ДВР=ВДР 2,1 3,6 1,4 4,8 3,0 5,3 ВДР=ВРД 1,0 0,9 1,1 1,4 3,0 0,9 ДВР=ДРВ 0,3 – 0,4 – – – ица – Коммуникативные позиции студентов 2 и 6 курсов лечебного факультета (%) Примечание: 1) О – общее количество студентов; Ю – юноши; Д – девушки; 2) Д – детская позиция; В – взрослая позиция; Р – родительская позиция; 3) «=» – количественное равенство двух формул. 81 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 мнению, именно родительская позиция облада- ет наибольшей конфликтогенностью, что делает коммуникацию малоэффективной. мость врачебной профессии и не менее высокая требовательность к подготовке будущих врачей обусловливают доминирование «патерналист- ской» (покровительственной) модели взаимоот- ношений в диаде «преподаватель – студент» над моделью «сотрудничества». Полученные нами в результате анализа гендерные различия в представленности КП сту- дентов ЛФ с учетом пола и их динамики (более выраженный динамический рост формулы ВДР и снижение формулы ВРД у девушек в сравнении с юношами) подтверждают обнаруженные ранее нами гендерные особенности мотивационных ориентаций в межличностных коммуникациях. Доминирование взрослой позиции у сту- дентов 6ЛФ может быть обусловлено, прежде всего, изменением характера отношений между преподавателем и студентом: преподаватель рас- сматривает студента-старшекурсника (тем более студента выпускного курса) как «завтрашнего» врача и старается выстраивать с ним доверитель- ные отношения, используя диалогическое обще- ние в рамках модели «сотрудничества». У девушек была выявлена более выражен- ная следующая тенденция: «по шкалам «Ориен- тация на принятие партнера» и «Ориентация на адекватность восприятия и понимание партнера» преобладает высокий уровень, а по шкале «Ори- ентация на достижение компромисса» и общей гармоничности – средний уровень» [6]. Это, по нашему мнению, обеспечивает им более эффек- тивную КД. Результаты и обсуждение Это способствует тому, что на старших курсах «студент: 1) осознает и реализует цели, задачи, уста- новки образовательно-научно-профессионально- воспитательного процесса в ВГМУ; 2) овладевает основными процедурами и общей культурой интеллектуального труда, само- стоятельной деятельности; Выявленные общие и гендерные особен- ности КП в общении свидетельствуют о том, что созданная в ВГМУ информационная и образова- тельная среда в целом оказывает благоприятное влияние на формирование коммуникативных на- выков будущих врачей, что проявляется в гибком использовании студентами лечебного факультета КП в общении, адекватных требованиям образо- вательного процесса. 3) овладевает функциями своей социаль- ной роли, стремится к самообразованию, приоб- щению к профессии на основе разностороннего саморазвития личности в качестве будущего спе- циалиста-интеллигента; 4) целесообразно организует личный бюд- жет времени для успешной реализации системы функций своей социальной роли; Литература 3. Наименьшая представленность в изуча- емой выборке студентов родительской КП обу- словлена естественным «вытеснением» ее други- ми КП, так как она рассматривается студентами наименее эффективной при организации КД. 3. Наименьшая представленность в изуча- емой выборке студентов родительской КП обу- словлена естественным «вытеснением» ее други- ми КП, так как она рассматривается студентами наименее эффективной при организации КД. 1. Особенности коммуникативной толерантности студен- тов лечебного факультета ВГМУ / А. Л. Церковский [и др.] // Вестн. ВГМУ. – 2021. – Т. 20, № 6. – С. 110–117. 2. О подготовке к коммуникативной деятельности студен- тов ВГМУ / А. Л. Церковский [и др.] // Вестн. фармации. – 2020. – № 4. – С. 100–104. 4. Более выраженный динамический рост формулы ВДР и снижение формулы ВРД у деву- шек в сравнении с юношами подтверждают об- наруженные ранее нами гендерные особенности мотивационных ориентаций в межличностных коммуникациях, которые обеспечивают девуш- кам более эффективную КД. 3. Берн, Э. Трансактный анализ / Э. Берн. – Москва : Ака- дем. проект : Трикста, 2004. – 187 с. 4. Ильин, Е. П. Психология общения и межличностных от- ношений / Е. П. Ильин. – Санкт-Петербург : Питер, 2009. – 576 с. 5. Курпатов, А. В. Счастливый ребенок. Универсальные правила / А. В. Курпатов. – Санкт-Петербург : ИД КА- ПИТАЛ, 2019. – 350 с. 5. Выявленные общие и гендерные особен- ности КП в общении свидетельствуют о том, что созданная в ВГМУ информационная и образова- тельная среда в целом оказывает благоприятное влияние на формирование коммуникативных на- выков будущих врачей, что проявляется в гибком использовании студентами лечебного факультета КП в общении, адекватных требованиям образо- вательного процесса. 6. Об этапах формирования конкурентоспособности вы- пускника ВГМУ / А. Л. Церковский [и др.] // Достижения фундаментальной, клинической медицины и фармации [Электронный ресурс] : материалы 75-й науч. сес. ВГМУ (29-30 янв. 2020 г.) / М-во здравоохранения Республики Беларусь, УО «Витебский гос. ордена Дружбы народов мед. ун-т». – Витебск : ВГМУ, 2020. – С. 500–501. – 1 электрон. опт. диск (CD-ROM). – Загл. с этикетки диска. 7. О мотивационных ориентациях в межличностных ком- муникациях студентов-шестикурсников / С. А. Петрович [и др.] // Достижения фундаментальной, клинической ме- дицины и фармации [Электронный ресурс] : материалы 76-й науч. сес. ВГМУ, 28-29 янв. 2021 г., Витебск / М-во здравоохранения Республики Беларусь, УО «Витебский гос. ордена Дружбы народов мед. ун-т» ; [под ред. А. Т. Щастного]. – Витебск : ВГМУ, 2021. – С. 378–379. – 1 электрон. опт. диск (CD-ROM). – Загл. с этикетки диска. Заключение 5) достигает высокой академической успе- ваемости; 1. Более высокая представленность детской («зависимой») позиции у студентов 2ЛФ можно объяснить личностными особенностями поколе- ния Z, сложностью образовательного процесса в медицинском университете, а также доминирова- нием «патерналистской» (покровительственной) модели взаимоотношений в диаде «преподава- тель – студент» над моделью «сотрудничества». 6) достигает в учебе чувства удовлетворен- ности; 7) развивает оптимистические представ- ления о возможностях успешного овладения из- бранной профессией» [6]. Доминирование взрослой КП у студентов 6ЛФ обусловлено также коммуникативными ори- ентациями «на принятие партнера, на адекват- ность восприятия и понимание его, на достиже- ние компромисса в общении с ним…» [7]. Доминирование взрослой КП у студентов 6ЛФ обусловлено также коммуникативными ори- ентациями «на принятие партнера, на адекват- ность восприятия и понимание его, на достиже- ние компромисса в общении с ним…» [7]. 2. Доминирование взрослой позиции у сту- дентов 6ЛФ может быть обусловлено: 1) измене- нием характера отношений между преподавателем и студентом, при котором преподаватель рассма- тривает студента-старшекурсника (тем более сту- дента выпускного курса) как «завтрашнего» вра- ча и старается выстраивать с ним доверительные отношения, используя диалогическое общение в рамках модели «сотрудничества»; 2) коммуника- тивными ориентациями «на принятие партнера, на адекватность восприятия и понимание его, на достижение компромисса в общении с ним…». Наименьшая представленность в изучае- мой выборке студентов родительской КП находит свое подтверждение в отсутствии этой позиции среди трех полученных количественных равенств двух формул. По нашему мнению, родительская КП есте- ственным образом «вытесняется» другими КП, так как рассматривается студентами наименее эффективной при организации КД. По нашему 82 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Литература 6. Результаты исследования могут быть ис- пользованы в образовательном процессе ВГМУ, деятельности социально-педагогической и пси- хологической службы, работе кураторов акаде- мических групп и тьюторов. Поступила 31.01.2022 г. Принята в печать 21.04.2022 г. Поступила 31.01.2022 г. Принята в печать 21.04.2022 г. References Skorikova EA, Vozmitel II. On the stages of competitiveness formation of a graduate of the Higher Medical School. V: M-vo zdravookhraneniia Respubliki Belarus', UO "Vitebskii gos ordena Druzhby narodov med un-t". Dostizheniia fundamental'noi, klinicheskoi meditsiny i farmatsii [Elektronnyi resurs]: materialy 75-i nauch ses VGMU (29- 30 ianv 2020 g.). Vitebsk, RB: VGMU; 2020. R. 500-1. 1 elektron opt disk (CD-ROM). Zagl s etiketki diska. (In Russ.) Petrovich SA, Tcerkovskii AL, Gapova OI, Kasian OA, Vozmitel II, Skorikova EA. On motivational orientations in interpersonal communications of sixth-year students. V: M-vo zdravookhraneniia Respubliki Belarus', UO "Vitebskii gos ordena Druzhby narodov med un-t", Shchastnyi AT, red. Dostizheniia fundamental'noi, klinicheskoi meditsiny i farmatsii [Elektronnyi resurs]: materialy 76-i nauch ses VGMU, 28-29 ianv 2021 g, Vitebsk. Vitebsk, RB: VGMU; 2021. R. 378-9. 1 elektron opt disk (CD-ROM). Zagl s etiketki diska. (In Russ.) Skorikova EA, Vozmitel II. On the stages of competitiveness formation of a graduate of the Higher Medical School. V: M-vo zdravookhraneniia Respubliki Belarus', UO "Vitebskii gos ordena Druzhby narodov med un-t". Dostizheniia fundamental'noi, klinicheskoi meditsiny i farmatsii [Elektronnyi resurs]: materialy 75-i nauch ses VGMU (29- 30 ianv 2020 g.). Vitebsk, RB: VGMU; 2020. R. 500-1. 1 elektron opt disk (CD-ROM). Zagl s etiketki diska. (In Russ.) 1. Tcerkovskii AL, Gapova OI, Skorikova EA, Petrovich SA, Kasian OA, Muzhichenko VA. Features of communicative tolerance of students of the medical faculty of VSMU. Vestn VGMU. 2021;20(6):110-7. (In Russ.) 2. Tcerkovskii AL, Skorikova EA, Gapova OI, Petrovich SA, Vozmitel II, Kasian OA. O About preparation for communicative activity of VSMU students. Vestn Farmatsii. 2020;(4):100-4. (In Russ.) 7. 3. Bern E. Transactional Analysis. Moscow, RF: Akadem proekt: Triksta; 2004. 187 р. (In Russ.) 4. Ilin EP. Psychology of communication and interpersonal relationships. Saint Petersburg, RF: Piter; 2009. 576 р. (In Russ.) 5. Kurpatov AV. The Happy Child. Universal rules. Saint Petersburg, RF: ID KAPITAL; 2019. 350 р. (In Russ.) 6. Tcerkovskii AL, Gapova OI, Petrovich SA, Kasian OA, Submitted 31.01.2022 Accepted 21.04.2022 Submitted 31.01.2022 Accepted 21.04.2022 Submitted 31.01.2022 Accepted 21.04.2022 83 ESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Сведения об авторах: Церковский А.Л. – к.м.н., доцент, заведующий кафедрой психологии и педагогики с курсом ФПК и ПК, Витеб- ский государственный ордена Дружбы народов медицинский университет; Церковский А.Л. – к.м.н., доцент, заведующий кафедрой психологии и педагогики с курсом ФПК и ПК, Витеб- ский государственный ордена Дружбы народов медицинский университет; Гапова О.И. – старший преподаватель кафедры психологии и педагогики с курсом ФПК и ПК, Витебский государ- ственный ордена Дружбы народов медицинский университет; Скорикова Е.А. – старший преподаватель кафедры психологии и педагогики с курсом ФПК и ПК, Витебский го- сударственный ордена Дружбы народов медицинский университет; Петрович С.А. – старший преподаватель кафедры психологии и педагогики с курсом ФПК и ПК, Витебский госу- дарственный ордена Дружбы народов медицинский университет. р ру р у р тарший преподаватель кафедры психологии и педагогики с курсом ФПК и ПК, Витебский государ- на Дружбы народов медицинский университет; ственный ордена Дружбы народов медицинский университет; Скорикова Е.А. – старший преподаватель кафедры психологии и педагогики с курсом ФПК и ПК, Витебский го- сударственный ордена Дружбы народов медицинский университет; р ру р у р Скорикова Е.А. – старший преподаватель кафедры психологии и педагогики с курсом ФПК и ПК, Витебский го- сударственный ордена Дружбы народов медицинский университет; у р р ру р у р Петрович С.А. – старший преподаватель кафедры психологии и педагогики с курсом ФПК и ПК, Витебский госу- дарственный ордена Дружбы народов медицинский университет. Петрович С.А. – старший преподаватель кафедры психологии и педагогики с курсом ФПК и ПК, Витебский госу- дарственный ордена Дружбы народов медицинский университет. Резюме. езю е. Цель работы состоит в оценке возможностей повышения качества преподавания клинической дисциплины на ан- глийском языке путем разработки учебно-методического комплекса (УМК) для студентов 6 курса факультета под- готовки иностранных граждан по дисциплине «Доказательная и персонализированная медицина. Доказательная база диагностики и лечения COVID-19». В статье обсуждаются теоретические и практические аспекты разработ- ки УМК для методического обеспечения учебного процесса на английском языке. «Доказательная и персонали- зированная медицина» является новой дисциплиной, преподаваемой в университете. Авторы статьи представили свой оригинальный опыт создания системы научно-методического обеспечения с учетом отсутствия стандарти- зированного подхода к разработке УМК клинической дисциплины медицинского университета на английском языке. Актуальность изучения вопросов доказательной и персонализированной медицины, в том числе касаю- щихся диагностики, лечения и профилактики новой коронавирусной инфекции, определяется современными по- требностями системы здравоохранения и медицинского образования. В статье определено место дисциплины в системе клинических дисциплин, преподаваемых на английском языке, представлены основные структурные компоненты и содержание УМК. Авторами статьи предложено применение элементов технологии проблемного обучения с использованием метода case-study при проведении практических занятий в рамках изучения дисци- плины, в том числе в режиме on-line, а также определена роль тестовых заданий в системе контроля знаний. Ключевые слова: педагогика высшей школы, качество образования, учебно-методическое обеспечение, доказа- тельная медицина. а: педагогика высшей школы, качество образования, учебно-методическое обеспечение, доказа- ина. HALIUCHENKA V.A., ZHYLTSOU I.V., SKREBLO Y.I., ADAMENKO G.P., KALIADKA Y.I. Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus Vestnik VGMU. 2022;21(2):85-93. ВЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 DOI: https://doi.org/10.22263/2312-4156.2022.2.85 РАЗРАБОТКА УЧЕБНО-МЕТОДИЧЕСКОГО КОМПЛЕКСА ПО УЧЕБНОЙ ДИСЦИПЛИНЕ «ДОКАЗАТЕЛЬНАЯ И ПЕРСОНАЛИЗИРОВАННАЯ МЕДИЦИНА. ДОКАЗАТЕЛЬНАЯ БАЗА ДИАГНОСТИКИ И ЛЕЧЕНИЯ COVID-19» (НА АНГЛИЙСКОМ ЯЗЫКЕ) ДЛЯ СТУДЕНТОВ МЕДИЦИНСКОГО УНИВЕРСИТЕТА ГОЛЮЧЕНКО О.А., ЖИЛЬЦОВ И.В., СКРЕБЛО Е.И., АДАМЕНКО Г.П., КОЛЯДКО Е.И. Витебский государственный ордена Дружбы народов медицинский университет, г. Витебск, Республика Беларусь Вестник ВГМУ. – 2022. – Том 21, №2. – С. 85-93. THE DEVELOPMENT OF EDUCATIONAL AND METHODOLOGICAL COMPLEX IN THE DISCIPLINE «EVIDENCE-BASED AND PERSONALIZED MEDICINE. EVIDENCE BASE FOR DIAGNOSING AND TREATMENT OF COVID-19» (IN THE ENGLISH LANGUAGE) FOR THE STUDENTS OF THE MEDICAL UNIVERSITY HALIUCHENKA V.A., ZHYLTSOU I.V., SKREBLO Y.I., ADAMENKO G.P., KALIADKA Y.I. Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus Vestnik VGMU. 2022;21(2):85-93. ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Information about authors: f Tserkovsky A.L. – Candidate of Medical Sciences, associate professor, head of the Chair of Psychology and Pedagogics with the course of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Tserkovsky A.L. – Candidate of Medical Sciences, associate professor, head of the Chair of Psychology and Pedagogics with the course of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Gapova O.I. – senior lecturer of the Chair of Psychology and Pedagogics with the course of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Skorikova E.A. – senior lecturer of the Chair of Psychology and Pedagogics with the course of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Petrovich S.A. – senior lecturer of the Chair of Psychology and Pedagogics with the course of the Faculty for Advanced Training & Retraining Vitebsk State Order of Peoples’Friendship Medical University g g f p p y Petrovich S.A. – senior lecturer of the Chair of Psychology and Pedagogics with the course of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University. Адрес для корреспонденции: Республика Беларусь, 210009, г. Витебск, пр. Фрунзе, 27, Витебский государствен- ный ордена Дружбы народов медицинский университет, кафедра психологии и педагогики с курсом ФПК и ПК. E-mail: Tserkovsky.vsmu@gmail.com – Церковский Александр Леонидович. Адрес для корреспонденции: Республика Беларусь, 210009, г. Витебск, пр. Фрунзе, 27, Витебский государствен- ный ордена Дружбы народов медицинский университет, кафедра психологии и педагогики с курсом ФПК и ПК. E-mail: Tserkovsky.vsmu@gmail.com – Церковский Александр Леонидович. Correspondence address: Republic of Belarus, 210009, Vitebsk, 27 Frunze ave., Vitebsk State Order of Peoples’ Friendship Medical University, Chair of Psychology and Pedagogics with the course of the Faculty for Advanced Training & Retraining. E-mail: Tserkovsky.vsmu@gmail.com – Alexander L. Tserkovsky. Correspondence address: Republic of Belarus, 210009, Vitebsk, 27 Frunze ave., Vitebsk State Order of Peoples’ Friendship Medical University, Chair of Psychology and Pedagogics with the course of the Faculty for Advanced Training & Retraining. E-mail: Tserkovsky.vsmu@gmail.com – Alexander L. Tserkovsky. 84 Abstract. The aim of this article is to assess the possibilities of improving the quality of teaching a clinical discipline in English by developing an educational and methodological complex (EMC) for the 6th-year students of the Overseas Students Training Faculty in the discipline «Evidence-Based and Personalized Medicine. Evidence base for diagnosing and treatment of COVID-19». The theoretical and practical aspects of the teaching materials development for the methodological support of the educational process in English are discussesed in this article. «Evidence-Based and Personalized Medicine» is a new discipline taught at the university. The authors of the article have presented their original experience in creating a system 85 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 of scientific and methodological support, taking into account the lack of a standardized approach to the development of teaching materials for the clinical discipline in English. The relevance of studying the issues of evidence-based and personalized medicine, including those related to the diagnosis, treatment and prevention of a new coronavirus infection, is determined by the modern needs of the healthcare system and medical education. The article defines the place of the discipline in the system of clinical disciplines taught in English, presents the main structural components and content of EMC. The authors have proposed to apply problem-based learning technology elements using the case-study method while conducting practical classes (including on-line ones), and they have also determined the role of MCQ in the knowledge control system. Key words: pedagogy of higher education, quality of education, educational and methodological support, evidence-based medicine. которые способствовали бы эффективному про- фессиональному развитию студента. В условиях расширения экспорта образо- вательных услуг в медицинских вузах Республи- ки Беларусь сохраняет актуальность проблема повышения качества подготовки иностранных студентов, одним из решений которой является преподавание учебных дисциплин на английском языке. В связи с этим возникает необходимость в использовании специальных инструментов на- учно-методического обеспечения учебного про- цесса, важнейшим из которых является учеб- но-методический комплекс (УМК). На кафедре доказательной медицины и клинической диагно- стики ФПК и ПК Витебского государственного медицинского университета (ВГМУ) впервые разработан УМК на английском языке для мето- дического сопровождения новой учебной дисци- плины: «Доказательная и персонализированная медицина. Доказательная база диагностики и ле- чения COVID-19». Данная работа посвящена тео- ретическим и практическим аспектам разработки УМК для методического обеспечения учебного процесса на английском языке. Abstract. В научной литературе описаны различные подходы к разработке УМК учебных дисциплин, однако на текущий момент не существует стан- дартизированного подхода к разработке УМК клинической дисциплины медицинского универ- ситета на английском языке. Теоретические аспекты разработки учебно-методического комплекса по професси- ональной учебной дисциплине на английском языке Комплексное методическое обеспечение образовательного процесса на английском языке – это система информационного, методического и материально-технического обеспечения обра- зовательного процесса, направленная на подго- товку специалиста, отвечающего требованиям, установленным образовательным стандартом специальности высшего образования. Инстру- ментом системного методического обеспечения образовательного процесса является УМК [1]. Актуальность работы определяется уни- кальностью и новизной учебной дисциплины (УО «ВГМУ» является первым медицинским универ- ситетом в Республике Беларусь, в учебный план которого включена доказательная и персонализи- рованная медицина как учебная дисциплина для изучения на русском и английском языках), а так- же наличием особенностей преподавания клини- ческих дисциплин англоговорящим студентам. Использование языка-посредника при соз- дании УМК позволяет более полно реализовать его функцию инструмента системно-методиче- ского обеспечения образовательного процесса, объединяющего в единое целое различные ди- дактические средства обучения, подчиняя их целям обучения и обеспечения самостоятельной работы англоговорящих студентов. Такой УМК более полно фиксирует и раскрывает требования к содержанию изучаемой дисциплины, умениям, навыкам и компетенциям подготовки выпускни- ков, которые установлены образовательным стан- дартом. Создание оптимального комплекса учеб- но-методического обеспечения образовательного процесса для иностранных студентов на англий- ском языке – весьма сложная и трудоемкая зада- ча. Такой подход определяет для современного педагога необходимость владения навыками на- учно-исследовательской и научно-методической работы, разработки средств и методов обучения, Кроме того, возможность использовать в процессе обучения учебно-методические мате- риалы на английском языке стимулирует обуча- 86 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 ющихся к саморегуляции учебно-познавательной деятельности, активизирует их общепрофессио- нальные и специальные (предметные) умения и навыки, повышает роль самостоятельной работы при подготовке к занятиям, создает условия для успешной сдачи экзамена или зачета по дисци- плине [2]. позволяющих отличать эффективные диагно- стические и лечебные вмешательства от неэф- фективных; методах анализа индивидуальных особенностей пациента с целью подбора тера- пии, наилучшим образом подходящей для него, а также с целью разработки персонализированных лекарственных средств, предназначенных для ле- чения конкретной формы заболевания данного пациента, базирующихся на новейших достиже- ниях протеомики, геномики, молекулярной гене- тики и генетической инженерии. В соответствии с Кодексом Республики Беларусь об образовании и Постановлением Ми- нистерства образования Республики Беларусь от 26.07.2011 №167, а также согласно Положению об учебно-методическом комплексе, определен по- рядок создания и структура УМК. УМК для пре- подавания учебной дисциплины в учреждении высшего медицинского образования разрабаты- вается авторским коллективом преподавателей, ведущих занятия по данной дисциплине в стро- гом соответствии с характеристиками, отражён- ными в учебном плане (название, трудоёмкость, семестры, формы учебной работы, виды кон- трольных мероприятий и т.д.). Теоретические аспекты разработки учебно-методического комплекса по професси- ональной учебной дисциплине на английском языке Содержание учеб- ной программы по учебной дисциплине должно соответствовать требованиям министерства об- разования Республики Беларусь к обязательному минимуму содержания дисциплины и отражать все дидактические единицы, представленные в государственном образовательном стандарте по специальности/направлению подготовки, а ло- гика и порядок их представления могут варьиро- вать. При этом содержание программы должно опираться на современные достижения науки, образовательной практики и реализовывать ав- торский подход к объекту изучения. Изучение англоязычными студентами учебной дисциплины «Доказательная и персо- нализированная медицина. Доказательная база диагностики и лечения COVID-19» определяется необходимостью целенаправленного овладения знаниями о методах объективной оценки клини- ческой эффективности лекарственных средств, лечебных и диагностических вмешательствах, а также о подходах к повышению эффективности терапии различных заболеваний путем исполь- зования различных вариантов ее индивидуали- зации. Будущему врачу важно овладеть умением использовать различные источники доказатель- ной медицинской информации для принятия решений о выборе оптимальной стратегии диа- гностики и лечения, что чрезвычайно актуально для практикующего врача любой специальности [4]. Помимо этого, в условиях продолжающейся пандемии COVID-19 медицинские специалисты должны быть в курсе последних достижений ме- дицинской науки и использовать в ходе диагно- стики, лечения и профилактики данного заболе- вания средства и методы с объективно доказанной эффективностью, а также уметь самостоятельно находить публикации о результатах клинических исследований, доказывающих либо опровергаю- щих эффективность и безопасность очередных предлагаемых вмешательств, и оценивать их ме- тодологическое качество. Компонентами УМК являются средства нормативного обеспечения, средства учебно- методического обеспечения, средства обучения, средства текущей и итоговой аттестации обучаю- щихся [3]. Только соблюдая вышеперечисленные принципы, можно создавать высокоэффективный УМК, позволяющий сформировать необходимые профессиональные компетенции. Кроме того, процесс подготовки компе- тентного врача на уровне базового медицинского образования предполагает создание у специали- ста основы для дальнейшего профессионального развития в любой области медицины, а также в области научных исследований в медицине. Пре- подавание с использованием научного метода, в том числе доказательной медицины, опреде- ляется в качестве обязательной составляющей процесса обучения в медицинской организации образования с точки зрения международной си- стемы аккредитации [5]. Учебная дисциплина «Доказательная и персонализированная медицина. Доказатель- ная база диагностики и лечения COVID-19» в системе клинических дисциплин, преподавае- мых на английском языке «Доказательная и персонализированная медицина. Доказательная база диагностики и лечения COVID-19» – учебная дисциплина, со- держащая систематизированные научные знания о строгих объективных критериях и методах, Учебная дисциплина «Доказательная и персонализированная медицина. Доказатель- ная база диагностики и лечения COVID-19» в системе клинических дисциплин, преподавае- мых на английском языке «Доказательная и персонализированная медицина. Теоретические аспекты разработки учебно-методического комплекса по професси- ональной учебной дисциплине на английском языке Доказательная база диагностики и лечения COVID-19» – учебная дисциплина, со- держащая систематизированные научные знания о строгих объективных критериях и методах, 87 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Основные структурные компоненты и содержание УМК по дисциплине «Доказа- тельная и персонализированная медицина. Доказательная база диагностики и лечения COVID-19» на английском языке УМК содержит такие разделы, как «норма- тивные документы», «теоретический», «практи- ческий», «блок контроля знаний», «справочные и вспомогательные материалы». В соответствии с положением об УМК раздел «Нормативные до- кументы» включает в себя следующие элементы: пояснительную записку, учебный план, учебно- тематический план, учебную программу, распи- сание занятий и график отработок пропущенных занятий. УМК по учебной дисциплине «Доказатель- ная и персонализированная медицина. Доказа- тельная база диагностики и лечения COVID-19» для студентов 6 курса факультета подготовки иностранных граждан (ФПИГ) на английском языке разработан в соответствии с образователь- ным стандартом высшего медицинского обра- зования и учебной программой данной учебной дисциплины. В пояснительной записке отражены цели УМК, сущность изучения дисциплины, реализуе- мые межпредметные связи, указаны особенности структурирования учебного материала, требова- ния к знаниям, умениям и навыкам студентов ме- дицинского вуза после изучения дисциплины, а также определено количество часов, выделенных на изучение дисциплины с учетом самостоятель- ной работы, в соответствии с учебным планом, определена форма контроля знаний (зачет). Структура УМК соответствует нормам, определенным Положением об учебно-методиче- ском комплексе для научно-методического обе- спечения преподавания дисциплины при получе- нии высшего образования и представляет собой единую учебно-методическую базу, состоящую из систематизированных учебных и методических материалов, а также дидактических средств обуче- ния, переведенных на английский язык. План учебной дисциплины включает на- звание разделов, содержание изучаемых вопро- сов, а также объем отведенного учебного време- ни (табл. 1). Учебно-методические материалы на ан- глийском языке, входящие в УМК, полностью соответствуют содержанию аналогичного УМК на русском языке. Лексико-грамматическая экс- пертиза, выполненная специалистами Отдела международных связей УО «Витебский государ- ственный медицинский университет», показала высокое качество перевода текста входящих в УМК документов на английский язык. Календарно-тематический план по дисци- плине «Доказательная и персонализированная медицина. Доказательная база диагностики и лечения COVID-19» для студентов с английским языком обучения, расписание занятий, графики отработок утверждаются заведующим кафедрой и являются ежегодно обновляемыми норматив- ными материалами. у Структура и содержание основных разде- лов УМК «Доказательная и персонализирован- ная медицина. Доказательная база диагностики и лечения COVID-19» получили положительные отзывы рецензентов как отражающие совре- менный уровень развития медицинской науки и ориентирующие на использование активных форм и методов обучения, современных инфор- мационно-коммуникативных технологий. Теоретические аспекты разработки учебно-методического комплекса по професси- ональной учебной дисциплине на английском языке УМК рекомендован к использованию для обеспечения учебного процесса при получении высшего обра- зования 1 ступени студентов ФПИГ УО «ВГМУ» (с английским языком обучения). Теоретический раздел УМК содержит те- оретические материалы, раскрывающие основ- ные понятия по теме занятий, касающиеся дока- зательной и персонализированной медицины, а также доказательной базы диагностики, профи- лактики и лечения COVID-19. Материалы теоретического раздела пред- ставлены в виде презентаций Power Point, содер- жащих по 80-100 слайдов, в которых раскрыты вопросы, соответствующие учебной программе и методическим рекомендациям для подготовки к занятиям. Презентации подготовлены и обновля- ются преподавателями кафедры, имеющими вы- сокий уровень квалификации и аттестованными для ведения занятий со студентами на английском языке. Содержание презентаций соответствует материалам, предлагаемым к изучению на рус- ском языке. Объем и содержание теоретического материала обсуждаются на заседаниях кафедры и корректируется с участием всех преподавателей Содержание учебно-методического ком- плекса по учебной дисциплине «Доказательная и персонализированная медицина. Доказатель- ная база диагностики и лечения COVID-19» для студентов медицинского университета с английским языком обучения 88 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Таблица 1 – Распределение тем, включенных в план изучения дисциплины «Доказательная и пер- сонализированная медицина. Доказательная база диагностики и лечения COVID-19» № п/п Name of the section, theme In total Audience classes Independent work Lectures Practical classes Lab. classes 1. 1. Basics of evidence-based medicine 1.1. Evidence-based medicine: basic deﬁ nitions and principles 1.2. Sources of evidence-based medical information and work with them. Evaluation of the quality of scientiﬁ c and medical publications in accor- dance with the principles of evidence-based medicine 2. Eﬀ ectiveness of drugs and therapeutic interventions from the stand- point of evidence-based medicine 2.1. Clinical trials 2.2. Treatment methods and drugs with unproven eﬃ cacy from the point of view of evidence-based medicine 2.3. Medical advertising and evidence-based medicine 3. Basics of personalized medicine 3.1. Development and application of clinical guidelines 3.2. Principles of creation and applying of personalized medicines 7 7 2. 4. Evidence base for the diagnosis and treatment of COVID-19 4.1. Modern data about the origin and structure of SARS-CoV-2 4.2. Current views on the epidemiology of COVID-19 4.3. Current views on the pathogenesis of COVID-19 4.4. The clinical picture of COVID-19 and its complications 4.5. Principles of laboratory and instrumental diagnostics of COVID-19 4.6. Eﬀ ective treatments for COVID-19, their evidence base 4.7. Теоретические аспекты разработки учебно-методического комплекса по професси- ональной учебной дисциплине на английском языке К преимуществам метода case-study можно отнести получение навыков решения реальных проблем с использованием принципов проблемного обучения. При этом про- цесс обучения имитирует механизм принятия ре- шения, он более адекватен жизненной ситуации, поскольку требует не только знания и понимания терминов, но и умения оперировать ими, выстра- ивая логические схемы решения проблемы, аргу- ментировать свое мнение. Обучение посредством метода case-study направлено в большей степени на формирование продуктивного клинического мышления будущего врача, его интеллектуально- го и коммуникативного потенциала. Explain the administration scheme. Ground your opinion. 3. A 48-old man without any chronic diseases seeks medical attention of a physician complaining of general malaise, febrile fever, dry cough, and dyspnea. These symptoms are present for 6 days. Patient’s wife is sick with COVID-19 pneumonia. Using evidence-based recommendations propose a plan of diagnosing, treatment and complications prevention. Support your opinion with scientiﬁ c data. В процессе case-study под руководством преподавателя студенты систематизируют, обоб- щают изученный материал, развивают умение выделять главное, устанавливать причинно-след- ственные связи между отдельными элементами содержания, приобретают навыки эффективного поиска достоверной медицинской информации с учетом уровня доказательности и ее применения в практической деятельности. Во время практических занятий студен- там предлагается решить ряд предложенных за- даний, касающихся правил поиска медицинской информации об эффективности медицинских вмешательств в сети Интернет с учетом уровня доказательности, а также посвященных состав- лению индивидуального плана диагностики, ле- чения и профилактики COVID-19 у пациентов в различных клинических ситуациях. В зависи- мости от уровня подготовки студентов и уровня коммуникации студентов в группе предлагается индивидуальное выполнение заданий или работа в малых группах. После составления ответа сту- дентам предлагается обсудить результат и сде- лать итоговые выводы об эффективности лекар- ственных средств и медицинских вмешательств. При выполнении данного задания используются технические средства обучения (компьютер, под- ключенный к сети Интернет, индивидуальные гаджеты, мультимедийный экран). Использование элементов случай-ориенти- рованного обучения в виде решения клинических ситуационных заданий с последующим составле- нием ответов на вопросы или в виде выводов с обоснованием возможно также в рамках дистан- ционного обучения и в процессе самоподготовки студентов [9]. Подготовка занятия посредством кейс- метода является весьма трудоемким процессом, требующим больших затрат времени и продук- тивного интеллектуального труда преподавателя. Однако его применение позволяет реализовывать принципы проблемного обучения, активизиро- вать мыслительную деятельность обучающихся, актуализировать познавательную активность и познавательную самостоятельность, «погрузить» их в атмосферу самостоятельного поиска знаний. Теоретические аспекты разработки учебно-методического комплекса по професси- ональной учебной дисциплине на английском языке Eﬀ ective methods of prevention of COVID-19, their evidence base 7 7 In total: 14 14 а 1 – Распределение тем, включенных в план изучения дисциплины «Доказательная и пер- нная медицина. Доказательная база диагностики и лечения COVID-19» кафедры. Предлагаемые к изучению материалы раскрывают основные принципы доказательной и персонализированной медицины, касаются во- просов методологии организации клинических исследований и критической оценки представ- ляемых результатов с учетом дизайна и качества исследований. Так, к изучению предлагаются результаты исследований, оценивающих эффек- тивность различных терапевтических подходов и лекарственных средств, применяемых для ле- чения COVID-19 (IVERCOR-COVID19, Adaptive COVID-19 Treatment Trial, SIMPLE, RECOVERY и др.), а также систематические обзоры и мета- анализы. В процессе обсуждения на занятиях затрагиваются вопросы ограничений и проблем использования результатов обширных много- центровых исследований, а также вопросы воз- можности использования результатов новых ис- следований в клинической практике после их включения в национальные рекомендации по лечению COVID-19. Вопросы диагностики, ле- кафедры. Предлагаемые к изучению материалы раскрывают основные принципы доказательной и персонализированной медицины, касаются во- просов методологии организации клинических исследований и критической оценки представ- ляемых результатов с учетом дизайна и качества исследований. Так, к изучению предлагаются результаты исследований, оценивающих эффек- тивность различных терапевтических подходов и лекарственных средств, применяемых для ле- чения COVID-19 (IVERCOR-COVID19, Adaptive COVID-19 Treatment Trial, SIMPLE, RECOVERY и др.), а также систематические обзоры и мета- анализы. В процессе обсуждения на занятиях затрагиваются вопросы ограничений и проблем использования результатов обширных много- центровых исследований, а также вопросы воз- можности использования результатов новых ис- следований в клинической практике после их включения в национальные рекомендации по лечению COVID-19. Вопросы диагностики, ле- чения и профилактики COVID-19 обсуждаются с учетом современных международных рекоменда- ций [6, 7]. Преимуществом представления учебного материала в виде презентаций является нагляд- ность и структурированность, а также возмож- ность его быстрой актуализации и коррекции. В практическом разделе представлены ме- тодические указания для подготовки к занятиям, перечень практических навыков, которыми дол- жен овладеть обучающийся при изучении учеб- ной дисциплины, а также задания для организа- ции случай-ориентированного обучения. Для решения одной из главных задач – фор- мирование практических навыков и умений у сту- дентов медицинского вуза – высокорезультатив- ным методом обучения является метод case-study. Суть этого метода заключается в осмыслении, критическом анализе и практическом решении конкретных проблем или случаев. В методологи- ческом контексте, в рамках проблемного обуче- 89 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 ния, кейс-метод можно представить как сложную систему, в которую интегрированы различные методы познания [8]. Примеры тестовых заданий по разделу «Доказательная медицина» на английском языке Примеры тестовых заданий по разделу «Доказательная медицина» на английском языке «Доказательная медицина» на англи Choose the correct answer Choose the correct answer 1) Which of the following does the evidence- based medicine (EBM) include? A. best external evidence Раздел справочных и вспомогательных ма- териалов УМК содержит список рекомендуемой литературы, которая имеется в библиотеке уч- реждения образования «Витебский государствен- ный медицинский университет». Также предло- жены электронные ресурсы, которые могут быть полезны и интересны студентам при подготовке к занятиям и при разработке проблемы в case-study (Cochrane Library, PubMed, WHO и др.). Одним из литературных источников, рекомендованных к прочтению, является монография по органи- зации, выполнению и статистической обработке результатов клинических исследований, одним из соавторов которой является заведующий ка- федрой доказательной медицины и клинической диагностики ФПК и ПК, д.м.н. И.В. Жильцов. B. individual clinical experience C. patient values +D. all of the above +D. all of the above 2) What is the highest level of evidence that can be used in making clinical decisions? A. case studies + B. randomized controlled trials with high power C. randomized controlled trials with low power D. non-randomized concurrent cohort comparisons between contemporaneous patients 3) Which of the following data bases is a collection of systematic reviews? A. CINAHL B. PubMed A. CINAHL A. CINAHL B PubMed B. PubMed + C. Cochrane D. SPORTDiscus 4) Which of the following statements oﬀ ers the best rationale for selecting the treatment for a patient? Заключение Современное медицинское образование невозможно без применения инновационных подходов, направленных на формирование про- блемно-исследовательского учебного процес- са, обеспечивающего эффективность познава- тельной деятельности, выработку комплексного клинического стиля мышления. УМК позволяет сконцентрировать необходимые методические, информационные и дидактические ресурсы для профессиональной подготовки иностранных граждан, удовлетворяющей конечным результа- там обучения и требованиям системы медицин- ского обеспечения в современном обществе. + A. Treatment theory is unknown but clinical beneﬁ ts have been demonstrated in the literature B. Treatment theory is known but clinical beneﬁ ts have not been demonstrated in the literature C. Treatment theory is unknown and clinical beneﬁ ts have not been demonstrated in the literature 5) The ideal diagnostic test has a _ 5) The ideal diagnostic test has a ________ + A. high sensitivity and high speciﬁ city + A. high sensitivity and high speciﬁ city B. high sensitivity and low speciﬁ city C. low sensitivity and high speciﬁ city D. low sensitivity and low speciﬁ city Тестирование как одна из форм аттестации представляет собой процедуру, позволяющую объективно установить уровень учебных дости- жений студентов, и является одной из наиболее технологичных форм проведения автоматизиро- ванного контроля с управляемыми параметра- ми качества. В настоящее время тестирование широко применяется на всех этапах дидактиче- ского процесса. С его помощью можно эффек- тивно обеспечить предварительный, текущий и итоговый контроль знаний, учет успеваемости и академических достижений. Актуальность ис- Теоретические аспекты разработки учебно-методического комплекса по професси- ональной учебной дисциплине на английском языке Метод case-study обеспечивает оптимизацию взаимодействия, сотрудничества и сотворчества преподавателя и студента, а также позволяет ре- ализовать один из значимых методологических принципов обучения – единство теории и прак- тики [8, 10, 11]. Сложность предлагаемых клинических си- туаций варьирует в зависимости от личностных качеств студентов, их профессиональных интере- сов и предпочтений. Ниже представлены примеры заданий в рамках метода case-study, используемых при про- ведении практических занятий по дисциплине: В разделе контроля знаний УМК представ- лены материалы для проведения текущей и ито- говой аттестации по дисциплине «Доказательная и персонализированная медицина. Доказательная база диагностики и лечения COVID-19». Пред- ложены контрольные вопросы для текущей и итоговой аттестации, а также тестовые задания, обеспечивающие возможность самоконтроля об- учающихся. Представленные вопросы состав- лены в соответствии с содержанием учебной 1. Find information about the eﬃ cacy of vitamin D for prevention and treatment of COVID-19 using recommended sources of evidence-based medical information. Present the used sources of information. Ground your opinion. 2. Find information about the eﬃ cacy of molnupiravir for COVID-19 treatment using recommended sources of evidence-based medical information. Present the used sources of information. 90 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 программы. Каждый вопрос включает в себя не- большую четко очерченную часть учебного мате- риала, последовательно раскрывая его. пользования тестирования в учебном процессе возросла и в связи с необходимостью организа- ции элементов дистанционного обучения в усло- виях пандемии COVID-19. Однако данный метод контроля имеет и свои недостатки, в частности, не позволяет оценить умение излагать материал логически, строить ответ, не демонстрирует на- выки клинического мышления. Это определяет необходимость сочетания тестирования с други- ми формами и методами контроля. Литература Принята в печать 21.04.2022 г. References https://iaar.agency/iaar/standarty-naar. [Accessed 13th Apr 2022]. (In Russ.) https://iaar.agency/iaar/standarty-naar. [Accessed 13th Apr 2022]. (In Russ.) 1. Shchastnyi AT, Glushanko VS, Konevalova NIu, Alferova MV, Gerberg AA, Lappo VA, i dr; M-vo zdravookhraneniia Resp Belarus', UO "Viteb gos ordena Druzhby narodov med un-t". Innovative components of the modernization of the educational process: [monografiia]. Vitebsk, RB: [VGMU]; 2016. 168 р. (In Russ.) 6. COVID-19. Clinical management: Living guidance, 25 January 2021. Avaiable from: https://www.who.int/ publications/i/item/WHO-2019-nCoV-clinical-2021-2. 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Submitted 21.02.2022 Accepted 21.04.2022 Submitted 21.02.2022 Accepted 21.04.2022 Литература 1. Инновационные составляющие модернизации образо- вательного процесса : [монография] / А. Т. Щастный [и др.] ; М-во здравоохранения Респ. Беларусь, УО «Ви- теб. гос. ордена Дружбы народов мед. ун-т». – Витебск : [ВГМУ], 2016. – 168 с. 2. Фоминых, И. В. Роль учебно-методического комплек- са в обеспечении качества образования / И. В. Фоми- ных // Теория и практика образования в современном 91 ESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 мире : материалы VI Междунар. науч. конф. (г. Санкт- Петербург, дек. 2014 г.) / гл. ред. И. Г. Ахметова. – Санкт-Петербург : Занев. 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Ситуационные задачи как средство по- вышения эффективности образовательного процесса в ВПО при дистанционном обучении / Е. И. Тупикин // Междунар. журн. приклад. и фундам. исслед. – 2017. – № 4-1. – С. 266–267. 5. Стандарты и руководство IAAR по международной ак- кредитации медицинских организаций образования за рубежом (на основе стандартов WFME/AMSE) [Элек- тронный ресурс]. – Режим доступа: https://iaar.agency/ iaar/standarty-naar. – Дата доступа: 13.04.2022. 10. Добротин, Д. Ю. Применение кейс-метода в обучении студентов педагогических вузов / Д. Ю. Добротин, И. Н. Добротина // Вестн. Моск. гос. обл. ун-та. Сер. Педаго- гика. 2019. – № 1. – С. 62–70. y у 6. COVID-19. Clinical management: Living guidance, 25 January 2021 [Electronic resource]. – Mode of access: https://www.who.int/publications/i/item/WHO-2019- nCoV-clinical-2021-2. – Date of access: 13.04.2022. 11. Петров, В. В. Проблемное обучение в медицинском университете / В. В. Петров // Бюл. мед. Интернет- конф. – 2016. – Т. 6, № 7. – С. 1383–1384. 7. Interim Clinical Guidance for Management of Patients Поступила 21.02.2022 г. Принята в печать 21.04.2022 г. Information about authors: Haliuchenka V.A. – Candidate of Medical Sciences, associate professor of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University, ORCID: https://orcid.org/0000-0003-4025-9589; Zhyltsou I.V. – Doctor of Medical Sciences, professor, head of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University, Zhyltsou I.V. – Doctor of Medical Sciences, professor, head of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University, ORCID: https://orcid.org/0000-0002-4912-2880; ORCID: https://orcid.org/0000-0002-4912-2880; ORCID: https://orcid.org/0000-0002-4912-2880; p g ; Skreblo Y.I. – senior lecturer of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Adamenko G.P. – Doctor of Medical Sciences, professor of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Kaliadka Y.I. – lecturer of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University. Skreblo Y.I. – senior lecturer of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Adamenko G.P. – Doctor of Medical Sciences, professor of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Kaliadka Y.I. – lecturer of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University. g g, f p p y; Adamenko G.P. – Doctor of Medical Sciences, professor of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; Kaliadka Y.I. – lecturer of the Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University. Адрес для корреспонденции: Республика Беларусь, 210009, г. Витебск, пр-т Фрунзе, 27, Витебский государ- ственный ордена Дружбы народов медицинский университет, кафедра доказательной медицины и клинической диагностики ФПК и ПК. E-mail: goluchenkoolga@gmail.com – Голюченко Ольга Анатольевна. Сведения об авторах: Сведения об авторах: Сведения об авторах: Голюченко О.А. – к.м.н., доцент кафедры доказательной медицины и клинической диагностики ФПК и ПК, Ви- тебский государственный ордена Дружбы народов медицинский университет, ORCID: https://orcid.org/0000-0003-4025-9589; Жильцов И.В. – д.м.н., профессор, заведующий кафедрой доказательной медицины и клинической диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет, ORCID: https://orcid.org/0000-0002-4912-2880; д р Голюченко О.А. – к.м.н., доцент кафедры доказательной медицины и клинической диагностики ФПК и ПК, Ви- тебский государственный ордена Дружбы народов медицинский университет, p g ; Жильцов И.В. – д.м.н., профессор, заведующий кафедрой доказательной медицины и клинической диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет, ORCID: https://orcid.org/0000-0002-4912-2880; 92 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Скребло Е.И. – старший преподаватель кафедры доказательной медицины и клинической диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет; Адаменко Г.П. – д.м.н., профессор кафедры доказательной медицины и клинической диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет; Колядко Е.И. – ассистент кафедры доказательной медицины и клинической диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет. Скребло Е.И. – старший преподаватель кафедры доказательной медицины и клинической диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет; у у у Адаменко Г.П. – д.м.н., профессор кафедры доказательной медицины и клинической диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет; Колядко Е.И. – ассистент кафедры доказательной медицины и клинической диагностики ФПК и ПК, Витебский государственный ордена Дружбы народов медицинский университет. VASILENKO N.I., KADUSHKO R.V., MYASOEDOV A.M., POGOTSKY A.K. VASILENKO N.I., KADUSHKO R.V., MYASOEDOV A.M., POGOTSKY A.K. Vitebsk State Order of Peoples’ Friendship Medical University, Vitebsk, Republic of Belarus Резюме. Данная статья на примере деятельности сотрудников Витебского государственного медицинского института в годы Великой Отечественной войны способствует формированию у студенческой молодежи гражданственности, патриотизма, общечеловеческих ценностей, прививает уважение к профессии медицинских работников, внесших огромный вклад в победу в Великой Отечественной войне, показывает их героизм и самопожертвование. По- казана роль музея ВГМУ в воспитании студенческой молодежи в период обучения в университете, раскрываются формы и методы работы общественных организаций (профсоюз, Совет ветеранов, БРСМ, Белорусский Союз женщин) учреждения образования «Витебский государственный ордена Дружбы народов медицинский универ- ситет», кураторов студенческих групп. Ключевые слова: воспитательный процесс, гражданско-патриотическое воспитание, патриотизм, Великая Отечественная война, патриоты-медики, героизм, музей, общественные организации. 2022 год в Республике Беларусь объявлен Годом исторической памяти. Большое внимание Information about authors: Correspondence address: Republic of Belarus, 210009, Vitebsk, 27 Frunze ave., Vitebsk State Order of Peoples’ Friendship Medical University, Chair of Evidence-Based Medicine and Clinical Diagnostics of the Faculty for Advanced Training & Retraining. E-mail: goluchenkoolga@gmail.com – Volha A. Haliuchenka. 93 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Abstract. Патриотами не рождаются, общечеловече- ские ценности, такие как справедливость, добро, достоинство, надежда, любовь к человеку, миру, своей стране развиваются на протяжении всей жизни человека. Особенно важно формировать эти качества в семье, школе, учреждениях сред- него и высшего образования, при освоении про- фессии на рабочем месте. За годы оккупации на территории БССР фашистами было создано более 260 нацистских лагерей смерти, более 2,2 миллиона человек по- гибло от рук фашистских оккупантов, более 9,2 тысячи населенных пунктов было разрушено и сожжено, 209 из 270 городов превращено в руи- ны, 628 деревень уничтожено вместе с жителями, 188 деревень так и не смогли возродиться [5]. По- гиб каждый третий житель Белоруссии. Для стабильного существования общества, государства важны многие профессии и специ- альности, но профессия медицинского работника необходима во все времена. Её значение и роль особенно возрастают в критические, поворот- ные периоды истории, такие как эпидемии, во- йны. В силу этого очень важно сформировать у медицинских работников развитую гражданскую позицию, чувство патриотизма, высокой нрав- ственности, ответственности за жизнь, здоровье людей. Беларусь – республика-партизанка. Мно- гие медики сражались в партизанских отрядах, в подполье. Источником широко распространен- ного в республике партизанского движения были патриотизм наших людей, преданность своей От- чизне и ненависть к врагу. С первых дней войны (с 9 июня 1941 года – по 26 июня 1944 года) дей- ствовало витебское подполье. В его деятельности можно выделить два периода: первый – от начала оккупации города Витебска до открытия Витеб- ских (Суражских) ворот в феврале 1942 года, а второй – с февраля 1942 года до освобождения г. Витебска [6]. С первых шагов получения медицинско- го образования студенты-медики активно вклю- чаются в непривычную после школы деятель- ность – адаптируются к новым условиям жизни и обучения вдали от семьи, школы, друзей, малой родины, осваивают большие объемы учебной нагрузки, привыкают к новой атмосфере и тем- пу университетской жизни, получают полную самостоятельность и начинают понимать меру собственной ответственности в принятии реше- ний. Они также становятся участниками после- довательного, систематически осуществляемого воспитательного процесса, что не менее важно, чем получение знаний по профилю своей специ- альности и профессиональных навыков. Курато- ры студенческих групп, общественные организа- ции университета (профсоюз, Совет ветеранов, БРСМ, Белорусский Союз женщин) работают не- посредственно со студентами, начиная с первого курса, организуют посещение музеев, театров, выставочных залов, концертов, знаковых мест, Прифронтовое положение города Витеб- ска в течение трёх лет обусловило специфику деятельности патриотов витебского подполья. Борьба проходила почти в непосредственной близости от войск противника, в его ближайшем тылу. Abstract. The given article on the example of the activities of the staff members of Vitebsk State Medical Institute during the years of the Great Patriotic War facilitates the formation of citizenship, patriotism, universal human values in the student youth, cultivates respect for the profession of medical workers who have made a huge contribution to the victory gained in the Great Patriotic War, shows their heroism and self-sacrifice. The role of VSMU museum in the education of student youth during their studying at the university is defined. Forms and methods of the work of public organizations (Trade Union, Veterans Council, Belarusian Republican Youth Union, Belarusian Union of Women) of the Educational Establishment «Vitebsk State Order of Peoples’ Friendship Medical University» and those of student academic group tutors are revealed. Key words: educational process, civil-patriotic education, patriotism, Great Patriotic War, patriots-doctors, heroism, public organizations. «От живых донесенье погибшим несу: Нет, ничто не забыто, Нет, никто не забыт, Даже тот, кто в безвестной могиле лежит». «От живых донесенье погибшим несу: Нет, ничто не забыто, Нет, никто не забыт, Даже тот, кто в безвестной могиле лежит». Ю. Друнина 2022 год в Республике Беларусь объявлен Годом исторической памяти. Большое внимание у нас в стране уделяется работе с молодёжью как главному стратегическому ресурсу развития на- 94 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 шей Родины. Актуальность гражданско-патри- отического воспитания молодёжи выражается в необходимости поступательного развития суве- ренного белорусского государства и формирова- ния в Республике Беларусь гражданского обще- ства. Одна из основных задач государственной молодёжной политики – воспитание граждани- на, патриота, одухотворенного идеалами добра, социальной справедливости, ответственности, способного творить и созидать во имя своего От- ечества, готового к его защите вплоть до самопо- жертвования. посвященных героям-медикам, а также встречи с ветеранами Великой Отечественной войны, малолетними узниками, их родственниками, про- водят тематические встречи на диалоговых пло- щадках, поздравляют ветеранов с праздниками и памятными датами, оказывают им волонтёрскую помощь, проводят благотворительные акции по наведению порядка на мемориалах и памятниках времён Великой Отечественной войны [1-4]. Сегодня, в Год исторической памяти, рас- сказать молодёжи о подвиге, героизме медиков в годы Великой Отечественной войны – наш святой долг. Это позволит приблизить их немер- кнущие подвиги, примеры патриотизма и граж- данского долга к чувствам нашего студенчества, приобщить его к истокам героизма дедов и пра- дедов, укрепить тем самым живую связь времен и поколений. Abstract. Героическую борьбу против фашистских захватчиков в рядах витебских подпольщиков вели медицинские работники. Десятки врачей, медсестёр, санитарок, фармацевтов остались в оккупированном городе, чтобы оказывать меди- цинскую помощь больным и раненым бойцам, не успевшим эвакуироваться из Витебска. С по- 95 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Махнову, с помощью которых были устроены на работу в витебских больницах А.И. Богдано- ва, В.М. Величенко, Н.П. Круглова, зав. аптекой М.Д. Виниченко и др. мощью наших врачей-патриотов сотни бойцов и командиров Красной Армии ушли из больниц в партизанские отряды, чтобы продолжить войну с врагом. Витебские медики снабжали партизан лекарствами, хирургическими инструментами, проводили большую работу по спасению молодё- жи от угона в Германию, собирали ценные раз- ведывательные данные. мощью наших врачей-патриотов сотни бойцов и командиров Красной Армии ушли из больниц в партизанские отряды, чтобы продолжить войну с врагом. Витебские медики снабжали партизан лекарствами, хирургическими инструментами, проводили большую работу по спасению молодё- жи от угона в Германию, собирали ценные раз- ведывательные данные. За их деятельность в подполье М.Л. Му- рашко (бывший заведующий кафедрой ВГМИ) и Р.Ф. Махнов (врач, выпускник ВГМИ) были звер- ски замучены фашистами в 1943 году. Гитлеровцы регулярно проводили кара- тельные операции против партизан и подполь- щиков с целью подавления сопротивления, пора- бощения жителей оккупированной территории, разграбления имущества. Партизанам требова- лось большое количество лекарственных средств и перевязочного материала для оказания помощи раненым. Из витебской аптеки, расположенной на площади Свободы, неоднократно получала ле- карства для партизан комсомолка Валя Лебедева. Врач-гинеколог А.И. Богданова обеспечивала ле- карствами и перевязочным материалом партизан- ский отряд А.А. Погорелова. Врач Н.И. Чертков снабжал партизан не только разведывательными данными, но и необходимыми медикаментами. Сёстры Дунке и Липатова шефствовали над отря- дом «Моряк» бригады Алексея, партизаны кото- рой дважды получали большое количество лекар- ственных средств с витебского аптечного склада. Особенно активно действовала подпольная группа медиков под руководством врача туберку- лёзной больницы Ксении Сергеевны Околович. Надёжными помощниками руководителя меди- цинского подполья стали врач Т.М. Широчен- ко, медсестры Т.К. Вишневская, Л.А. Полянина, А.Я. Лукашенко, Тамара Бигус, Игорь Ольшанко и другие [7]. Медицинские работники витебского под- полья поддерживали тесную связь с разведчика- ми партизанских бригад. На территорию Лиоз- ненского района Витебской области в конце 1941 года был заброшен советский разведчик В.И. Чирков. В марте 1942 года под видом бежавшего военнопленного он проник в Витебск. Вскоре он познакомился с К.С. Околович. От нее получил документы на имя Владимира Белицкого, офор- мил городскую прописку. Abstract. До конца 1942 года он успешно вёл разведывательную деятельность, имел адреса конспиративных квартир в Витеб- ске, пользовался помощью медиков подпольной группы. Через витебских подпольщиков снабжа- лись медикаментами и партизанские отряды, дей- ствовавшие на территории Смоленской области. К.С. Околович 7 и 20 мая 1942 года вывела две группы (около 30 человек) из Витебска. Мно- гие из состава этих групп имели документы и ме- дицинские справки, сделанные ею [7]. Смоленские партизаны были предупрежде- ны о готовящейся крупной карательной операции фашистов на основании разведывательных дан- ных витебского подполья [8]. Медики-подпольщики К.С. Околович, М.Л. Мурашко, Р.Ф. Махнов в апреле 1942 г. вывели к партизанам группу военнопленных во главе с полковником П.Н. Тищенко, кадровым военным Красной Армии, начальником штаба артилле- рийского корпуса. Раненый полковник лежал в госпитале в г. Велиже, наладил связь с местными патриотами и получал с их помощью информа- цию о положении на фронтах. Продолжил лече- ние полковник П.Н. Тищенко в г. Витебске в пер- вой больнице по улице Лабазной. Главный врач В.Н. Чертков, врачи М.Б. Мурашко, М.Л. Мураш- ко, Р.Ф. Махнов и др. помогали оформить ему и группе других раненых документы, чтобы выйти из города. С двумя маленькими детьми расстреляна фашистами выпускница Витебского государ- ственного медицинского института (ВГМИ), врач Анна Николаевна Мамонова, спасшая жизнь раненому лётчику. Выпускник Витебского госу- дарственного медицинского института Антон Чернецкий был заживо сожжён фашистскими ка- рателями вместе с жителями села Карпиловка за оказание им медицинской помощи. Ненависть фашистов, жестокость, желание истребить белорусское население поистине не знали предела. Почти 1100 дней и ночей длилась муже- ственная борьба за освобождение Витебска. Мы не вправе предать забвению имена медиков, со- трудников ВГМИ, участвовавших в нём. Своей сплоченностью, целенаправленно- стью в подпольной работе медицинские работ- ники во многом обязаны М.Л. Мурашко, Р.Ф. Преподаватели института, бывшие студен- ты, добровольцами ушедшие на фронт, в парти- 96 ТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, №2 свободу и независимость Отечества. Материалы музея используются авторами конкурсных иссле- довательских работ, посвященных подвигу бело- русских медиков в годы Великой Отечественной войны, учащимися средних школ города, студен- тами, научными сотрудниками, кураторами ака- демических групп [9]. занские отряды и подполье, навсегда оставили память о своей молодости, овеянной героиче- ской борьбой за освобождение родной белорус- ской земли от фашистского гнета, порабощения и уничтожения. Благодаря мужеству медиков, спасавших раненых, большое количество воинов было вновь возвращено в боевой строй. Abstract. На протяжении учебного года ежегодно проводятся экскурсии в музей истории ВГМУ не только для отечественных студентов, которые уже проходят обучение в нём, но и для школьни- ков (профориентационного характера), а также обзорные экскурсии для гостей университета, участников методических семинаров, научных и научно-практических конференций. На базе му- зея систематически проходят уроки-экскурсии с англоговорящими студентами факультета под- готовки иностранных граждан с обеспечением синхронного перевода эмоциональной речи экс- курсовода – заведующим и сотрудниками кафе- дры иностранных языков. Такие мероприятия не оставляют побывавших в музее равнодушными, способствуют формированию гражданско-патри- отических качеств средствами музея, сохранению преемственности поколений на основе историче- ской памяти, примеров героического прошлого. Доказательством тому служит книга отзывов и предложений, где посетители оставили свои наи- лучшие впечатления о музее. В освобождении Витебска от фашистов принимали участие преподаватели, выпускники ВГМИ. Участники военных событий навсегда останутся в сердцах благодарных потомков. Важную роль в гражданско-патриотиче- ском воспитании студентов нашего университе- та играет музей его истории. Музей – это Книга памяти всего ценного из того, что было, которая передаётся, как эстафета, поколениям нынеш- ним и будущим. У музея свой язык – язык му- зейных реликвий. Сама атмосфера музея, музей- ные экспонаты имеют уникальную возможность воздействовать на интеллектуальные, волевые и эмоциональные сферы студентов одновременно. Потенциал музея позволяет нашим студентам со- вершить путешествие в минувшие десятилетия, увидеть прошлое и день сегодняшний, сопоста- вить, сравнить события разных времён, позна- комиться с подлинными документами, увидеть бережно хранимые экспонаты, которые дают наглядную информацию о трудовых и ратных подвигах предыдущих поколений. Знакомство с содержанием экспозиций музея, посвящен- ных подвигам медицинских работников, вра- чей-подпольщиков и партизан в годы Великой Отечественной войны: «В первые дни войны», «Родина-мать зовёт», «Они сражались за Роди- ну», «Плакаты военного времени», «Медицин- ская династия Котович–Мартовы–Подолинские», «Антонов Игнатий Петрович», «Белов Сергей Иванович», «Узники фашистских концлагерей» и других, оказывает огромное влияние на по- сетителей музея, воспитывает патриотическое чувство верности и долга, готовности прийти на помощь в трудную минуту испытаний, учит лю- бить Родину, быть верными клятве Гиппократа в любой, самой сложной жизненной ситуации, беречь мир, взаимопомощь и свободу. Abstract. Оно даёт сознание беспримерного мужества, оставленного нам в наследство обыкновенными людьми, став- шими героями, так как их жизнь была освещена высокой целью; понимание того, какой ценой до- сталась победа советскому народу; глубокое ува- жение к памяти тех, кто в трудные годы воевал за Особенно эффективны в плане граждан- ско-патриотического воспитания студенческой молодёжи медицинского университета встречи- беседы с ветеранами Великой Отечественной войны, малолетними узниками, где студенты из первых уст слышат жуткую правду о годах лише- нья, геноциде белорусского народа, миллионах жертв, всех ужасах войны, тяжёлых годах после- военного лихолетья, восстановления народного хозяйства и развития нашей страны. Однако их – живых свидетелей страшных, но великих со- бытий, руками которых свершилась Победа – с каждым годом, к горькому сожалению, остаётся всё меньше и меньше [10, 11]. В ВГМУ опубликованы воспоминания ве- теранов. Главный посыл изданной книги – доне- сти правду о войне до молодежи, чтобы она бе- режно сохранялась, передавалась из поколения в поколение. Забыть ужасы войны – значит предать прошлое и настоящее, допустить возможность повторения тех страшных лет. Значимым событием в жизни ВГМУ стало открытие мемориальной доски военному врачу, 97 ESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 ветерану Великой Отечественной войны, ветера- ну труда ВГМУ, лауреату Государственной пре- мии СССР Белову Сергею Ивановичу, бывшему заведующему кафедрой военной подготовки, до- центу курса истории на кафедре общественного здоровья и здравоохранения. На данное знаковое мероприятие приезжали родственники С.И. Бе- лова, в том числе и правнук ветерана. В Витеб- ском государственном ордена Дружбы народов медицинском университете всегда помнят и чтят подвиг своих сотрудников и студентов в годы Великой Отечественной войны. Их гуманизм, патриотизм и сила духа – путеводная звезда для студентов-медиков / фармацевтов, всех работни- ков здравоохранения наших дней. Преемствен- ность поколений продолжается. 3. Мясоедов, А. М. Нравственная культура как основа фор- мирования личности врача / А. М. Мясоедов, Н. Г. Харке- вич // Проблемы врачебной этики в современном мире : XIII Междунар. мед. конф. (Витебск, 8–9 июня 2017 г.) : тез. докл. / Витеб. епархия Римско-катол. Церкви в Респ. Беларусь [и др.]. – Минск : ПРО ХРИСТО, 2017. – С. 39– 41. 4. Мясоедов, А. М. Роль первичной профсоюзной органи- зации студентов УО « ВГМУ» в гражданско-патриотиче- ском воспитании студентов-медиков / А. М. Мясоедов // Медицинское образование XXI века: практикоориентиро- ванность и повышение качества подготовки специалистов : сб. материалов Респ. науч.-практ. конф. с междунар. уча- стием / М-во здравоохранения Респ. Беларусь, УО «Ви- теб. гос. ордена Дружбы народов мед. ун-т» ; [гл. ред. Литература Принята в печать 21.04.2022 г. Abstract. А. Т. Щастный ; редкол.: Н. Ю. Коневалова и др.]. – Витебск : ВГМУ, 2018. – С. 349–351. Годовщина Хатынской трагедии : [по данным БЕЛТА] // Витеб. вести. – 2022. – 25 марта. – С. 11. Годовщина Хатынской трагедии : [по данным БЕЛТА] // Витеб. вести. – 2022. – 25 марта. – С. 11. 6. Подвигу народа в Великой Отечественной войне – па- мять и благодарность потомков : материалы науч.-практ. конф. студентов и сотр. ВГМУ, посвящ. 60-летию Победы в Великой Отечественной войне / Витеб. гос. мед. ун-т ; редкол.: А. Н. Косинец (председатель) [и др.]. – Витебск : ВГМУ, 2005. – 158 с. «И пусть летят года, Проносятся столетья, И изменился мир, И общество не то. Вы в памяти людей В любые лихолетья, И ваши имена Не умертвит никто». «И пусть летят года, Проносятся столетья, И изменился мир, И общество не то. Вы в памяти людей В любые лихолетья Вы в памяти людей 7. Памяць. Вiцебск : гiст.-дакум. хронiка : y 2 кн. Кн. 1 / [рэд- кал.: Г. П. Пашкоў (гал. рэд.) i iнш.]. – Мінск : БелЭн, 2002. – 648 с. В любые лихолетья, И ваши имена Не умертвит никто». 8. Пахомов, Н. И. Витебское подполье / Н. И. Пахомов, Н. И. Дорофеенко, Н. В. Дорофеенко. – 2-е изд., перераб. и доп. – Минск : Беларусь, 1974. – 223 с. student public organizations: metod material v pomoshch’ kuratoram ucheb grupp, studench aktivu, proforgam grupp, vospitateliam obshchezhitii. Vitebsk, RB: VGMU; 2019. 43 р. (In Russ.) 2. Miasoedov AM. Civil and patriotic education of students (on the example of the primary trade union organization Литература 9. Патриотическое воспитание современной молодежи на примерах мужества и героизма сотрудников ВГМИ в годы Великой Отечественной войны / Н. И. Василенко [и др.] // Достижения фундаментальной, клинической медицины и фармации : материалы 69-й науч. сес. сотр. ун-та, 29-30 янв. 2014 г. / М-во здравоохранения Респ. Беларусь, Витеб. гос. мед. ун-т ; [ред.: В. П. Дейкало, С. А. Сушков]. – Ви- тебск : ВГМУ, 2014. – С. 376–377. 1. Формирование гражданственности и патриотизма в мо- лодежной медицинской среде: роль студенческих обще- ственных организаций : метод. материал в помощь ку- раторам учеб. групп, студенч. активу, профоргам групп, воспитателям общежитий / [сост.: Кулик С. П., Терехов Е. А., Мясоедов А. М. ; под ред. О. А. Сыродоевой] ; М-во здравоохранения Респ. Беларусь, УО «Витеб. гос. ордена Дружбы народов мед. ун-т». – Витебск : [ВГМУ], 2019. – 43 с. 1. Формирование гражданственности и патриотизма в мо- лодежной медицинской среде: роль студенческих обще- ственных организаций : метод. материал в помощь ку- раторам учеб. групп, студенч. активу, профоргам групп, воспитателям общежитий / [сост.: Кулик С. П., Терехов Е. А., Мясоедов А. М. ; под ред. О. А. Сыродоевой] ; М-во здравоохранения Респ. Беларусь, УО «Витеб. гос. ордена Дружбы народов мед. ун-т». – Витебск : [ВГМУ], 2019. – 43 с. 10. Это забыть нельзя: воспоминания ветеранов Витебского государственного медицинского университета о Великой Отечественной войне : материалы в помощь кураторам учеб. групп, студенч. активу, профоргам групп, воспитате- лям общежитий / [сост.: Мясоедов А. М., Назарук А. А.] ; М-во здравоохранения Респ. Беларусь [и др.]. – Витебск : [ВГМУ], 2018. – 28 с. 2. Мясоедов, А. М. Гражданско-патриотическое воспита- ние студенческой молодежи (на примере первичной про- фсоюзной организации студентов УО «ВГМУ») / А. М. Мясоедов // Инновационные обучающие технологии в медицине [Электронный ресурс] : сб. материалов Между- нар. Респ. науч.-практ. конф. с междунар. участием / М-во здравоохранения Респ. Беларусь, УО «Витеб. гос. ордена Дружбы народов мед. ун-т» ; [гл. ред. А. Т. Щастный ; ред- кол.: Н. Ю. Коневалова и др.]. – Витебск : ВГМУ, 2017. – С. 353–356. – 1 электрон. опт. диск (CD-ROM). – Загл. с этикетки диска. 11. Патриотизм и мужество, гуманизм и самоотверженность (посвящается подвигу медиков в годы Великой Отече- ственной войны) : темат. сб. / М-во здравоохранения Респ. Беларусь, УО «Витеб. гос. ордена Дружбы народов мед. ун-т» ; [авт.-сост.: С. П. Кулик и др.]. – Витебск : [ВГМУ], 2021. – 104 с. Поступила 21.02.2022 г. Принята в печать 21.04.2022 г. Поступила 21.02.2022 г. Поступила 21.02.2022 г. Принята в печать 21.04.2022 г. References student public organizations: metod material v pomoshch’ kuratoram ucheb grupp, studench aktivu, proforgam grupp, vospitateliam obshchezhitii. Vitebsk, RB: VGMU; 2019. 43 р. (In Russ.) student public organizations: metod material v pomoshch’ kuratoram ucheb grupp, studench aktivu, proforgam grupp, vospitateliam obshchezhitii. Vitebsk, RB: VGMU; 2019. 43 р. (In Russ.) student public organizations: metod material v pomoshch’ kuratoram ucheb grupp, studench aktivu, proforgam grupp, vospitateliam obshchezhitii. Vitebsk, RB: VGMU; 2019. 43 р. (In Russ.) 1. 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Submitted 21.02.2022 Accepted 21.04.2022 Submitted 21.02.2022 Accepted 21.04.2022 Сведения об авторах: Василенко Н.И. – председатель Совета ветеранов ВГМУ, лаборант кафедры общественного здоровья и здравоох- ранения с курсом ФПК и ПК, Витебский ордена Дружбы народов медицинский университет; К РВ ф ф й В б й й Василенко Н.И. – председатель Совета ветеранов ВГМУ, лаборант кафедры общественного здоровья и здравоох- ранения с курсом ФПК и ПК, Витебский ордена Дружбы народов медицинский университет; Кадушко Р.В. – к.филолог.н., доцент, заведующая кафедрой иностранных языков, Витебский государственный ордена Дружбы народов медицинский университет; Кадушко Р.В. – к.филолог.н., доцент, заведующая кафедрой иностранных языков, Витебский государственный ордена Дружбы народов медицинский университет; Кадушко Р.В. – к.филолог.н., доцент, заведующая кафедрой иностранных языков, Витебский государственный ордена Дружбы народов медицинский университет; Мясоедов А.М. – м.пед.н., старший преподаватель кафедры социально-гуманитарных наук, председатель профко- ма студентов, Витебский государственный ордена Дружбы народов медицинский университет; Мясоедов А.М. – м.пед.н., старший преподаватель кафедры социально-гуманитарных наук, председатель профко- ма студентов, Витебский государственный ордена Дружбы народов медицинский университет; Погоцкий А.К. – старший преподаватель кафедры челюстно-лицевой хирургии и хирургической стоматологии с курсом ФПК и ПК, председатель профкома сотрудников, Витебский государственный ордена Дружбы народов медицинский университет. у р Information about authors: Information about authors: f Vasilenko N.I. – Chairwoman of the VSMU Council of Veterans, laboratory assistant of the Chair of Public Health & Health Service with the course of the Faculty for Advanced Training & Retraining, Vitebsk State Order of Peoples’ Friendship Medical University; p y Kadushko R.V. – Candidate of Philological Sciences, associate professor, head of the Chair of Foreign Lan shko R.V. – Candidate of Philological Sciences, associate professor, head of the Chair of Foreign Language Order of Peoples’ Friendship Medical University; adushko R.V. Candidate of Philological Sciences, associate professor, head of the Chair of Foreign Langu ate Order of Peoples’ Friendship Medical University; State Order of Peoples’ Friendship Medical University; State Order of Peoples’ Friendship Medical University; ov A.M. – Master of Pedagogical Sciences, senior lecturer of the Chair of Social Sciences & Huma n of the students trade union committee Vitebsk State Order of Peoples’Friendship Medical University; Myasoedov A.M. – Master of Pedagogical Sciences, senior lecturer of the Chair of Social Sciences & Humanities, chairman of the students trade union committee, Vitebsk State Order of Peoples’Friendship Medical University; Myasoedov A.M. Master of Pedagogical Sciences, senior lecturer of the Chair of Social Sciences & Humanities, chairman of the students trade union committee, Vitebsk State Order of Peoples’ Friendship Medical University; Pogotsky A.K. – senior lecturer of the Chair of Maxillofacial Surgery & Operative Dentistry with the course of the Faculty for Advanced Training & Retraining, chairman of the employees trade union committee, Vitebsk State Order of Peoples’ Friendship Medical University. Pogotsky A.K. – senior lecturer of the Chair of Maxillofacial Surgery & Operative Dentistry with the course of the Faculty for Advanced Training & Retraining, chairman of the employees trade union committee, Vitebsk State Order of Peoples’ Friendship Medical University. Pogotsky A.K. – senior lecturer of the Chair of Maxillofacial Surgery & Operative Dentistry with the course of the Faculty for Advanced Training & Retraining, chairman of the employees trade union committee, Vitebsk State Order of Peoples’ Friendship Medical University. Адрес для корреспонденции: Республика Беларусь, 210009, г. Витебск, пр-т Фрунзе, 27, Витебский государ- ственный ордена Дружбы народов медицинский университет, кафедра иностранных языков. E-mail: regina. kadushko@mail.ru – Кадушко Регина Владимировна. @ ду д р Correspondence address: Republic of Belarus, 210009, Vitebsk, 27 Frunze ave., Vitebsk State Order of Peoples’ Friendship Medical University, Chair of Foreign Languages. E-mail: regina.kadushko@mail.ru – Regina V. Kadushko. Information about authors: 99 VESTNIK VITE ЮБИЛЕЙ К ЮБИЛЕЮ ОЛЕГА ДАНИИЛОВИЧА МЯДЕЛЬЦА 70 ЛЕТ СО ДНЯ РОЖДЕНИЯ Мяделец Олег Даниилович родился 18 апреля 1952 году в д. Красная Гора Шарковщинского рай- она Витебской области в семье рабочего. Учился в местной школе, в 1968 году окончил 9 классов и в этом же году поступил в Островецкое медицинское училище (Гродненская область). В 1970 году в связи с расформированием данного медучилища продолжил обучение в Юратишковском медицин- ском училище (Гродненская область), которое окончил с отличием в 1972 году. Работал фельдшером в психиатрической больнице, затем с мая 1972 года по июнь 1974 года служил в Советской Армии (г. Москва, фельдшер роты-прапорщик). В 1974 году поступил в Витебский государственный медицинский институт который окончил с К ЮБИЛЕЮ ОЛЕГА ДАНИИЛОВИЧА МЯДЕЛЬЦА 70 ЛЕТ СО ДНЯ РОЖДЕНИЯ К ЮБИЛЕЮ ОЛЕГА ДАНИИЛОВИЧА МЯДЕЛЬЦА 70 ЛЕТ СО ДНЯ РОЖДЕНИЯ Мяделец Олег Даниилович родился 18 апреля 1952 году в д. Красная Гора Шарковщинского рай- она Витебской области в семье рабочего. Учился в местной школе, в 1968 году окончил 9 классов и в этом же году поступил в Островецкое медицинское училище (Гродненская область). В 1970 году в связи с расформированием данного медучилища продолжил обучение в Юратишковском медицин- ском училище (Гродненская область), которое окончил с отличием в 1972 году. Работал фельдшером в психиатрической больнице, затем с мая 1972 года по июнь 1974 года служил в Советской Армии (г. Москва, фельдшер роты-прапорщик). Мяделец Олег Даниилович родился 18 апреля 1952 году в д. Красная Гора Шарковщинского рай- она Витебской области в семье рабочего. Учился в местной школе, в 1968 году окончил 9 классов и в этом же году поступил в Островецкое медицинское училище (Гродненская область). В 1970 году в связи с расформированием данного медучилища продолжил обучение в Юратишковском медицин- ском училище (Гродненская область), которое окончил с отличием в 1972 году. Работал фельдшером в психиатрической больнице, затем с мая 1972 года по июнь 1974 года служил в Советской Армии (г. Москва, фельдшер роты-прапорщик). Мяделец Олег Даниилович родился 18 апреля 1952 году в д. Красная Гора Шарковщинского рай- она Витебской области в семье рабочего. Учился в местной школе, в 1968 году окончил 9 классов и в этом же году поступил в Островецкое медицинское училище (Гродненская область). В 1970 году в связи с расформированием данного медучилища продолжил обучение в Юратишковском медицин- ском училище (Гродненская область), которое окончил с отличием в 1972 году. Работал фельдшером в психиатрической больнице, затем с мая 1972 года по июнь 1974 года служил в Советской Армии (г. Information about authors: Москва, фельдшер роты-прапорщик). ( ф р р р р ) В 1974 году поступил в Витебский государственный медицинский институт, который окончил с отличием в 1980 году. Был направлен в качестве стажера-исследователя на кафедру гистологии ВГМИ. С тех пор трудовая деятельность связана с Витебским медицинским университетом. С 1980 по 1982 г.г. О.Д. Мяделец стажер-исследователь, с 1982 по 1985 – аспирант, с 1985 по 1989 – ассистент, с 1989 по 1990 – старший преподаватель, с 1990 по 1993 – докторант, с 1993 по 1994 – старший преподаватель, с 1994 по 1996 – профессор по специальности «медицинские науки», с 1996 по настоящее время – за- ведующий кафедрой гистологии, цитологии и эмбриологии ВГМУ. ( ф р р р р ) В 1974 году поступил в Витебский государственный медицинский институт, который окончил с отличием в 1980 году. Был направлен в качестве стажера-исследователя на кафедру гистологии ВГМИ. С тех пор трудовая деятельность связана с Витебским медицинским университетом. С 1980 по 1982 г.г. О.Д. Мяделец стажер-исследователь, с 1982 по 1985 – аспирант, с 1985 по 1989 – ассистент, с 1989 по 1990 – старший преподаватель, с 1990 по 1993 – докторант, с 1993 по 1994 – старший преподаватель, с 1994 по 1996 – профессор по специальности «медицинские науки», с 1996 по настоящее время – за- ведующий кафедрой гистологии, цитологии и эмбриологии ВГМУ. В 1987 году защитил кандидатскую (специальность «медицинские науки»), а в 1994 – докторскую (специальность «медицинские науки») диссертации. В 1996 году присвоено ученое звание профессо- ра. В этом же году избран по конкурсу заведующим кафедрой гистологии, цитологии и эмбриологии Витебского государственного медицинского университета. Сферой научных интересов Мядельца О.Д. является изучение структуры и функции кожного покрова и иммунокомпетентных органов, а также патоморфологии морфологии хронических дерматозов: псориаза, атопического дерматита, буллезных дерматозов, псориатической и других видов эритродермий, паранеопластических дерматозов, алопе- ции, эозинофильных и нейтрофильных дерматозов и других видов кожной патологии. Данные научных разработок широко внедряются в практику здравоохранения, а также в учебный процесс ряда кафедр ВГМУ, на кафедрах гистологии, цитологии и эмбриологии Белорусского, Украинского и Гродненского медицинских университетов, Гомельского медицинского университета, Смоленской и Ярославской ме- дицинских академий. Соредактор 2 переводов иностранных монографий. Автор 520 научных и учебно-методических трудов, из них 4 учебников, 9 монографий, 40 учеб- Автор 520 научных и учебно-методических трудов, из них 4 учебников, 9 монографий, 40 учеб- 100 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № ных пособий монографического типа с грифом Министерства образования и УМО, 3 инструкций на метод. Information about authors: Подготовил 13 кандидатов наук и готовит 1 доктора наук. Им создана научная школа, зани- мающаяся вопросами разработки и внедрения в практику здравоохранения новых морфологических методов диагностики кожных заболеваний. О.Д. Мяделец награжден знаками «Отличник здравоохра- нения» и «Отличник образования» Республики Беларусь. В разное время награждался грамотами Ми- нистерства здравоохранения СССР, Республики Беларусь, Министерства образования Республики Бе- ларусь, Витебского облисполкома, Витебского горисполкома, райисполкома, Витебского медицинского университета, медалью ВГМУ. Он являлся председателем Витебского областного общества «Знание», членом Президиума Республиканского научного общества анатомов, гистологов, эмбриологов, членом специализированного совета по защите диссертаций, членом экспертного совета Высшей аттестацион- ной комиссии РБ. Является членом совета по защите диссертаций при Витебской академии ветеринар- ной медицины. ных пособий монографического типа с грифом Министерства образования и УМО, 3 инструкций на метод. Подготовил 13 кандидатов наук и готовит 1 доктора наук. Им создана научная школа, зани- мающаяся вопросами разработки и внедрения в практику здравоохранения новых морфологических методов диагностики кожных заболеваний. О.Д. Мяделец награжден знаками «Отличник здравоохра- нения» и «Отличник образования» Республики Беларусь. В разное время награждался грамотами Ми- нистерства здравоохранения СССР, Республики Беларусь, Министерства образования Республики Бе- ларусь, Витебского облисполкома, Витебского горисполкома, райисполкома, Витебского медицинского университета, медалью ВГМУ. Он являлся председателем Витебского областного общества «Знание», членом Президиума Республиканского научного общества анатомов, гистологов, эмбриологов, членом специализированного совета по защите диссертаций, членом экспертного совета Высшей аттестацион- ной комиссии РБ. Является членом совета по защите диссертаций при Витебской академии ветеринар- ной медицины. Мядельца О.Д. отличают большое трудолюбие и жизнелюбие. Он пользуется большим уважени- ем у студентов, сотрудников кафедры, университета. Ректорат, профессорско-преподавательский состав, сотрудники кафедры гистологии, цитологии и эмбриологии 101 VESTNIK VITEB НЕКРОЛОГ ПАВЕЛ ДМИТРИЕВИЧ НОВИКОВ (14.06.1971 г. – 07.04.2022 г.) 7 апреля 2022 года на 51-м году жизни ушёл из жизни профессор кафедры клинической имму- нологии и аллергологии с курсом ФПК и ПК, доктор медицинских наук, профессор Новиков Павел Дмитриевич. ПАВЕЛ ДМИТРИЕВИЧ НОВИКОВ (14.06.1971 г. – 07.04.2022 г.) ПАВЕЛ ДМИТРИЕВИЧ НОВИКОВ (14.06.1971 г. – 07.04.2022 г.) 7 апреля 2022 года на 51-м году жизни ушёл из жизни профессор кафедры клинич нологии и аллергологии с курсом ФПК и ПК, доктор медицинских наук, профессор Но Дмитриевич. 7 апреля 2022 года на 51-м году жизни ушёл из жизни профессор кафедры клинической имму- нологии и аллергологии с курсом ФПК и ПК, доктор медицинских наук, профессор Новиков Павел Дмитриевич. 7 апреля 2022 года на 51-м году жизни ушёл из жизни профессор кафедры клинической имму- нологии и аллергологии с курсом ФПК и ПК, доктор медицинских наук, профессор Новиков Павел Дмитриевич. Ректорат, профессорско-преподавательский состав, профком сотрудников ВГМУ Information about authors: Этот научно-практический комплекс, оборудо- ванный по всем современным требованиям, – самый настоящий и долгожданный подарок для студентов и преподавателей университета и на- шего города. На празднике присутствовали почетные гости: Владимир Петрович Пенин, заместитель председателя Витебского областного исполнительного комитета; Михаил Леонтьевич Вишневецкий, начальник Главного управления по здравоохранению Витебского областного исполнительного комите- та. Также перед собравшимися выступили ректор университета Анатолий Тадеушевич Щастный, декан стоматологического факультета Иван Юрьевич Карпук, главный врач университетской стоматологиче- ской поликлиники Анастасия Алексеевна Пожарицкая и студент 3 курса стоматологического факульте- та Никита Богословский. В открывшейся клинике помимо учебного процесса будет вестись лечебная работа по оказанию Information about authors: р Павел Дмитриевич родился в г. Витебске в 1971 году. С юности увлекался научной деятельно- стью. Со студенческой скамьи работал вместе с выдающимися родителями – профессорами Валенти- ной Ивановной и Дмитрием Кузьмичом Новиковыми. После окончания Витебского медицинского ин- ститута в 1994 году трудовую деятельность начал врачом-стажёром по педиатрии Витебской областной детской клинической больницы. С 1995 по 1997 г. на базе Витебской областной детской клинической больницы проходил клиническую ординатуру по специальности «педиатрия». С 1997 по 2003 год рабо- тал ассистентом кафедры детских болезней ВГМУ. Защитил кандидатскую диссертацию «Клиническое значение нарушений иммунного статуса при бронхитах у детей». С 2003 г. работал на кафедре клини- ческой иммунологии и аллергологии с курсом ФПК и ПК ассистентом, с 2003 г. – доцентом, а с 2005 г. профессором В 2004 г защитил докторскую диссертацию на тему «Диагностика иммунодефицитных – профессором. В 2004 г. защитил докторскую диссертацию на тему «Диагностика иммунодефицитных и аллергических болезней». Опубликовал 180 научных работ, соавтор более 10 монографий, учебных пособий, учебника «Клиническая иммунология и аллергология» (2019 г.). Имеет 8 патентов, подгото- вил 4 кандидата медицинских наук и одного доктора медицинских наук. – профессором. В 2004 г. защитил докторскую диссертацию на тему «Диагностика иммунодефицитных и аллергических болезней». Опубликовал 180 научных работ, соавтор более 10 монографий, учебных пособий, учебника «Клиническая иммунология и аллергология» (2019 г.). Имеет 8 патентов, подгото- вил 4 кандидата медицинских наук и одного доктора медицинских наук. Павел Дмитриевич стоял у истоков создания Белоруской ассоциации аллергологов и клиниче- ских иммунологов (БААКИ) – члена Всемирной организации по аллергии (WAO). Последние годы работал исполнительным директором общества. Профессор П.Д. Новиков входил в редколлегию международного научно-практического журнала «Иммунопатология, аллергология, инфектология». С 2021 года, после смерти профессора Д.К. Новико- ва, исполнял обязанности главного редактора. Павел Дмитриевич всю жизнь был предан педиатрии и аллергологии-иммунологии роко известно как в Республике Беларусь, так и за её пределами. Ректорат, профком сотрудников, профессорско-преподавательский состав, коллектив сотрудни- ков университета глубоко скорбит в связи с безвременной кончиной Павла Дмитриевича. В нашей па- мяти он останется как талантливый, доброжелательный, открытый к общению коллега, высокий про- фессионал. Мы выражаем свои соболезнования семье, родным и близким, скорбим вместе с ними. Светлая память о нём сохранится в наших сердцах навсегда. Ректорат, профессорско-преподавательский состав, профком сотрудников ВГМУ 102 ВЕ НОВОСТИ ОТКРЫТИЕ УНИВЕРСИТЕТСКОЙ СТОМАТОЛОГИЧЕСКОЙ ПОЛИКЛИНИКИ 24 февраля 2022 года состоялось торже- ственное открытие клиники «Университетская стоматологическая поликлиника» Витебского государственного медицинского университета. ОТКРЫТИЕ УНИВЕРСИТЕТСКОЙ СТОМАТОЛОГИЧЕСКОЙ ПОЛИКЛИНИКИ ОТКРЫТИЕ УНИВЕРСИТЕТСКОЙ СТОМАТОЛОГИЧЕСКОЙ ПОЛИКЛИНИКИ 24 февраля 2022 года состоялось торже- ственное открытие клиники «Университетская стоматологическая поликлиника» Витебского государственного медицинского университета. Этот научно-практический комплекс, оборудо- ванный по всем современным требованиям, – самый настоящий и долгожданный подарок для студентов и преподавателей университета и на- шего города. На празднике присутствовали почетные гости: Владимир Петрович Пенин, заместитель председателя Витебского областного исполнительного комитета; Михаил Леонтьевич Вишневецкий, начальник Главного управления по здравоохранению Витебского областного исполнительного комите- та. Также перед собравшимися выступили ректор университета Анатолий Тадеушевич Щастный, декан стоматологического факультета Иван Юрьевич Карпук, главный врач университетской стоматологиче- ской поликлиники Анастасия Алексеевна Пожарицкая и студент 3 курса стоматологического факульте- та Никита Богословский. В открывшейся клинике помимо учебного процесса будет вестись лечебная работа по оказанию стоматологической помощи студентам витебских вузов, что, безусловно, станет весомой помощью практическому здравоохранению нашего города. Распоряжением Президента Республики Беларусь №45рп от 01 марта 2022 года назначены гран- ты Президента Республики Беларусь на 2022 год: В области образования: НАЗНАЧЕНИЕ ГРАНТОВ ПРЕЗИДЕНТА РЕСПУБЛИКИ БЕЛАРУСЬ ЗНАЧЕНИЕ ГРАНТОВ ПРЕЗИДЕНТА РЕСПУБЛИКИ БЕЛАРУСЬ Распоряжением Президента Республики Беларусь №45рп от 01 марта 2022 года назначены гран- ты Президента Республики Беларусь на 2022 год: Распоряжением Президента Республики Беларусь №45рп от 01 марта 2022 года назначены гран- ты Президента Республики Беларусь на 2022 год: В б б Распоряжением Президента Республики Беларусь №45рп от 01 марта 2022 года назначены гран- ты Президента Республики Беларусь на 2022 год: В б б у В области образования: Кирпиченко Андрею Александровичу, заведующему кафедрой психиатрии и наркологии с кур- сом факультета повышения квалификации и переподготовки кадров, д.м.н., профессору. р р Карпук Наталье Анатольевне, доценту кафедры общей и ортопедической стоматологии с курсом факультета повышения квалификации и переподготовки кадров, к.м.н., доценту. Карпук Наталье Анатольевне, доценту кафедры общей и ортопедической стоматол факультета повышения квалификации и переподготовки кадров, к.м.н., доценту. Ректорат, научная часть поздравляют Андрея Александровича и Наталью Анатольевну с назначе- нием грантов Президента Республики Беларусь и желают им дальнейших творческих успехов! Ректорат, научная часть поздравляют Андрея Александровича и Наталью Анатольевну с назначе- нием грантов Президента Республики Беларусь и желают им дальнейших творческих успехов! 103 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 ФИНАНСОВАЯ ПОДДЕРЖКА ЛАБОРАТОРИИ ПРОФЕССИОНАЛЬНОГО МАСТЕРСТВА «ХИРУРГИЧЕСКИЕ БОЛЕЗНИ» На основании решения Cовета специального фонда Президента Республики Беларусь по соци- альной поддержке одаренных учащихся и студентов (Распоряжение Президента Республики Беларусь №65рп от 31.03.2022) Лаборатории профессионального мастерства «Хирургические болезни» УО «Ви- тебский государственный ордена Дружбы народов медицинский университет» оказана финансовая поддержка в размере 58000 рублей в целях укрепления материально-технической базы. Лаборатория профессионального мастерства была создана на основании приказа ректора от 06.10.2017 № 455-уч. с целью повышения эффективности практико-ориентированного обучения и повы- шения конкурентоспособности выпускников УО ВГМУ. Руководитель Лаборатории профессионального мастерства «Хирургические болезни» – заведующая кафедрой оперативной хирургии и топографической анатомии, к.м.н. Купченко Анна Михайловна. В Лаборатории занимаются 45 студентов лечебного фа- культета ВГМУ, из них: 20 студентов – 4 курса, 16 студентов – 5 курса, 9 студентов – 6 курса. Студенты Лаборатории профессионального мастерства «Хирургические болезни» являются по- бедителями внутривузовских и международных олимпиад по хирургии (в г.Смоленске, г.Рязани, г. Нур- Султане), активно занимаются научно-исследовательской работой, которая нашла отражение в 20 ста- тьях, опубликованных в материалах научно-практических конференций студентов и молодых ученых, получили 2 диплома I степени на VII Международном молодежном медицинском конгрессе в г. Санкт- Петербурге. Работы студентов Лаборатории на Республиканском конкурсе научных работ получили I и II категории. Выделенные денежные средства предполагается использовать на приобретение оборудования Студенты Лаборатории профессионального мастерства «Хирургические болезни» являются по- бедителями внутривузовских и международных олимпиад по хирургии (в г.Смоленске, г.Рязани, г. Нур- Султане), активно занимаются научно-исследовательской работой, которая нашла отражение в 20 ста- тьях, опубликованных в материалах научно-практических конференций студентов и молодых ученых, получили 2 диплома I степени на VII Международном молодежном медицинском конгрессе в г. Санкт- Петербурге. Работы студентов Лаборатории на Республиканском конкурсе научных работ получили I и II категории. Выделенные денежные средства предполагается использовать на приобретение оборудования, необходимого для улучшения качества практической подготовки студентов по специальности 1-79 01 01 «Лечебное дело», а также развития у студентов интереса к практической хирургии. Данное оборудо- вание дополнит техническое оснащение Лаборатории профессионального мастерства «Хирургические болезни» и позволит расширить подготовку студентов с использованием современных технологий и эндовидеохирургических методик оперирования, что отразится на их профессиональных компетен- циях. Широкое внедрение в хирургическую практику малоинвазивных методов лечения требует их предварительной отработки с использованием симуляционных тренажеров, максимально воспроизво- дящих реальную профессиональную среду. Использование данного оборудования позволит повысить качество профессиональной подготовки будущих врачей, увеличить объем научно-исследовательской и экспериментальной работы студентов, а также качественно подготовиться к участию в республикан- ских и международных олимпиадах по хирургии. 104 ВЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 2 ОЛИМПИАДНОЕ ДВИЖЕНИЕ ВГМУ В 2022 году студенты ВГМУ успешно выступили на международных олимпиадах. 21 января 10 студентов ВГМУ приняли участие во II Международной студенческой предметной онлайн-олимпиаде студентов медицинских вузов «От учения Абу Али ибн Сино – до Третьего ренессанса», проведенной Бухарским государственным медицинским институтом им. Абу Али ибн Сино (Республика Узбекистан), где студент 4-го курса лечебного факультета, участник лаборатории профессионального мастерства «Хи- рургические болезни» Неверовский С.О. завоевал диплом «За лучшее решение ситуационной задачи». рур р у р у В апреле студентки 6-го курса лечебного факультета Шафранская В.А., Винникова А.А., Лазарь М.С. отмечены дипломом за победу в номинации «Лучшая практическая подготовка», а также дипло- мом победителя в общекомандном зачете видеоприветствий и заняли седьмое общекомандное место (из 18) в V Всероссийской студенческой олимпиаде по оториноларингологии с международным уча- стием, проведенной Первым Санкт-Петербургским государственным медицинским университетом им. академика И.П.Павлова (Российская Федерация). Центром профессионального мастерства ВГМУ в текущем учебном году, несмотря на сложную эпидемиологическую ситуацию, проведены две внутривузовские олимпиады. 21-25 марта проведена олимпиада «Шаг в профессию» по направлениям «Анестезиология и реаниматология», «Хирургические болезни», «Врач общей практики», «Педиатрия», «Акушерство и гинекология», «Стоматология», «Фар- мация», в которой приняли участие более 100 студентов лечебного, педиатрического, стоматологическо- го и фармацевтического факультетов университета. Победителями по направлению «Анестезиология и реаниматология» стали: 1 место – Комленок Е.Н. (5 курс, ЛФ), 2 место – Щербинин В.И. (5 курс, ЛФ), 3 место – Молчанская А.Д. (6 курс, ЛФ), по направлению «Хирургические болезни»: 1 место – Жихарев Е.А. (5 курс, ЛФ), 2 место – Игнатович В.А. (4 курс, ЛФ), 3 место – Багрова Л.С. (5 курс, ЛФ); по на- правлению «Врач общей практики»: 1 место – Супоненко З.С. (5 курс, ЛФ), 2 место – Ермолицкий А.В. (5 курс, ЛФ), 3 место – Терентьева Е.А. (5 курс, ЛФ); по направлению «Педиатрия»: 1 место – Волк А. (1 курс, педиатрический факультет), 2 место – Сафончик А. (5 курс, ЛФ), 3 место – Манукян Г.(1 курс, педиатрический факультет); по направлению «Акушерство и гинекология»: 1 место – Толкачёва П.С. (4 курс, ЛФ), 2 место – Сычевич М.В. (4 курс, ЛФ), 3 место – Мозговая А.А. (5 курс, ЛФ); по направлению «Стоматология»: 1 место поделили Дорофеенко Е.В. (4 курс, стом. фак.) и Амелевич А.Д. (4 курс, стом. фак.), 3 место поделили Колодинская Е.Г. (4 курс, стом. фак.) и Пархимович А.А. (4 курс, стом. фак.); по направлению «Фармация»: 1 место – Гатило В.А. (4 курс, фарм. фак.), 2 место – Заруба Д.А. (4 курс, фарм. фак.), 3 место поделили Мхитарян Д.К. ОЛИМПИАДНОЕ ДВИЖЕНИЕ ВГМУ (4 курс, фарм. фак.) и Климова Ю.С. (3 курс, фарм. фак.) Центром профессионального мастерства ВГМУ в текущем учебном году, несмотря на сложную эпидемиологическую ситуацию, проведены две внутривузовские олимпиады. 21-25 марта проведена олимпиада «Шаг в профессию» по направлениям «Анестезиология и реаниматология», «Хирургические болезни», «Врач общей практики», «Педиатрия», «Акушерство и гинекология», «Стоматология», «Фар- мация», в которой приняли участие более 100 студентов лечебного, педиатрического, стоматологическо- го и фармацевтического факультетов университета. Победителями по направлению «Анестезиология и реаниматология» стали: 1 место – Комленок Е.Н. (5 курс, ЛФ), 2 место – Щербинин В.И. (5 курс, ЛФ), 3 место – Молчанская А.Д. (6 курс, ЛФ), по направлению «Хирургические болезни»: 1 место – Жихарев Е.А. (5 курс, ЛФ), 2 место – Игнатович В.А. (4 курс, ЛФ), 3 место – Багрова Л.С. (5 курс, ЛФ); по на- правлению «Врач общей практики»: 1 место – Супоненко З.С. (5 курс, ЛФ), 2 место – Ермолицкий А.В. (5 курс, ЛФ), 3 место – Терентьева Е.А. (5 курс, ЛФ); по направлению «Педиатрия»: 1 место – Волк А. (1 курс, педиатрический факультет), 2 место – Сафончик А. (5 курс, ЛФ), 3 место – Манукян Г.(1 курс, педиатрический факультет); по направлению «Акушерство и гинекология»: 1 место – Толкачёва П.С. (4 курс, ЛФ), 2 место – Сычевич М.В. (4 курс, ЛФ), 3 место – Мозговая А.А. (5 курс, ЛФ); по направлению «Стоматология»: 1 место поделили Дорофеенко Е.В. (4 курс, стом. фак.) и Амелевич А.Д. (4 курс, стом. фак.), 3 место поделили Колодинская Е.Г. (4 курс, стом. фак.) и Пархимович А.А. (4 курс, стом. фак.); по направлению «Фармация»: 1 место – Гатило В.А. (4 курс, фарм. фак.), 2 место – Заруба Д.А. (4 курс, фарм. фак.), 3 место поделили Мхитарян Д.К. (4 курс, фарм. фак.) и Климова Ю.С. (3 курс, фарм. фак.) Центром профессионального мастерства ВГМУ в текущем учебном году, несмотря на сложную эпидемиологическую ситуацию, проведены две внутривузовские олимпиады. 21-25 марта проведена олимпиада «Шаг в профессию» по направлениям «Анестезиология и реаниматология», «Хирургические болезни», «Врач общей практики», «Педиатрия», «Акушерство и гинекология», «Стоматология», «Фар- мация», в которой приняли участие более 100 студентов лечебного, педиатрического, стоматологическо- го и фармацевтического факультетов университета. Победителями по направлению «Анестезиология и реаниматология» стали: 1 место – Комленок Е.Н. (5 курс, ЛФ), 2 место – Щербинин В.И. (5 курс, ЛФ), 3 место – Молчанская А.Д. (6 курс, ЛФ), по направлению «Хирургические болезни»: 1 место – Жихарев Е.А. (5 курс, ЛФ), 2 место – Игнатович В.А. (4 курс, ЛФ), 3 место – Багрова Л.С. ПОБЕДА В КОНКУРСЕ «MEDICALSTARTUP» ПОБЕДА В КОНКУРСЕ «MEDICALSTARTUP» 18 марта 2022 г. в Гомеле прошел заключи- тельный этап конкурса стартап-проектов в сфе- ре здравоохранения «MedicalStartup». Органи- заторами конкурса выступили Республиканский молодежный совет при Министерстве здравоох- ранения Республики Беларусь и учреждение об- разования «Гомельский государственный меди- цинский университет». Конкурс проводился в три этапа: первый этап – прием заявок для участия в конкурсе, вто- рой этап – заочный отбор финалистов экспертны- ми комиссиями, 3 этап – финал. В финал прошло 19 стартап-проектов, в числе которых были работы молодых ученых Витебского государственного медицинского университета: старшего преподавателя кафедры психиатрии и наркологии с курсом ФПК и ПК Уселенка Глеба Олеговича и ассистента кафе- дры клинической иммунологии и аллергологии с курсом ФПК и ПК Юпатовой Татьяны Геннадьевны. Финал конкурса заключался в очном представлении стартап-проектов участниками. В результате были определены 3 лауреата конкурса и победитель – Усиленок Г.О. с проектом «Разработка, валидация и оценка программного комплекса нейропсихологической диагностики». Лучшие проекты конкурса «MedicalStartup» будут представлены на выставке «Здравоохранение Беларуси». Все участники заклю- чительного этапа получили дипломы финалиста конкурса. Но самое главное – опыт, общение с едино- мышленниками и построение новых планов. 18 марта 2022 г. в Гомеле прошел заключи- тельный этап конкурса стартап-проектов в сфе- ре здравоохранения «MedicalStartup». Органи- заторами конкурса выступили Республиканский молодежный совет при Министерстве здравоох- ранения Республики Беларусь и учреждение об- разования «Гомельский государственный меди- цинский университет». Конкурс проводился в три этапа: первый этап – прием заявок для участия в конкурсе, вто- рой этап – заочный отбор финалистов экспертны- этап прием заявок для участия в конкурсе, вто рой этап – заочный отбор финалистов экспертны- ми комиссиями, 3 этап – финал. В финал прошло 19 стартап-проектов, в числе которых были работы молодых ученых Витебского государственного медицинского университета: старшего преподавателя кафедры психиатрии и наркологии с курсом ФПК и ПК Уселенка Глеба Олеговича и ассистента кафе- дры клинической иммунологии и аллергологии с курсом ФПК и ПК Юпатовой Татьяны Геннадьевны. Финал конкурса заключался в очном представлении стартап-проектов участниками. В результате были определены 3 лауреата конкурса и победитель – Усиленок Г.О. с проектом «Разработка, валидация и оценка программного комплекса нейропсихологической диагностики». Лучшие проекты конкурса «MedicalStartup» будут представлены на выставке «Здравоохранение Беларуси». Все участники заклю- чительного этапа получили дипломы финалиста конкурса. Но самое главное – опыт, общение с едино- мышленниками и построение новых планов. ОЛИМПИАДНОЕ ДВИЖЕНИЕ ВГМУ (5 курс, ЛФ); по на- правлению «Врач общей практики»: 1 место – Супоненко З.С. (5 курс, ЛФ), 2 место – Ермолицкий А.В. (5 курс, ЛФ), 3 место – Терентьева Е.А. (5 курс, ЛФ); по направлению «Педиатрия»: 1 место – Волк А. (1 курс, педиатрический факультет), 2 место – Сафончик А. (5 курс, ЛФ), 3 место – Манукян Г.(1 курс, педиатрический факультет); по направлению «Акушерство и гинекология»: 1 место – Толкачёва П.С. (4 курс, ЛФ), 2 место – Сычевич М.В. (4 курс, ЛФ), 3 место – Мозговая А.А. (5 курс, ЛФ); по направлению «Стоматология»: 1 место поделили Дорофеенко Е.В. (4 курс, стом. фак.) и Амелевич А.Д. (4 курс, стом. фак.), 3 место поделили Колодинская Е.Г. (4 курс, стом. фак.) и Пархимович А.А. (4 курс, стом. фак.); по направлению «Фармация»: 1 место – Гатило В.А. (4 курс, фарм. фак.), 2 место – Заруба Д.А. (4 курс, фарм. фак.), 3 место поделили Мхитарян Д.К. (4 курс, фарм. фак.) и Климова Ю.С. (3 курс, фарм. фак.) Во внутривузовской олимпиаде «Первая помощь», состоявшейся в апреле этого года, активно принимали участие студенты стоматологического факультета и факультета подготовки иностранных граждан. Олимпиада проводилась в учебном Центре практической подготовки и симуляционного об- учения с использованием технологии ОСКЭ, использовалась командная форма соревнований. Первое место заняла команда «Стомики» 4-го курса третьей группы стоматологического факультета: Суднико- ва Е.П., Матюшко В.А., Пролеско К.А., Волонге А.Р. (капитан команды), Плоская А.В., Подъелец К.С. р ( ) Поздравляем победителей! Желаем успехов и дальнейшего профессионального 105 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 ВИТЕБСК – МОЛОДЁЖНАЯ СТОЛИЦА РЕСПУБЛИКИ БЕЛАРУСЬ 12 марта 2022 года прошло торжественное открытие Республиканского проекта «Витебск – мо- лодёжная столица Республики Беларусь». В концертной программе, посвящённой этому знаковому для нашего города событию, приняли участие коллективы художественной самодеятельности учреждений высшего и профессионально-технического образования Витебска, а также Минска и Гродно. Наш университет на сцене концертного зала «Витебск» достойно представила самая большая твор- ческая команда: студия эстрадной песни «Тандем», ансамбль спортивного бального танца «Квикстеп», ансамбль народного танца «Миллениум», «INFINITY DANCE GROUP», хореографический коллектив «Existence», вокальный ансамбль «Панацея», хоровая капелла ВГМУ, студенты факультета подготовки иностранных граждан – всего более 130 человек. Ещё в процессе подготовки программы организатора- ми и режиссёрско-постановочной группой Национального центра художественного творчества детей и молодёжи был отмечен высокий профессиональный и организационный уровень наших творческих кол- лективов, в результате чего они заняли в программе значительное место. Профессионализм творческих коллективов ВГМУ был отмечен и присутствовавшими в зале, в том числе и почётными гостями празд- ника, среди которых были Министр образования Республики Беларусь А.И.Иванец, руководители города и области, ректоры вузов Витебской области и проректоры всех вузов страны. 106 106 ВЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ЭКСПЕРТНО-МЕДИЙНЫЙ ФОРУМ «СОЮЗНОЕ ГОСУДАРСТВО: ЭКОНОМИЧЕСКАЯ ИНТЕГРАЦИЯ – ЗАДАЧИ РАЗВИТИЯ 31 марта 2022 года в г.Минске во Дворце Республики состоялся экспертно-медийный форум «Со- юзное государство: экономическая интеграция – задачи развития». В форуме принимали участие пред- ставители Витебского государственного медицинского университета Земко Виктория Юрьевна, доцент кафедры анестезиологии и реаниматологии с курсом ФПК и ПК, член Республиканского молодёжного совета при Министерстве здравоохранения Республики Беларусь, и Будницкий Максим, студент 2 кур- са фармацевтического факультета, член Общественного республиканского студенческого совета при Министерстве образования Республики Беларусь. Министр образования Республики Беларусь Андрей Иванович Иванец встретился с молодыми учёными и членами Общественного республиканского студенческого совета. Рассматривались вопро- сы интеграции образования в пределах Союзного государства, также обсудили планы на дальнейшее сотрудничество и перспективы развития. Также в программе форума состоялось очередное очное заседание Республиканского молодеж- ного совета при Министерстве здравоохранения Республики Беларусь. С приветственным словом вы- ступила первый заместитель министра Елена Николаевна Кроткова. В ходе заседания прошла диало- говая площадка членов совета с Еленой Кротковой, во время которой активно обсуждались вопросы здравоохранения и дальнейший план работы Республиканского молодежного совета. Члены совета вы- ступили с докладами о том, какие мероприятия проводятся с целью формирования активной граждан- ской позиции врача, внесли ряд предложений по расширению и углублению понимания этой важной тематики среди молодежи. ОБЛАСТНАЯ НАУЧНО-ПРАКТИЧЕСКАЯ КОНФЕРЕНЦИЯ «АКТУАЛЬНЫЕ ВОПРОСЫ АНЕСТЕЗИОЛОГИИ И РЕАНИМАТОЛОГИИ» 1 апреля 2022 года на базе учреждения здравоохранения «Витебская областная клиническая боль- ница» состоялась областная научно-практическая конференция «Актуальные вопросы анестезиологии и реаниматологии», приуроченная к празднованию 50-летнего юбилея отделения анестезиологии и ре- анимации Витебской областной клинической больницы. В конференции приняли участие заместитель начальника Главного управления по здравоохране- нию Витоблисполкома Л.Ф. Ковалева, ректор ВГМУ А.Т. Щастный, проректор по учебной и лечебной работе А.Н. Щупакова, главный врач учреждения здравоохранения «Витебская областная клиническая больница» Е.А. Матусевич и другие почетные гости. Открыл пленарное заседание главный внештатный анестезиолог-реаниматолог главного управ- ления по здравоохранению Витоблисполкома А.В. Гончаров. Он рассказал об основных этапах ста- новления службы анестезиологии и реаниматологии Витебской области, обобщил опыт и достижения пятидесятилетней работы, обозначил задачи и приоритеты развития отделения и областной службы в целом. Заместитель начальника главного управления по здравоохранению Витоблисполкома Л.Ф. Кова- лева высоко оценила работу специалистов службы в период пандемии. Ректор ВГМУ А.Т. Щастный, отмечая высокий профессионализм специалистов службы, обратил внимание на авангардные тенденции в продвижении реанимационной и анестезиологической помощи в регионе. Руководитель учреждения здравоохранения «Витебская областная клиническая больница» Е.А. Матусевич пожелал коллективу здоровья и успешной реализации задуманных проектов. Пленарное заседание завершилось торжественным награждением Почетными грамотами луч- ших работников, среди которых были и сотрудники университета. Пленарное заседание завершилось торжественным награждением Почетными грамотами луч- ших работников, среди которых были и сотрудники университета. 107 VESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 Структура рукописи Структура рукописи Рукопись статьи должна включать следующие части: 1. Титульный раздел 2. Структурированное резюме и ключевые слова на русском и английском языках 3. Введение 2. Структурированное резюме и ключевые слова на русском и английском языках 3. Введение 3. Введение 3. Введение 4. Материал и методы 5. Результаты 6. Обсуждение 7. Заключение 8. Литература 9. Рисунки и таблицы 1. Титульный раздел должен содержать: 1. Титульный раздел должен содержать: ПРАВИЛА ДЛЯ АВТОРОВ Журнал «Вестник ВГМУ» публикует статьи на русском и английском языках по следующим от- раслям науки: – медицинским; – биологическим (медико-биологические аспекты); – фармацевтическим; – психологии и педагогике. Вне очереди публикуются научные статьи аспирантов последнего года обучения (включая статьи, подготовленные ими в соавторстве), при условии их полного соответствия требованиям, предъявляе- мым к научным публикациям издания. Статья должна быть тщательно отредактирована и выверена. Рукопись должна быть визирована всеми авторами. Это означает, что за правильность приведенных данных ответственность несут ав- торы. В исключительных случаях, для оценки достоверности результатов, редакция может запросить копии документов, подтверждающих представляемые материалы. Объем полноразмерной оригинальной статьи должен составлять не менее 14 000 печатных зна- ков, включая пробелы между словами, знаки препинания, цифры и другие. При подготовке текста статьи на компьютере необходимо использовать программу Microsoft Word. Размеры полей: сверху – 2 см; снизу – 2 см; слева – 2 см; справа – 2 см. Рукопись печатается че- рез двойной интервал с выделенными жирным заголовками и подзаголовками. Все страницы, начиная с титульной, должны быть последовательно пронумерованы. В статье следует применять только общепринятые символы и сокращения. При необходимости их использования аббревиатуру в тексте необходимо расшифровывать при первом упоминании (это от- носится также и к резюме). Сокращения в названии можно использовать только в тех случаях, когда это абсолютно необходимо. Все величины выражаются в единицах Международной Системы (СИ). При- меняются только международные непатентованные названия лекарственных средств. 1. Титульный раздел должен содержать: ие статьи – должно быть максимально кратким, информативным и точно определять со- тьи. Фамилию и инициалы автора (авторов) – при написании авторов статьи фамилию следует указы- вать до инициалов имени и отчества; Официальное название учреждений, в которых выполнялась работа. Сведения об авторах – указываются полностью фамилии, имена, отчества авторов, ученые сте- пени и звания, должности, место работы (название учреждения, кафедры, отдела), ORCID (если есть). Все лица, обозначенные как авторы, должны соответствовать критериям этого понятия (см. рекомен- дации ICJME). Адрес для корреспонденции – приводятся рабочий почтовый адрес места работы или домашний адрес, телефоны, электронный адрес того автора, с кем следует вести редакционную переписку. Адрес для корреспонденции публикуется вместе со статьей. 108 ЕСТНИК ВИТЕБСКОГО ГОСУДАРСТВЕННОГО МЕДИЦИНСКОГО УНИВЕРСИТЕТА, 2022, ТОМ 21, № Благодарности – авторы могут выразить благодарности людям или организациям, способство- вавшим публикации рукописи в журнале, но не являющимся её авторами (научное руководство или консультация, критический анализ исследования, сбор данных, финансирование, техническое и линг- вистическое редактирование, предоставление пациентов для участия в исследовании и их лечение, предоставленные данные, в том числе рисунки и пр.). Хорошим тоном считается выражение благодар- ности анонимным рецензентам. Информацию об источнике поддержки в виде грантов, оборудования, лекарственных препаратов: указывается источник финансирования как научной работы, так и процесса публикации статьи (фонд, коммерческая или государственная организация, частное лицо и др.). Наличие / отсутствие конфликта интересов. Наиболее частая причина возникновения конфликта интересов – финансовые отношения. Возможны и другие причины: личные отношения, научное со- перничество. Количество рисунков и таблиц. Если количество рисунков и таблиц не указано на титульной стра- нице, редакции и рецензентам бывает трудно определить, все ли рисунки и таблицы, которые должны сопровождать рукопись, были в неё включены. 2. Структурированное резюме оригинальной научной статьи должно точно отражать содержа- ние статьи и быть пригодным для опубликования отдельно от нее, содержать ключевые слова, позволя- ющие индексировать данную статью. 2. Структурированное резюме оригинальной научной статьи должно точно отражать содержа- ние статьи и быть пригодным для опубликования отдельно от нее, содержать ключевые слова, позволя- ющие индексировать данную статью. Резюме должно включать разделы «Цель», «Материал и методы», «Результаты», «Заключение», «Ключевые слова» (не менее 6) и «Источники финансирования» и быть представленным на двух язы- ках: русском и английском. Объем резюме должен составлять около 200-250 слов. Резюме других видов статей (краткие сообщения, обзоры, случаи из практики) не структуриру- ются, объем их должен составлять не менее 100-150 слов. Резюме других видов статей (краткие сообщения, обзоры, случаи из практики) не структуриру- ются, объем их должен составлять не менее 100-150 слов. Подписано в печать 21.04.2022 г. Формат 1/8. Бумага офсетная. Гарнитура «Таймс». Усл.печ.л. 12.,79. Тираж 200 экз. Заказ Издатель и полиграфическое исполнение УО «Витебский государственный ордена Дружбы народов медицинский университет». Лицензия ЛП № 02330/453 от 30.12.2013. Адрес: пр-т Фрунзе, 27, г. Витебск, Республика Беларусь, 210009. При перепечатке материалов ссылка на «Вестник ВГМУ» обязательна. 1. Титульный раздел должен содержать: В резюме на английском языке обязательно указываются фамилии и инициалы авторов на ан- глийском языке. Резюме статей, ключевые слова на русском и английском языках, информация об ав- торах, а также пристатейные библиографические списки размещаются на сайте журнала и отсылаются редакцией в электронные информационные базы для индексации. 3. В разделе «Введение» статьи описывается состояние изучаемой проблемы и её актуальность. Указывается цель исследования либо гипотеза, проверяемая исследованием или наблюдением и, если необходимо, указана ее связь с важными научными и практическими направлениями. Анализ источ- ников, использованных при подготовке научной статьи, должен свидетельствовать о знании автором (авторами) статьи научных достижений в соответствующей области. Обязательными являются ссылки на работы других авторов. При этом должны присутствовать ссылки на публикации последних лет, включая зарубежные публикации в данной области. 4. Раздел «Материал и методы» должен содержать детальную характеристику объектов иссле- дований, описание использованных методов, оборудования, диагностических и лечебных технологий. На методики исследований должны быть представлены ссылки. 4. Раздел «Материал и методы» должен содержать детальную характеристику объектов иссле- дований, описание использованных методов, оборудования, диагностических и лечебных технологий. На методики исследований должны быть представлены ссылки. При описании экспериментов, проводившихся на людях, авторы должны указать, соответство- вала ли процедура этическим стандартам локального и национального комитета, отвечающего за экс- перименты на людях, а также требованиям Хельсинкской Декларации Всемирной медицинской ас- социации. При описании экспериментов на животных авторы должны указать, действовали ли они в соответствии с локальными и национальными требованиями к использованию и обращению с лабора- торными животными. 5. Раздел «Результаты» должен подробно освещать содержание исследований и их результаты, которые следует отражать, максимально используя рисунки и таблицы. Важно, чтобы проиллюстри- рованная информация не дублировала уже приведенную в тексте. При необходимости раздел может делиться на подразделы (с разъяснительными заголовками). 5. Раздел «Результаты» должен подробно освещать содержание исследований и их результаты, которые следует отражать, максимально используя рисунки и таблицы. Важно, чтобы проиллюстри- рованная информация не дублировала уже приведенную в тексте. При необходимости раздел может делиться на подразделы (с разъяснительными заголовками). Представленные в статье результаты желательно сопоставить с предыдущими работами в этой области как автора, так и других исследователей. Такое сравнение дополнительно раскроет новизну проведенной работы, придаст ей объективности. Формулы, уравнения и сноски, встречающиеся в статье, должны быть пронумерованы в соответ- ствии с порядком цитирования в тексте. 109 ESTNIK VITEBSKOGO GOSUDARSTVENNOGO MEDITSINSKOGO UNIVERSITETA, 2022, VOL. 21, N2 6. В разделе «Обсуждение» полученные результаты должны быть обсуждены с точки зрения их научной новизны и сопоставлены с соответствующими известными данными. 6. 110 Подписано в печать 21.04.2022 г. Формат 1/8. Бумага офсетная. Гарнитура «Таймс». Усл.печ.л. 12.,79. Тираж 200 экз. Заказ Издатель и полиграфическое исполнение УО «Витебский государственный ордена Дружбы народов медицинский университет». Лицензия ЛП № 02330/453 от 30.12.2013. Адрес: пр-т Фрунзе, 27, г. Витебск, Республика Беларусь, 210009. При перепечатке материалов ссылка на «Вестник ВГМУ» обязательна. р р ру у ру При перепечатке материалов ссылка на «Вестник ВГМУ» обязательна. у р Лицензия ЛП № 02330/453 от 30.12.2013. Адрес: пр-т Фрунзе, 27, г. Витебск, Республика Беларусь, 210009. 1. Титульный раздел должен содержать: В разделе «Обсуждение» полученные результаты должны быть обсуждены с точки зрения их научной новизны и сопоставлены с соответствующими известными данными. 6. В разделе «Обсуждение» полученные результаты должны быть обсуждены с точки зрения их научной новизны и сопоставлены с соответствующими известными данными. 7. Заключение. Должны быть четко сформулированы выводы и в сжатом виде отразить основ- ные полученные результаты с указанием их новизны, преимуществ и возможностей применения. Вы- воды необходимо сопоставить с целями исследования. 7. Заключение. Должны быть четко сформулированы выводы и в сжатом виде отразить основ- ные полученные результаты с указанием их новизны, преимуществ и возможностей применения. Вы- воды необходимо сопоставить с целями исследования. 8. Литература оформляется в соответствии с ГОСТом – 7.1-2003. Ссылки нумеруются согласно порядку цитирования в тексте. Порядковые номера ссылок должны быть написаны внутри квадратных скобок, например: [1, 2]. В оригинальных статьях желательно цитировать не более 15 источников, в обзорах литературы – не более 50. Желательно цитировать источники, опубликованные в течение последних 5-7 лет. В ста- тье не допускаются ссылки на авторефераты диссертационных работ или сами диссертации, т.к. они являются рукописями. Ссылки на тезисы и статьи в малотиражных региональных сборниках можно использовать только при крайней необходимости. Авторы несут полную ответственность за точность и полноту всех ссылок, и точность цитирова- ния первоисточников. Авторы несут полную ответственность за точность и полноту всех ссылок, и точность цитирова- ния первоисточников. Редакция с целью максимального снижения неполноты или неточности информации в приводи- мых пристатейных списках литературы проводит в обязательном порядке проверку всех ссылок и сама оформляет References (литературу на английском языке) в формате Vancouver. Редакция с целью максимального снижения неполноты или неточности информации в приводи- мых пристатейных списках литературы проводит в обязательном порядке проверку всех ссылок и сама оформляет References (литературу на английском языке) в формате Vancouver. 9. Таблицы, иллюстрации и рисунки должны быть набраны в отдельном файле, через один интервал, иметь название и подстрочные примечания (если необходимо). Убедитесь, что каждая табли- ца и рисунок процитированы в тексте. В названиях таблиц и рисунков не должно быть сокращений. Непосредственно в таблицах (в заголовках строк или столбцов) или в их названии указывается, какие статистические показатели приводятся. Формат рисунка может быть TIFF, JPEG, CDR; разрешение не менее 300 dpi. Диаграммы, вы- полненные в приложении MS Excel, необходимо представлять в формате .xls, что позволит провести их допечатную подготовку. Диаграммы печатаются при помощи монохромной печати, поэтому при их оформлении предпочтительно использовать узорную заливку объектов и различный характер линий. 110
https://openalex.org/W3199955237	https://www.frontiersin.org/articles/10.3389/fmed.2021.729300/pdf	English	null	Stimuli-Responsive Nanoplatform-Assisted Photodynamic Therapy Against Bacterial Infections	Frontiers in medicine	2,021	cc-by	6,527	You Zhou 1, Wenmin Deng 2, Mulan Mo 1, Dexu Luo 1, Houhe Liu 1, Yuan Jiang 1,3, Wenjie Chen 1,4,5* and Chuanshan Xu 1* You Zhou 1, Wenmin Deng 2, Mulan Mo 1, Dexu Luo 1, Houhe Liu 1, Yuan Jiang 1,3, Wenjie Chen 1,4,5* and Chuanshan Xu 1* 1 Key Laboratory of Molecular Target & Clinical Pharmacology and the State & National Medical Products Administration Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, The Fifth Afﬁliated Hospital, Guangzhou Medical University, Guangzhou, China, 2 Department of Clinical Pharmacy, The People’s Hospital of Dianbai District, Maoming, China, 3 Department of Rehabilitation Medicine, The First Afﬁliated Hospital of Chengdu Medical College, Chengdu, China, 4 State Key Laboratory of Respiratory Disease, Guangdong-Hongkong-Macao Joint Laboratory of Respiratory Infectious Disease, Guangzhou, China, 5 Sydney Vital Translational Cancer Research Centre, Sydney, NSW, Australia MINI REVIEW published: 13 September 2021 doi: 10.3389/fmed.2021.729300 MINI REVIEW Bacterial infections are common diseases causing tremendous deaths in clinical settings. It has been a big challenge to human beings because of the antibiotics abuse and the newly emerging microbes. Photodynamic therapy (PDT) is a reactive oxygen species- based therapeutic technique through light-activated photosensitizer (PS). Recent studies have highlighted the potential of PDT as an alternative method of antibacterial treatment for its broad applicability and high efﬁciency. However, there are some shortcomings due to the low selectivity and speciﬁcity of PS. Growing evidence has shown that drug delivery nanoplatforms have unique advantages in enhancing therapeutic efﬁcacy of drugs. Particularly, stimuli-responsive nanoplatforms, as a promising delivery system, provide great opportunities for the effective delivery of PS. In the present mini-review, we brieﬂy introduced the unique microenvironment in bacterial infection tissues and the application of PDT on bacterial infections. Then we review the stimuli-responsive nanoplatforms (including pH-, enzymes-, redox-, magnetic-, and electric-) used in PDT against bacterial infections. Lastly, some perspectives have also been proposed to further promote the future developments of antibacterial PDT. Edited by: Yolanda Gilaberte, Hospital Universitario Miguel Servet, Spain Reviewed by: Tim Maisch, University of Regensburg, Germany Francisco Galindo, University of Jaume I, Spain Reviewed by: Tim Maisch, University of Regensburg, Germany Francisco Galindo, University of Jaume I, Spain Correspondence: Chuanshan Xu xcshan@163.com Wenjie Chen wenjie.chen1@hdr.mq.edu.au Correspondence: Chuanshan Xu xcshan@163.com Wenjie Chen wenjie.chen1@hdr.mq.edu.au Specialty section: This article was submitted to Infectious Diseases - Surveillance, Prevention and Treatment, a section of the journal Frontiers in Medicine Keywords: bacterial infections, photodynamic therapy, stimuli-responsive, nanoplatforms, drug delivery systems INTRODUCTION Received: 22 June 2021 Accepted: 13 August 2021 Published: 13 September 2021 According to epidemiological reports, infections are a dominant contributor to the global disease burden. The mortality rates of bacterial infections are very high especially in developing countries, where medical resources such as vaccines and anti-infection therapeutics are less accessible (1). The use of antibiotics is a great milestone in ﬁghting against bacterial infections. There are more than 38 antibiotics in clinical setting and 45 antibiotics have been undergoing clinical trials up to 2019. These antibiotics are developed to kill or inhibit the bacteria contagion through diﬀerent mechanisms, including damage to the cell walls of bacteria, increase of the cell membrane permeability, According to epidemiological reports, infections are a dominant contributor to the global disease burden. The mortality rates of bacterial infections are very high especially in developing countries, where medical resources such as vaccines and anti-infection therapeutics are less accessible (1). The use of antibiotics is a great milestone in ﬁghting against bacterial infections. There are more than 38 antibiotics in clinical setting and 45 antibiotics have been undergoing clinical trials up to 2019. These antibiotics are developed to kill or inhibit the bacteria contagion through diﬀerent mechanisms, including damage to the cell walls of bacteria, increase of the cell membrane permeability, Citation: Zhou Y, Deng W, Mo M, Luo D, Liu H, Jiang Y, Chen W and Xu C (2021) Stimuli-Responsive Nanoplatform-Assisted Photodynamic Therapy Against Bacterial Infections. Front. Med. 8:729300. doi: 10.3389/fmed.2021.729300 Zhou Y, Deng W, Mo M, Luo D, Liu H, Jiang Y, Chen W and Xu C (2021) Stimuli-Responsive Nanoplatform-Assisted Photodynamic Therapy Against Bacterial Infections. Front. Med. 8:729300. doi: 10.3389/fmed.2021.729300 September 2021 \| Volume 8 \| Article 729300 Frontiers in Medicine \| www.frontiersin.org Stimuli-Responsive Nanoplatform in Antibacterial PDT Zhou et al. and inhibition of the nucleic acid or protein synthesis (2). Due to antibiotics abuse, more and more bacteria have evolved the antibiotic resistance, which leads to the ineﬀectiveness of antibacterial therapy (3). Increasing antibiotic resistance among pathogenic bacteria is one of the most challenging issues in the present medical ﬁeld; It is estimated that around 0.7 million people die every year, and it is predicted that maybe 10 million people every year will die from drug-resistant bacterial infections by 2050 (4). Thus, it is highly imperative to understand the underlying mechanisms of drug resistant infections and overcome them. In general, resistance to antibiotics occurs mainly through drug inactivation, composition and permeability modiﬁcation, drug eﬄux, and acquired genetic resistance (5). There are increasing evidences revealing that many infections are caused by polybacteria, either in terms of origins or in manifestation. This may lead to limiting the therapeutic eﬃcacy of a single antibiotic (6). To overcome the shortcomings of antibiotics, photodynamic therapy (PDT) has been developed as a promising alternative to treat bacterial infections with broad- spectrum and multitarget features (7, 8). (P. aeruginosa) infection (23). Among the interaction between bacteria and hosts, a complicated inﬂammatory response is activated to combat infections (24–26). Interestingly, the excessive production of reactive oxygen species (ROS) by activated immune cells plays an essential role in the host immune defenses against pathogens, indicating a participation of oxidative stress in the pathology of bacterial infections (27). Application of PDT on Bacterial Infections Photodynamic therapy is a clinically approved technique and mainly applied in treating cancerous and non-cancer diseases on the basis of ROS that generates from light-activated PS. Recently, PDT has also been employed to eliminate pathogens for treating the bacterial infections because it is less aﬀected by the known antibiotic-resistance pathway (28). The successful applications of PDT are dependent on three essential factors, including light sources, PSs, and oxygen. Citation: When a PS is irradiated by light with a speciﬁc wavelength, it can be excited from the ground state to a triplet state. Then the excited PS can transfer electrons to molecular oxygen to generate superoxide anion and hydroxyl radicals, and hydrogen peroxide subsequently (Type I reaction). In another pathway, the excited PS transmits energy to ground triplet state oxygen to generate excited singlet oxygen and ﬁnally stimulates the bursting production of ROS (Type II reaction) (29). ROS in PDT induce a lethal oxidative damage to biological macromolecules like membrane lipids and nucleic acids (30). Beyond the direct killing of pathogenic bacteria through structural disruption, studies found that PDT treatment can induce inactivation of physiologic function-relative protein in bacteria (31, 32). Furthermore, bacterial virulence factors can also be inactivated under PDT treatment (33). Limited by a short lifespan and diﬀusion distance of ROS, the cellular targets of ROS are mainly dependent on the cellular localization of the PS (34, 35). Innate immunity in host may also aﬀect the therapy eﬀect since the attraction and accumulation of neutrophils into the infection regions were required for PDT-mediated bacteria killing and infection clearance (36). The multitargets of ROS oxidation to biomacromolecules make bacteria hard to develop a resistance to PDT, but studies indicate that the eﬃciency of PDT could be aﬀected by bacteria strains, genetic background, and its surrounding microenvironment (37–39). Mechanism underlying such a phenomenon is quite complex. Nevertheless, the correlations between the responses of diﬀerent strains to PDT and the antioxidative systems, cell membrane contents, bioﬁlm production ability, quorum sensing signaling systems have been previously reviewed (40–43). Photodynamic therapy eﬃcacies rely on eﬃcient delivery of photosensitizers (PSs). Stimuli-responsive nanoplatforms are a smart and promising delivery system that respond to endogenous stimuli (changes in pH, enzyme concentration, and redox gradients) or exogenous stimuli (magnetic ﬁeld, ultrasound intensity, light, temperature, and electric pulses). These on- demand properties render the control of drug release in spatial– temporal and dosage-dependent manner (9). In this mini- review, we brieﬂy introduced the unique microenvironment in bacterial infection tissues and the application of PDT on bacterial infections. Then we focus on the progress of stimuli-responsive nanoplatform-assisted antibacterial PDT. The potential opportunities and challenges will be also outlook to boost the developments of antibacterial PDT. Frontiers in Medicine \| www.frontiersin.org Stimuli-Responsive Nanoplatform-Assisted PDT on Bacterial Infections bioﬁlm infection (49). Aggregation-induced emission (AIE) PSs exhibit potential application prospect in the PDT because of their aggregation-enhanced ROS production (50). Bibo et al. fabricated an AIE PS loaded in the zwitterionic polyurethane nanomicelles. The AIE PS aggregated around bacteria when zwitterionic moiety acquired positive charge by acid protonation, thus the nanomicelle achieved a superior antibacterial activity (51). In addition to the delivery of PS, low pH also oﬀers possibilities for overcoming the limitations of low oxygen level. Since manganese dioxide (MnO2) can produce oxygen by its catalytic activity at low pH and high H2O2 in the infected sites, Deng et al. synthesized a multicomponent nanoparticle by coencapsulating ultrasmall-sized Hf (IV)-porphyrin Metal- Organic Framework (MOF) and MnO2 in human serum albumin. In this nanoplatform, the production rate of ROS was much higher than porphyrin-based MOF treatment alone by in situ O2 generation. With the alleviated hypoxia, it realizes great therapeutic outcomes in S aureus–infected models (52). Meanwhile, the magnetic resonance signal of Mn2+ provides the detection of bacteria, which is favorable to build up the theranostic platform (53, 54). The PS delivery is developed to improve eﬃcacy of the conventional antibacterial PDT approaches. Owing to the advantages in both pharmacokinetics and pharmacodynamics, stimuli-responsive nanoplatforms have been applied to overcome the issues of poor delivery performance (45). In the following section, we will review stimuli-responsive Nanoplatform- Assisted PDT in ﬁghting against bacterial infections. These speciﬁc stimuli include pH, enzymes, redox gradients, magnetic and electric ﬁeld, and will be presented systematically below. pH-Responsive Nanoplatform-Assisted PDT in Bacterial Infections Chemical reactions, protonation, or degradation of administrated compounds can occur under acidic circumstances. Therefore, the pH-responsive strategy has been applied in antibacterial PDT. Polyacrylic acid (PAA) is often used in the delivery of PS for its pH responsive property. Hao et al. synthesized zeolitic imidazolate framework-8 (ZIF-8) for the local delivery of ammonium methylbenzene blue. Then PAA is incorporated for pH responsiveness and higher drug loading capacity. This nanoplatform had long blood circulation in physiological environment and pH responsive drug release in bacterial infection site. The in vitro and in vivo experiments showed better therapeutic eﬃcacy than PS treatment alone (46). Similar to the above mentioned platform, Perni et al. constructed a silica–toluidine blue O (TBO) nanoconjugate by the formation of amide bonds between silica nanoparticles and TBO. The controlled delivery of TBO released from the conjugates because of the amide bond cleavage in bacterial infection tissues. In this research, TBO shows an enhanced photosensitive activity in eliminating methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis (S. epidermidis), and Escherichia coli (E. coli) (47). Protonation in low pH environment often results in a charge change, which is conducive to the accumulation of PS in bacteria surface with negative charge. Thus, Wang et al. used chlorin e6 (Ce6)-linked supramolecule to develop a self-assembled micelle to treat bacterial infections. The negatively-charged micelles could change to positive charge in bacterial infection sites, subsequently adhering to bacteria membranes. This micelle signiﬁcantly enhanced the inhibition eﬀect of Ce6-based PDT on a variety of bacteria including MRAS, and showed a great anti-infection activity in the subcutaneous infection model (48). The strategy was also implemented in Ce6 loaded SiO2-polymer nanoparticles (named SiO2-PCe6−IL). Ce6 COO- and 1-vinyl imidazole with dodecyl were assembled by anion exchange reaction, and SiO2 nanoparticles were introduced to control the density of Ce6-IL polymers. With the protonation of Ce6 in bacterial infection sites, the charge of SiO2-PCe6−IL was inverted from negative to positive. And the acquired positive charge of polymer led to an interaction of SiO2- PIL+ with negative charge extracellular polymeric substances, which induced a rapid release of Ce6 from nanoparticles and dramatically improved the PDT eﬃcacy against MRSA Frontiers in Medicine \| www.frontiersin.org UNIQUE MICROENVIRONMENT IN BACTERIAL INFECTION TISSUES The unique microenvironment in bacterial infection tissues provides a prerequisite for designing stimuli- responsive nanoplatforms (10). The anaerobic fermentation, acidogenic/acid-tolerant bacteria metabolism, and local accumulation of organic acids such as lactic and acetic acids create an acidic microenvironment (pH 4.5–6.5) in infectious sites (11–14). Additionally, bacteria can secrete several protein virulence factors including lipases, esterases, proteases, hyaluronidases, alpha toxins, and chemotactic factors to protect themselves (15–17). These enzymes are highly expressed in Gram-positive bacteria, and they are likely to help bacteria to obtain energy, promote spread, escape from immune detection, etc. (18–20). Metalloproteinase 9 (MMP9) expressed in local infection acts as a synergistic virulence factor (21, 22). At the same time, surrounding tissues increase the detoxication components synthesis and induce the activation of defense response. For instance, an increase of glutathione (GSH) in the epithelial lining ﬂuid was found in the Pseudomonas aeruginosa The wide spectrum and credible eﬀects have already promoted the clinical trials of PDT in antibacterial treatment (Supplementary Table 1). But there are still some shortcomings attracting an attention, such as the water-insolubility of PS, insuﬃcient uptakes of PS by pathogenic bacteria, the oxygen shortage in infection lesions, and the phototoxicity-induced side-eﬀects (44). New PS and drug delivery systems are under development, in which stimuli-responsive nanoplatforms show an important role in promoting ROS production and enhancing antibacterial PDT eﬃcacy (8). September 2021 \| Volume 8 \| Article 729300 2 Stimuli-Responsive Nanoplatform in Antibacterial PDT Zhou et al. Stimuli-Responsive Nanoplatform-Assisted PDT on Bacterial Infections Enzymes-Responsive Nanoplatform-Assisted PDT in Bacterial Infections The speciﬁc high expression and selective catalytic activity endow enzymes with an excellent trigger for controlled delivery of drugs. Based on the cleavage of ester linkage by lipase, more than one team have reported lipase-responsive nanoplatforms for anti- bacterial PDT (55–57). For example, nanoliposomes were used to deliver PS pheophorbide A. Erythromycin-loaded liposomes were coated with pullulan-pheophorbide A conjugates. Once the nanoplatform reached the infection site, pheophorbide A and erythromycin release were triggered by the lipase- dependent cleavage of ester linkage in lipid as well as the one between pullulan and pheophorbide A. This formulation fulﬁlled the synergistic therapy in skin infection by PS and erythromycin codelivery (55). In another study, pheophorbide A was conjugated with DSPE-PEG to form the nanoliposome, and the photoactivity of pheophorbide A quenched in spherical shape was gradually recovered by the cleavage of ester linkage by P. acnes lipases in infection foci, and the formation of nanoliposome enhanced the skin penetration of pheophorbide A (56). Songhee et al. generated a hypocrellin A-loaded methoxy poly (ethylene glycol)-block-poly(ε-caprolactone) polymer micelle. This lipase-sensitive micelle not only overcomes the aggregation and low water solubility of hypocrellin A in vivo, but also enhances the eﬃciency of PDT in a MRSA-induced acute peritonitis model (57). For developing MMP-responsive nanoplatform, MMP9 sensitive peptide (YGRKKKRRQRRR- GPLGVRG-EEEEEE) was conjugated with Ce6 to construct a polypeptide nanoparticle. Negatively charged surface by EEEEEE peptide shell was removed by overexpressed MMP9 in the keratitis microenvironment. Subsequently, the exposed cationic peptides helped the nanoparticles to penetrate and accumulate in bioﬁlms as well as bind to Gram-negative bacteria, thereby improving the antibacterial PDT eﬃcacy (58). Hyaluronidase secreted by MRSA was also applied as an endogenous stimulus September 2021 \| Volume 8 \| Article 729300 3 Stimuli-Responsive Nanoplatform in Antibacterial PDT Zhou et al. GURE 1 \| Scheme illustration of stimuli-responsive Nanoplatforms-Assisted PDT in ﬁghting against bacterial infections. Internal (A) and external (B) muli-responsive platform assisted PDT are showed. PAA, polyacrylic acid; HA, hyaluronic acid; BSA, bovine serum albumin. FIGURE 1 \| Scheme illustration of stimuli-responsive Nanoplatforms-Assisted PDT in ﬁghting against bacterial infections. Internal (A) and external (B) stimuli-responsive platform assisted PDT are showed. PAA, polyacrylic acid; HA, hyaluronic acid; BSA, bovine serum albumin. linker in conjugates could signiﬁcantly improve the phototoxicity of porphyrin against S. aureus, despite the lack of additional targeting groups (60). Mao et al. reported a metabolic labeling strategy for precise delivery of AIE PS. Enzymes-Responsive Nanoplatform-Assisted PDT in Bacterial Infections They used MIL- 100 (Fe), a MOF composed of iron (III) metal centers and trimesic acid ligand nanoparticles, as the carrier for 3-azido- D-alanine delivery ﬁrstly. MIL-100 (Fe) would be dissociated as a result of coordination breaking between trimesic acid and iron (III). In this way, the speciﬁc release of the encapsulated 3-azido-D-alanine can be achieved. When MIL- 100 (Fe) accumulated and degraded within the infection environment with high levels of H2O2, 3-azido-D-alanine was released and selectively integrated into the cell walls of bacteria. Then the dibenzocyclooctyne-modiﬁed AIE PS nanoparticles can subsequently react with the 3-azido-d-alanine labeled bacteria. Through this modiﬁcation, the implementation of in antibacterial PDT. Yuwen et al. prepared a MoS2@HA- Ce6 nanosheet. MoS2 nanosheets served as a ﬂuorescence quencher, and hyaluronic acid conjugated with Ce6 (HA–Ce6) was assembled on the surface of MoS2 nanosheets. These nanosheets could restore the photodynamic activity of Ce6 after the hyaluronic acid shell degraded by hyaluronidase, and in vivo study shows an excellent MRSA eradication eﬀect (59). Redox-Responsive Nanoplatform-Assisted PDT in Bacterial Infections Ce6, Chlorin e6; TBO, Toluidine blue O; DHTPY, Pyridinium, 1-(2,3-dihydroxypropyl)-4-[2-[4-(diphenylamino)phenyl]ethenyl]; ICG, Indocyanine green; MB, Methylbenzene blue; Pheo A, Pheophorbide A; HA, Hypocrellin A; TPETM, 2-(1-(5-(4-(1,2,2-tris(4-methoxyphenyl)vinyl)phenyl)th-iophen-2-yl)ethylidene)malononitrile; C6, Coumarin 6; MgPc, Mg (II) or Al (III) phthalocyanine; t-PtCP, [5,15-bisphenyl-10,20-bis(4-methoxy-carbonylphenyl)-porphyrin] platinum; TMP, meso-Tetra (N-methyl-4-pyridyl) porphine tetra tosylate. PDT in the infected tissue can signiﬁcantly reduce bacteria growth (61). magnetic properties to deliver PS. They enhanced bacterial killing signiﬁcantly by achieving targeting and combination therapy (65–68). In addition to the magnetic ﬁeld, the electric ﬁeld has also served as a novel switch for controlled delivery in PDT on infection. Steven et al. designed an electric-responsive hydrogel carrying PS to treat wound infections. The hydrogel was composed of polyelectrolyte poly (methyl vinyl ether-co- maleic acid) (PMVE-co-MA), which can regulate the ionic conductivities of the hydrogel by PMVE/MA concentration ratio. The hydrogel acquired a rapid release of excess PS in a PMVE/MA ratio-dependent manner upon electric stimulation. Thus, this electric responsive hydrogel is a potential option in antibacterial PDT at an open wound (69). External Stimuli-Responsive Nanoplatforms-Assisted PDT in Bacterial Infections Diﬀerent from the internal stimuli, external stimuli can control the drug release more precisely, and external stimuli can be controlled accurately either by the local or the intensity to meet the treatment requirements (62, 63). For example, magnetic ﬁeld has been used in the targeted delivery of magnetic materials. Sun et al. developed a Ce6-and C6- loaded Fe3O4-silane core-shell nanoparticle to ﬁght against periodontal bioﬁlms growing in dentin disks. Under the magnetically driven force, this nanoparticle could increase the penetration of the PS into bioﬁlms. The results demonstrated that the magnetic nanoparticle had a strong antibioﬁlm activity with excellent biocompatibility, real-time monitoring, and magnetically-targeting capacities (64). Several other magnetic nanoparticles have constructed on the basis of the iron or Fe3O4 Frontiers in Medicine \| www.frontiersin.org Redox-Responsive Nanoplatform-Assisted PDT in Bacterial Infections The high levels of GSH and/or H2O2 in bacteria-infected lesions provides alternatives for the design of redox-responsive systems. Michael et al. conjugated porphyrin to hyperbranched polyglycerol nanoparticles with disulﬁde linker with GSH- responsive property. Experiments in vitro showed that disulﬁde September 2021 \| Volume 8 \| Article 729300 Frontiers in Medicine \| www.frontiersin.org 4 Stimuli-Responsive Nanoplatform in Antibacterial PDT Zhou et al. TABLE 1 \| Summary of stimuli-responsive Nanoplatforms-Assisted PDT in anti-bacterial. Source of respond Responsive method Bacteria species PS Vehicle Advantages References pH Proton; Amide bond break; PAA; MnO2 catalysis MRSA; S. epidermidis; E. coil; S. aureus Ce6; TBO; DHTPY; ICG; Porphyrin; MB Micelle; Nanoparticle; MOF; ZIF-8; Nanosheet; Enhance bacterial afﬁnity; Control delivery; Enhance biocompatibility; Prolong circulation; Targeting property; High ROS generation; Bioﬁlm penetration; Hypoxia alleviation; Bioimaging functionality; Synergistic effect (46–54) Enzyme Ester linkage; Hyaluronic acid; MMP-9- sensitive peptides P. acnes; MRSA; P. aeruginosa; Pheo A; HA; Ce6; Liposome; Micelle; Nanosheet; Nanoparticle; Prevent PS aggregation; Enhance water solubility; High selectivity and penetration property; Enhance retention time (55–59) Redox MIL-100 (Fe); Disulﬁde bond MRSA TPETM; Porphyrin Nanoparticle Precise detection and therapy; Multivalent targeting (60, 61) Magnetic Fe3O4; Fe S. sanguinis; P. gingivalis; F. nucleatum; Salmonella DT104; MRSA; VRE; E. faecalis; S. aureus; B. cereus; MSSA; E. coli; S. typhimurium Ce6/C6; MB; MgPc; t-PtCP Nanoparticle Good biocompatibility; Real-time monitoring; Magnetically-targeting; Multimodel treatment; Improve ROS generation (64–68) Electric Hydrogel lysis MRSA TMP; MB Hydrogel Rapid release (69) MRSA, Methicillin-resistant Staphylococcus aureus; S. epidermidis, Staphylococcus epidermidis; E. coil, Escherichia coli; S. aureus, Staphylococcus aureus; P. acnes, Propionibacterium acnes; P. aeruginosa, Pseudomonas aeruginosa; S. sanguinis, Streptococcus sanguinis; P. gingivalis, Porphyromonas gingivalis; F. nucleatum, Fusobacterium nucleatum; VRE, (VAN)- resistant Enterococcus; E. faecalis, Enterococcus faecalis; B. cereus, Bacillus cereus; MSSA, Methicillin-sensitive Staphylococcus aureus; S. typhimurium, Salmonella typhimurium. Ce6, Chlorin e6; TBO, Toluidine blue O; DHTPY, Pyridinium, 1-(2,3-dihydroxypropyl)-4-[2-[4-(diphenylamino)phenyl]ethenyl]; ICG, Indocyanine green; MB, Methylbenzene blue; Pheo A, Pheophorbide A; HA, Hypocrellin A; TPETM, 2-(1-(5-(4-(1,2,2-tris(4-methoxyphenyl)vinyl)phenyl)th-iophen-2-yl)ethylidene)malononitrile; C6, Coumarin 6; MgPc, Mg (II) or Al (III) phthalocyanine; t-PtCP, [5,15-bisphenyl-10,20-bis(4-methoxy-carbonylphenyl)-porphyrin] platinum; TMP, meso-Tetra (N-methyl-4-pyridyl) porphine tetra tosylate. (69) MRSA, Methicillin-resistant Staphylococcus aureus; S. epidermidis, Staphylococcus epidermidis; E. coil, Escherichia coli; S. aureus, Staphylococcus aureus; P. acnes, Propionibacterium acnes; P. aeruginosa, Pseudomonas aeruginosa; S. sanguinis, Streptococcus sanguinis; P. gingivalis, Porphyromonas gingivalis; F. nucleatum, Fusobacterium nucleatum; VRE, (VAN)- resistant Enterococcus; E. faecalis, Enterococcus faecalis; B. cereus, Bacillus cereus; MSSA, Methicillin-sensitive Staphylococcus aureus; S. typhimurium, Salmonella typhimurium. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed. 2021.729300/full#supplementary-material ACKNOWLEDGMENTS The authors express their sincere gratitude to Mr. Ruchao Jiang for his generous backup supporting. Summary and Outlook Stimuli-responsive nanoplatforms have unique advantages in spatial and temporal manipulations of drug release and elongation of blood retention, which provide a promising strategy for drug delivery (70). Currently, regarding unique September 2021 \| Volume 8 \| Article 729300 5 Stimuli-Responsive Nanoplatform in Antibacterial PDT Zhou et al. microenvironment of bacterial infections, various responsive strategies have been widely developed on the basis of pH, enzymes, and redox gradients to improve the eﬃciency of antibacterial PDT. In addition, external factors such as magnetic and electric ﬁeld were also applied as trigger sources (Figure 1). Compared with already recent developments, stimuli-responsive platforms can not only improve the solubility of PS, but also confer other advantages (Table 1). On one hand, stimuli- responsive platforms triggered by the unique microenvironment in bacterial-infected tissues confer the selective drug release. On the other hand, these nanoplatforms can provide targeting ability for PS, for example, by stimuli-responsive charge converting and magnetically-driven force. Therefore, stimuli-responsive platforms avoided potential side-eﬀects and enhanced the PDT eradication of bacterial infections. What is more, the codelivery of stimuli-responsive catalytic activity components like manganese dioxide (MnO2) can lead to an in situ oxygen generation in bacterial infection sites for hypoxia alleviation (52). Lastly, stimuli-responsive nanoplatforms with excellent loading performance exhibit broad prospects for synergetic therapies (55). These properties make stimuli- responsive nanoplatforms a great option in assisting PDT for ﬁghting against bacterial infections. Among them, the external stimuli-responsive platforms may be superior to those responsive to internal stimuli since they can be designed to artiﬁcially control the drug release more easily. Besides, a dual-responsive nanoplatform with more smart features may have greater potential to further enhance the antibacterial PDT eﬃcacy. encouraging stimuli-responsive Nanoplatforms-Assisted PDT in bacterial infections are all reported by in vitro and in vivo experimental studies, but no clinical trials are undergoing so far. The curative eﬀect of these nanoplatforms needs to be demonstrated by more extensive clinical data. Furthermore, there are challenges such as uncertainty of the in vivo fate of nanoplatforms, limiting the development of stimuli-responsive nanoplatforms. Therefore, further addressing the above shortcomings should be an important task for translating stimuli-responsive nanoplatform-assisted PDT to clinical antibacterial infections. AUTHOR CONTRIBUTIONS YZ, CX, and WC contributed to the conception and structure of this review. YZ prepared the manuscript. WD did the literature search and wrote the introduction. CX and WC reviewed and revised the manuscript. MM, DL, HL, and YJ provided written comments. All authors contributed to the article and approved the submitted version. FUNDING This work was supported by the grants of High-level University Construction Fund of Guangdong Province (Nos. 06-410- 2106154, 06-410-2106153, and 06-410-2107229) and the fund of Guangdong-Hongkong-Macao Joint Laboratory of Respiratory Infectious Disease for WC. Recent evidences have shown that the stimuli-responsive nanoplatform-assisted PDT is a promising way to combat bacterial infections in vitro and in vivo. However, a stimuli- responsive property often means more tedious preparation and complicated characterization. Additionally, there are heterogenicity among bacterial species and patient population in the application process, these diversities may make the stimuli-responsive elements ineﬀective and ultimately aﬀect the drug-releasing and PDT eﬃcacy. 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Nanoparticles having amphiphilic silane containingChlorin e6 with strong anti-bioﬁlm Frontiers in Medicine \| www.frontiersin.org September 2021 \| Volume 8 \| Article 729300 8

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